Bertrand Lefebvre
email: bertrand.lefebvre@univ-lyon1.fr

Thomas E Guensburg

Emmanuel L O Martin

Rich Mooi

Elise Nardin

Martina Nohejlova

Farid Saleh

Khaoula Kouraïss

Khadija El Hariri

Bruno David

Exceptionally preserved soft parts in fossils from the Lower Ordovician of Morocco clarify stylophoran affinities within basal deuterostomes

Keywords: deuterostomes, echinoderms, Fezouata Lagerstätte, Morocco, phylogeny deuterostomes, echinoderms, Fezouata Lagerstätte, Morocco, phylogeny

Exceptionally preserved

Introduction

Phylogenetic relationships among the main clades of deuterostomes [START_REF] Swalla | Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives[END_REF][START_REF] Erwin | The Cambrian conundrum: early divergence and later ecological success in the early history of animals[END_REF][START_REF] David | How Hox genes can shed light on the place of echinoderms among the deuterostomes[END_REF] and in particular, the earliest chordates (including the vertebrates) [START_REF] Jefferies | The Ancestry of the Vertebrates[END_REF][START_REF] Holland | Scenarios for the making of vertebrates[END_REF][START_REF] Janvier | Facts and fancies about early fossil chordates and vertebrates[END_REF] represent long-debated issues.

In recent years, however, the combination of molecular, embryological, and anatomical data has made it possible to establish a robust phylogeny for extant deuterostomes. With the Hemichordata (acorn worms), the echinoderms (sea lilies, starfish, sea urchins, and related forms) are now considered to belong to a clade, the Ambulacraria, as the sister-group of chordates (cephalochordates, tunicates and vertebrates) [START_REF] Bottjer | Paleogenomics of echinoderms[END_REF][START_REF] Swalla | Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives[END_REF][START_REF] David | How Hox genes can shed light on the place of echinoderms among the deuterostomes[END_REF][START_REF] Holland | Scenarios for the making of vertebrates[END_REF][START_REF] Janvier | Facts and fancies about early fossil chordates and vertebrates[END_REF][START_REF] Lowe | The deuterostome context of chordate origins[END_REF]. This phylogenetic scenario provides insights not only into the distinctive morphological features (apomorphies) acquired within each clade of deuterostomes, but also into the primitive characters (plesiomorphies) inherited from their common ancestors [START_REF] Bottjer | Paleogenomics of echinoderms[END_REF][START_REF] Swalla | Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives[END_REF][START_REF] David | How Hox genes can shed light on the place of echinoderms among the deuterostomes[END_REF][START_REF] Lowe | The deuterostome context of chordate origins[END_REF]. For example, the occurrence of gill slits in both extant hemichordates and chordates strongly supports the view that these structures were very likely present in earliest deuterostomes [START_REF] Smith | The pre-radial history of echinoderms[END_REF][START_REF] Bottjer | Paleogenomics of echinoderms[END_REF][START_REF] Swalla | Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives[END_REF][START_REF] Lowe | The deuterostome context of chordate origins[END_REF]. Their absence in echinoderms is most parsimoniously regarded as a secondary loss during the course of echinoderm evolution [START_REF] Smith | The pre-radial history of echinoderms[END_REF][START_REF] Bottjer | Paleogenomics of echinoderms[END_REF][START_REF] Swalla | Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives[END_REF][START_REF] Zamora | Deciphering the early evolution of echinoderms with Cambrian fossils[END_REF], or perhaps at the origin of the phylum itself (Mooi andDavid, 1998, 2008).

Integrating fossil data into the phylogeny of deuterostomes offers the opportunity to document the sequential acquisition of key characters through time and to provide minimum dates for lineage divergences. The interpretation of the earliest known deuterostomes [START_REF] Shu | Primitive deuterostomes from the Chengjiang Lagerstätte (Lower Cambrian, China)[END_REF][START_REF] Shu | Ancestral echinoderms from the Chengjiang deposits of China[END_REF][START_REF] Caron | Tentaculate fossils from the Cambrian of Canada (British Columbia) and China (Yunnan) interpreted as primitive deuterostomes[END_REF][START_REF] Caron | Tubicolous enteropneusts from the Cambrian period[END_REF] is particularly challenging because their morphologies are often bizarrely unlike those of modern forms, with unexpected combinations of anatomical features. Along with cambroernids [START_REF] Caron | Tentaculate fossils from the Cambrian of Canada (British Columbia) and China (Yunnan) interpreted as primitive deuterostomes[END_REF], conodonts [START_REF] Briggs | Conodonts -a major extinct group added to the vertebrates[END_REF], vetulicolians [START_REF] Shu | Primitive deuterostomes from the Chengjiang Lagerstätte (Lower Cambrian, China)[END_REF] and vetulicystids [START_REF] Shu | Ancestral echinoderms from the Chengjiang deposits of China[END_REF], stylophorans belong to the bestiary of extinct, enigmatic early deuterostomes.

Stylophorans (cornutes and mitrates), a group of unusual middle Cambrian-Pennsylvanian (509-300 Myr) fossils, are small (typically one or two centimeters in length), bipartite organisms consisting of a single, articulated appendage inserted into a flattened, asymmetric to bilaterally symmetric body made of a multiplated, internal skeleton, or test [START_REF] Ubaghs | Stylophora[END_REF][START_REF] Clausen | Palaeoanatomy and biological affinities of a Cambrian deuterostome (Stylophora)[END_REF][START_REF] Smith | The pre-radial history of echinoderms[END_REF][START_REF] David | Are homalozoans echinoderms? An answer from the extraxial-axial theory[END_REF][START_REF] Dominguez | Paired gill slits in a fossil with a calcite skeleton[END_REF][START_REF] Lefebvre | Functional morphology of stylophoran echinoderms[END_REF] (Fig. 1A,C). The appendage comprises two distinct parts: a highly flexible proximal region (partially inserted into the test) of telescopic, imbricate rings; and a relatively stiff distal region consisting of opposing, delicate biserial platelets mounted on massive, uniserial elements (ossicles). The two platelet biseries form a roof over a longitudinal median groove running along the upper surfaces of the ossicles. Some interpretations suggest that the biserial platelets are fixed (Fig. 1F), but others show them as hinged, movable elements, much like cover plates in modern sea lilies and their kin (crinoids) (Fig. 1C). In life, the test likely contained the viscera, and in some taxa, bears a series of pores interpreted either as respiratory [START_REF] Ubaghs | Stylophora[END_REF][START_REF] David | Are homalozoans echinoderms? An answer from the extraxial-axial theory[END_REF][START_REF] Lefebvre | Functional morphology of stylophoran echinoderms[END_REF] or pharyngeal structures [START_REF] Jefferies | The Ancestry of the Vertebrates[END_REF][START_REF] Dominguez | Paired gill slits in a fossil with a calcite skeleton[END_REF][START_REF] Shu | Ancestral echinoderms from the Chengjiang deposits of China[END_REF][START_REF] Clausen | Palaeoanatomy and biological affinities of a Cambrian deuterostome (Stylophora)[END_REF][START_REF] Smith | The pre-radial history of echinoderms[END_REF][START_REF] Bottjer | Paleogenomics of echinoderms[END_REF] (Fig. 1A,C). Both the appendage and the test consist of numerous calcitic skeletal elements possessing the same microstructure (called stereom) found in all echinoderms [START_REF] Ubaghs | Stylophora[END_REF][START_REF] Jefferies | The Ancestry of the Vertebrates[END_REF][START_REF] David | Are homalozoans echinoderms? An answer from the extraxial-axial theory[END_REF][START_REF] Dominguez | Paired gill slits in a fossil with a calcite skeleton[END_REF][START_REF] Lefebvre | Functional morphology of stylophoran echinoderms[END_REF][START_REF] Clausen | Palaeoanatomy and biological affinities of a Cambrian deuterostome (Stylophora)[END_REF][START_REF] Smith | The pre-radial history of echinoderms[END_REF][START_REF] Bottjer | Paleogenomics of echinoderms[END_REF][START_REF] Zamora | Deciphering the early evolution of echinoderms with Cambrian fossils[END_REF].

The phylogenetic position of stylophorans within deuterostomes has been a contentious matter for decades. In the early 1960s, the stylophoran appendage was interpreted as a feeding arm, comparable in morphology to the feeding structures of various extant echinoderms [START_REF] Ubaghs | Stylophora[END_REF][START_REF] David | Are homalozoans echinoderms? An answer from the extraxial-axial theory[END_REF][START_REF] Lefebvre | Functional morphology of stylophoran echinoderms[END_REF]. In this hypothesis, stylophorans are considered bona fide echinoderms, with their appendage bearing a single ambulacral ray of the water vascular system (Figs. 1A-C, 2). In the late 1960s, the stylophoran appendage was reinterpreted as a chordate-like tail, containing a notochord and serially arranged muscle blocks [START_REF] Jefferies | The Ancestry of the Vertebrates[END_REF][START_REF] Dominguez | Paired gill slits in a fossil with a calcite skeleton[END_REF]. Thereby, stylophorans were designated calcichordates, ancestral members of all three modern chordate lineages. This interpretation implies that the stereom skeleton of extant echinoderms was an apomorphy of all deuterostomes and therefore secondarily and independently lost in all lineages with the exception of echinoderms. Most recently, the stylophoran appendage has been interpreted as a muscular, hemichordate-like stalk [START_REF] Shu | Ancestral echinoderms from the Chengjiang deposits of China[END_REF][START_REF] Clausen | Palaeoanatomy and biological affinities of a Cambrian deuterostome (Stylophora)[END_REF][START_REF] Smith | The pre-radial history of echinoderms[END_REF][START_REF] Bottjer | Paleogenomics of echinoderms[END_REF][START_REF] Swalla | Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives[END_REF]Caron et al., 2010) (Figs. 1D-F, 2). In this interpretation, stylophorans remained echinoderms (because of the presence of the distinctive stereom), but are among the earliest members of the phylum, because they retain plesiomorphic features lost in all more derived taxa (e.g., locomotory stalk), and lack apomorphies present in more derived taxa (e.g., water vascular system).

Two basic assumptions of the calcichordate interpretation have previously been falsified.

First, the purported sister-group relationship between echinoderms and chordates was not confirmed by molecular phylogenetics, which indicated that echinoderms are more closely related to hemichordates than they are to chordates [START_REF] Bottjer | Paleogenomics of echinoderms[END_REF][START_REF] Swalla | Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives[END_REF][START_REF] Erwin | The Cambrian conundrum: early divergence and later ecological success in the early history of animals[END_REF][START_REF] Lowe | The deuterostome context of chordate origins[END_REF]. Second, available evidence supports the view that stereom is an echinoderm synapomorphy that first appeared in the early Cambrian [START_REF] Bottjer | Paleogenomics of echinoderms[END_REF][START_REF] Kouchinsky | Chronology of early Cambrian biomineralization[END_REF][START_REF] Zamora | Deciphering the early evolution of echinoderms with Cambrian fossils[END_REF]. The last two remaining, current hypotheses both agree that stylophorans are echinoderms (Fig. 2), but differ markedly in the interpretation of their appendage: feeding arm constructed like a typical echinoderm ray [H1] versus tail constructed like a hemichordate-like stalk [H2].

Here we report the first evidence for soft part preservation in stylophorans. These data make it possible to test, for the first time, soft anatomies predicted by H1 and H2. The feeding arm hypothesis (H1) requires that: (1) the longitudinal median groove contained a water vascular canal, with lateral tube feet; (2) biserial platelets are cover plates that opened to expose the tube feet; (3) the mouth was located at the proximal end of the food groove; and (4) the fore-gut (esophagus) was located within the proximal rings. The locomotory stalk scenario (H2) implies that: (1) the longitudinal median groove contained a peduncular nerve; and (2) biserial platelets are integrated, contiguous body wall elements sutured to each other so as to enclose a coelom.

Material and methods

All

Results

Stylophorans with soft parts were collected in the Lower Ordovician Fezouata Konservat-Lagerstätte, in a small excavation along the western flank of Bou Izargane hill, approximately 18 km north of Zagora (central Anti-Atlas, Morocco; Fig. 3). Here, the Fezouata Shale corresponds to a 1,000 m-thick, repetitive succession of argillaceous siltstones, unconformable over the middle Cambrian sandstones of the Tabanite Group [START_REF] Destombes | Lower Palaeozoic rocks of Morocco[END_REF][START_REF] Martin | The Lower Ordovician Fezouata Konservat-Lagerstätte from Morocco: age, environment and evolutionary perspectives[END_REF].

The excavation is approximately 270 m above the base of the Ordovician series and within the lower stratigraphic interval (about 80 m thick) yielding soft-bodied faunas typical of the Fezouata Biota [START_REF] Vinther | Machaeridians are Palaeozoic armoured annelids[END_REF][START_REF] Vinther | Ancestral morphology of crown-group molluscs revealed by a new Ordovician stem aculiferan[END_REF][START_REF] Van Roy | Ordovician faunas of Burgess Shale type[END_REF][START_REF] Janvier | Facts and fancies about early fossil chordates and vertebrates[END_REF][START_REF] Van Roy | A giant Ordovician anomalocaridid[END_REF][START_REF] Lefebvre | The Fezouata Shale (Lower Ordovician, Anti-Atlas, Morocco): a historical review[END_REF][START_REF] Martin | The Lower Ordovician Fezouata Konservat-Lagerstätte from Morocco: age, environment and evolutionary perspectives[END_REF] (Fig. 3). Nearby graptolites indicate a late Tremadocian age (A. murrayi Zone) (Gutiérrez-Marco and Martin, 2016;[START_REF] Lefebvre | The Fezouata Shale (Lower Ordovician, Anti-Atlas, Morocco): a historical review[END_REF][START_REF] Lefebvre | Age calibration of the Lower Ordovician Fezouata Lagerstätte, Morocco[END_REF][START_REF] Martin | The Lower Ordovician Fezouata Konservat-Lagerstätte from Morocco: age, environment and evolutionary perspectives[END_REF].

All specimens were collected from a single, small, 3 cm-thick lens of dark blue mudstones.

Three fossiliferous intervals occur within the lens: a lower layer with many densely packed, complete, and fully articulated marrellomorphs (Furca sp.; Fig. 4A), an intermediate horizon with a low-diversity assemblage of trilobites (Anacheirurus sp., Bavarilla sp.) with soft parts (Fig. 4B-C), and an upper, low-diversity assemblage comprising almost exclusively cornute stylophorans (more than 300 specimens belonging to two taxa, Hanusia n. sp. and Thoralicystis n. sp.; Figs 56789). Deposition is interpreted as rapid burial of autochthonous communities by distal storm deposits in relatively shallow environmental conditions [START_REF] Martin | The Lower Ordovician Fezouata Konservat-Lagerstätte from Morocco: age, environment and evolutionary perspectives[END_REF][START_REF] Vaucher | A genetic link between Konzentratand Konservat-Lagerstätten in the Fezouata Shale (Lower Ordovician, Morocco)[END_REF]. In this level, arthropods and echinoderms are typically preserved as faint, flattened impressions on the dark rock. As in the Burgess Shale, echinoderm skeletons are indicated as impressions without preservation of actual stereom. In contrast, their soft tissues appear as colorful structures, resulting from the oxidation of original pyrite into yellow, red to purple iron oxide pseudomorphs (Figs. 5A, 6A, 7A, 8A, C). This type of preservation is not unique to the Bou Izargane cornutes [START_REF] Vinther | Machaeridians are Palaeozoic armoured annelids[END_REF][START_REF] Vinther | Ancestral morphology of crown-group molluscs revealed by a new Ordovician stem aculiferan[END_REF][START_REF] Van Roy | Ordovician faunas of Burgess Shale type[END_REF][START_REF] Janvier | Facts and fancies about early fossil chordates and vertebrates[END_REF][START_REF] Lefebvre | The Fezouata Shale (Lower Ordovician, Anti-Atlas, Morocco): a historical review[END_REF]. Similarly preserved soft parts (e.g. guts) have been described in arthropods [START_REF] Gutiérrez-Marco | Digestive and appendicular soft-parts, with behavioural implications, in a large trilobite from the Fezouata Lagerstätte, Morocco[END_REF], hyolithids (Marti Mus, 2016), and machaeridian annelids [START_REF] Vinther | Machaeridians are Palaeozoic armoured annelids[END_REF] occurring in the same stratigraphic interval of the Fezouata Shale. In the cornutes, the identification and precise distribution of Fe-rich soft tissues were obtained by chemical analyses and elemental mapping under SEM (Figs. 5B,E,6).

At least one specimen of Thoralicystis n. sp. shows clear evidence of soft-bodied structures in the distal part of its appendage (Figs. 56). Preserved soft tissues consist of: (1) two superimposed longitudinal canals housed in the continuous, median groove on the upper surface of ossicles, and extending from the stylocone cavity (proximally) to the distal end of the appendage; (2) numerous small, lateral tube-like extensions branching regularly from the external longitudinal canal and protruding outward from between widely open, hinged plates.

Several specimens of Thoralicystis n. sp. and Hanusia n. sp. exhibit strong evidence of soft tissues preserved within the proximal part of their appendage (Figs. 78). All these specimens display a spindle-shaped, elongate cavity extending throughout the proximal rings of the appendage from the stylocone cavity (distally), to the main body cavity (proximally). All these soft-bodied structures are described for the first time in stylophorans.

Discussion

The stylophoran appendage is a feeding arm

On the basis of this new evidence, it is possible to evaluate critically the two current interpretations of the stylophoran appendage. In the stalk model (H2), the outermost of the two longitudinal canals could be interpreted as the peduncular nerve housed in a fully enclosed body cavity, or coelom. However, H2 must be rejected because of the presence of open plates that precludes such a coelom, and the presence of lateral extensions that branch regularly from the external longitudinal canal and protrude externally in between the open plates. This organisation is incompatible with that of a stalk-, or tail-like structure, but fits requisites of the feeding-arm model (H1). The evidence strongly supports H1, in which the external longitudinal canal is interpreted as an ambulacral canal of the water vascular system, and the lateral, tube-like structures as ambulacral tube feet (Figs. 5-6, 9), the morphology of which is strikingly similar to that of exceptionally preserved tube feet described from Paleozoic arm-bearing forms [START_REF] Glass | Preservation of tube feet in an ophiuroid (Echinodermata) from the Lower Devonian Hunsrück Slate of Germany and a redescription of Bundenbachia beneckei and Palaeophiomyxa grandis[END_REF][START_REF] Sutton | A starfish with three-dimensionally preserved soft parts from the Silurian of England[END_REF][START_REF] Glass | Pyritized tube feet in a protasterid ophiuroid from the Upper Ordovician of Kentucky, U.S.A[END_REF][START_REF] Clark | Water vascular system architecture in an Ordovician ophiuroid[END_REF]. As a consequence, the mouth of stylophorans is most parsimoniously located at the proximal end of this ambulacral groove (Fig. 5C), the spindle-shaped cavity extending from the mouth (distally) and connected to the main body cavity (proximally) representing the anterior part of the gut (Figs. 5,(7)(8). Comparison with the situation in the arms and pinnules of modern crinoids suggests that the internal longitudinal canal internal to the space above the water vascular system is a coelomic canal (Figs. 56). There is no evidence that what are interpreted here as tube feet represent some sort of musculature, because they project as free-standing structures extending from the longitudinal canal, and protrude outside of the body wall into the external medium. Labyrinthic meshwork has been described in the proximal cavity of the stylocone (large, cone-shaped, uniserial ossicle located at the proximal end of the distal part of the appendage) of the primitive stylophoran Ceratocystis (middle Cambrian) [START_REF] Clausen | Palaeoanatomy and biological affinities of a Cambrian deuterostome (Stylophora)[END_REF]. Similar observations in extant echinoderms suggest that this labyrinthic microstructure was associated with muscle insertion, implying that the stylocone cavity once housed musculature with functionality similar to that in hemichordates. However, living echinoderms express muscles as well, so this trait is compatible with both H1 (Fig. 1B) and H2 (Fig. 1E), and cannot be used to provide evidence for or against either.

Finally, the putative locomotory function of the stylophoran appendage has been frequently considered as incompatible with its interpretation as a feeding arm and the presence of a watervascular canal [START_REF] Rahman | Ichnological insights into mitrate palaeobiology[END_REF][START_REF] Rozhnov | A new cornute (Homalozoa: Echinodermata) from the uppermost middle Cambrian (Stage 3, Furongian) from northern Iran: its systematics and functional morphology[END_REF]. As this is the case in many extant arm-bearing echinoderms (e.g. asteroids, ophiuroids, comatulids), it is very likely that stylophorans used their appendage not only for nutrition, but also locomotion [START_REF] Lefebvre | Functional morphology of stylophoran echinoderms[END_REF].

Implications for echinoderm phylogeny

Preserved soft tissues in cornutes from the Fezouata Shale provide strong evidence suggesting the existence of ambulacral structures in the stylophoran appendage. The evidence supports the view that this extinct group of ambulacrarians possessed at least two of the major apomorphies supporting recognition of the phylum Echinodermata: (1) a multi-element endoskeleton, made of calcitic plates displaying the typical, three-dimensional, stereomic microstructure; and (2) a water vascular system. However, the precise phylogenetic position of stylophorans within echinoderms remains so far unresolved because they do not display any evidence of the radial, five-fold symmetry generally considered a synapomorphy of the phylum [START_REF] Shu | Ancestral echinoderms from the Chengjiang deposits of China[END_REF][START_REF] Smith | The pre-radial history of echinoderms[END_REF][START_REF] Bottjer | Paleogenomics of echinoderms[END_REF][START_REF] Swalla | Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives[END_REF][START_REF] Zamora | Deciphering the early evolution of echinoderms with Cambrian fossils[END_REF]. If this absence is original, stylophorans would then represent a relatively early branch of pre-radial echinoderms [START_REF] Ubaghs | Stylophora[END_REF][START_REF] Ubaghs | Early Paleozoic echinoderms[END_REF][START_REF] Shu | Ancestral echinoderms from the Chengjiang deposits of China[END_REF][START_REF] Smith | The pre-radial history of echinoderms[END_REF][START_REF] Bottjer | Paleogenomics of echinoderms[END_REF][START_REF] Swalla | Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives[END_REF][START_REF] Zamora | Deciphering the early evolution of echinoderms with Cambrian fossils[END_REF]. Such an interpretation requires that anatomically similar, arm-like, feeding structures were acquired independently in stylophorans in the middle Cambrian, and again, in later forms such as asteroids and crinoids, in the Early Ordovician [START_REF] Ubaghs | Stylophora[END_REF][START_REF] Ubaghs | Early Paleozoic echinoderms[END_REF].

Alternatively, the late appearance of stylophorans in the fossil record 10-12 m.y. after radial taxa (e.g., eocrinoids, helicoplacoids, and edrioasteroid-like forms such as stromatocystitoids) [START_REF] Kouchinsky | Chronology of early Cambrian biomineralization[END_REF][START_REF] Zamora | Cambrian echinoderm diversity and palaeobiogeography[END_REF] and their unusual, flattened morphology more parsimoniously suggest that stylophorans are relatively derived echinoderms descended from pentaradial ancestors [START_REF] David | Are homalozoans echinoderms? An answer from the extraxial-axial theory[END_REF]. This interpretation is supported by embryological data, which provide no evidence suggesting that 5-part radial symmetry is preceded by stages with fewer rays [START_REF] Mooi | Radial symmetry, the anterior/posterior axis, and echinoderm Hox genes[END_REF]. In this context, the possession of arm-like, ambulacral structures containing coelomic extensions other than the hydrocoel could represent an apomorphy uniting stylophorans with forms such as crinoids [START_REF] David | Are homalozoans echinoderms? An answer from the extraxial-axial theory[END_REF][START_REF] Lefebvre | Functional morphology of stylophoran echinoderms[END_REF].

Implications for early deuterostome phylogeny

The exceptionally preserved cornute specimens from Morocco bring revolutionary new evidence to consider. That evidence actually reverses the anterior-posterior axis suggested by some very recent interpretations seeking to revive a modified version of the calcichordate interpretation [START_REF] Shu | Primitive deuterostomes from the Chengjiang Lagerstätte (Lower Cambrian, China)[END_REF][START_REF] Shu | Ancestral echinoderms from the Chengjiang deposits of China[END_REF][START_REF] Clausen | Palaeoanatomy and biological affinities of a Cambrian deuterostome (Stylophora)[END_REF][START_REF] Smith | The pre-radial history of echinoderms[END_REF][START_REF] Bottjer | Paleogenomics of echinoderms[END_REF][START_REF] Swalla | Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives[END_REF][START_REF] Zamora | Deciphering the early evolution of echinoderms with Cambrian fossils[END_REF]. Since the early 2000s, molecular phylogenies have strongly supported an ambulacrarian clade that unites hemichordates and echinoderms as sister-group to the chordates [START_REF] Bottjer | Paleogenomics of echinoderms[END_REF][START_REF] Swalla | Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives[END_REF][START_REF] David | How Hox genes can shed light on the place of echinoderms among the deuterostomes[END_REF][START_REF] Holland | Scenarios for the making of vertebrates[END_REF][START_REF] Janvier | Facts and fancies about early fossil chordates and vertebrates[END_REF]Lower et al., 2015). As a consequence of this new phylogenetic framework, several authors reinterpreted stylophorans as early members of the Echinodermata, morphologically extremely close to the last common ancestor of both hemichordates and echinoderms [START_REF] Shu | Primitive deuterostomes from the Chengjiang Lagerstätte (Lower Cambrian, China)[END_REF][START_REF] Shu | Ancestral echinoderms from the Chengjiang deposits of China[END_REF][START_REF] Clausen | Palaeoanatomy and biological affinities of a Cambrian deuterostome (Stylophora)[END_REF][START_REF] Smith | The pre-radial history of echinoderms[END_REF][START_REF] Bottjer | Paleogenomics of echinoderms[END_REF][START_REF] Swalla | Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives[END_REF]. In this interpretation, stylophorans would possess a single echinoderm apomorphy: the calcite, stereom skeleton. They would not yet have acquired two other significant echinoderm apomorphies: the water-vascular system and the radial, fivefold symmetry.

In a surprising parallel to the calcichordate hypothesis, stylophorans would share with other (early) ambulacrarians and deuterostomes (1) a bipartite body organization, with a head and a muscular, locomotory tail (or stalk); and (2) the presence (plesiomorphic retention) of gill slits [START_REF] Shu | Primitive deuterostomes from the Chengjiang Lagerstätte (Lower Cambrian, China)[END_REF][START_REF] Shu | Ancestral echinoderms from the Chengjiang deposits of China[END_REF][START_REF] Morris | The Cambrian "explosion" of metazoans and molecular biology: would Darwin be satisfied?[END_REF][START_REF] Clausen | Palaeoanatomy and biological affinities of a Cambrian deuterostome (Stylophora)[END_REF][START_REF] Smith | The pre-radial history of echinoderms[END_REF][START_REF] Bottjer | Paleogenomics of echinoderms[END_REF][START_REF] Swalla | Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives[END_REF][START_REF] Caron | Tentaculate fossils from the Cambrian of Canada (British Columbia) and China (Yunnan) interpreted as primitive deuterostomes[END_REF][START_REF] Morris | Pikaia gracilens Walcott, a stem-group chordate from the Middle Cambrian of British Columbia[END_REF][START_REF] Zamora | Deciphering the early evolution of echinoderms with Cambrian fossils[END_REF][START_REF] Morris | Rare primitive deuterostomes from the Cambrian (Series 3) of Utah[END_REF]. As a consequence, this interpretation of stylophorans as hemichordate-like, early echinoderms with a bipartite organization (anterior head and posterior, tail-like appendage) has in the last 15 years caused many comparisons of early deuterostomes to stylophorans, i.e. Herpetogaster and cambroernids [START_REF] Caron | Tentaculate fossils from the Cambrian of Canada (British Columbia) and China (Yunnan) interpreted as primitive deuterostomes[END_REF], Pikaia and other putative early chordates [START_REF] Donoghue | The origin and early evolution of chordates: molecular clocks and the fossil record[END_REF][START_REF] Gee | Careful with that amphioxus[END_REF][START_REF] Morris | Pikaia gracilens Walcott, a stem-group chordate from the Middle Cambrian of British Columbia[END_REF], vetulicolians [START_REF] Shu | Primitive deuterostomes from the Chengjiang Lagerstätte (Lower Cambrian, China)[END_REF][START_REF] Morris | The Cambrian "explosion" of metazoans and molecular biology: would Darwin be satisfied?[END_REF][START_REF] Vinther | Vetulicolians from the Lower Cambrian Sirius Passett Lagerstätte, North Greenland, and the polarity of morphological characters in basal deuterostomes[END_REF][START_REF] Ou | Evidence for gill slits and a pharynx in Cambrian vetulicolians: implications for the early evolution of deuterostomes[END_REF], and vetulicystids [START_REF] Shu | Ancestral echinoderms from the Chengjiang deposits of China[END_REF][START_REF] Morris | Rare primitive deuterostomes from the Cambrian (Series 3) of Utah[END_REF].

Our study demonstrates that: (1) stylophorans had a water-vascular system; and (2) that their appendage was not a locomotory, muscular tail. By bringing new, unequivocal evidence that the stylophoran appendage is an echinoderm feeding arm (comparable in morphology to a crinoid feeding arm) and not a hemichordate-like tail (or stalk), our discovery shows that the interpretation of these fossils as early echinoderms retaining features of basal ambulacrarians and/or hemichordates can be definitively rejected. In fact, any suggestion that stylophorans can tell us something about common ancestry of echinoderms with other deuterostomes is seriously compromised by the solid evidence that such conclusions rely on a reconstruction that has the animal the wrong way around. The bipartite body organization of stylophorans (theca and feeding arm) is not homologous to the bipartite body organization of Herpetogaster, vetulicolians, vetulicystids, early chordates and/or hemichordates (head and post-anal tail).

Whatever the precise phylogenetic position of stylophorans within the Echinodermata, the significance of the Fezouata fossils lies in the picture they give of the remarkable diversity within the phylum throughout its evolution, and not of the supposed origins of the phylum itself.

Figure captions

  figured specimens are housed in the collections of Cadi-Ayyad University (Faculté des Sciences et Techniques, Guéliz), Marrakesh, Morocco. Locality details are indicated on specimen labels, and can be provided on request. Specimens were prepared with scalpels and needles under high magnification using a Zeiss SteREO Discovery.V8 stereomicrocope. Interpretative drawings were made using a camera lucida attached to a Zeiss SteREO Discovery.V8 stereomicroscope. Photographs were made with a Zeiss AxioCam MRc5 digital camera mounted on a Zeiss SteREO Discovery.V8 stereomicroscope. Digital photographs were processed in Adobe Photoshop Elements 9. Figs. 3A-B and Figs. S2A, S4A-B are composite images stitched together using Adobe Photoshop Elements 9. Elemental composition was analysed in a FEI Quanta 250 scanning electron microscope (SEM) equipped with an energy Xray analyser (EDX) at the Centre Technologique des Microstructures, plateforme de l'Université Claude Bernard Lyon 1.

Fig. 1 .

 1 Fig. 1. The two current interpretations of stylophoran morphology and their implications for softtissue anatomy, based on the cornute Phyllocystis blayaci (Lower Ordovician, France). A-C. H1, stylophorans as typical echinoderms with a single feeding arm. D-F. H2, stylophorans as preradial echinoderms with a hemichordate-like stalk. A, D. Basic anatomical features deduced from skeletal morphology. B, E. Reconstructions of soft tissue anatomy along a longitudinal

Fig. 2 .

 2 Fig.2. The two current interpretations of stylophoran phylogenetic position within ambulacrarian deuterostomes. H1, stylophorans as typical echinoderms with a single feeding arm. H2, stylophorans as pre-radial echinoderms with a hemichordate-like stalk.

Fig. 3 .

 3 Fig. 3. Geographic and geologic context of Bou Izargane locality. On the maps (left), Ordovician outcrops in north-western Africa and in the Zagora area are indicated in green. The red star shows the location of Bou Izargane hill. On the right, partial stratigraphic column of the Fezouata Shale, showing the position of the Bou Izargane excavation. The left column indicates sea level changes. Numbers along the right column indicate the height (in meters) above the Cambrian/Ordovician boundary. The colors used in the log correspond to those of outcropping rocks.

Fig. 4 .

 4 Fig. 4. Arthropods with soft-tissue preservation collected in the same lens as cornute stylophorans with soft parts, late Tremadocian (Lower Ordovician), Bou Izargane, Zagora area (Morocco). A. Two associated specimens of the marrellomorph Furca sp. (AA.BIZ15.OI.364). B-C. Two specimens of the trilobite Bavarilla zemmourensis. B. Arrows indicate four locomotory appendages below the thorax (AA.BIZ15.OI.181). C. The arrow shows one frontal appendage at the anterior extremity of the cephalon (AA.BIZ15.OI.16). Scale bars, 5 mm (A, C); 2 mm (B).

Fig. 5 .

 5 Fig. 5. Thoralicystis n. sp. from the Fezouata Shale, Lower Ordovician, Morocco (AA.BIZ15.OI.259). A-C. Complete specimen in lateral view. A. Composite photographic reconstruction. B. Composite SEM elemental map of Fe. C. Composite reconstruction of soft part anatomy based on camera lucida drawings, SEM elemental maps (Fe) and back-scattered electron micrographs. D-F. Magnified views of parts of the distal appendage outlined in (A-C). D. Back-scattered electron micrograph. The arrows indicate the position of tube feet. E. SEM elemental map showing the distribution of Fe. F. Reconstruction of the soft parts based on camera lucida drawings, SEM elemental maps (Fe) and back-scattered electron micrographs. pr. cavity, proximal cavity; pr. rings, proximal rings; styl., stylocone; wvs, water vascular system. Scale bars, 5 mm (A-C); 1 mm (D-F).

Fig. 6 .

 6 Fig. 6. Soft-tissue preservation in the distal appendage of the cornute stylophoran Thoralicystis n. sp. (AA.BIZ15.OI.259), late Tremadocian (Lower Ordovician), Bou Izargane, Zagora area (Morocco). A. Photographic view of the magnified area. B. Back-scattered electron micrograph showing the water vascular system (ambulacral canal, tube feet) and the second, more internal, longitudinal canal. Skeletal elements (biserial platelets, ossicles) are not visible. C-J. SEM elemental maps of Al (C), C (D), Fe (E), K (F), Mg (G), Na (H), O (I) and Si (J). Scale bars, 1 mm.

Fig. 7 .

 7 Fig. 7. Soft-tissue preservation in the test and proximal appendage of the cornute stylophoran Hanusia n. sp. (AA.BIZ15.OI.110), late Tremadocian (Lower Ordovician), Bou Izargane, Zagora area (Morocco). A. Composite photographic reconstruction of the specimen in lateral view. B. Composite reconstruction based on back-scattered electron micrographs. C. Reconstruction of

Fig. 8 .

 8 Fig. 8. Soft-tissue preservation in the proximal appendage of the cornute stylophoran Hanusia n. sp. (AA.BIZ15.OI.80), late Tremadocian (Lower Ordovician), Bou Izargane, Zagora area (Morocco). A. Specimen in upper view. B. Reconstruction of (A), based on camera lucida drawings. C. Closer view of the proximal region of the appendage outlined in (A). D.Reconstruction of (C), based on camera lucida drawings. pr. cavity, proximal cavity; pr. rings, proximal rings. Scale bars, 1 mm.

Fig. 9 .

 9 Fig. 9. Reconstruction of the cornute stylophoran Thoralicystis n. sp., late Tremadocian (Lower Ordovician), Zagora area (Morocco), with the tube feet protruding beyond the cover plates. The appendage is about 20 mm long.

Acknowledgements

This paper is a contribution of the ANR (Agence Nationale de la Recherche) research project entitled "The Rise of Animal Life (Cambrian-Ordovician): organisation and tempo" (grant number ANR-11-BS56-0025), the CNRS-CNRST cooperation project VALORIZ (grant number 52943) and the CNRS-INSU TelluS-INTERRVIE 2018 project TAPHO FEZOUATA. Abel Prieur (Lyon 1 University, Villeurbanne) is recognized for discovering the level with exceptional preservation in the field, Jean Vannier (Lyon 1 University, Villeurbanne), for technical assistance with SEM, and both Bernard Pittet and Romain Vaucher (Lyon 1 University, Villeurbanne) for making available detailed sedimentological information on the locality. Ronald L. Parsley (Tulane University, New Orleans), Imran Rahman (Oxford University Museum of Natural History), Sergei Rozhnov (Russian Academy of Sciences, Moscow), Samuel Zamora (Instituto Geológico y Minero de España, Zaragoza) and five anonymous reviewers provided insightful comments on several versions of the MS.


Sylvain Brun

Vincent Rincheval

Sebastien Gaumer

Bernard Mignotte

Dr Isabelle Guenal
email: guenal@genetique.uvsq.fr

reaper and bax initiate two different

Keywords: Apoptosis, Drosophila, bcl-2, reaper, bax, mitochondria

Abstract:

bcl-2 was the first regulator of apoptosis shown to be involved in oncogenesis.

Subsequent studies in mammals, in the nematode and in Drosophila revealed wide evolutionary conservation of the regulation of apoptosis. Although dbok/debcl, a member of the bcl-2 gene family described in Drosophila, shows pro-apoptotic activities, no anti-apoptotic bcl-2 family gene has been studied in Drosophila. We have previously reported that the human anti-apoptotic gene bcl-2 is functional in Drosophila, suggesting that the fruit fly shares regulatory mechanisms with vertebrates and the nematode, involving anti-apoptotic members of the bcl-2 family. We now report that bcl-2 suppresses rpr-induced apoptosis in Drosophila. Additionally, we have compared features of bax-and rpr-induced apoptosis.

Flow cytometry analysis of wing disc cells demonstrate that both killers trigger mitochondrial defects. Interestingly, bcl-2 suppresses both bax-and rpr-induced mitochondrial defects while the caspase-inhibitor p35 is specific to the rpr pathway. Finally, we show that the inhibition of apoptosis by bcl-2 is associated with the down-regulation of rpr expression.

Introduction:

Programmed cell death by apoptosis is essential to metazoan development, metamorphosis and tissue homeostasis (Vaux & Korsmeyer, 1999). Apoptosis is a powerful biological process that removes potentially dangerous cells like self-reactive lymphocytes, virus-infected cells and tumor cells. Genetic and biochemical studies, in mammals as well as in invertebrates, reveal a high evolutionary conservation of the regulation of apoptosis. The principal executioners of apoptosis belong to a highly conserved family of proteases, the cysteinyl-aspartases (caspases). Caspases are constitutively present in cells as zymogenes, and their activation by proteolysis or auto-proteolysis provokes the cleavage of other caspases in a proteolytic activation cascade that ultimately cleaves and inactivates specific targets essential for cell structure and DNA integrity (Nunez et al., 1998;Thornberry & Lazebnik, 1998).

Genetic studies in Caenorhabditis elegans identified the genes ced-3, ced-4, egl-1, which are essential to trigger cell death, and ced-9 which inhibits cell death. In cells triggered to die, the protein EGL-1 inhibits CED-9, which provokes the activation of the caspase CED-3 by CED-4 (Conradt & Horvitz, 1998). Although this model has been a scaffold for understanding the regulation of caspase activation in other species, the regulation of apoptosis in the fruit fly and in mammals shows an increased complexity compared to that in the nematode.

First, while only one caspase (CED-3) is involved in apoptosis in the nematode, at least ten caspases in mammals and seven in Drosophila melanogaster are involved in apoptosis (Chen et al., 1998;Dorstyn et al., 1999a;Dorstyn et al., 1999b;Doumanis et al., 2001;Fraser & Evan, 1997;[START_REF] Harvey | [END_REF]Song et al., 1997).

Second, in C. elegans, the regulation of apotosis by the bcl-2 family is mediated by the anti-apoptotic gene ced-9 and the pro-apoptotic BH3-only gene egl-1. In Drosophila as in mammals, pro-apoptotic members of the bcl-2 family are not limited to BH3-only genes (Gross et al., 1999). Indeed, both bcl-2 family genes identified in the fly genome are homologous to the mammalian pro-apoptotic gene bok, and present different BH interaction domains [START_REF] Brachmann | [END_REF]Colussi et al., 2000;[START_REF] Igaki | Proc. Natl. Acad. Sci. USA[END_REF]Zhang et al., 2000). However, while debcl/drob-1/dborg-1/dbok has been shown to be pro-apoptotic, whether the second predicted gene of the family will be pro-or anti-apoptotic remains to be addressed.

Third, in contrast with CED-4 which constitutively activates the caspase CED-3, Apaf-1 in mammals as well as DAPAF-1/DARK/HAC-1 in the fruit fly both need ATP and cytochrome c (cyt c) as co-factors to activate caspases [START_REF] Kanuka | [END_REF][START_REF] Li | [END_REF]Rodriguez et al., 1999;[START_REF] Zhou | [END_REF]. In mammals, Bax triggers the release of cyt c and other key pro-apoptotic factors from the inter-membrane space of mitochondria like the Apoptosis Inducing Factor (AIF) and several caspases [START_REF] Zamzami | [END_REF], while Bcl-2 inhibits this release and apoptosis induced by Bax or many other death stimuli (Gross et al., 1999). In Drosophila, cyt c may regulate apoptosis without being released [START_REF] Dorstyn | [END_REF]Varkey et al., 1999;Zimmermann et al., 2002). Indeed, DAPAF-1 and cyt c seem to cooperate with DRONC and DRICE in an apoptosome-like complex localized to mitochondria and responsible for the processing of both caspases DRONC and DRICE in Drosophila cells [START_REF] Dorstyn | [END_REF][START_REF] Kanuka | [END_REF][START_REF] Li | [END_REF]Rodriguez et al., 1999;[START_REF] Zhou | [END_REF]. Interestingly, dBOK/DEBCL, which localizes to mitochondria, triggers cyt c release and apoptosis when expressed in mammalian cells, while in Drosophila cells, it triggers the translocation of DRONC to mitochondria and caspase activation without cyt c release.

In Drosophila, mitochondria might be targets of the three RHG domain protein RPR, HID and GRIM. Although the three genes reaper (rpr), head involution defective (hid) and grim are unknown in mammals, they are functional in vertebrates, and HID and GRIM localize to mitochondria during apoptosis in mammalian cells (Claveria et al., 1998;Evans et al., 1997;[START_REF] Haining | Proc. Natl. Acad. Sci. USA[END_REF]. Furthermore, in Xenopus cells extracts, rpr induces cyt c release from mitochondria (Thress et al., 1999), and bcl-2 inhibits rpr-dependent caspase-activation (Evans et al., 1997). Zimmermann et al. show that rpr-dependent apoptosis can be independent of dapaf-1 and cyt c in vitro (Zimmermann et al., 2002). However, although rpr-dependent cyt c release is not detected in Drosophila cells, dapaf-1 mutants partially suppress rpr-induced apoptosis, suggesting a requirement for active DAPAF-1 in the rpr apoptotic pathway in vivo.

Whether an anti-apoptotic bcl-2 family member acts on mitochondria and cyt c to protect cells from apoptosis in Drosophila remains to be addressed. To our knowledge, no anti-apoptotic member of the bcl-2 gene family has been found in the fruit fly. Nevertheless, we have shown in our laboratory that the murine pro-apoptotic gene bax and the human anti-apoptotic gene bcl-2 are functional in Drosophila melanogaster (Gaumer et al., 2000).

Our current study demonstrates that bax and rpr induce two different apoptotic pathways, both affecting mitochondria. We show here that bcl-2 suppresses rpr-and bax-induced cell death as well as rpr-and bax-induced mitochondrial defects, while the protective effect of the caspase-inhibitor p35 is specific to the rpr death pathway.

Although in mammals most of the components of the cell death program are constitutively present, many apoptosis regulators in Drosophila are controlled at the transcriptional level. Among others, rpr is transcriptionally induced to trigger apoptosis during embryonic development, metamorphosis and after X-ray irradiation (Baehrecke, 2000;[START_REF] Nordstrom | [END_REF]. In particular, after X-ray irradiation rpr expression is up-regulated by dmp53, the homologue of the mammalian oncosuppressor p53. Additionally, the imd/rip pathway, which is involved in the immune response to bacterial infection [START_REF] Lemaitre | Proc. Natl. Acad. Sci. U S A[END_REF], can also trigger rpr expression (Georgel et al., 2001). Strikingly, we also show in this paper that inhibition of apoptosis by expression of bcl-2 during embryonic development (Gaumer et al., 2000) is correlated with the down-regulation of rpr expression, suggesting the existence of an amplification loop in rpr-induced apoptosis.

Results:

A semi-quantitative approach to detect the genetic suppression of wing phenotypes.

We have used the vg-GAL4 transgenic Drosophila strain to drive the expression of UAS-rpr, UAS-bax and several other transgenes in the wing margin during larval development. Flies expressing rpr or bax in the wing show two principal phenotypes: notches and wing fusion defects. We have focused on the notch phenotype to screen the flies. Surprisingly, expressivity and penetrance of the notch phenotype are variable in a population of flies of the same genotype. At 25°C, flies expressing rpr in the wing exhibit a distribution of phenotypes that can be classified into four categories according to their strength: strong, intermediate, weak and wildtype (figure 1). At 18°C, flies expressing bax in the wing exhibit a distribution of phenotypes that can be classified into three categories: strong, intermediate and weak (figure 2). Therefore, we have set a semi-quantitative approach to study genetic suppression between flies co-expressing rpr or bax and several other transgenes. To compare distributions of phenotypes between two different lineages, we have used the statistical Wilcoxon test (Wilcoxon, 1945). This test defines a Ws value between two populations and allows assessment of whether their distributions, which correspond to gradual phenotypes, are significantly different or not, and which population is composed of stronger phenotypes. We define the statistically significant limit as <10 -3 . bcl-2 and p35 suppress rpr-induced cell death in the wing.

The Drosophila pro-apoptotic gene rpr plays a key role in apoptosis. Since bcl-2 and rpr have antagonistic effects on apoptosis, we tested whether the human bcl-2 gene could suppress phenotypes induced by expression of rpr in the wing. Our results show that the phenotypes induced by rpr are weaker when bcl-2 is co-expressed. Although the suppression effect of bcl-2 is partial, distributions of phenotypes clearly shift to weaker phenotypes as the number of UAS-bcl-2 copies increases (figure 3A and table 1). Indeed, Ws values between control flies expressing only rpr and flies co-expressing rpr and one to three copies of bcl-2 decrease from -4.1 to -9.6 (<10 -3 ) as shown in table 1. Furthermore, almost 60% of flies co-expressing rpr and three copies of bcl-2 show a wildtype phenotype compared to 4% of control flies. We did not find a statistical difference between distributions of phenotypes observed in flies co-expressing rpr and one or two copies of bcl-2. However, there is a significant difference between distributions of flies co-expressing rpr and one or two copies of bcl-2 and the phenotype distribution of flies co-expressing rpr and three copies of bcl-2 (Ws3-4=-6.89; 3-4=4.6x10 -11 ). These results clearly show that bcl-2 suppresses the phenotypes induced by rpr in the wing in a dose-dependent manner.

Since it has been shown that rpr-induced apoptosis is caspase-dependent in the developing eye (Hay et al., 1995), we asked whether the caspase-inhibitor p35 could inhibit rpr-induced apoptosis in the wing. Our results show that expression of p35 partially suppresses the notches induced by rpr expression (figure 3B). Phenotypes of flies co-expressing rpr and p35 are weaker than phenotypes of control flies, and almost 70% of flies co-expressing rpr and one copy of p35 display weak or wildtype phenotypes compared to 37% of control flies. The Ws1-5 value between the distributions is -5.36 and 1-5<10 -3 (table 1). A second independent p35 strain was also used to suppress rpr-induced cell death and both p35 strains display similar protective activity (data not shown).

Since bcl-2 can prevent caspase-independent cell death (Okuno et al., 1998) in mammals, we have studied the co-expression of bcl-2 and p35 in Drosophila. The data presented in figure 3B and table 1 show no statistical difference between phenotype distributions of flies co-expressing rpr and one copy of bcl-2, flies co-expressing rpr and one copy of p35 and flies co-expressing rpr and one copy of p35 plus one copy of bcl-2.

Consistent with this observation, we also observe no differences between the effects of two copies of bcl-2, one copy of p35, two copies of bcl-2 plus one copy of p35 on rpr-induced phenotypes (figure 3C table 1). Strikingly, while the jump from two to three copies of UAS-bcl-2 has a great suppressive effect on the phenotypes induced by rpr, adding a UAS-p35 transgene in the same genetic background has no effect. Taken together, these data show that the co-expression of both inhibitors of apoptosis has no additive effect on the phenotypes induced by rpr, suggesting that their inhibitory effects are somehow redundant.

bcl-2 down-regulates rpr expression during embryonic development.

Since many death signals transcriptionally induce rpr in cells committed to die, we asked whether bcl-2 could only inhibit events downstream of rpr, or whether bcl-2 also prevents the transcriptional induction of rpr. In order to determine if bcl-2 can act upstream of rpr, we have used the rpr-11-LacZ construct [START_REF] Nordstrom | [END_REF] to visualize rpr expression during embryonic development in embryos expressing bcl-2. We have analyzed the X-gal staining pattern in situ in embryos until stage 14 of embryonic development (figure 4).

In control embryos (rpr-11-LacZ/da-GAL4), LacZ staining patterns are in agreement with those described by Nordstrom et al.. Interestingly, in embryos co-expressing bcl-2 and the LacZ reporter (UAS-bcl-2/+;rpr-11-LacZ/da-GAL4), rpr-11-LacZ expression does not begin until stage 9 (figure 4C) while it is already detected at stage 8 in control embryos (figure 4B). At stage 9, LacZ staining in embryos expressing bcl-2 is weak and only detectable in the posterior end of the extending germ band. In contrast, control embryos at a similar stage display staining at the posterior end of the extending germ band, dorsally in the region adjacent to the cephalic furrow, and more anteriorely at the clypeolabrum as well as gnathal buds (figure 4D). Subsequently, light staining is detected in segmental stripes in control embryos at stage 9 while this segmental staining does not appear until stage 10/11 in embryos expressing bcl-2 (figure 4E). Finally, for both types of embryos, LacZ staining in these zones persists and intensifies from stage 11 to stage 14 (figure 4E, 4F, 4G and 4H), but rpr expression remains weaker in embryos expressing bcl-2.

In order to quantify variations of LacZ staining observed in situ, we have measured enzymatic activity of -galactosidase in embryonic extracts at different times of development for control embryos (rpr-11-LacZ/da-GAL4), embryos expressing bcl-2

(UAS-bcl-2/+;rpr-11-LacZ/da-GAL4) and embryos expressing p35

(UAS-p35/+;rpr-11-LacZ/da-GAL4) (table 2). Activity assays were performed nine times independently, and then analyzed with the statistical Student test. For embryos of the three genotypes analyzed, our data clearly show that -galactosidase activities are very low and not statistically different before 3h A.E.L (table 2) (>0.05). Subsequently, -galactosidase activities slightly increase 3-6h A.E.L., and become much stronger at later stages (6-9h).

Indeed, for each genotype analyzed, activities measured are statistically different between the three developing times (data not shown). Strikingly, as previously observed in situ, -galactosidase activity detected is statistically lower in embryos expressing bcl-2 than in control embryos at 3-6h and at 6-9h A.E.L. Furthermore, the activity in 3-6h-embryos expressing bcl-2 is statistically comparable to the activity in 0-3h-control embryos (data not shown). Therefore, the 3-6h-embryos expressing bcl-2 (including embryos at stage 8 and 9 of development) exhibit a delay in rpr induction, and at later stages, rpr expression is down-regulated in embryos expressing bcl-2. In contrast, expression of p35 has no effect on rpr expression during embryonic development. At all developmental stages analyzed, -galactosidase activity in embryos expressing p35 is statistically comparable to that in control embryos (table 2). Taken together, these data argue that bcl-2 affects the transcriptional induction of rpr during development, and this activity is specific to bcl-2 since the expression of p35 has no effect on the rpr-11-LacZ transgene.

bax-induced cell death is sensitive to bcl-2 level but insensitive to p35.

Since p35 inhibits many Drosophila cell death pathways, we have tested whether p35 could also suppress bax-induced phenotypes. As previously described, bax-induced phenotypes are variable at 18°C. The severity of wing notches ranges from strong to weak phenotypes (figure 2). To study genetic interactions between bax and bcl-2 or p35, females carrying both the vg-GAL4 driver and UAS-bax (vg-GAL4,UAS-bax/CyO-gfp) were crossed to males carrying copies of the UAS-bcl-2 or UAS-p35 transgenes. Results are presented in figure 5, and distributions of phenotypes are compared using the statistical Wilcoxon test (table 3 and4).

Distributions of phenotypes between females co-expressing bax and p35 and females expressing bax but not p35 are not statistically different (Ws1-2=-0.79; 1-2=0.43). This result is comparable to that in male lineages: Ws5-6=2.55; 5-6=0.01. A similar result was obtained using a different UAS-p35 transgenic strain as well as a third strain carrying two copies of UAS-p35 (data not shown). Note that the different p35 strains suppress rpr-induced cell death in the wing (figure 3 and data not shown). In contrast, bax is sensitive to bcl-2 level and the strength of bax-induced phenotypes decreases as the number of bcl-2 copies increases.

Indeed, 73% of females and 41% of males co-expressing bax and one copy of bcl-2 have weak phenotypes compared respectively to 10% of females and 15% of males expressing only bax. Moreover, females co-expressing bax and three copies of bcl-2 have weaker phenotypes than those co-expressing a single copy of bcl-2 (Ws3-4=-3.76; 3-4<10 -3 ). Similarly, two copies of bcl-2 in male lineage suppress phenotypes induced by bax more effectively than one copy (Ws7-8=-5.97; 7-8<10 -3 ). In these experiments, bcl-2 suppresses phenotypes induced by bax in a dose-dependent manner while p35 has no effect. These data suggest that bcl-2 inhibits apoptosis triggered through a p35-insensitive pathway in the fly.

Wing discs are differently affected by bax and rpr expression

As shown in figure 6, acridine orange (AO) staining of wing discs of third instar larvae expressing rpr or bax driven by vg-GAL4 shows prominent apoptosis, while in wildtype discs, apoptosis is very low and appears preferentially in the notum (figure 6). Staining in discs expressing rpr is intense and foreshadows vg-GAL4 expression. More central labeling is detectable in discs expressing bax, though most of AO staining is also found in the area of vg-GAL4 expression. This discrepancy of staining pattern suggests that although massive apoptosis occurs in discs expressing rpr like in discs expressing bax, the wing pouch of the disc is more affected by bax expression. Additionally, expression of bax or rpr triggers a dramatic decrease of disc size. Thus, both reduced size of discs and AO staining pattern show that rpr-and bax-induced apoptosis occur at least before as well as within the third instar larval stage of development.

bax and rpr induce mitochondrial membrane potential collapse, cell shrinkage but no necrosis t has been shown in mammalian cells and recently in Drosophila cells that mitochondrial membrane potential (m) drop is an early event in apoptosis when compared to DNA fragmentation and plasma membrane injuries [START_REF] Vayssière | Proc. Natl. Acad. Sci. USA[END_REF]Zimmermann et al., 2002). Therefore, we asked whether rpr-and bax-induced cell death in vivo in Drosophila provoke m collapse. Flow cytometry analyses were carried out to study the integrity of the plasma membrane, variations of m and cell shrinkage in third instar larvae wing discs. We Therefore, since experimental procedures as well as markers used for in situ labeling and in vitro measurements are different, the results from both types of experiments are not quantitatively comparable.

However, it is clear that discs expressing rpr or bax exhibit a significant increase in the portion of apoptotic cells. Thus, in order to estimate apoptosis triggered by expression of rpr or bax, we have subtracted the background level of apoptosis occurring in control discs (window L) from apoptosis measured in discs expressing rpr or bax (figure 7C). This analysis shows that expression of rpr induces m drop and cell shrinkage. Indeed, only 63% of cells in discs expressing rpr maintain high DiOC6(3) fluorescence (window H), versus 84% in control discs (figure 7Ab andd). In discs expressing rpr, 21% more cells undergo apoptosis than in control discs (figure 7C), while almost none become PI-positive (figure 7Ac).

Consistent with the in situ observations, equivalent results are obtained when bax is expressed (figure 7B andC). We observe 8% of PI positive cells in bax expressing discs and 7% in bax control discs, showing that bax expression does not induce necrosis. Moreover, bax induces 22% more apoptosis than in control discs (figure 7C). This quite low apoptogenic effect of rpr and bax is likely due to the restricted vg-GAL4 expression in the wing disc (figure 6A).

Indeed, flow cytometry analysis of (vg-GAL4/UAS-gfp) third instar larvae shows that only 30% of cells in wing discs are positive for Green Fluorescent Protein (data not shown).

bcl-2 suppresses m collapse induced by bax and rpr, while p35 activity is specific to rpr

In order to characterize the effect of bcl-2 and p35 on m collapse specifically triggered by bax and rpr, we have studied the co-expression of bax or rpr with p35 or bcl-2

(figure 7B andC). Control discs and discs co-expressing rpr and bcl-2 display similar numbers of apoptotic cells (e.i. cells with a low m and a decrease in size) (16% and 15% respectively) (figure 7B). When rpr and p35 are co-expressed, only 13% more cells than in controls undergo apoptosis (figure 7C) compared to 21% more when only rpr is expressed.

These data argue that p35 protects more than half of the cells triggered to die by rpr, and bcl-2 completely restores wildtype mitochondrial phenotype.

In wing discs over-expressing bax, 22% more cells undergo apoptosis than in bax control discs. When bcl-2 and bax are co-expressed, only 6% more cells undergo apoptosis than in controls (figure 7C). In contrast, the percentage of cells undergoing apoptosis in discs co-expressing bax and p35 remains similar to discs expressing bax but not p35 (38% and 40% respectively) (figure 7B).Therefore, p35 has no effect on bax-induced cell shrinkage and m drop. Taken together these results demonstrate that both bcl-2 and p35 can protect cells from m drop and cell shrinkage, but while bcl-2 is effective against both bax-and rpr-induced defects, p35 protection is specific to rpr-induced effect.

Discussion:

To further characterize the genetic regulation of apoptosis, we have used the Additionally, the effects observed in our experiments are not likely to be mediated by the titration of the GAL4 activator by UAS elements. Indeed, we show that there is no additive effect on the phenotypes induced by UAS-rpr when the UAS-p35 transgene is added to one or two UAS-bcl-2 transgenes. Furthermore, two independent UAS-p35 insertions, alone or together, have no effect on the phenotypes induced by UAS-bax (data not shown). This method has proven to be very powerful in detecting gradual suppression effects that a classical dichotomy screen can not detect.

In this study, we have focused on the involvement of mitochondria as well as the bcl-2 gene family in apoptosis in Drosophila melanogaster. In mammals, bax triggers apoptosis by permeabilizing the outer mitochondrial membrane. This permeabilization is correlated with mitochondrial membrane potential (m) drop and cyt c release (Desagher & Martinou, 2000). rpr is also thought to act on mitochondria. Indeed, in Xenopus cells extracts, RPR binds to Scythe to induce cyt c release from purified mitochondria (Thress et al., 1999). We report here that expression of bax or rpr in the wing margin during development induces apoptosis, and flow cytometry experiments performed on dissociated wing disc cells show that bax and rpr trigger m drop and cell shrinkage in vivo in Drosophila. These results point to mitochondrial changes as common events in bax and rpr death pathways in Drosophila (figure 8). Moreover, bax and rpr do not induce necrosis and late apoptotic cells or bodies are likely phagocytosed in vivo as previously reported [START_REF] Milan | Proc. Natl. Acad. Sci. USA[END_REF].

Nevertheless, several features differentiate bax-and rpr-induced apoptosis. Although bax and rpr induce comparable decrease in wing disc size, massive AO staining, as well as comparable apoptogenic effect (about 20%), adult phenotypes are much stronger after bax expression. One explanation could be that bax-induced apoptosis is irreversible while some of the cells initially triggered by rpr and showing m collapse ultimately do not undergo apoptosis. Since AO staining is prominent in rpr as well as bax expressing discs, and given that AO likely stains cells which are committed to apoptosis, this hypothesis appears unlikely.

In addition, as shown by AO staining in situ, the wing pouch of third instar larvae is more affected by bax expression than by rpr expression. This result suggests that earlier during development, cells in the wing disc respond differently to both death inducers. Since cells of the wing pouch give rise to the adult wing blade (Bate & Martinez Arias, 1993), it is likely that adult flies expressing bax present stronger wing phenotypes. Finally, when cell death is induced in the wing during larval and pupal development, the adult phenotype is also dependent on compensation mechanisms triggered in order to recover normal wing [START_REF] Milan | Proc. Natl. Acad. Sci. USA[END_REF]. Therefore, we can not exclude that this process respond differently to bax-and rpr-induced apoptosis.

It has already been shown that rpr-induced apoptosis in the eye is caspase-dependent (Hay et al., 1995). To test the requirement of caspases in bax and rpr apoptotic pathways in the wing, we have studied their genetic interactions with the caspase-inhibitor p35. We show here that cell death triggered by rpr in the wing involves cell shrinkage and mitochondrial defects, and that this process is caspase-dependent. In contrast, p35 has no effect on mitochondrial changes triggered by bax. Moreover, we have never observed any inhibitory effect of p35 on bax-induced apoptosis in the wing by using two independent p35-transgenic lines, and a third line carrying two copies of p35. The inability of p35 to suppress the bax death pathway may be due to the activation of p35-insensitive caspases or to the activation of caspase-independent events. In mammals, it remains controversial whether bax-induced cell death is caspase-dependent (Okuno et al., 1998;[START_REF] Vekrellis | [END_REF][START_REF] Xiang | Proc. Natl. Acad. Sci. USA[END_REF]. In Drosophila, it is also not clear whether the pro-apoptotic bcl-2 family gene dbok/debcl acts in a caspase-dependent death pathway [START_REF] Chen | [END_REF]Zimmermann et al., 2002). In our experiments, bcl-2 inhibits bax-as well as rpr-induced phenotypes in a dose-dependent manner, and bcl-2 inhibits m drop and cell shrinkage induced by bax and rpr in vivo.

Taken together, our data suggest that bax and rpr induce two different death pathways, though mitochondrial defects as well as the inhibition by bcl-2 constitute two common features of rpr-and bax-induced apoptosis (figure 8).

Taking into account what occurs in vitro, we hypothesize that bcl-2 can inhibit rprand bax-induced apoptosis by regulating cyt c-dependent DAPAF-1 activation. But in this case, since we have shown that p35 does not counteract bax, the caspases regulated by DAPAF-1 would be p35-insensitive. DREDD cleavage (possibly by auto-activation), DRONC activity in the eye as well as STRICA-induced cell death are not inhibited by p35 (Chen et al., 1998;Doumanis et al., 2001;Hawkins et al., 2000). Therefore, these caspases are good candidates for potential members of a bax-induced cell death pathway. In addition,

consistent with what occurs in mammals (Okuno et al., 1998), we show that in Drosophila, bcl-2 suppresses caspase-dependent and -independent apoptotic mitochondrial events and apoptosis.

Our results are the first evidence of a protective activity of bcl-2 against rpr-induced apoptosis in vivo in Drosophila. Since bcl-2 can prevent caspase-independent cell death in mammals (Okuno et al., 1998), we have studied the effect of the co-expression of bcl-2 and p35 on rpr-induced adult wing phenotypes. We show that there is no additive effect of p35 and bcl-2 when they are co-expressed. This data suggests that bcl-2 and p35 do not inhibit completely different pathways, and that bcl-2 could affect the activation of p35-sensitive caspases downstream of rpr expression. Furthermore, we show that p35 suppresses m collapse induced by rpr, which suggests that p35-sensitive caspases act on mitochondria (figure 8). This data is in agreement with Varkey et al. who have shown that rpr-induced cyt c modifications are caspase-dependent (Varkey et al., 1999). Nevertheless, we do not know whether the action of these p35-sensitive caspases initiates a mitochondrial apoptotic pathway, or whether it is the result of positive feedback involving these caspases, which would accelerate mitochondrial dysfunction.

Finally, while bcl-2 displays anti-apoptotic properties related to an activity downstream of rpr, we also show here that bcl-2 can act by regulating rpr transcriptional induction. This is the first evidence of the action of the bcl-2 gene family on the expression of rpr. In regards to the current knowledge of the genetic regulation of apoptosis, and considering our results, it is likely that the human gene bcl-2 acts on mitochondria in Drosophila to inhibit apoptosis triggered by rpr or by bax (figure 8). However, exactly how bcl-2 down-regulates rpr expression remains to be addressed. One can ask whether this effect is cell-autonomous. Indeed, in embryos, cells committed to die may sensitize neighbor cells and increase their level of rpr product. By decreasing the number of apoptotic cells, bcl-2 would reduce rpr expression. Nevertheless, since p35 has no effect on rpr expression during embryonic development, it is unlikely that the effect of bcl-2 is due to such a non cell-autonomous activity.

Until recently, two death signals were known to be involved in the regulation of rpr expression. First, rpr is a transcriptional target of the steroid hormone ecdysone during metamorphosis (Baehrecke, 2000), but to our knowledge, no interaction between bcl-2 and steroid-induced gene expression has been described. Second, it has been shown that rpr is a transcriptional target of dmp53 after X-ray irradiation (Brodsky et al., 2000). Similarly, in mammals, the transcription factor NF-kappaB is involved in cell proliferation as well as in oncogenesis (Rayet & Gelinas, 1999), and among the many target genes of NF-kappaB are p53 and c-myc. In Drosophila, a death domain protein that promotes apoptosis, imd/rip, and a homologue of a mammalian member of the death domain receptor pathway, dfadd, have been recently characterized (Georgel et al., 2001;Hu & Yang, 2000). Both of them interact with dredd/caspase-8, which suggests that a death domain receptor pathway that utilizes caspase-mediated NF-kappaB activation of rpr transcription could regulate apoptosis (figure 7). Our results suggest that this feedback control of rpr expression should be sensitive to bcl-2 but not to p35. Whether bcl-2 activates or inhibits NF-kappaB to regulate apoptosis remains controversial (De Moissac et al., 1998;Grimm et al., 1996;Hour et al., 2000). Since rpr expression is affected by imd/rip, dmp53 and bcl-2, genetic interaction studies between these genes will be of great interest to better understand the link between the rel/NF-B gene family and the regulation of apoptosis by p53, bcl-2 and death domain receptors such as the TNF-receptors in Drosophila, and in mammals. More generally, since no anti-apoptotic bcl-2 has been studied in Drosophila, further studies of the human anti-apoptotic bcl-2 gene in the fly will give new insights into the regulation of apoptosis by the bcl-2 family, and delineate new strategies for the design of chemotherapeutic apoptosis inducers which are insensitive to the bcl-2 oncogene.

Materials and methods:

Fly stocks:

Flies were raised on standard medium. Both driver strains used in this study, vestigial-GAL4 (vg-GAL4) and daughterless-GAL4 (da-GAL4) as well as the (UAS-gfp) strain (UAS: Upstream Activating Sequence) were described previously (Gaumer et al., 2000). The strains carrying UAS-p35, UAS-bax and UAS-bcl-2 transgenes have been generated in our laboratory and were described previously (Gaumer et al., 2000). The rpr-11-LacZ construct was first described by Nordstrom et al [START_REF] Nordstrom | [END_REF]. The fly stock carrying the UAS-rpr transgene was generated by recombination of the X-linked (UAS-rpr, UAS-hid) strain described previously [START_REF] Zhou | Proc. Natl. Acad. Sci. USA[END_REF]. We used w 1118 Canton S fly stock as the reference strain.

rpr suppression study:

Males (UAS-rpr/Y), (UAS-rpr/Y;vg-GAL4/CyO) or (UAS-rpr/Y;UAS-p35) were crossed at 25°C to females of the following strains: (vg-GAL4), (vg-GAL4;UAS-bcl-2), (UAS-bcl-2;vg-GAL4;UAS-bcl-2), (UAS-bcl-2;UAS-bcl-2;UAS-bcl-2). The progeny were then raised at 25°C and females expressing UAS-rpr were analyzed within the first day after eclosion to prevent wings from being damaged.

bax suppression study:

Females (vg-GAL4,UAS-bax/CyO-gfp) (GFP: Green fluorescent protein) were crossed at 18°C to males of the following strains: (w 1118 ), (UAS-bcl-2), (UAS-p35),

(UAS-bcl-2/Y;UAS-bcl-2;UAS-bcl-2). The progeny were raised at 18°C and analyzed every day.

Classification of the phenotypes and the Wilcoxon test:

The principle of the phenotype classification was different in the rpr suppression study and in the bax suppression study. Note that in both sets of experiments, all the lineages were analyzed in parallel and by a blind observer. In the rpr suppression study, flies were classified in four different phenotypic categories according to their wing phenotype: strong, intermediate, weak and wildtype (figure 1). For flies showing two different wings, the strongest phenotype of both wings was used for the classification. In the bax suppression study, flies were classified in the «strong» category when both wings had strong phenotypes, in the «weak» category when both wings had weak phenotypes and in the «intermediate» category when it did not belong to either the «strong» or «weak» category (figure 2).

In our experiments we have analyzed the distribution of graded phenotypes based on their expressivity. The Wilcoxon test was used to compare the distributions of the phenotypes between two lineages A and B (Wilcoxon, 1945) (table 1, 3 and4). The sign of WsA-B value determines whether the distribution A is stronger than B (Ws<0) or whether the distribution B is stronger than A (Ws>0). We considered the difference between A and B significant when

A-B<10 -3 .

Staining of embryos with X-gal:

Females (UAS-bcl-2;da-GAL4) or (da-GAL4) were crossed to males (rpr-11-LacZ) at 25°C. Embryos were dechorionated 5 min in 1X bleach and rinsed in water. Embryos were fixed 10 min in 1X PBS pH 7.6, 3.7% formaldehyde/heptane (1:2) and washed four times in 1X PBS pH 7.6, 0.3% triton X-100. The embryos were stained for 15 to 25 min in 1X PBS pH 7.6, K4Fe2 4 mM, K3Fe3 4 mM, MgCl2 1 mM, 1% triton X-100, X-gal 560 µg.ml -1 (Eurogentec), fixed 3 min in 1X PBS pH 7.6, 3.7% formaldehyde and washed three times in 1X PBS pH 7.6, 0.05% triton X-100. The embryos were swelled over night in 1X PBS pH 7.6, 0.05% triton X-100/glycerol (40:60) and finally mounted in glycerol.

Embryos were staged according to Campos-Ortega and Hartenstein (Campos-Ortega & Hartenstein, 1985). Samples were observed with a conventional research microscope DMRHC Leica.

-galactosidase activity assay:

Females (UAS-bcl-2;da-GAL4), (UAS-p35;da-GAL4) or (da-GAL4) were crossed to males (rpr-11-LacZ) at 25°C. Embryos were collected at different development times A.E.L and harvested in 500µl buffer Z (described by Tzou et al. (Tzou et al., in press)). The final concentration of proteins in extracts was 138 ± 87 µg.ml -1 (n=81), and the final concentration of ONPG (Sigma) for the -galactosidase activity assay was 0.35 mg.ml -1 . The kinetics of ONPG hydrolysis were measured over 6h. The activity assays for each genotype analyzed and at all development time segments were performed nine times independently (table 2). The measured activities were compared using the statistical Student test.

Detection of vg-GAL4 expression in wing discs:

Females (vg-GAL4) were crossed to (UAS-gfp) males. (vg-GAL4/UAS-gfp) lineage was raised at 25°C and third instar larvae were dissected and mounted in 1X PBS pH 7.6. Discs were then observed with a conventional research microscope DMRHC Leica using the L5 filter to detect green fluorescence.

AO staining of wing discs:

Females (UAS-rpr) or (UAS-bax,vg-GAL4/CyO-gfp) were crossed respectively to (vg-GAL4) or (w 1118 ) males. (UAS-rpr/+;vg-GAL4/+) lineage was raised at 25°C and (UAS-bax,vg-GAL4/+) as well as (w 1118 ) lineage were raised at 21°C. Third instar larvae were dissected in 1X PBS pH 7.6 and stained for 2 minutes in (100 ng.ml -1 ) Acridine Orange (Molecular Probes). Discs were then mounted in PBS and observed with a conventional research microscope DMRHC Leica using the I3 filter to detect green fluorescence.

Flow cytometry analysis:

Males (UAS-rpr/Y) or (UAS-bax/CyO-gfp) were crossed respectively at 25°C and 21°C to females of the following strains: (w 1118 ), (vg-GAL4), (UAS-bcl-2;vg-GAL4;UAS-bcl-2), (vg-GAL4;UAS-p35). Progeny were raised at the same temperatures. Wing discs from third instar female larvae were dissected in 1X PBS pH 7.6, and pooled in Schneider's Drosophila medium (GibcoBRL). Six discs were pooled per sample. The discs were then incubated in 150 µL Trypsine (GibcoBRL) for 1h30 at 28°C. The reaction was stopped by adding 10 µL Fetal Calf Serum (FCS). The samples were stained at room temperature in Schneider's Drosophila medium for 30 min in 0.1 µM DiOC6(3), and for 5 min in 50 µg.mL -1 Propidium Iodide (PI) (final volume of 500 µL). Samples were preserved on ice before analysis. The cationic lipophilic fluorochrome DiOC6(3) (Molecular Probes) is a cell permeable marker which at low doses (0.1 µM), specifically accumulates in mitochondria in mammalian cells in proportion to m (Petit et al., 1990). PI (Sigma) is a non specific DNA intercalating agent which is excluded by the plasma membrane of living cells.

Flow cytometry measurements were performed on a XL3C flow cytometer (Beckman-Coulter France). Fluorescence excitation was obtained using the blue wavelength The -galactosidase activity has been measured 9 times independently (n=9).  values presented here are given by the comparison of the -galactosidase activity of each genotype analyzed with the activity of the control genotype of the same development time laps.  values noted with * are superior to the statistical significant limit fixed at 0.05.  values noted with * are superior to the significant limit fixed at 1x10 -3 . The genotypes of the considered distributions are numbered from 1 to 4 (n, grey cells). N specifies the number of flies analyzed. Ws6-8= -10.3 6-8<1x10 -13

Ws7-8= -6.0 7-8= 4.9 x10 -9

 values noted with * are superior to the significant limit fixed at 1x10 -3 . The genotypes of the considered distributions are numbered from 5 to 8 (n, grey cells). N specifies the number of flies analyzed. All flies considered express one copy of rpr in the wing as in control (UAS-rpr/X;vg-GAL4/+), and one copy of bcl-2 (UAS-rpr/X;vg-GAL4/+;UAS-bcl-2/+), two copies of bcl-2 (UAS-rpr/UAS-bcl-2;vg-GAL4/+;UAS-bcl-2/+) or three copies of bcl-2 (UAS-rpr/UAS-bcl-2;vg-GAL4/UAS-bcl-2;UAS-bcl-2/+) in (A); one copy of p35 (UAS-rpr/X;vg-GAL4/UAS-p35), one copy of bcl-2 or one copy of bcl-2 plus one copy of p35 (UAS-rpr/X;vg-GAL4/UAS-p35;UAS-bcl-2/+) in (B); two copies of bcl-2, one copy of p35 or two copies of bcl-2 plus one copy of p35 (UAS-rpr/UAS-bcl-2;vg-GAL4/UAS-p35;UAS-bcl-2/+) in (C). The statistical Wilcoxon test was used to compare the phenotype distributions (table 1). (A) bcl-2 partially suppresses the phenotypes induced by rpr in a dose-dependent manner. The strength of the phenotypes induced by rpr using the vg-GAL4 driver decreases as the number of bcl-2 copies increases. (B) and (C), p35 suppresses rpr-induced phenotypes in the wing, but no additive effect on the suppression of rpr-induced phenotypes is observed when bcl-2 and p35 are co-expressed. We have used the rpr-11-LacZ reporter gene [START_REF] Nordstrom | [END_REF] to study rpr expression during embryonic development in embryos expressing bcl-2. (A), (C), (E) and (G), embryos expressing the UAS-bcl-2 transgene under da-GAL4 control (ubiquitous expression). The approximate times of development and the corresponding stages of development are mentioned on the left and on the right of the figure respectively. The -galactosidase activity in these embryos is compared to that in control embryos at similar stages (B), (D), (F) and (H). The appearance of -galactosidase activity is delayed in bcl-2 embryos until stage 9. At stage 11 and 14, the pattern of rpr-11-LacZ is comparable in bcl-2 embryos (E,G) and in control embryos (F,H), but the -galactosidase activity is weaker in embryos expressing bcl-2. All views are sagittal with anterior to the left and dorsal towards the top. Scale bar, 100 µm. 5A and 5B. All flies considered express one copy of bax in the wing as in control (vg-GAL4,UAS-bax/+) and one copy of bcl-2 (vg-GAL4,UAS-bax/UAS-bcl-2), two copies of bcl-2 in males (Y/+;vg-GAL4,UAS-bax/UAS-bcl-2;UAS-bcl-2/+), three copies of bcl-2 in females (UAS-bcl-2/X;vg-GAL4,UAS-bax/UAS-bcl-2;UAS-bcl-2/+) or one copy of p35 (vg-GAL4,UAS-bax/UAS-p35).The phenotype distributions for the different lineages are compared to each other with the statistical Wilcoxon test (table 3 and4 can be separated into two populations: one with both high fluorescence and large cell size (noted H) and the other with lower fluorescence and reduced cell size (noted L). The percentage of cells in each population is specified. Note that while rpr expression induces a loss of mitochondrial membrane potential and a reduction of cell size, PI fluorescence is not affected.

(B) The same analyses have been performed with (UAS-rpr/X;vg-GAL4/+;UAS-p35/+) and

(UAS-rpr/UAS-bcl-2;vg-GAL4/+;UAS-bcl-2/+), bax control (UAS-bax/+), (UAS-bax/vg-GAL4), (UAS-bax/vg-GAL4;UAS-p35/+) and (UAS-bcl-2/X;UAS-bax/vg-GAL4;UAS-bcl-2/+) larvae. The percentage of cells in populations described in (A) are presented in the table (B) for the different cell samples.

(C) The histograms present the percentage of cells showing a mitochondrial membrane potential decay in window L (low DiOC6(3) fluorescence) after percentage of the control has been deducted. In these experiments, bcl-2 can clearly block loss of mitochondrial membrane potential induced by both rpr and bax whereas p35 only affects changes in mitochondrial potential triggered by rpr. The results obtained have been successfully reproduced. (Georgel et al., 2001;Hu & Yang, 2000) and 5) Bcl-2 down-regulates rpr expression. This last effect of Bcl-2 might be mediated through the indirect inhibition of Rel/NF-B.

  have used Propidium Iodide (PI) which penetrates only cells with a loss of membrane integrity, like necrotic cells or late apoptotic cells, as a specific indicator of cell membrane integrity. We have assessed mitochondrial membrane potential using DiOC6(3), a lipophilic cation taken up by mitochondria in proportion to m, and cell shrinkage was analyzed by observing cell size reduction. Results presented for the study of bax-induced apoptosis and for the study of rpr-induced apoptosis are taken from independent experiments. In both studies, assays on cells isolated from discs carrying the death inducer without a driver (control discs) or from discs expressing the death inducer alone or co-expressing the death inducer and p35 or bcl-2 have been performed in parallel. Only cytograms corresponding to cells of genotype (UAS-rpr/X) and (UAS-rpr/X;vg-GAL4/+) are presented (figure7A). For each sample analyzed, cell debris and aggregates as well as PI-positive cells are excluded from the DiOC6(3) analysis.In rpr control discs (figure7Aa), 6% of cells show high PI fluorescence (window P) and PI-negative cells with no major membrane injuries split into two distinct populations based on their DiOC6(3) fluorescence and their size (figure7Ab): 84% of cells have a large size and high DiOC6(3) fluorescence (figure7Ab, window H), while other dots in window L characterize cells with a reduced size and m drop. These 16% of cells in window L exhibit characteristics of apoptotis: they undergo m collapse and cell shrinkage while their plasma membrane is not permeable. Moreover, as demonstrated below, expression of each death inducer rpr or bax increases this apoptotic population. Thus, we observe a background level of apoptotic cells in control discs, which is in agreement with in situ observation of normal developmental apoptosis by AO staining of wildtype wing discs (figure6B). In addition, the dissociation process, performed to obtain individual disc cells, probably deprives cells of survival signals, thus accelerating commitment to apoptosis (see materials and methods).

  UAS/GAL4 system to express mammalian regulators of apoptosis in Drosophila. Specifically, we have studied the genetic interactions between the human anti-apoptotic gene bcl-2 with the Drosophila death pathway controlled by rpr. In addition, we have compared apoptosis induced by the Drosophila gene rpr and the murine gene bax in the wing margin during development. Since both killers induce a notch phenotype with variable expressivity, we have set a semi-quantitative approach to compare distributions of gradual phenotypes between different genotypes. As the classification of the phenotypes might depend on the observer, the suppression analysis has only been performed on progeny from crosses performed in parallel and carefully analyzed by a single blind observer. Therefore, we have not compared distributions from independent sets of experiments. However, we have analyzed the distributions of wing phenotypes of flies expressing bax in six independent experiments, and 5/6 distributions compared using the Wilcoxon test were statistically similar. Only one distribution analyzed was statistically different from four others (data not shown).

(

  488nm) of an argon ion laser operating at 15mW. Green fluorescence of DiOC6(3) was collected with a 525nm band pass filter and red fluorescence of PI with a 620nm band pass filter. Analyses were performed on 10 4 cells and data were stored in listmode. Light scattering values were measured on a linear scale of 1024 channels and fluorescence intensities on a logarithmic scale of fluorescence.

Figure 1 :

 1 Figure 1: rpr induces variable phenotypes in the wing. At 25°C, the phenotypes induced by UAS-rpr driven by vg-GAL4 are variable. We classified flies into four categories based on the strength of their wing phenotype: wildtype (A), weak (B), intermediate (C) and strong (D) phenotypes. In our experiment, the three mutant categories are defined as follows: (A) wildtype wings are similar to these of the Canton S w 1118 reference strain. Note that the wing in (A) is from the Canton S w 1118 strain. (B) Wings with weak phenotypes miss only bristles on the proximal posterior margin (arrow head). (C) Wings with intermediate phenotype bear small notches, generally localized on the proximal posterior part of the wing margin. (D) The notches on wings with strong phenotypes are larger extending from the posterior proximal part to the distal part of the wing. (A) Scale bar, 500 µm.

Figure 2 :

 2 Figure 2: bax induces variable phenotypes in the wing. At 18°C, the phenotypes induced by UAS-bax driven by vg-GAL4 are variable. Considering the notches appearing on altered wings, we classified flies into three categories (B,C and D) according to the strength of their wing phenotypes. (A) wildtype Canton S w 1118 fly wing. (B) The size of a wing with a weak phenotype is almost conserved when compared to wildtype, and the notches are scattered along the margin. (C) The size of a wing with an intermediate phenotype is reduced, and an important part of the margin has disappeared. (D) The strong phenotype is comparable to the vestigial phenotype. The wing is partially absent or completely absent, sometimes the flies show thorax abnormalities. Scale bar, 500 µm.

Figure 3 :

 3 Figure 3: Analyses of the distributions of the phenotypes induced by rpr. The percentages of flies listed in each phenotypic category (strong, intermediate, weak and wildtype) are reported in (A), (B) and (C). All flies considered express one copy of rpr in the wing as in control(UAS-rpr/X;vg-GAL4/+), and one copy of bcl-2 (UAS-rpr/X;vg-GAL4/+;UAS-bcl-2/+), two copies of bcl-2 (UAS-rpr/UAS-bcl-2;vg-GAL4/+;UAS-bcl-2/+) or three copies of bcl-2 (UAS-rpr/UAS-bcl-2;vg-GAL4/UAS-bcl-2;UAS-bcl-2/+) in (A); one copy of p35 (UAS-rpr/X;vg-GAL4/UAS-p35), one copy of bcl-2 or one copy of bcl-2 plus one copy of p35 (UAS-rpr/X;vg-GAL4/UAS-p35;UAS-bcl-2/+) in (B); two copies of bcl-2, one copy of p35 or two copies of bcl-2 plus one copy of p35 (UAS-rpr/UAS-bcl-2;vg-GAL4/UAS-p35;UAS-bcl-2/+) in (C). The statistical Wilcoxon test was used to compare the phenotype distributions (table 1). (A) bcl-2 partially suppresses the phenotypes induced by rpr in a dose-dependent manner. The strength of the phenotypes induced by rpr using the vg-GAL4 driver decreases as the number of bcl-2 copies increases. (B) and (C), p35 suppresses rpr-induced phenotypes in the wing, but no additive effect on the suppression of rpr-induced phenotypes is observed when bcl-2 and p35 are co-expressed.

Figure 4 :

 4 Figure 4: rpr repression during embryonic development.We have used the rpr-11-LacZ reporter gene[START_REF] Nordstrom | [END_REF] to study rpr expression during embryonic development in embryos expressing bcl-2. (A), (C), (E) and (G), embryos expressing the UAS-bcl-2 transgene under da-GAL4 control (ubiquitous expression). The approximate times of development and the corresponding stages of development are mentioned on the left and on the right of the figure respectively. The -galactosidase activity in these embryos is compared to that in control embryos at similar stages (B), (D), (F) and (H). The appearance of -galactosidase activity is delayed in bcl-2 embryos until stage 9. At stage 11 and 14, the pattern of rpr-11-LacZ is comparable in bcl-2 embryos (E,G) and in control embryos (F,H), but the -galactosidase activity is weaker in embryos expressing bcl-2. All views are sagittal with anterior to the left and dorsal towards the top. Scale bar, 100 µm.

Figure 5 :

 5 Figure 5: Analyses of the distributions of the phenotypes induced by bax. The percentages of flies listed in each phenotypic category (strong, intermediate and weak) are reported in figure5Aand 5B. All flies considered express one copy of bax in the wing as in control (vg-GAL4,UAS-bax/+) and one copy of bcl-2 (vg-GAL4,UAS-bax/UAS-bcl-2), two copies of bcl-2 in males (Y/+;vg-GAL4,UAS-bax/UAS-bcl-2;UAS-bcl-2/+), three copies of bcl-2 in females (UAS-bcl-2/X;vg-GAL4,UAS-bax/UAS-bcl-2;UAS-bcl-2/+) or one copy of p35 (vg-GAL4,UAS-bax/UAS-p35).The phenotype distributions for the different lineages are compared to each other with the statistical Wilcoxon test (table3 and 4).(A) In females, bcl-2 partially suppresses the phenotypes induced by bax in a dose-dependent manner while p35 has no effect. (B) Similarly, in males, bcl-2 suppresses the phenotypes induced by bax in a dose-dependent manner while p35 has no effect.

  Figure 5: Analyses of the distributions of the phenotypes induced by bax. The percentages of flies listed in each phenotypic category (strong, intermediate and weak) are reported in figure5Aand 5B. All flies considered express one copy of bax in the wing as in control (vg-GAL4,UAS-bax/+) and one copy of bcl-2 (vg-GAL4,UAS-bax/UAS-bcl-2), two copies of bcl-2 in males (Y/+;vg-GAL4,UAS-bax/UAS-bcl-2;UAS-bcl-2/+), three copies of bcl-2 in females (UAS-bcl-2/X;vg-GAL4,UAS-bax/UAS-bcl-2;UAS-bcl-2/+) or one copy of p35 (vg-GAL4,UAS-bax/UAS-p35).The phenotype distributions for the different lineages are compared to each other with the statistical Wilcoxon test (table3 and 4).(A) In females, bcl-2 partially suppresses the phenotypes induced by bax in a dose-dependent manner while p35 has no effect. (B) Similarly, in males, bcl-2 suppresses the phenotypes induced by bax in a dose-dependent manner while p35 has no effect.

Figure 6 :

 6 Figure 6: Apoptosis induced by bax and rpr in wing discs. (A) vg-GAL4 drives the expression of GFP in an extended zone of (vg-GAL4/UAS-gfp) wing disc that corresponds essentially to the future hinge and the future margin of the wing. (B), (C), (D), AO staining of wing discs of third instar larvae. (B), low apoptosis is observed in (w 1118 ) wildtype discs, and most of this apoptosis is localized in the notum. (C), Massive apoptosis is induced in the area of vg-GAL4 expression in (UAS-rpr/+;vg-GAL4/+) wing discs, and (D) in (UAS-bax,vg-GAL4/+) wing discs. However, cells in the wing pouch are differently affected by bax and rpr. All discs are shown with anterior to the top. Scale bar, 100 µm.

Figure 7 :

 7 Figure 7: p35 and bcl-2 affect mitochondrial changes induced by bax or rpr expression in wing discs. (A) Multiparametric analysis by flow cytometry of cells extracted from third instar larvae discs. Each dot represents one cell. The data shown are relative to cells extracted from rpr control (UAS-rpr/X) larvae (a, b) or from (UAS-rpr/X;vg-GAL4/+) larvae (c, d). For each sample analyzed, size and membrane integrity, as measured by PI fluorescence (reported in cytograms PI versus size) as well as the mitochondrial membrane potential, as measured by DiOC6(3) fluorescence (reported in cytogram DiOC6(3) versus size) were recorded. PI cytograms are separated into two windows, P (upper window) and N which designate PI-positive and PI-negative fluorescent cells respectively. For analysis of DiOC6(3) fluorescence, only PI-negative cells are considered in multiparametric cytograms, and cells

Figure 8 :

 8 Figure 8: Hypothetical model of apoptosis control in Drosophila. This model takes into account the following points : 1) mitochondria are a target of Bax and RPR in Drosophila, 2) unlike rpr, bax induces apoptosis through a p35-insensitive pathway 3) Bcl-2 probably acts by counteracting mitochondrial defects triggered by bax and rpr, 4) a pathway homologous to the TNF death pathway may exist in the fly(Georgel et al., 2001;Hu & Yang, 2000) and 5) Bcl-2 down-regulates rpr expression. This last effect of Bcl-2 might be mediated through the indirect inhibition of Rel/NF-B.

Table 1 : Ws values and  values of the Wilcoxon test in the rpr suppression study.

 1

	genotypes analyzed	n	1	2	3	4	5	6
	control (N=217)
	UAS-rpr/X;vg-GAL4/+	1
	1 copy bcl-2 (N=210)		Ws1-2= -4.1
	UAS-rpr/X;vg-GAL4/+;UAS-bcl-2/+	2	1-2= 4.9x10 -5
	2 copies bcl-2 (N=176)
	UAS-rpr/UAS-bcl-2;vg-GAL4/+;
	UAS-bcl-2/+

Table 2 :

 2 Quantification of the -galactosidase activity during embryonic development.

	development	genotypes analyzed	-galactosidase activity	standard	 value
	time laps			(mmol ONPG/min/µg prot)	deviation
		control	(rpr-11-lacZ/da-GAL4)	0.9	0.8	1*
	0-3h	1 copy p35	(UAS-p35/+;rpr-11-lacZ/da-GAL4)	0.7	0.8	0.48*
		1 copy bcl-2 (UAS-bcl-2/+;rpr-11-lacZ/da-GAL4)	0.6	0.5	0.31*
		control		2.8	0.9	1*
	3-6h	1 copy p35		3.2	1.4	0.46*
		1 copy bcl-2		1.6	0.7	0.01
		control		17.4	6.4	1*
	6-9h	1 copy p35		18.1	8.3	0.85*
		1 copy bcl-2		7.5	5.7	3.2x10 -3

Table 3 : Ws values and  values of the Wilcoxon test for the female lineages in the bax suppression study.

 3

Table 4 : Ws values and  values of the Wilcoxon test for the male lineages in the bax suppression study.

 4

	males analyzed	n	5	6	7
	control (N=67)
	vg-GAL4,UAS-bax/+	5
	1 copy p35 (N=64)		Ws5-6= 2.6
	vg-GAL4,UAS-bax/UAS-p35	6	5-6= 0.01*
	1 copy bcl-2 (N=88)		Ws5-7= -4.4	Ws6-7= -6.6
	vg-GAL4,UAS-bax/UAS-bcl-2	7	-= 1.3x10 -5	-= 1.6 x10 -10
	2 copies bcl-2 (N=117)		Ws5-8= -9.0
	vg-GAL4,UAS-bax/UAS-bcl-2;	8	5-8<1x10 -13
	UAS-bcl-2/+

Acknowledgements:

We are grateful to Dr G. Lecellier for expert help with statistics. We also thank E.

Blanchardon who provided us the (UAS-rpr) strain, Imène Seninet for technical help, Dr L.

Theodore for helpful discussions and M.R. Watson for her critical reading of the manuscript. This work was supported in part by grants from the Association pour la Recherche contre le Cancer (#4480) and from the Ligue Contre le Cancer (Comité des Yvelines). S. Brun was a recipient of a fellowship from the Ministère de l'Education Nationale et de l'Enseignement Supérieur et de la Recherche. S. Gaumer was supported by a fellowship from the Association pour la Recherche contre le Cancer.