Filipe Pinto-Teixeira

Clara Koo

Anthony Michael Rossi

Nathalie Neriec

Claire Bertet

Xin Li

Alberto Del-Valle-Rodriguez

Claude Desplan

Development of concurrent retinotopic maps in the fly motion detection circuit

Keywords: Drosophila, retinotopy, pattern formation, direction selective neurons, optic lobe, neural development, neural connectivity, Notch

Understanding how complex brain wiring is produced during development is a daunting challenge. In Drosophila, information from 800 retinal ommatidia is processed in distinct brain neuropiles, each subdivided into 800 matching retinotopic columns. In the Lobula Plate there are four T4 and four T5 neuronal subtypes. T4 neurons respond to bright edge motion while T5 neurons to dark edge motion, and each is tuned to motion in one of the four cardinal directions, effectively establishing eight concurrent retinotopic maps to support wide-field motion. We discovered a mode of neurogenesis where two sequential Notch-dependent divisions of either a horizontal or a vertical progenitor produces matching sets of two T4 and two T5 neurons retinotopically coincident with pair-wise opposite direction selectivity. We show that retinotopy is an emergent characteristic of this neurogenic program and derives directly from neuronal birth order. Our work illustrates how simple developmental rules can implement complex neural organization.

In Brief

The circuit for motion perception emerges out of the developmental program that specifies the identity of the neurons

Introduction

A central question in neuroscience is how individual neurons are assembled into functional neural networks. In both vertebrates and invertebrates, visual input from the retina is retinotopically mapped onto the brain such that neighboring pixels are represented as neighboring regions on the retinotopic map. An example of such complex organization is found in the Drosophila optic lobe, which receives retinotopic input from photoreceptors in the eye [START_REF] Fischbach | The optic lobe of Drosophila melanogaster. I. A Golgi analysis of wild-type structure[END_REF][START_REF] Morante | The color-vision circuit in the medulla of Drosophila[END_REF]. The compound eye of the fly is made up of ~800 ommatidia (unit eyes). Photoreceptors from each ommatidium send their axons into the optic lobe, which is comprised of four neuropiles that each contains ~800 columns: the Lamina, Medulla, Lobula and Lobula Plate (Figure 1A-C) [START_REF] Bausenwein | The optic lobe of Drosophila melanogaster. II. Sorting of retinotopic pathways in the medulla[END_REF][START_REF] Fischbach | The optic lobe of Drosophila melanogaster. I. A Golgi analysis of wild-type structure[END_REF][START_REF] Morante | The color-vision circuit in the medulla of Drosophila[END_REF][START_REF] Takemura | Synaptic circuits of the Drosophila optic lobe: the input terminals to the medulla[END_REF]. Each column processes visual information from one point in space, maintaining retinotopy (Morante andDesplan, 2004, 2008).

The retinotopic organization of the fly visual system is crucial for circuit function, as exemplified by motion detection circuits. Within the optic lobe, visual motion information is processed in two parallel pathways, the ON pathway detecting bright edge motion, and the OFF pathway that processes dark edge motion [START_REF] Clark | Defining the computational structure of the motion detector in Drosophila[END_REF][START_REF] Eichner | Internal structure of the fly elementary motion detector[END_REF][START_REF] Joesch | ON and OFF pathways in Drosophila motion vision[END_REF][START_REF] Joesch | Functional specialization of parallel motion detection circuits in the fly[END_REF]. The two pathways bifurcate early since distinct lamina neurons, the first to make contact with photoreceptors, connect to different sets of Medulla neurons, which themselves then synapse onto dendrites of T4 neurons (ON) in the Medulla, or T5 neurons (OFF) in the Lobula (Figure 1A-C) [START_REF] Behnia | Processing properties of ON and OFF pathways for Drosophila motion detection[END_REF][START_REF] Clark | Defining the computational structure of the motion detector in Drosophila[END_REF][START_REF] Rister | Dissection of the peripheral motion channel in the visual system of Drosophila melanogaster[END_REF][START_REF] Shinomiya | Candidate neural substrates for off-edge motion detection in Drosophila[END_REF][START_REF] Silies | Modular use of peripheral input channels tunes motion-detecting circuitry[END_REF][START_REF] Takemura | A visual motion detection circuit suggested by Drosophila connectomics[END_REF]. T4 and T5 neurons are the first neurons in each pathway that are direction selective [START_REF] Maisak | A directional tuning map of Drosophila elementary motion detectors[END_REF]. They process the visual signal originating from one main column and integrate it with ~7 neighboring columns to compute local motion [START_REF] Fisher | Orientation Selectivity Sharpens Motion Detection in Drosophila[END_REF][START_REF] Leong | Direction Selectivity in Drosophila Emerges from Preferred-Direction Enhancement and Null-Direction Suppression[END_REF][START_REF] Strother | The Emergence of Directional Selectivity in the Visual Motion Pathway of Drosophila[END_REF][START_REF] Takemura | A visual motion detection circuit suggested by Drosophila connectomics[END_REF][START_REF] Takemura | The comprehensive connectome of a neural substrate for 'ON' motion detection in Drosophila[END_REF].

Both T4 and T5 neurons exist in four subtypes (termed a,b,c,d), directionally tuned to one of the four cardinal directions (front-to-back, back-to-front, upward, downward) (Figure 1B). Thus, for each column, four T4 and four T5 neurons, one of each subtype, represent eight independent motion detectors. T4 (ON) and T5 (OFF) neurons with the same directional tuning project retinotopically to one of the four layers of the Lobula Plate [START_REF] Maisak | A directional tuning map of Drosophila elementary motion detectors[END_REF] that is organized into two layers for horizontal motion (layer a: front-to-back; layer b: back-to-front) and two layers for vertical motion (layer c: upward; layer d: downward) (Figure 1B). Within each layer, T4 and T5 neurons synapse with the dendrites of Lobula Plate Tangential Cells [START_REF] Mauss | Optogenetic and pharmacologic dissection of feedforward inhibition in Drosophila motion vision[END_REF][START_REF] Schnell | Columnar cells necessary for motion responses of wide-field visual interneurons in Drosophila[END_REF] that integrate the retinotopic local motion signals from T4 and T5 neurons to produce direction selective wide-field motion responses. Thus, the retinotopy of the T4/T5 circuit is crucial for detecting broad field motion: at the level of T4 (Medulla) and T5 (Lobula) dendrites, where the retinotopic organization of the inputs onto T4/T5 dendrites allows direction selectivity to first emerge [START_REF] Fisher | Orientation Selectivity Sharpens Motion Detection in Drosophila[END_REF][START_REF] Strother | The Emergence of Directional Selectivity in the Visual Motion Pathway of Drosophila[END_REF][START_REF] Takemura | A visual motion detection circuit suggested by Drosophila connectomics[END_REF], and at the level of their axons, where retinotopic organization allows for efficient, selective coding of specific global motion patterns [START_REF] Barnhart | Multilayered non-linear integration of local motion cues underpins efficient coding of global motion patterns in Drosophila[END_REF][START_REF] Mauss | Neural Circuit to Integrate Opposing Motions in the Visual Field[END_REF]. It is therefore critical that the correct number of each T4 and T5 neuronal subtype be produced so that each Medulla column is innervated by the four T4 neuronal subtypes and each Lobula column by the four T5 subtypes. Furthermore, all eight subtypes of T4 and T5 neurons must project retinotopically to individual layers of the Lobula Plate.

The four neuropiles of the optic lobes develop during the larval and early pupal stages from two crescent-shaped neuroepithelial domains: the Outer Proliferation Center (OPC), which produces neurons of the Lamina and Medulla, and the Inner Proliferation Center (IPC), which generates neurons of the Lobula and Lobula plate (Figure 1D,E,F and Movie 1) [START_REF] Apitz | A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system[END_REF][START_REF] Egger | Regulation of spindle orientation and neural stem cell fate in the Drosophila optic lobe[END_REF][START_REF] Hofbauer | Proliferation pattern and early differentiation of the optic lobes in Drosophila melanogaster. Roux's archives of developmental biology : the official organ of the[END_REF][START_REF] Nassif | Early development of the Drosophila brain: III. The pattern of neuropile founder tracts during the larval period[END_REF][START_REF] Ngo | Spatio-temporal pattern of neuronal differentiation in the Drosophila visual system: A user's guide to the dynamic morphology of the developing optic lobe[END_REF]. The IPC crescent is localized between the OPC and the developing central brain (Figure 1D,E and Movie 1). It is divided into three domains: the surface IPC (sIPC) marked by Wingless expression (which will not be discussed further in this work) that is attached to the proximal IPC (pIPC), and a distal domain (dIPC) (Figure 1D). T4/T5 neurons are produced by progenitors that originate from the pIPC [START_REF] Apitz | A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system[END_REF]. Unlike the OPC neuroepithelium that is sequentially converted by a proneural wave into neuroprogenitors (neuroblasts (NBs) in the fly) [START_REF] Egger | Regulation of spindle orientation and neural stem cell fate in the Drosophila optic lobe[END_REF][START_REF] Egger | Notch regulates the switch from symmetric to asymmetric neural stem cell division in the Drosophila optic lobe[END_REF][START_REF] Reddy | Influence of fat-hippo and notch signaling on the proliferation and differentiation of Drosophila optic neuroepithelia[END_REF], the pIPC-neuroepithelium produces Dichaete + migrating progenitors that move distally to generate the dIPC (Figures 1D,E,G,H andMovie 2) [START_REF] Apitz | A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system[END_REF]. Once migrating progenitors reach the dIPC, they acquire a NB identity and divide to produce neurons (Figure 1E,I,J,K,L).

dIPC NBs progress through two temporal windows [START_REF] Apitz | A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system[END_REF]. First, Dichaete + NBs divide to self-renew and produce the distal C2, C3, T2 and T3 neurons (C/T neurons) to the outside of the dIPC crescent (Figure 1H, J, L) [START_REF] Apitz | A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system[END_REF]. In the second temporal window, these NBs express Atonal (Ato) and Dachshund (Dac), and produce T4 and T5 neurons to the inside of the crescent (Figure 1I,J,K,L) [START_REF] Apitz | A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system[END_REF][START_REF] Oliva | Proper connectivity of Drosophila motion detector neurons requires Atonal function in progenitor cells[END_REF].

We investigated the developmental program that establishes the identity of the four T4 and four T5 neuronal subtypes and how this program leads to their eight coincident retinotopic maps. We identified a causal link between a mode of neurogenesis and retinotopy in which a single NB produces two ON and two OFF neurons with opposite motion direction selectivity (along the horizontal or the vertical axis) that innervate a single column in three neuropiles. We also show that vertical and horizontal T4/T5 motion detectors are produced by different NBs distinguished by Decapentaplegic (Dpp) activity. We conclude that retinotopy results from the features of this neurogenic program, which depends on neuronal birth order and a unique mode of NB division to pattern a complex and highly organized neural network. Thus, simple developmental rules can generate a complex neural organization across three neuropils of the optic lobes.

Results

Vertical and horizontal motion sensitive T4 and T5 neurons originate from two distinct populations of progenitors

The dIPC is formed by migrating progenitors produced by distinct pIPC-neuroepithelial domains expressing either Dpp or Brinker (Brk) [START_REF] Apitz | A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system[END_REF]. The two Dppneuroepithelial domains are labeled by dpp-Gal4 [START_REF] Apitz | A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system[END_REF] (Figure 2A,B) and by R45H05-Gal4 (termed Dpp-pIPC-Gal4) (Figure S1). The complementary Brk (a negative regulator of the Dpp pathway) domain sits between the two Dpp domains and is labeled by brk-lacZ and brk-Gal4, but does not express dpp-Gal4 (Figure 2A,B and Figure S1). Mosaic analysis with a repressible cellular marker (MARCM) and live imaging revealed that, even though cells delaminated from specific pIPC-neuroepitelial locations (Figure 1D,E,G and Movie 1), they spread along the entire dorso-ventral axis when reaching the dIPC such that Brk and Dpp derived NBs distributed along the entire dIPC dorsal-ventral axis (Figure 2A-D).

We hypothesized that Dpp signaling compartmentalizes the neuroepithelium and that the Brk and Dpp domains produce distinct subsets of T4 and T5 neurons. Upon binding Dpp, its receptor Thickveins (Tkv) phosphorylates the transcriptional effector Mad (pMad) that in turn regulates target gene expression. A lacZ reporter for Tkv was expressed within both the Dpp-and Brk-positive neuroepithelial domains (Figure 2E,F). However, pMad was only present in the Dpp domains, showing that the Dpp pathway is only active in the Dpp compartments of the neuroepithelium (Figure 2G,H). To test the role of Dpp in the specification of neuroepithelial identity, we used dpp-Gal4 to express a UAS-dpp-RNAi transgene; this led to the loss of pMad and to the expansion of the brk-lacZ reporter into the Dpp domains (Figure 2I). We also overexpressed a constitutively active form of the Tkv receptor (UAS-Tkv*) in the entire pIPC-neuroepithelium using the pIPC-specific driver line R35B01-Gal4 (termed pIPC-Gal4) (Figure S1). This resulted in the loss of brk-lacZ expression and the expansion of pMad to the Brk domain (Figure 2J).

To test the contribution of the Brk-and Dpp-neuroepithelial domains to the production of T4 and T5 subtypes, we used memory cassettes [START_REF] Bertet | Temporal patterning of neuroblasts controls Notch-mediated cell survival through regulation of Hid or Reaper[END_REF] in combination with the T4/T5 specific line R42F06-lexA (termed T4/T5-lexA) [START_REF] Schnell | Columnar cells necessary for motion responses of wide-field visual interneurons in Drosophila[END_REF]. Activation of the memory cassette in early larvae (L2) within the Brk-neuroepithelial domain (using brk-Gal4) labeled all T4/T5 neurons of the horizontal system (subtypes a and b) (Figure 3A,C), while activation in the Dpp-neuroepithelial domains (using dpp-pIPC-Gal4) labeled all T4/T5 neurons of the vertical system (subtypes c and d) (Figure 3B,C). This demonstrates that the motion-detecting T4/T5 neurons of the horizontal and vertical systems derive from distinct neuroepithelial domains.

Given that Dpp signaling established neuroepithelial identity, we hypothesized that Dpp signaling specifies the identity of horizontal and vertical system neurons. To test this hypothesis we overexpressed Tkv* in the entire pIPC-neuroepithelium from late L2 until early pupae using the pIPC-specific driver line R17B05-Gal4 (Figure S1) and visualized the neurons produced using T4/T5-lexA. Expression of Tkv* resulted in pMad expression in the entire pIPC neuroepithelium and in all migrating chains (Figure 3D,E), and in the production of a Lobula Plate consisting of only c and d layers (compare Figure 3F to 3G) that are specifically labeled by the cell adhesion molecule Connectin (compare Figure 3H to 3I) [START_REF] Gao | The neural substrate of spectral preference in Drosophila[END_REF].

Altogether, these observations show that Dpp signaling establishes the identity of the Brkand Dpp-neuroepithelial domains, which give rise to horizontal (Brk) and vertical (Dpp) motion sensitive T4/T5 neurons, respectively.

Notch signaling specifies T4 (OFF) versus T5 (ON) neuronal identity

Upon delamination, migrating progenitors reach the dIPC where they assume a NB identity (Figure 1L). dIPC NBs first express Dichaete and divide to self-renew and produce Pros + GMCs that generate neurons with C/T identity (Figure 1H,J,L) [START_REF] Apitz | A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system[END_REF]. These NBs then age and express Ato and Dac, and exclusively produce T4 and T5 neurons (Figure 1,H,I,J,K,L) [START_REF] Apitz | A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system[END_REF][START_REF] Oliva | Proper connectivity of Drosophila motion detector neurons requires Atonal function in progenitor cells[END_REF]. We observed that newborn Ato + /Dac + /Pros + GMCs were distributed in a salt and pepper manner surrounding T4/T5 Ato + /Pros -NBs (Figure 4A, Figure S3). The GMCs lost Ato expression by the time they divided to produce T4 and T5 neurons (Figure S3). During a typical GMC division, Notch signaling triggers a binary fate decision establishing the distinct identity of its two neuronal progeny: a Notch ON and a Notch OFF neuron [START_REF] Bertet | Temporal patterning of neuroblasts controls Notch-mediated cell survival through regulation of Hid or Reaper[END_REF][START_REF] Buescher | Binary sibling neuronal cell fate decisions in the Drosophila embryonic central nervous system are nonstochastic and require inscuteable-mediated asymmetry of ganglion mother cells[END_REF]Li et al., 2013b;[START_REF] Truman | Role of Notch signaling in establishing the hemilineages of secondary neurons in Drosophila melanogaster[END_REF]. To test the involvement of Notch in the specification of T4 versus T5 neuronal identity, we first determined the Notch status of developing neurons by monitoring the expression of the bHLH-O protein Hairy/enhancer-ofsplit-related with YRPW motif (Hey), which is a reliable sensor of Notch activity in optic lobe neurons [START_REF] Bertet | Temporal patterning of neuroblasts controls Notch-mediated cell survival through regulation of Hid or Reaper[END_REF]. We found that Hey was expressed in a salt and pepper manner in early born T4/T5 neurons (Figure 4B). This suggests that dIPC NBs that are Ato + /Dac + produce GMCs that divide once to produce two neurons with alternative identities dictated by their Notch status. To test this hypothesis, we generated MARCM and Twin-Spot MARCM clones during the larval and early pupal stages that labeled the two progeny of Ato + /Dac + GMCs with distinct fluorescent proteins [START_REF] Yu | Twin-spot MARCM to reveal the developmental origin and identity of neurons[END_REF]. We visualized them in the adult using R42F06-Gal4 (termed T4/T5-Gal4). All 2-cell clones (N=15/15) contained one T4 and one T5 neuron of the same subtype, i.e. they projected to the same Lobula Plate layer at the same retinotopic position (Figure 4C,D and Figure S2).

To further test the role of Notch in the specification of T4 vs. T5 identity, we analyzed Ato + /Dac + GMC Notch mutant MARCM clones, which consistently showed two T4 neurons of the same subtype projecting to the same retinotopic position (N=32/32). This was true for GMCs derived from both horizontal and vertical system NBs (Figure 4E,F,S2). This shows that T4 and T5 neurons of the same subtype (a, b, c, or d) are siblings and that T4/T5 neurogenesis relies on four distinct GMC populations, each producing a T4 and a T5 neuron of a unique subtype: a or b originating from the Brk region, and c or d originating from the Dpp region (Figure 4G).

Atonal NBs undergo a terminal division to produce two GMCs that generate T4 and T5 neurons detecting motion in opposite directions

To investigate how T4/T5a neurons are distinguished from T4/T5b neurons within the horizontal system, and how T4/T5c neurons are distinguished from T4/T5d neurons within the vertical system, we characterized the mode of division of dIPC NBs. NBs can proliferate in distinct modes during development [START_REF] Kohwi | Temporal fate specification and neural progenitor competence during development[END_REF]Li et al., 2013a): Most divide to self-renew and to produce a smaller GMC that divides once to produce two daughter cells ("type I" NBs) [START_REF] Buescher | Binary sibling neuronal cell fate decisions in the Drosophila embryonic central nervous system are nonstochastic and require inscuteable-mediated asymmetry of ganglion mother cells[END_REF]. Others self-renew and generate a ganglion mother cell (GMC) that does not divide, but differentiates directly into a neuron ("type 0" NBs) [START_REF] Bertet | Temporal patterning of neuroblasts controls Notch-mediated cell survival through regulation of Hid or Reaper[END_REF][START_REF] Karcavich | Drosophila neuroblast 7-3 cell lineage: a model system for studying programmed cell death, Notch/Numb signaling, and sequential specification of ganglion mother cell identity[END_REF][START_REF] Ulvklo | Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression[END_REF]. "Type II" NBs selfrenew and produce intermediate precursors that divide multiple times themselves to generate GMCs that then divide only once [START_REF] Bello | Amplification of neural stem cell proliferation by intermediate progenitor cells in Drosophila brain development[END_REF][START_REF] Boone | Identification of Drosophila type II neuroblast lineages containing transit amplifying ganglion mother cells[END_REF][START_REF] Bowman | The tumor suppressors Brat and Numb regulate transit-amplifying neuroblast lineages in Drosophila[END_REF].

To divide asymmetrically to self-renew and produce a GMC, NBs rely on the asymmetric localization of cell fate determinants that are differentially segregated between the two progeny [START_REF] Homem | Drosophila neuroblasts: a model for stem cell biology[END_REF]. Prospero is a determinant of GMC identity that represses the expression of NB determinants (i.e. Deadpan) and of cell cycle genes [START_REF] Choksi | Prospero acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells[END_REF][START_REF] Lai | Transient nuclear Prospero induces neural progenitor quiescence[END_REF][START_REF] Southall | Neural stem cell transcriptional networks highlight genes essential for nervous system development[END_REF] to signal terminal differentiation [START_REF] Doe | The prospero gene specifies cell fates in the Drosophila central nervous system[END_REF]. Prospero expression initiates in NBs, where it is kept inactive by the adaptor protein Miranda that tethers it to the cortex. During a NB division, Miranda and Prospero form a crescent in the basal cell cortex: In this manner, Miranda and Prospero are inherited by the GMC after cell division [START_REF] Shen | Miranda is required for the asymmetric localization of Prospero during mitosis in Drosophila[END_REF]. Once in the GMC, Miranda is degraded, allowing Prospero to enter the nucleus to promote cell cycle exit and differentiation. The GMC then divides only once to produce two mitotically inactive cells [START_REF] Choksi | Prospero acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells[END_REF][START_REF] Hirata | Asymmetric segregation of the homeodomain protein Prospero during Drosophila development[END_REF][START_REF] Ikeshima-Kataoka | Miranda directs Prospero to a daughter cell during Drosophila asymmetric divisions[END_REF][START_REF] Southall | Neural stem cell transcriptional networks highlight genes essential for nervous system development[END_REF].

NB proliferation ends either when the NB undergoes apoptosis, or when the NB itself starts expressing Prospero and terminally divides. To test how Ato + NBs end their life, we inhibited apoptosis using a mutation of the initiator caspase-9, dronc. This did not lead to NB accumulation (Figure S4), suggesting that Ato + NBs stop proliferation by a terminal division.

To assess the mode of dIPC NB divisions, we examined the expression of the proneural factor Asense (Ase), which is expressed in "type I" NBs and their GMCs, as well as the cellular localization of both Miranda and Prospero. C/T dIPC NBs (Dichaete + ) express Dpn and Ase (Figure S4), and always showed typical asymmetric cortically localized Miranda and Prospero crescents (N=15) (Figure 5A,B, Figure S4, Figure S5) [START_REF] Apitz | A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system[END_REF]. These NBs self-renewed and produced a smaller GMC that gave rise to C/T neurons (Figure 5C, Movie 3). In contrast, Ato + /Dac + NBs do not express Ase but instead express another proneural protein, Ato (Figure 1I, Figure S4) [START_REF] Apitz | A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system[END_REF][START_REF] Oliva | Proper connectivity of Drosophila motion detector neurons requires Atonal function in progenitor cells[END_REF]. Ato + /Dac + NBs showed lower levels of Miranda expression as compared to Dichaete + NBs (Figure S4). Nevertheless, they formed crescents of both Miranda and Prospero, although of weaker intensity than Dichaete + NBs (Figure 5A,B, Figure S4, Figure S5). To unambiguously assess for Prospero crescents, we analyzed NBs at prometaphase/metaphase in both the Dichaete + and Ato + temporal windows, when crescents are easiest to observe [START_REF] Doe | The prospero gene specifies cell fates in the Drosophila central nervous system[END_REF][START_REF] Shen | Miranda is required for the asymmetric localization of Prospero during mitosis in Drosophila[END_REF]. Virtually all Dichaete + (N=15/15) and Ato + NBs produced Prospero crescents (N=14/15) (Figure 5B). The formation of Prospero cortical crescents in Ato + NBs was further confirmed with a Pros::GFP line (Figure S6). However, these Ato + NBs did not self-renew: their progeny consisted of two Ato + /Dac + cells of similar size (Figure 5C, Movie 4), a cell that inherited cortical Prospero (and Numb, see below), and a cell that transiently retained Dpn but also expressed nuclear Prospero immediately after cell division (Figure 5D), becoming the second GMC product of the Ato + NB. This shows that Ato + NBs divide once to effectively produce two GMCs (Figure 5F).

To identify the progeny of single, terminally dividing Ato + NBs, we analyzed MARCM and Twin-spot-MARCM clones marked by T4/T5-Gal4. In all analyzed clones (N=9/9), two pairs of T4/T5 neurons, either from the horizontal (a and b) or from the vertical (c and d) subtypes, projecting to the same retinotopic location, were observed (Figure 5E and Figure S2). This demonstrates that when horizontal (Brk) and vertical (Dpp) system dIPC NBs reach the Ato + temporal window, they divide once to end their life, producing two sibling GMCs with different identities: a or b for Brk and c or d for Dpp (Figure 5F).

Atonal NBs divide to produce a Notch ON and a Notch OFF GMC When NBs divide, they not only segregate Prospero to the GMC, but also the Notch signaling inhibitor Numb [START_REF] Knoblich | Asymmetric segregation of Numb and Prospero during cell division[END_REF][START_REF] Rhyu | Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells[END_REF][START_REF] Wang | Polo inhibits progenitor self-renewal and regulates Numb asymmetry by phosphorylating Pon[END_REF] (Figure 6A). As a consequence, the NB remains Notch ON while the GMC becomes Notch OFF . We hypothesized that the distinct identities of sibling T4/T5 GMCs could be the result of a Notch-mediated binary fate decision: The terminally dividing NB would produce one Notch OFF GMC and another cell that retains its Notch ON identity and becomes the second GMC. This would establish the subtype identity (a vs. b or c vs. d) of the T4/T5 neurons from each GMC.. Consistent with this hypothesis, we observed that Ato + NBs asymmetrically segregated Numb together with Prospero only one progeny cell (Figure 6A). Furthermore, we found that among the Ato + /Pros + GMC population, we could identify GMCs that transiently expressed the Notch activity reporter E(spl)mγGFP [START_REF] Almeida | Regulation of post-embryonic neuroblasts by Drosophila Grainyhead[END_REF], corresponding to the Notch ON GMC formed from the Ato + NB (Figure 6B). To further test this hypothesis, we generated MARCM clones of Notch mutant NBs, which consistently produced clones of four T4 neurons (see above) that were all either of subtype b (from Brk + NBs) (N=5) or of subtype c (from Dpp + NBs) (N=9) (Figure 6C,D,E). These observations are consistent with a mode of neurogenesis in which a T4/T5 NB stops proliferation by dividing to produce two GMCs with a different Notch status: Notch first specifies the identity of the two sister GMCs (a vs. b or d vs. c), and then later the identity of sister neuronal progeny (T4 vs. T5) (Figure 6E).

Neuronal birth location and mode of neurogenesis establish a precise retinotopic map

During development, each T4 or T5 neuron must find its correct target location along the dorsal-ventral and anterior-posterior axes of its target neuropiles: the Medulla and Lobula Plate for T4 neurons, the Lobula and Lobula Plate for T5 neurons. In the medulla, all unicolumnar neurons from a single column, such as the Medulla intrinsic 1 (Mi1) neuron, are produced by a single NB located on top of this developing column [START_REF] Erclik | Integration of temporal and spatial patterning generates neural diversity[END_REF]Li et al., 2013b). This facilitates establishment of retinotopy as neurons are born within the column in which they will project. Indeed, the proneural wave that converts the OPC neuroepithelium into NBs is tightly synchronized with the morphogenetic furrow in the eye disc such that all NBs along one dorsal-ventral position are produced at the same time, matching the development of the eye [START_REF] Egger | Regulation of spindle orientation and neural stem cell fate in the Drosophila optic lobe[END_REF][START_REF] Egger | Notch regulates the switch from symmetric to asymmetric neural stem cell division in the Drosophila optic lobe[END_REF][START_REF] Ngo | Concomitant requirement for Notch and Jak/Stat signaling during neuro-epithelial differentiation in the Drosophila optic lobe[END_REF][START_REF] Sato | Notch-mediated lateral inhibition regulates proneural wave propagation when combined with EGF-mediated reaction diffusion[END_REF].

How T4 and T5 neurons project retinotopically to their targets is more difficult to understand. In contrast to OPC-derived neurons, T4 and T5 neurons are born far away from their first target neuropile without an obvious wave that could be synchronized with the eye morphogenetic furrow or OPC proneural wave: NBs delaminate from the pIPC, either from the Dpp or from the Brk domain, and migrate to establish the dIPC-NB crescent along the dorsal-ventral axis (Figure 2A-D). Furthermore, T4 and T5 axons have to cross the Lobula Plate, then project to the Medulla (T4) or Lobula (T5), and then come back to the Lobula Plate, terminating at the correct retinotopic location.

To address how retinotopy is established by the eight T4 and T5 neuronal subtypes, we investigated how they synchronize their targeting with target neuropile formation. We hypothesized that the mode of neurogenesis of Ato + NBs, which we showed synchronously produces the full complement of T4 and T5 neurons with opposite motion direction selectivity, allows T4/T5 neurons to establish retinotopic projections. We observed that all four T4/T5 neurons produced by one NB projected to the same retinotopic position in their target neuropiles (Figure 5E Figure S2). To understand how a complex system of eight coincident retinotopic maps (one for each T4 and each T5 of the a, b, c, or d subtypes) comprising a total of 6400 neurons between three neuropiles is established, we visualized how T4/T5 neurons project along the dorsal-ventral and antero-posterior axes of the developing target neuropiles. We stochastically labeled developing T4/T5 neurons with different fluorophore combinations using Multicolor FlpOut (MCFO) [START_REF] Nern | Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system[END_REF] in combination with ato-Gal4 that labels T4/T5 neurons during development. This revealed that newborn T4/T5 neurons project to newly forming columns of their target neuropiles according to their relative birth position along the dorsal-ventral axis (Figure 7A,B and Movie 5). Based on these observations, we used ato>GFP to perform live imaging of developing T4/T5 neurons and visualized how innervation along the dorsal-ventral and antero-posterior axes of the neuropiles is coordinated. We focused on T4 neuronal projections and found that newborn T4 neurons targeted newly formed Medulla columns along the entire dorsal-ventral axis, a process that is reiterated along the anterior-posterior axis of the developing Medulla (Figure 7C and Movie 6).

Consistent with these observations, activation of a memory cassette in the pIPC neuroepithelium using brk-Gal4 or dpp-pIPC-Gal4 in early L2 marked all T4/T5 neurons, while activation at progressively later development stages marked neurons at more anterior parts of the visual field (Figure 7D and S7). Thus, NB time of birth establishes a temporal axis: NBs born early in development produce neurons targeting columns corresponding to the posterior eye (older columns), while later born NBs produce neurons that process information from more anterior parts of the eye (younger columns) (Figure 7E). We note that dIPC NBs do not exhibit spatial organization along the temporal axis since dIPC NBs are continuously replaced by new NBs originating from the pIPC. In contrast, the proneural wave in the OPC converts neuroepithelium crescent to NBs from its outer edge towards its inner edge, building the Medulla along a temporal axis (Figure 1K,L and Figure 7E). Because the dIPC is located distally to the developing Medulla, newborn T4 neurons find the youngest Medulla column that was just formed. Therefore, the time of birth of a dIPC-NB establishes a temporal axis that is synchronous with the conversion of the OPC neuroepithelium. Because each T4/T5 NB produces the full complement of T4/T5 neurons for either the horizontal or vertical system, this ensures that as new target columns are added through time they are supplied with the correct number of each T4/T5 neuronal subtype. Our results are consistent with a model in which matching of neuronal birth location and a specific mode of neurogenesis establishes a retinotopic, coherent connection across all optic lobe neuropiles.

Discussion

As neurons are produced and their identities are specified, they must be precisely incorporated into neuronal circuits. Understanding how neurons are specified, how the developing brain orchestrates the correct targeting of a myriad of individual neurons, and in which way these two developmental processes are related, are difficult problems to solve. We addressed these by studying how each of the eight T4/T5 neuronal subtypes is specified and how their eight retinotopic maps are precisely established.

Typically, NBs change their transcription factor identity at each division. Neuronal progeny inherit this identity through an intermediate GMC to dictate their fate [START_REF] Doe | Temporal Patterning in the Drosophila CNS. Annual review of cell and developmental biology[END_REF][START_REF] Rossi | Timing temporal transitions during brain development[END_REF]. Here we identified a mode of neurogenesis that relies on two consecutive Notch binary fate decisions to produce four distinct T4/T5 neurons from a single NB temporal window. Because T4/T5 neurons with opposite motion direction selectivity for one retinotopic position are produced by a single NB at the same time, these four neurons innervate their target neuropiles synchronously, connecting with the same, newly-produced target column to establish retinotopy. If each of the four T4 and T5 neurons were produced independently, synchronization of their projection patterns between three neuropiles could be much more difficult to achieve. This would require the establishment of a deterministic spatiotemporal molecular code, such that each column would use a unique molecular code recognized by all the neurons that are supposed to target it. The stepwise, synchronous production of sibling retinotopic neurons described here reduces the target possibilities at each time point since the progeny of one NB always find the newest column produced in the Medulla or Lobula. Our results illustrate how the developmental program that specifies T4/T5 fate meets the functional requirements of the motion circuit by establishing coherent retinotopic maps within horizontal and vertical systems.

Such successive divisions that rely on the reutilization of the Notch pathway are reminiscent of the divisions of Drosophila Sensory Organ Precursors (SOPs). Although these cells are not bona fide NBs, SOPs divide in a Notch-dependent manner multiple times to first produce two distinct cells (pIIa and pIIb) that divide once (pIIa) or twice (pIIb) more to give rise to the full complement of cells that form the sensory organ, only one of which is a neuron [START_REF] Schweisguth | Asymmetric cell division in the Drosophila bristle lineage: from the polarization of sensory organ precursor cells to Notch-mediated binary fate decision[END_REF]. In olfactory sensilla, a similar precursor also appears to divide several times in a Notch-dependent fashion to produce up to four olfactory neurons, as well as sensilla cells. In this case, some of the four progeny die, producing 1, 2, 3 or in some rare cases four neurons per sensillum [START_REF] Endo | Notch signal organizes the Drosophila olfactory circuitry by diversifying the sensory neuronal lineages[END_REF].

In the case of T4/T5 neurogenesis, we demonstrated that Notch signaling is used in two consecutive divisions: After the final NB division, the Notch target in one of the two GMCs is E(spl)my (but not Hey), while in the GMC division, Hey (but not E(spl)my) marks only one of the early born neurons (T5). How Notch differs in these distinct contexts, and how such precise temporal control is established is not known. However, the observation that Notch signalling activates different reporters in different contexts and cell types [START_REF] Zacharioudaki | D. melanogaster: T4/T5-Gal4 (R42F06) BDSC Stock #41253 Fly: D. melanogaster: Ato-Gal4 B. Hassan N/A Fly: D. melanogaster: 20XUAS-6XGFP BDSC Stock #52262 Fly: D. melanogaster: pIPC-Gal (R35B01) BDSC Stock #49898 Fly: D. melanogaster: brk x47 A. Tomlinson N/A Fly: D. melanogaster: DPP-pIPC-Gal4 (R45H05) BDSC Stock #46529 Fly: D. melanogaster: Brk-Gal4(brk 3sB ) BDSC Stock #53707 Fly: D. melanogaster: tkv k16713 BDSC Stock #11191 Fly: D. melanogaster: UAS-Dcr-2 BDSC Stock #24650 Fly: D. melanogaster: UAS-dppRNAi BDSC Stock #36779 Fly: D. melanogaster[END_REF] supports the notion that differential transcriptional programs are activated in different cell types. Furthermore, Notch activity is rather transient, which helps explain how Notch signalling instructs different gene expression programs at each round of division.

A recent preprint on a similar topic [START_REF] Apitz | Spatio-temporal relays control layer specificity of motion-direction sensitive neurons in Drosophila[END_REF] is in line with our findings on the role of Dpp and Notch in the specification of the eight T4/T5 subtypes and shows that both Dac and Ato are required for the transition between neuroblasts competence states in the dIPC and for the switch to T4/T5 neuron formation. Another upcoming report [START_REF] Mora | A Temporal transcriptional switch governs stem cell division, neuronal numbers and maintenance of differentiation[END_REF] addresses the role of the temporal transition from Ase + to Ato + in dIPC neuroblasts and shows how Ato expression is required for subsequent neuronal differentiation of T4/T5 neurons. It further suggests that Ato + neuroblasts divide symmetrically to self-amplify before producing the T4/T5 progeny. However, the data reported above, including our precise lineage analysis, do not support such an amplification step that would disrupt the stoichiometry of production of the C/T neurons and eight T4/T5 subtypes.

The lineage of the T4/T5 direction-selective neurons suggests how motion circuitry and the optic lobe neuropiles themselves might have evolved. We found that horizontal and vertical motion selective neurons originate from two distinct pIPC-neuroepithelial domains whose identity is established by Dpp signaling. In the absence of Dpp signaling, Brk expression was expanded to the Dpp domains, suggesting that the default status of the neuroepithelium is to express Brk. Horizontal and vertical motion-selective neurons were produced by distinct progenitor pools and both rely on the special type of neurogenesis described above to produce their complement of T4/T5 neurons. The most parsimonious evolutionary history for this developmental program is that the Notch-mediated binary fate decisions that specify layers of the Lobula Plate with opposite tuning, as well as T4 (moving bright edges) vs. T5 (moving dark edges) fate, was implemented before the specification of horizontal and vertical motion-selective subtype identity. The ancestor might have only responded to horizontal motion (Brk) before splitting of the neuroepithelium occurred, allowing the acquisition of vertical motion vision (Dpp), perhaps when the animals developed the capacity for flight.

T4 and T5 neurons share morphological and functional similarities [START_REF] Haag | A common directional tuning mechanism of Drosophila motion-sensing neurons in the ON and in the OFF pathway[END_REF], but also important differences, such as the organization of their dendritic processes in the Medulla (T4) vs. Lobula (T5) where each subtype (a,b,c, and d) must be oriented according to its local motion direction preference [START_REF] Fisher | Orientation Selectivity Sharpens Motion Detection in Drosophila[END_REF][START_REF] Strother | The Emergence of Directional Selectivity in the Visual Motion Pathway of Drosophila[END_REF]. Dpp signaling and the two Notch binary fate decisions establish the specification of the four T4 and four T5 subtypes. Future studies will be required to understand how the dendrites of each subtype are properly organized.

Sensory maps and neural circuits are largely genetically "hard-wired" in Drosophila and are usually activity-independent [START_REF] Hassan | Beyond Molecular Codes: Simple Rules to Wire Complex Brains[END_REF][START_REF] Hiesinger | Activity-independent prespecification of synaptic partners in the visual map of Drosophila[END_REF][START_REF] Jefferis | Target neuron prespecification in the olfactory map of Drosophila[END_REF][START_REF] Kolodkin | Wiring visual systems: common and divergent mechanisms and principles[END_REF][START_REF] Langen | The Developmental Rules of Neural Superposition in Drosophila[END_REF]. Despite this developmental rigidity, we have a very limited understanding of how genetic programs drive developmental processes that are able to establish precise neural circuits. This study shows that the neurogenic program that specifies the identity of the eight T4/T5 neuron subtypes is also sufficient to establish the coherent retinotopy that supports global motion perception in the fly. It provides an example of how the establishment of connectivity within a neural circuit can only be fully understood in its developmental context. The existence of a causal link between the genetic program that specifies cell fate and the circuit these cells build provides an example of how a complex hard-wired neuronal circuit can be built from simple developmental rules.

STAR Methods

Contact for reagent and resource sharing

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Claude Desplan (cd38@nyu.edu).

Experimental Model and Subject Details

Fly Stocks-Flies were raised at 25°C and 60% humidity on standard cornmeal agar medium at a 12h light/dark cycle. Male and Female L3 larvae and 2-5 day old adult flies were analysed. No differences were observed between sexes. We used the following transgenic flies in this study (See Table S1 for more details); {} enclose individual genotypes, separated by commas: {yw;;}, {;UAS-CD8::GFP; T4/T5-Gal4 (R42F06)}, {;Ato-Gal4;} a gift from Bassem Hassan; {;Ato-Gal4; UAS-redStinger}, {;Ato-Gal4; 20XUAS-6XGFP}, {; UAS-redStinger; pIPC-Gal (R35B01)}, {; UAS-nls-GFP; pIPC-Gal Immunostaining Protocol-Larval or adult brains were dissected in 1XPBS and fixed in 4% Formaldehyde at room temperature for 20 min. Brains were then rinsed 3X times in PBX (1% PBS, 0.3% triton) and left to wash for at least 2 hours and then incubated in primary antibody solution overnight at 4°C. Brains were then rinsed 3X times and left to wash for at least 2 hours and then incubated in secondary antibody solution in PBX 2 hours (with added 2% horse serum) at room temperature. They were finally rinsed 3X and left to wash overnight in PBX and mounted in Slowfade.

For the staining of Miranda, Prospero and Numb crescents, an optimized fixation protocol and immunostaining was used:

Larval brains were fixed in 4% formaldehyde with 0.1 M PIPES (pH = 6.9), 0.3% Triton X-100, 20 mM EGTA, and 1 mM MgSO4 for 20 min at room temperature. Brains were then rinsed 3X times in PBX (1% PBS, 0.3% triton) and incubated in primary antibody solution in PBX (with added 2% horse serum) for 48 hours at 4°C. Brains were then rinsed 3X and left to wash for at least 8 hours and incubated in secondary antibody solution in PBX (with added 2% horse serum) overnight at 4°C. Brains where finally rinsed 3X wi th PBX and left to wash overnight at 4°C before mounting in Slowfade.

Multicolor Stochastic Labelling (MCFO)-"Multicolor FlpOut" (MCFO) labelling was carried out as described before [START_REF] Nern | Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system[END_REF]. Briefly, FLP recombinase expression was used to excise FRT-flanked transcriptional terminators from UAS reporter constructs carrying HA, V5 and FLAG epitope tags. L3 Larvae carrying Ato-Gal4 and MCFO-1 were raised at 25°C and heat shocked at 37°C for 15min, 8-12 hours before dissection and staining.

Live Imaging-Live imaging was performed in Eye-brain complexes from late L3 {;Ato-Gal4>20XUAS-6XGFP} or {;UAS-nls-GFP; pIPC-Gal (R35B01, BL49898)}. Larvae were dissected in imaging medium (Schneider's medium supplemented with 10% Fetal Bovine Serum, 1% Penicillin-Streptomycin and 0.25% Insulin) on ice. Eye-brain complexes were transferred to a cell culture petri dish, embedded in 0.4% low temperature gelling agarose. Once agarose solidified, eye-brain complexes were incubated in imaging medium for the duration of image acquisition. Image acquisition was done using an Olympus FV1000MPE multi-photon laser-scanning microscope (at 920nm).

Quantification and Statistical Analysis

Sample sizes were based on the standard of the field. Control or experimental specimens of the correct stage and genotype were randomly and independently selected from a pool of animals. Unless otherwise noted, we examined at least 10 brains for every genotype. In the main text "N" represents the number of samples analysed. Phenotypes were fully penetrant at all conditions. Data acquisition and analysis were not performed blinded as samples were identified based on their genotypes that were not limited in repeatability. When statistical analyses were performed the experimenter was blind to the genotypes analysed. The calculation of 95% confidence interval error bars and unpaired two-tailed Student's t test P values (Welch-corrected, not assuming equal s.d.) was performed using Prism 7.00 for Mac, GraphPad Software, La Jolla California USA. Data met the assumption of normality (D'Agostino-Pearson omnibus normality test).

Image Acquisition and Processing-Images were acquired using a Leica SP5 confocal using a 20X, 40X or 60X objective. When acquired, Z-stacks sections were of 0.6-2 μm intervals. Images were processed with Fiji [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF] software package. Image processing, when applied, was limited to correct global images brightness, global background subtraction and Despeckle. To allow the full visualization of the cell bodies and neuronal projections of adult MARCM and TWINSPOT T4/T5 Clones in a 2D image, stacks are shown as maximum intensity Z-projections. Z-projections were processed with the Fiji software package [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF]. Images were compiled in Illustrator (CC2014).

Highlights

•

A neuroblast produces 4 neurons detecting 2 directions of bright or dark edge motion

•

Distinct progenitors produce neurons processing vertical vs. horizontal motion.

• Notch specifies neurons responding to bright or dark edge motion.

•

This mode of neurogenesis establishes retinotopy in the fly global motion system. showed two pairs of sibling T4/T5 neurons of opposite motion direction sensitivity, projecting to the same retinotopic position in the neuropile. Arrow points to layer a and arrowhead to layer b of the Lobula Plate (See also Figure S2).

(F) Schematic illustrating dIPC neurogenesis. Ato + NBs divide to produce a GMC and a small cell that becomes a GMC.

Figure 1 .

 1 Figure 1. Development of the dIPC (A) Schematic of an adult optic lobe highlighting the ON (T4) and OFF (T5) pathways of the motion detection circuit. Visual input propagates retinotopically via T4 and T5 neurons to the four layers of the Lobula Plate. Dashed line shows detail in panel (B). (B) Four subtypes of T4 and of T5 neurons, each directionally tuned to one of the four cardinal directions, converge onto layers a and b of the horizontal system, or layers c and d of the vertical system in the Lobula Plate. (C) Dorsal cross-section of an adult brain in which R42F06-Gal4 (T4/T5-Gal4) drives membrane-GFP (green) expression in T4 and T5 neurons. T4 dendrites project to the Medulla and T5 to the Lobula (neuropiles labeled in grey by anti-Brp). (A, B, C) anterior is to the left. (D) Posterior view of a 3-D model of the developing larval Outer (purple) and Inner (grey) Proliferation Centers (OPC and IPC). Dashed arrow marks the boundary between the proximal-IPC (pIPC) and the surface IPC. Progenitors delaminate from the pIPC (grey) to become neuroblasts (NBs) in the distal-IPC (dIPC) (red). T4 and T5 neurons (green) are generated by dIPC NBs and form inside the dIPC crescent. See also Movie 1. (E, F) Red-Stinger driven by Ato-Gal4 labels T4/T5 neurons.(E) Posterior cross-section of a developing larval optic lobe. The pIPC-neuroepithelium is localized between the OPC and the central brain. Cells delaminating from the pIPC (arrowheads) migrate to form the dIPC-NBs (Dpn, red) that will generate T4 and T5 neurons

Figure 2 .

 2 Figure 2. IPC-derived migrating progenitors populate the dIPC (A) Lateral view of the pIPC-neuroepithelial Brk domain marked by brk-lacZ (red) flanked by Dpp domains marked by dpp-pIPC>GFP (green). Anterior is to the left. (B) Chains of migrating progenitors (arrows) emerging from the Dpp (green) or Brk domains (red). (C) Lateral view of the dIPC. Anterior is to the left. Dpp-derived NBs (arrows, green) and Brk-derived NBs (arrowheads, red) spread equally within the dIPC. T4/T5 neurons are located in the inside of the crescent. (D) Schematic summarizing the development of the dIPC. (E,F) The Dpp receptor Tkv is expressed in both the Dpp (E) and Brk domains (F). (G,H) Dpp signalling was active (pMad) in the Dpp (G), but not in the Brk domain (H). (I) Expression of dpp-RNAi in the Dpp domains lead to the expansion of brk-lacZ expression in the Dpp domain and loss of pMad. (J) Expression of an activated form of Tkv (Tkv*) downregulated brk-lacZ and leads to ectopic pMad in the Brk domain.

Figure 3 .

 3 Figure 3. Horizontal and Vertical motion detection systems originate from distinct neuroepithelial domains (A,B) Memory cassette for brk-Gal4 (A) and dpp-pIPC-Gal4 (B): The horizontal system (layers a and b of the Lobula Plate, arrowhead in (A)) emerged entirely from the Brk domain, while the vertical system (layers c and d, arrowhead in (B)) was generated from the Dpp domain. (C) Schematic of an adult brain representing horizontal, Brk-derived (red), and vertical sensing, Dpp-derived (green), T4 and T5 neurons. (D,E) R17B05-Gal4 was used to drive the expression of an activated form of Tkv (Tkv*) in the entire pIPC neuroepithelium, resulting in the expression of pMad (Red) in the entire pIPC neuroepithelium, migrating chains and dIPC progenitors. (F,G) Dorsal cross section of an adult wild type optic lobe (F) and an optic lobe where Tkv* was expressed in the pIPC neuroepithelium during development using R17B05-Gal4 (G). In (F) and (G) T4/T5-lexA drives the expression of membrane-GFP (green) in T4 and T5 neurons. (H,I) Overexpression of Tkv* in the pIPC produced a Lobula Plate neuropile with only two layers that express Connectin (Con) (arrowheads; magenta) (I), which specifically labels neurons innervating layers c and d (H).

Figure 4 .

 4 Figure 4. A binary, Notch-mediated cell fate decision specifies T4 vs. T5 neurons (A) Ato + /Pros + GMCs (arrowheads; blue and green) were distributed in a salt-and-pepper manner in the dIPC. (B) T4/T5 neurons marked by Acj6 (cyan) expressed the Notch sensor Hey (arrow; red) in a salt-and-pepper manner. (C,D) T4 and T5 neurons of the same subtype are sibling neurons and projected to the same retinotopic position in their target neuropiles (Brp, white). Twin-spot MARCM clone visualized with T4/T5-Gal4 of sibling T4 and T5 neurons of the horizontal (subtype b) [arrow in (C)], and vertical system (subtype d) [arrow in (D)]. See also Figure S2. (E,F) Notch mutant MARCM clones visualized with T4/T5-Gal4 of a dividing GMC produced two sibling T4 neurons of the same subtypes (arrow) for both the horizontal (E) and vertical systems (F). The two T4 neurons projected to the same retinotopic position. See also Figure S2.(G) Ato + NBs (yellow) produce GMCs that divide once to produce sibling T4 (Notch OFF ) and T5 (Notch ON ) neurons of the same subtype.

Figure 5 .

 5 Figure 5. Ato + /Dac + NBs divide symmetrically to produce two GMCs (A,B) First temporal window Dichaete + and second temporal window Ato + NBs localized Miranda (A) and Prospero (B) to the basal cortex during cell division. See also Figures S4 and S5. (C) Quantification of the mean size of the two progeny of Dichaete + and Ato + NBs at the end of telophase. Dichaete + NBs divided to self-renew (Bazooka, red) and produce a smaller GMC (Prospero, green)(see Movie 3). Ato + NBs produced two progeny (Bazooka, red and Prospero, green) of the same size that both assumed a GMC fate (see Movie 4). Data are represented as a scattered plot ± SD. Unpaired two-tailed Student's t test with Welch's correction. Dichaete + NBs progeny (t=10.79 df=7.338) and Ato + NBs progeny (t=1.164 df=9.994), Asterisk indicates P value<0.0001. (D) Division of an Ato + NB produced a GMC (not shown in image) and a cell that expressed perduring Deadpan and nuclear Prospero (arrowheads; green), becoming the second GMC. (E) A vertical system twin-spot MARCM clone of sister GMCs visualized with T4/T5-Gal4

Figure 6 .

 6 Figure 6. Ato + /Dac + neuroblasts terminally divide to produce two sister GMCs with different Notch identities (A) First temporal window Dichaete + and second temporal window Ato + NBs localized the Notch inhibitor Numb to the basal cortex during cell division. (B) Newborn Ato + /Pros + GMCs that did not inherit Numb transiently expressed the Notch reporter mγGFP (arrowheads). (C,D) Notch mutant MARCM clones visualized with T4/T5-Gal4 produced four T4 neurons projecting to the same retinotopic location, both of subtype b in the horizontal system (C), or subtype d for the vertical system (D) (see also Figure S2). (E) Schematic illustrating Ato + /Dac + neurogenesis. Two Notch binary fate decisions specify neuronal identity.

Figure 7 .

 7 Figure 7. Neurogenesis mode and neuronal birth location establish retinotopy (A,B) In a developing L3 larval brain, T4/T5 neurons projected to the neuropiles along the dorso-ventral axis according to their birth location. (A) Posterior view of MCFO labeled T4/T5 neurons. Ventral is to the left. (B) Anterior 3-D view of MCFO labeled T4/T5 neurons. Proximal is to the bottom. Neurons projected in the neuropiles along the dorsal-ventral axis according to their birth position. Ventrally born T4/T5 neurons projected ventrally in the neuropiles [arrow in (A)]. See also Movie 5. (C) Still pictures of a time-lapse movie highlighting T4 projections in the Medulla. Anterior view with proximal to the bottom. Neurons innervating columns along the same dorsoventral plane projected at the same time in the neuropiles. Dashed line follows the position of neurons that were targeting the medulla at T0 (see Movie 6). Arrows indicate the development along the anterior-posterior axis of the Medulla neuropile. (D) Activation of a memory cassette in the Brk-pIPC domain at late L2, mid-L3 and Late L3 and visualized in the Adult with T4/T5-Gal4 labeled neurons of the horizontal system along the antero-posterior axis (Arrowheads) depending on their time of birth (see also Figure S7). (E) 3-D Schematic illustrating the establishment of the retinotopy of T4 and T5 neurons.

Cell. Author manuscript; available in PMC 2019 April 05.

Acknowledgments

We would like to dedicate this manuscript to the memory of Jean-Philippe Grossier with whom we had extensive discussions on the establishment of retinotopy. We would like to acknowledge Laura Quintana-Rio for helping building the 3D model of the developing optic lobe; Chris Doe and Cheng-Yu Lee for sharing reagents and suggestions for improving the antibody staining protocol, and Sarah Bennert for help with the elaboration of the illustrations. We also thank the fly community, and especially the Doe lab and Janelia for gifts of antibodies and fly stocks, and the Desplan laboratory members for essential discussion and support. This work was supported by the Center for Genomics and Systems Biology of NYU Abu Dhabi and NIH grant R01 EY017916 to C.D. F. P-T., N.N. and A. D-V-R. were supported by the CGSB at NYUAD; C.B. by fellowships from EMBO (ALTF 680-2009) and HFSPO (LT000077/2010-L).

Code #713-545-147 Donkey Anti Chicken A488 Jackson Immunochemicals Code #703-545-155 Donkey Anti Rabbit A488 Jackson Immunochemicals Code #711-545-152 Donkey Anti Mouse A488 Jackson Immunochemicals Code #715-545-151 Dichaete Anti Guinea pig 488 Jackson Immunochemicals Code #706-545-148 Donkey Anti Mouse A555 Thermo Scientific Catalog #A-31570 Mouse Anti RFP MBL Code #M155-3 Rabbit Anti RFP MBL Code #PM005 Donkey Anti Rabbit A555 Thermo Scientific Catalog #A-31572 Donkey Anti Guinea pig Cy3 Jackson Immunochemicals Code #706-165-148 Donkey Anti Rat Cy3 Jackson Immunochemicals Code #712-165-153 Donkey Anti Guinea pig A647 Jackson Immunochemicals Code #706-605-148 Donkey Anti Rabbit A647 Jackson Immunochemicals Code #711-605-152 Donkey Anti Mouse A647 Jackson Immunochemicals Code #715-605-151 Donkey Anti Rat A647 Jackson Immunochemicals Code #712-605-153 Donkey Anti Rabbit 405 Jackson Immunochemicals Code #711-475-152 Cell. Author manuscript; available in PMC 2019 April 05.

The Supplementary Movie 2 depicting the development of the IPC was processed in Fiji [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF] by selecting the z-slice of interest for each time point and saving as an AVI file. The Supplementary Movies 3 and 4 depicting the division of Dichaete and Ato Neuroblasts, respectively, were processed in Fiji [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF]. For each movie the Z-slices of interest were concatenated as a stack and saved as an AVI file. The Supplementary Movie 5 depicting the 3-D view of MCFO labeled T4/T5 neurons was processed using Fiji 3D viewer [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF]. The Supplementary Movie 6 of a L3 larva optic lobe highlighting T4 projections in the developing Medulla was processed with Fluorender [START_REF] Wan | FluoRender: joint freehand segmentation and visualization for many-channel fluorescence data analysis[END_REF] and Fiji [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF] software packages. The Z-stack was initially opened in Fluorender to create a 4D view of the Data. The resulting TIFF File was opened in Fiji to add labels and saved as AVI file.

Generation of Three-dimensional Model of Developing Optic Lobe-Ato>Stinger optic lobes of third instar larvae were dissected and labeled with Dpn and E-cad. The optic lobe used for generating the three-dimensional model (Figure 1d) was mounted in a Posterior orientation. Sections at 0.6-μm intervals were collected with a Leica Sp5 Confocal. Isosurfaces were produced with Fiji/TrakEM2 by manually outlining the regions of interest throughout the stack: Opc neuroepithelium, IPC, progenitors cell streams, dIPC neuroblasts and T4/T5 neurons cell bodies. Segmented objects were visualized into a model using the Fiji 3D viewer [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF].

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

KEY RESOURCES


MA Dedee F Murrell
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MD Victoria P Werth

Diagnosis and management of pemphigus: Recommendations of an international panel of experts

Background-Several European countries recently developed international diagnostic and management guidelines for pemphigus, which have been instrumental in the standardization of pemphigus management.

Objective-We now present results from a subsequent Delphi consensus to broaden the generalizability of the recommendations.

Methods-A preliminary survey, based on the European Dermatology Forum and the European Academy of Dermatology and Venereology guidelines, was sent to a panel of international experts to determine the level of consensus. The results were discussed at the International Bullous Diseases Consensus Group in March 2016 during the annual American Academy of Dermatology conference. Following the meeting, a second survey was sent to more experts to achieve greater international consensus.

Results-The 39 experts participated in the first round of the Delphi survey, and 54 experts from 21 countries completed the second round. The number of statements in the survey was reduced from 175 topics in Delphi I to 24 topics in Delphi II on the basis of Delphi results and meeting discussion.

Limitations-Each recommendation represents the majority opinion and therefore may not reflect all possible treatment options available.

Conclusions-We present here the recommendations resulting from this Delphi process. This international consensus includes intravenous CD20 inhibitors as a first-line therapy option for moderate-to-severe pemphigus.

there is no defined standard of care owing to the paucity of large-scale clinical trials evaluating their efficacy. [START_REF] Mimouni | Differences and similarities among expert opinions on the diagnosis and treatment of pemphigus vulgaris[END_REF] There have been recent national attempts to standardize the diagnosis and management of pemphigus from individual countries, including in the United Kingdom, France, Japan, and Germany. [START_REF] Joly | Pemphigus. Guidelines for the diagnosis and treatment. Centres de reference des maladies bulleuses auto-immunes[END_REF][START_REF] Amagai | Committee for Guidelines for the Management of Pemphigus Disease[END_REF][START_REF] Eming | S2k guidelines for the treatment of pemphigus vulgaris/foliaceus and bullous pemphigoid[END_REF][START_REF] Harman | Guidelines for the management of pemphigus vulgaris[END_REF] However, it was the European Dermatology Forum and the European Academy of Dermatology and Venereology that passed the first international guidelines for the management of pemphigus. [START_REF] Hertl | Pemphigus. S2 guideline for diagnosis and treatment-guided by the European Dermatology Forum (EDF) in cooperation with the European Academy of Dermatology and Venereology (EADV)[END_REF] Although these efforts have been instrumental in the standardization of pemphigus management, the lack of involvement from countries outside of Europe may render these guidelines nongeneralizable to other countries.

In an attempt to garner greater international consensus, the International Bullous Diseases Consensus Group, convened by Dr Dedee Murrell and Dr Victoria Werth, met in March 2016 at the annual American Academy of Dermatology conference in Washington, DC, with the goal of developing international consensus guidelines for the diagnosis and management of pemphigus vulgaris and pemphigus foliaceus. Before the meeting, members of the group, which comprised experts in blistering diseases, completed a Delphi survey based on the European Dermatology Forum and European Academy of Dermatology and Venereology guidelines. Some of the tests and treatments mentioned may not be available or officially registered in all countries and have been assessed on the basis of their scientific usefulness rather than regulation status. The Delphi technique is a consensus-building process in which questionnaires are given to a group of experts in a series of rounds to ultimately achieve opinion convergence. [START_REF] Hsu | The Delphi technique: making sense of consensus[END_REF] The results of the questionnaire were discussed in the meeting and a follow-up survey was sent out to further consensus.

METHODS

The first round of surveys was delivered via email in February 2016 and completed by 39 expert participants. The results of the survey were tallied and delivered to the group. A median score of 70 percent or greater per question was used as the consensus threshold for agreement, and a median score of 30 or lower was established as the consensus threshold for disagreement. Statements that achieved median scores between 30 and 70 were determined as having reached no consensus among participants and discussed during the meeting. Afterward, these statements were revised according to the opinion of the participants and sent out and completed by 54 individuals in the subsequent round. The survey was designed and distributed using RedCAP software.

INITIAL CLINICAL PRESENTATION OF PEMPHIGUS

The initial evaluation of suspected pemphigus should seek to determine the signs or symptoms present that would corroborate the diagnosis of pemphigus, as well as to screen for possible comorbidities.

Major objectives

•

To verify the diagnosis of pemphigus

•

To evaluate possible risk factors, severity factors, and comorbidities

•

To specify the type of initial involvement (skin, mucosa) and its extent

•

To evaluate the prognosis depending on the age of the patient and general condition (Karnofsky score is optional)

•

There are 2 clinical scores, the Pemphigus Disease and Area Index (PDAI) and/or Autoimmune Bullous Skin Intensity and Severity Score (ABSIS), which are currently being used as clinical outcome parameters and in clinical trials for the evaluation of the extent and activity of pemphigus Presently, there are no agreed-on cutoff values to define mild, moderate, or severe disease for either the PDAI or the ABSIS; however, there have been 2 studies that have attempted to define these values. In 1 multicenter study based in Japan, researchers evaluated both patients with newly diagnosed and patients with relapsing pemphigus and determined PDAI cutoff values of 0 to 8 for mild, 9 to 24 for moderate, and 25 or higher for severe disease. [START_REF] Shimizu | Grading criteria for disease severity by pemphigus disease area index[END_REF] Another multicenter study, conducted internationally, assessed only patients with newly diagnosed pemphigus and determined cutoff values of 15 and 45 for PDAI and 17 and 53 for ABSIS to distinguish between mild, moderate, and severe (significant and extensive) forms of pemphigus. [START_REF] Pemphigus | Calculation of cut-off values based on the autoimmune bullous skin disorder intensity score (ABSIS) and pemphigus disease area index (PDAI) pemphigus scoring systems for defining moderate, significant, and extensive types of pemphigus[END_REF] Although these studies greatly add to our understanding of disease activity scoring, it is premature to definitively state cutoff values presently.

Specialists involved

The management of patients with pemphigus is the responsibility of dermatologists with experience in treating bullous diseases. If extensive, the initial management of the disease usually requires hospitalization until clinical control of the bullous eruption is achieved. In limited forms of pemphigus, additional diagnostic examinations and clinical monitoring can be done in either an inpatient or outpatient setting.

The overall disease management is coordinated by the dermatologist with the cooperation of the referring dermatologist/family practitioner, the general physician, and other medical specialists and hospital doctors from the center of reference and/or geographic area (if a reference center exists in the particular country).

Rarely, the disease can occur during childhood, and children should be managed by a multidisciplinary team, jointly by a reference center, a pediatric dermatology department, or a pediatrician.

Other health professionals who may serve as supportive adjuncts are as follows:

•

The referring dermatologist

•

The patient's primary care provider to manage comorbidities and monitor for treatment side effects The changes made to previous guidelines are summarized in the Supplemental Table I (available at http://www.jaad.org). The laboratory work-up is delineated in Table I.

THERAPEUTIC MANAGEMENT

See Fig 1.

Objectives

•

To promote healing of blisters and erosions

•

To improve functional status

•

To prevent or strictly limit development of new blisters and erosions

•

To improve the quality of life

MONITORING Objectives

•

To evaluate the efficacy and safety of treatment

•

To plan the gradual reduction of immunosuppressive treatment and the duration of maintenance therapy or its discontinuation

Definitions for disease outcome parameters

The following definitions have been developed by an international panel of experts.

Scheduling and content of consultations

The frequency of consultations (physical examination, additional examinations) depends on the following:

• The patient's clinical condition, including comorbidities

•

The severity and disease course specific to the patient's pemphigus during treatment

•

The therapeutics used (monitoring, tolerance, side effects)

•

The level of disease activity measure by the ABSIS and/or PDAI (optional)

Initially, follow-up visits should be offered every 2 weeks until clinical disease control is achieved. In the consolidation phase, patients should be seen every 1 to 2 weeks to determine how soon patients could be started on a steroid taper. Then, during the tapering phase, monthly clinical follow-ups are recommended for the next 3 months. Once the patient is in partial or complete remission while receiving minimal therapy, visits can be less frequent, such as every 3 months.

Clinical evaluation

The clinical follow-up should seek to clarify the following:

• Level of disease control

Serologic monitoring of disease activity

Determination of serum autoantibodies at the initiation of treatment, after 3 months, and every 3 to 6 months on the basis of the evolution or in cases of relapse by the following:

• ELISA: anti-desmoglein 1 (Dsgl) and/or desmoglein 3 (Dsg3) IgG

INFORMATION FOR PATIENTS AND THEIR FAMILIES

• Education about the disease, its clinical course and prognosis, treatment, relapse signs, and possible side effects of treatment

•

Awareness of self-support groups, which may help disseminate information regarding the disease, provide comfort, and share the experience of patients regarding daily life. Additionally, it may contribute to a better overall management of the disease by promoting cooperation between patients, patient associations, and health professionals

•

Information about referral centers

•

Education about triggers such as drugs, operations, radiation, and physical trauma

•

Counseling on dietary restrictions is not necessary owing to insufficient evidence

AREAS FOR FUTURE STUDIES

These recommendations are a working document whose purpose is to provide clinicians with the most up-to-date consensus on the diagnosis and management of pemphigus. Further studies are needed to clarify optimal therapeutic regimens and describe their safety and efficacy in the treatment of pemphigus. Some areas identified by the authors include the following:

Intravenous CD20 inhibitors

•

Although a recent clinical trial has demonstrated superior efficacy and safety of the intravenous CD20 inhibitor rituximab with short-term lower doses of corticosteroids than the standard dose of systemic corticosteroids initially with slow tapering, [START_REF] Shimizu | Grading criteria for disease severity by pemphigus disease area index[END_REF] the following questions remain about how best to use it:

-How should other medications be combined with intravenous CD20 inhibitors? -Should corticosteroids be used in combination with intravenous CD20 inhibitors from the start to gain disease control and reduce unnecessary iatrogenic morbidity for patients?

-

In some patients with comorbidities or mild disease, can CD20 inhibitors be used alone or with a topical corticosteroid? -What is the role of other immunosuppressives, intravenous immunoglobulins, immunoadsorption, etc, along with CD20 inhibitors?

• Dosing of CD20 inhibitors -Is there a specific disease activity level at which patients can be treated with only oral steroids and not necessarily with CD20 inhibitors? -What is the ideal threshold in patients receiving systemic corticosteroids or immune-suppressants to begin CD20 inhibitor therapy?

-What are the optimal dose, frequency, and total number of maintenance infusions to use? -Are these drugs indicated in patients who test for negative anti-DSG antibodies?

•

In cases of relapse, is a single dose of 1000 mg/infusion of rituximab (or 375 mg/m 2 in the lymphoma protocol) enough to achieve remission instead of a full dose cycle of rituximab (2 × 1000 mg 2 weeks apart or 4 × 375 m 2 /wk)?

•

Long-term side effects -Will more side effects occur when more patients are treated with multiple maintenance infusions of CD20 inhibitors?

Other treatment options

• What role do other treatment options, such as plasmapheresis, play in the treatment of pemphigus?

CONCLUSION

In summary, here we have presented the recommendations arising from a Delphi process involving 39 pemphigus experts. We have made recommendations for evaluation and treatment of pemphigus, including initial evaluation, diagnosis, and management, as well as regarding strategies for maintenance therapy and tapering of medications in remission.

CAPSULE SUMMARY

•

The European Dermatology Forum and the European Academy of Dermatology and Venereology passed management guidelines for pemphigus.

•

We present the recommendations of international experts, which have resulted from a Delphi consensus gathering exercise based on the European Dermatology Forum and the European Academy of Dermatology and Venereology guidelines.

•

This international consensus includes intravenous CD20 inhibitors as a firstline therapy option for moderate-to-severe pemphigus. Treatment options. The principal objective is to promote the healing of blisters and erosions, prevent development of new lesions, and minimize serious side effects of treatment. Table II.

Medication dosing

First-line treatment Corticosteroids

• Systemic corticosteroid therapy (predniso(lo)ne at 0.5 mg to 1.5 mg/kg/d) • Systemic corticosteroids (oral or intravenous pulses) can be combined with an immunosuppressive adjuvant at the onset of therapy, especially in cases of increased risk of corticosteroid therapy, complications due to expected prolonged use (>4 months), or dose dependency above minimal therapy (>10 mg/d). However, there is limited evidence that the addition of adjuvants is superior to treatment with corticosteroids alone

• Although limited, studies have not shown intravenous corticosteroid pulses to have an additional benefit on top of that of conventional first-line treatment with oral predniso(lo)ne and immunosuppressive adjuvants. Although more evidence is needed, steroid pulse therapy in addition to conventional treatment should be reserved for refractory cases of pemphigus

• Treat with the smallest dose for the shortest time possible to minimize risk of adverse events Anti-CD20 monoclonal antibodies Currently there are 2 intravenous CD20 inhibitors available, rituximab and ofatumumab. All the published trials so far have used rituximab • Treatment can be repeated in cases of clinical relapse or as early as 6 months after treatment. Lower doses are sometimes used for retreatment • Combine with short-term (<4 months) systemic corticosteroids and long-term (>12 months) immunosuppressive treatment, although the need for immunosuppressive adjuvants in rituximab therapy remains unclear

• The incidence of unforeseen fatal infections such as progressive multifocal leukoencephalopathy cannot be estimated due to the rarity of pemphigus Corticosteroid-sparing agents First-line corticosteroid-sparing agents

• Azathioprine (1-3 mg/kg/d) ■ Start 50 mg/d the first week to detect idiosyncratic reactions such as sudden-onset fevers, oral ulcers, elevated liver function test results and/or drug reaction with eosinophilia and systemic symptoms (and in that case stop immediately), and then raise to desired dose. Although not predictive for idiosyncratic reactions, TPMT activity should be evaluated in countries/ethnicities where there is a higher incidence of polymorphisms before commencing therapy because recommended azathioprine doses vary depending on TPMT activity. In general, adults with pemphigus and high TPMT activity are treated with normal doses of azathioprine (≥2.

•••••-

  Direct immunofluorescence (DIF) microscopy of perilesional skin • Serologic detection of serum autoantibodies against epithelial cell surface by indirect immunofluorescence (IIF) microscopy and/or enzyme-linked immunosorbent assay (ELISA) • Diagnosis requires clinical presentation and histopathology that are consistent with pemphigus and either a positive DIF microscopy or serologic detection of autoantibodies against epithelial cell surface antigens Functional symptoms (ie, pain; pruritus; intensity of dysphagia; ocular and ear, nose, and throat symptoms; dysuria; anogenital problems; and weight loss) Contraindications of systemic corticosteroid treatment and development of complications of immunosuppressive treatments • Contraception and plans for pregnancy in women of childbearing potential • Medication history, with special attention to causes of drug-induced pemphigus, including D-penicillamine, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and cephalosporins • Psychologic tolerance of possible side effects due to treatment, especially corticosteroid treatment Impact of disease burden on quality of life Physical examination Extent of skin and mucosal lesions and degree of disease damage • Patient's overall state of health and comorbidities -General condition (Karnofsky index) -Weight Vital signs, including blood pressure and temperature -Comorbidities (neoplastic, cardiovascular, musculoskeletal, etc)

Fig 1 .

 1 Fig 1.Diagnosis of pemphigus. Diagnosis requires clinical presentation and histopathology that are consistent with pemphigus and either a positive direct immunofluorescence (DIF) microscopy result or serologic detection of autoantibodies against epithelial cell surface antigens. ELISA, Enzyme-linked immunosorbent assay; PF, pemphigus foliaceus; PV, pemphigus vulgaris; WOCBP, women of childbearing potential.

Fig 2 .

 2 Fig 2.

  [START_REF] Joly | First-line use of rituximab combined with short-term-prednisone versus corticosteroid alone in the treatment of patients with pemphigus: a prospective multicenter randomized trial[END_REF]

  [START_REF] Murrell | Definitions and outcome measures for bullous pemphigoid: recommendations by an international panel of experts[END_REF]

  Complete remission off therapy: the absence of new and/or established lesions while the patient has not received any systemic therapy for at least 2 months•Relapse/flare: appearance of 3 or more new lesions in a month that do not heal spontaneously within 1 week, or by the extension of established lesions, in a patient who has achieved disease control

		-	If oral corticosteroids are already combined with an
			immunosuppressant, consider a change in immunosuppressant
	•	Control of disease activity: the time at which new lesions cease to form and
		established lesions begin to heal
	•	End of consolidation phase : the time at which no new lesions have developed for
		a minimum of 2 weeks and approximately 80% of lesions have healed. This is
		when most clinicians start to taper steroids
	•	Complete remission during therapy: the absence of new or established lesions
		while the patient is receiving minimal therapy
	•
	•	Go back to last dose if more than 3 lesions reappear during the tapering of oral
		corticosteroid therapy
	•	If relapse occurs (ie, the appearance of 3 or more new lesions in a month that do
		not heal spontaneously within 1 week, or if there is extension of established
		lesions), increase the oral corticosteroid dose by going back to the second-to-last
		dose until control of the lesions is achieved within 2 weeks and then resume
		taper
	•	If disease control is still not reached despite this, go back to the initial dose
		-	If oral corticosteroids are given alone, add an immunosuppressant
			(especially in cases of early-stage relapse occurring despite continued
			high-dose corticosteroid treatment)

•

Minimal therapy: prednisolone (or the equivalent) at a dose of 10 mg/d or less and/or minimal adjuvant therapy for at least 2 months

Approach to be maintained after consolidation phase

•

Expect slow clinical improvement, often requiring a period of 1 to 3 months for complete healing of lesions

•

Start tapering steroids as soon as disease control is reached or up to the end of the consolidation phase

•

Decrease predniso(lo)ne by 25% every 2 weeks, until 20 mg/d. Once a dose of 20 mg/d has been reached, decrease predniso(lo)ne by 2.5 mg/wk, and when 10 mg/d has been reached, decrease the dose by 1 mg/d thereafter

  The persistence of high levels of anti-Dsgl by ELISA has a positive predictive value for skin relapses, whereas the persistence of anti-Dsg3 IgG does not necessarily indicate a mucosal relapse Pemphigus may cause permanent sequelae not only as a result of the involvement of skin and mucosa but also owing to treatment side effects, justifying a request for recognition or help from departmental disability centers where available. The extent of immunosuppressive therapy increases the risk of side effects

	•
	Discontinuation of treatment
	•	Discontinuation of treatment is primarily based on the clinical symptoms but
		may also be supported by the findings of Dsg ELISA, IIF microscopy, and/or a
		negative result of DIF microscopy of a skin biopsy specimen
	•	Discontinuation of systemic corticosteroids may be proposed in patients in
		complete remission while receiving minimal therapy (prednisolone or equivalent
		at ≤10 mg/d). The adjuvants may be stopped 6 to 12 months after achievement of
		complete remission during minimal therapy with adjuvants only
	Possible sequelae
	•
	•	If ELISA is not available: IIF microscopy utilizing monkey esophagus
	•	Overall, serum concentrations of IgG autoantibodies against Dsg1 and Dsg3
		correlate with the clinical activity of pemphigus and may thus help in therapeutic
		decision making

•

  First-line treatment in new-onset moderate-to-severe pemphigus and/or for patients who do not achieve clinical remission with systemic corticosteroids and/or immunosuppressive adjuvants. 11

	Allows
		(1 week apart)
	for more rapid tapering of corticosteroid doses and a major corticosteroid-sparing effect	• A course of intravenous rituximab consists of 2 × 1000 mg (2 weeks apart) or 4 × 375 mg/m 2

  5 mg/kg/d). Patients with intermediate or low TPMT activity should receive a lower maintenance dose (≥0.5 to 1.5 mg/kg/d) depending on level of enzyme activity. Patients who lack TPMT activity should avoid treatment with azathioprine • Mycophenolate mofetil (30 mg/kg-45 mg/kg/d) or mycophenolic acid (1440 mg/d) Other corticosteroid-sparing agents • IVIG (2g/kg over 2-5 d/mo) ■ Treatment is generally combined with systemic corticosteroids (initially) and immunosuppressive adjuvants ■ Treatment should be performed over several days to avoid side effects ■ Aseptic meningitis is a rare but important side effect of IVIG treatment that needs to be kept in mind in patients who commonly experience episodes of migraine ■ Although uncommon, patients with IgA deficiency should receive IgA-depleted IVIG treatment

J Am Acad Dermatol. Author manuscript; available in PMC 2020 June 24.

Acknowledgments

Funding sources: Supported in part by the Office of Research and Development, Biomedical Laboratory Research and Development, Veterans Health Administration, US Department of Veterans Affairs. Dr Pena's clinical research is funded by a National Institutes of Health training grant.

Disclosure: Dr Murrell is an investigator and speaker for Roche; she is an investigator for and on an advisory board for Principia Biopharma and is also on an advisory board for Immune Pharmaceuticals and Lilly. Dr Joly is a consultant for Roche, Principia Biopharma, Lillyand Biogen. Dr Payne is a consultant for Syntimmune and TG Therapeutics and receives grants Sanofi. Dr Eming is an investigator for both Biotest AG and Fresenius Medical Care and is a speaker for Biotest AG and Novartis. Dr Jonkman is a monitor for Roche. Dr Prost is an investigator for Roche. Dr Yayli is an investigator for Roche. Dr Zillikens is on the advisory board for Roche and a consultant for Euroimmun, UCB, Fresenius, Almirall, and oGEN-x. Dr Zillikens has received grants and is a speaker for Euroimmun, Miltenyi/Biogen, Fresenius/Roche, Biotest AG, Almirall, and Dompe/Janssen. Dr Amagai receives speaker honoraria and grants from Nihon Pharmaceutical and research support from Medical & Biological Laboratories. Dr Hertl is on an advisory boards for Roche, Biogen, and Novartis; he has received grants from Biotest and Fresenius and is a speaker for Janssen. Dr Schmidt has received grants from Euroimmun and Fresenius and is a speaker for Biotest and Fresenius. Dr Hall is a consultant for Stieffel at GlaxoSmithKline Company, Eli Lilly, Syntimmune, and Immune Sciences and is on the data safety monitoring board for Roche and has received grants from Immune Sciences. Dr Peña, Dr Marinovic, Dr Hashimoto, Dr Diaz, Dr Sinha, Dr Daneshpazhooh, Dr Mimouni, Dr Borradori, Dr Kim, Dr Yamagami, Dr Lehman, Dr Saleh, Dr Culton, Dr Czernik, Dr Zone, Dr Fivenson, Dr Ujiie, Dr Wozniak, Dr Akman-Karakaş, Dr Bernard, Dr Korman, Dr Caux, Dr Drenovska, Dr Vassileva, Dr Feldman, Dr Cardones, Dr Bauer, Dr loannides, Dr Jedlickova, Dr Palisson, Dr Patsatsi, Dr Uzun, Dr Aoki, Dr Grando, Dr Shimizu, Dr Baum, Dr Cianchini, Dr Feliciani, Dr Iranzo, Dr Mascarό Jr, Dr Kowalewski, Dr Groves, Dr Harman, Dr Marinkovich, Dr Maverakis, and Dr Werth have no conflicts of interest to disclose.

Supplementary Material

Abbreviations used

ABSIS

Table I.

Laboratory work-up

Histopathology

• A biopsy specimen of a recent (<24 h) small vesicle consisting of the peripheral portion of a blister (1/3 of the sample) and perilesional skin (placed in 4% formalin solution) (2/3 of the sample) should be obtained for routine histopathologic analysis: intraepidermal suprabasal acantholysis in PV or acantholysis at the granular layer in PF DIF microscopy • Skin biopsy of perilesional skin (≤1 cm from a recent lesion), put into a cryotube for transportation in saline (delivery <36 h) in a cylinder of liquid nitrogen or Michel's fixative for DIF microscopy analysis:

■ DIF microscopy: IgG and/or C3 deposits at the surface of epidermal keratinocytes. The smooth and reticular staining pattern is also referred to as chicken wire, honeycomb, or fishnet-like ■ IgA deposits with an epithelial cell surface pattern in addition to IgG may be present in a subset of cases ■ Epithelial cell surface deposits may be associated with linear or granular deposits of IgG or C3 along the dermoepidermal junction, suggestive of other autoimmune blistering diseases (including paraneoplastic pemphigus or pemphigus erythematosus) or the coexistence of pemphigus and pemphigoid

Immune serologic tests Indirect immunofluorescence (IIF) microscopy

• IIF microscopy test on monkey esophagus or human skin to search for autoantibodies against surface proteins of epidermal keratinocytes, with a pattern similar to that seen on DIF microscopy • In cases of atypical presentation or the suspicion of another autoimmune bullous disorder, additional immunopathologic tests may be performed, such as IIF microscopy on rat bladder and immunoblot/ immunoprecipitation

• IIF microscopy on human cells with recombinant expression of desmoglein 1, desmoglein 3, or envoplakin (Euroimmun) is an alternative when desmoglein-or envoplakin-specific ELISA cannot be used ELISA

• Detection of anti-desmoglein 1 (PF/mucocutaneous PV) and/or anti-desmoglein 3 IgG autoantibodies (mucosal or mucocutaneous PV) by ELISA (Mannose-Binding Lectin, Euroimmun, Lübeck, Germany)

• The detection of IgG autoantibodies by ELISA is positive in more than 90% of cases J Am Acad Dermatol. Author manuscript; available in PMC 2020 June 24.