Elena Gonzalez-Gobartt

José Blanco-Ameijeiras

Susana Usieto

Guillaume Allio

Bertrand Benazeraf

Elisa Martí

Bertrand Bénazéraf

Cell intercalation driven by SMAD3 underlies secondary neural tube formation

Keywords: de novo lumen formation, lumenogenesis, epithelial polarization, mesenchymal-to-epithelial transition, TGF-beta, Smad3, spinal cord, body axis elongation

come

INTRODUCTION

Body axis elongation is operated by a proliferative cell population at the posterior end of the vertebrate embryo termed neuromesodermal progenitors (NMPs) [START_REF] Cambray | Two distinct sources for a population of maturing axial progenitors[END_REF][START_REF] Henrique | Neuromesodermal progenitors and the making of the spinal cord[END_REF][START_REF] Tzouanacou | Redefining the progression of lineage segregations during mammalian embryogenesis by clonal analysis[END_REF]. These cells have been found in zebrafish, chick, mouse, and human embryos, and they produce both the neural tissue that makes the caudal spinal cord, and mesodermal tissues such as muscle and bone [START_REF] Cambray | Two distinct sources for a population of maturing axial progenitors[END_REF][START_REF] Henrique | Neuromesodermal progenitors and the making of the spinal cord[END_REF][START_REF] Kölliker | Die embryonalen Keimblä tter und die Gewebe[END_REF]. NMPs have gain much recent attention since protocols for generating them from human pluripotent stem cells in vitro were established [START_REF] Gouti | In vitro generation of neuromesodermal progenitors reveals distinct roles for wnt signalling in the specification of spinal cord and paraxial mesoderm identity[END_REF]. This not only allowed to decipher the gene regulatory networks that determined NMPs fate specification and lineage restriction [START_REF] Gouti | A Gene Regulatory Network Balances Neural and Mesoderm Specification during Vertebrate Trunk Development[END_REF][START_REF] Metzis | Nervous System Regionalization Entails Axial Allocation before Neural Differentiation[END_REF], but also opens up a new experimental paradigm for studying the cellular and molecular basis of caudal spinal cord generation. However, despite their relevance to understand caudal skin-covered neural tube defects (NTDs) [START_REF] Greene | Neural tube defects[END_REF][START_REF] Morris | Prevention of neural tube defects in the UK: a missed opportunity[END_REF][START_REF] Saitsu | Involvement of the axially condensed tail bud mesenchyme in normal and abnormal human posterior neural tube development[END_REF] the signaling pathways and cellular events required for shaping a 3D secondary neural tube from NMPs, are poorly understood.

Morphogenesis of the secondary neural tube (SNT) includes the mesenchymal-toepithelial transition (MET) of the NMPs, concomitant to the lineage restriction and confinement of these cells within a solid medullary cord, surrounded by the growing basement membrane. The subsequent cavitation of this chord forms the SNT [START_REF] Catala | Organization and development of the tail bud analyzed with the quail-chick chimaera system[END_REF][START_REF] Colas | Towards a cellular and molecular understanding of neurulation[END_REF][START_REF] Griffith | The vertebrate tail bud: three germ layers from one tissue[END_REF][START_REF] Shimokita | Secondary neurulation: Fate-mapping and gene manipulation of the neural tube in tail bud[END_REF]. The limited availability of human tissue to perform histological analyses at different developmental stages [START_REF] Abbott | Tissue-bank shortage: Brain child[END_REF] reinforces the need to use animal models to understand the events shaping the SNT, particularly since NTDs rank among the most common categories of birth defects [START_REF] Greene | Neural tube defects[END_REF]. Here, we have used the chick embryo to gain understanding in the signalling proteins controlling the subcellular events and the 3D tissue rearrangements shaping the SNT. At early stages of development, the avian body plan is very similar to that of mammals and chick embryos display excellent optical properties, as they are thin and planar [START_REF] Le Douarin | Neurulation in amniote vertebrates: a novel view deduced from the use of quail-chick chimeras[END_REF][START_REF] Rupp | Culturing of avian embryos for time-lapse imaging[END_REF][START_REF] Shimokita | Secondary neurulation: Fate-mapping and gene manipulation of the neural tube in tail bud[END_REF]. Furthermore, the anatomical SNT region in the chick embryo extends up to the lumbar region [START_REF] Criley | Analysis of embryonic sources and mechanims of development of posterior levels of chick neural tubes[END_REF][START_REF] Dady | Junctional neurulation: a unique developmental program shaping a discrete region of the spinal cord highly susceptible to neural tube defects[END_REF], closely resembling human development [START_REF] O'rahilly | Neurulation in the normal human embryo[END_REF], 2002;[START_REF] Saitsu | Development of the posterior neural tube in human embryos[END_REF][START_REF] Saitsu | Involvement of the axially condensed tail bud mesenchyme in normal and abnormal human posterior neural tube development[END_REF], while SNT only occurs at the level of the tail in mice embryos [START_REF] Nievelstein | Embryonic development of the mammalian caudal neural tube[END_REF][START_REF] Schoenwolf | Histological and ultrastructural studies of secondary neurulation in mouse embryos[END_REF][START_REF] Shum | Lack of motor neuron differentiation is an intrinsic property of the mouse secondary neural tube[END_REF].

Here we found Transforming Growth Factor-beta (TGF-) signalling activity during SNT formation. Members of the TGF- superfamily have been implicated as major instructive signals of epithelial-to-mesenchymal (EMT) in many morphogenetic processes, including gastrulation and mesendodermal ingression from the embryonic epiblast [START_REF] Luxardi | Distinct Xenopus Nodal ligands sequentially induce mesendoderm and control gastrulation movements in parallel to the Wnt/PCP pathway[END_REF], and delamination of neural crest cells from the developing neural tube [START_REF] Theveneau | Neural crest delamination and migration: from epithelium-to-mesenchyme transition to collective cell migration[END_REF], among others. By in vivo loss-of-function experiments in the chick embryo, here we show that initial events in SNT formation such as the lineage restriction of dual fated NMPs, as well as the MET and full epithelialization of caudal neural progenitor cells, appeared to be independent of SMAD3 signalling. However, the resolution of a single, centrally positioned continuous lumen, which takes place through the intercalation of central cells, requires SMAD3 activity. Altogether, here we describe a novel activity for TGF-/SMAD3 that might be relevant to understand human caudal skin-covered NTDs.

RESULTS

Secondary neural tube (SNT) formation in the chick embryo requires TGF- mediated Smad3 activity

During vertebrate development, neuromesodermal progenitors (NMPs) are recruited to elongate the caudal body axis and drive the elongation of the neural tube (NT). These bipotential cells will generate neural progenitor cells (NPCs) of the caudal medullary cord by undergoing a mesenchymal to epithelial transition (MET) to form the secondary neural tube (SNT). The complete process, that requires the cavitation of the medullary cord, can be followed in HH15 chick embryo [START_REF] Hamburger | A series of normal stages in the development of the chick embryo[END_REF]) (50-55 hours post fertilisation) along the cranio-caudal axis (Fig. 1A-E). The first cells to epithelialize are confined at the periphery of the medullary chord, being surrounded by a developing basement membrane, while central cells remain mesenchymal until the very end of the process. It is between these two cell populations that small cavities, of varied size and shape, form and later resolve to form a single central lumen (Fig. 1A-E) [START_REF] Schoenwolf | Ultrastructure of secondary neurulation in the chick embryo[END_REF][START_REF] Schoenwolf | Characterization of intercellular junctions in the caudal portion of the developing neural tube of the chick embryo[END_REF].

Members of the Wnt and FGF signaling pathways have been shown to play relevant roles in the control of NMPs growth, maintenance, and differentiation along neural or mesodermal lineages (Reference), although very little is known about signals instructing the 3D shaping of the caudal neural tube. Here we have analysed the expression of ligands of the main developmental pathways in the tail bud of stage HH15 chick embryos. Whole mount in situ hybridization and transverse sections through these same embryos revealed the expression of BMP2 and TGF-1 potentially associate to the process of secondary neural tube formation (Supp.Fig. S1A-D). Interestingly, TGF- /BMP pathways are known to play key roles in anterior neural tube formation (reviewed in Le Dreau & Marti, 2012;[START_REF] Ulloa | Morphogens and the control of cell proliferation and patterning in the spinal cord[END_REF], however their roles in secondary neural tube formation are less understood. TGF- /BMP canonical signaling is substantially linear; ligands bind to a defined receptor complex composed of two transmembrane serine/threonine kinases, which then propagate the signal through the SMAD family of transcription factors (Fig. 1F,G). In situ hybridization experiments for SMAD mRNA, revealed the high expression of SMAD3 in polarizing NMPs at the dorsal periphery of the chord, later spreading to the entire developing SNT (Supp.Fig. S2A-D). We next in vivo assessed the endogenous activity of the TGF- /BMP pathways by electroporation of a BMP-(BRE:eGFP) (Le [START_REF] Dreau | The strength of SMAD1/5 activity determines the mode of stem cell division in the developing spinal cord[END_REF]Le Dreau et al., 2012) or a TGF- responsive (CAGA12-GFP) [START_REF] Miguez | Smad2 and Smad3 cooperate and antagonize simultaneously in vertebrate neurogenesis[END_REF] fluorescent reporter, together with a control H2B-RFP vector (Fig. 1H). Results showed 24hours post electroporation (hpe) that both pathways are active in the elongating chick embryo tail bud (Fig. 1I,J, see also Supp.Fig. S2J). However, electroporation of the TGF--responsive CAGA12:GFP fluorescent reporter, which is indeed specific to SMAD3 activity [START_REF] Miguez | Smad2 and Smad3 cooperate and antagonize simultaneously in vertebrate neurogenesis[END_REF], together with a control H2B-RFP vector showed 24hpe high caudal activity and within the SN region, while control H2B-RFP+ cells were found along the whole cranio-caudal axis (Supp.Fig S2J).

To test for the contribution of canonical TGF- /BMP signalling in SNT formation, we analysed the consequences of SMAD inhibition, following electroporation of shorthairpin RNA targeting specific chick SMAD mRNAs. BMP signalling appeared to be dispensable for the correct morphogenesis of the SNT, since inhibition of SMAD1/5 resulted in the formation of a normal SNT (Fig. 1M-O). Electroporation of sh-SMAD2, although efficiently inhibited endogenous SMAD2 expression [START_REF] Miguez | Smad2 and Smad3 cooperate and antagonize simultaneously in vertebrate neurogenesis[END_REF], was not sufficient to perturb SNT formation (Fig. 1P). However, inhibition of the TGF- effector SMAD3 resulted in an aberrant SNT, exhibiting multiple small lumens along the cranio-caudal (Fig. 1K,L) and the dorso-ventral axes (Fig. 1Q). While sh-SMAD3 electroporation efficiently decreased endogenous SMAD3 levels (median±IQR integrated density control=543.1±486.7 vs sh-SMAD3=148.6±113.5: Supp. Together, these observations prompted us to search for the precise cellular events regulated by TGF-/SMAD3 signalling, that might be instructive for SNT formation.

Cell confinement of NMPs and lineage restriction into NPCs are independent of Smad3 activity

Tail bud NMPs will generate neural precursor cells (NPCs) of the secondary neural tube and trunk mesodermal tissues. The co-expression of the transcription factors T/Brachyury (T/Bra) and Sox2 characterize NMPs, while NPCs emerging from dual fated NMPs downregulate T/Bra but maintain high Sox2 expression [START_REF] Gouti | In vitro generation of neuromesodermal progenitors reveals distinct roles for wnt signalling in the specification of spinal cord and paraxial mesoderm identity[END_REF][START_REF] Kondoh | Axial stem cells deriving both posterior neural and mesodermal tissues during gastrulation[END_REF][START_REF] Olivera-Martinez | Loss of FGFdependent mesoderm identity and rise of endogenous retinoid signalling determine cessation of body axis elongation[END_REF][START_REF] Tsakiridis | Assessing the bipotency of in vitro-derived neuromesodermal progenitors[END_REF][START_REF] Wymeersch | Position-dependent plasticity of distinct progenitor types in the primitive streak[END_REF]. The complete neural lineage restriction process can be followed in the same stage HH15 chick embryo, by analyzing T/Bra+ and Sox2+ expression at several axial levels from the posterior elongating tail bud to the level of the last formed somite (Fig. 2A-E). At initial stages, T/Bra is broadly expressed in the tail bud, including the lateral mesoderm here excluded from quantifications. Later on in development, it is going to be downregulated along SN and finally restricted to the notochord (Fig. 2A'-E'). Low levels of Sox2 expression appear in dorsal and central areas of the early tail bud (Fig. 2A'') and propagate ventrally (Fig. 2B'') to finally be confined to the neural tube (Fig. 2E''). This suggests that the differentiation of NPCs in the medullary cord advances in a dorsoventral direction.

Quantification of endogenous T/Bra and Sox2 within the region undergoing Secondary Neurulation (SN)-not only revealed a strong decrease of T/Bra expression along the process (median±IQR log10 of T/Bra CTCF axis elongation=3.6±0.2; cell confinement=3.3±0.5; lumen initiation=3.0±0.3; lumen resolution=2.3±0.4; SNT=0.8±0.5: Fig. 2F), but also an increase of Sox2 levels (median±IQR log10 of Sox2 CTCF axis elongation=2.5±0.2; cell confinement=3.0±0.3; lumen initiation=3.3±0.2; lumen resolution=3.4±0.2; SNT=3.4±0.2: Fig. 2F), consequent with the progressive generation of NPCs. The expression of T/Bra and Sox2 gradually changed over time in the developing SNT, suggesting the existence of a transitional state in the path from NMPs to NPCs. We also found a positive correlation between T/Bra expression and distance to the basement membrane, and a converse negative correlation between Sox2 expression and distance to the basement membrane (Supp.Fig S3A,B). Moreover, during cell confinement, lumen initiation and lumen resolution, the expression levels of Sox2 are high in peripheral cells, as compared to central cells (median±IQR log10 of Sox2 CTCF peripheral cells at cell confinement=3.1±0.2; lumen initiation =3.3±0.2; lumen resolution=3.5±0.2 vs central cells at cell confinement=2.9±0.4; lumen initiation=3.2±0.2; lumen resolution=3.3±0.2: Supp.Fig S3C). Conversely, levels of T/Bra expression are high in central cells as compared to peripheral cells (median±IQR log10 of T/Bra CTCF peripheral cells at cell confinement=3.1±0.3; lumen initiation=2.9±0.3; lumen resolution=2.2±0.4 vs central cells at cell confinement=3. 5±0.3;lumen initiation=3.1±0.3;lumen resolution=2.4±0.3: Supp.Fig S3D). In summary, bipotential NMPs convert to NPCs by downregulating T/Bra and upregulating Sox2; this event occurs concomitant to cell confinement by the basement membrane (Fig. 2G).

In order to test for a possible role of canonical TGF- signalling in NMPs lineage restriction, we analysed T/Bra and Sox2 expression, 24hpe of sh-SMAD3 electroporation (Fig. 2H-J). Results showed that T/Bra expression is progressively downregulated in NMPs electroporated with sh-SMAD3, although it remained highly expressed in the surrounding lateral mesodermal cells, as well as in axial notochord cells (Fig. 2J). Expression levels of Sox2 in sh-SMAD3 electroporated NPCs were comparable to surrounding non-electroporated cells (median±IQR Sox2 intensity ratio control=0.7±0.3 vs sh-SMAD3=0.8±0.4: Fig. 2H,I) and Sox2+ cells were progressively restricted to the SNT (Supp.Fig. S3E). Moreover, a basal lamina properly confines the SNT 24hpe of sh-SMAD3 electroporation (Fig. 1L). All these observations indicate that SMAD3 activity does not affect cell confinement and that it is dispensable for neural lineage restriction.

Lumen initiation occurs at a one cell distance from the basement membrane and it is independent of SMAD3 activity Concomitant to the neural lineage restriction of NMPs, morphogenesis of the secondary neural tube includes the epithelialization of these cells; a process that can be followed along the cranio-caudal axis of the stage HH15 chick embryo. The first cells to acquire epithelial polarity are those in contact with the forming basement membrane, the ones located dorsally and in the periphery of the cord (Fig. 3A-D), similar to the epithelialization process occurring in MDCK cells growing in 3D culture [START_REF] Bryant | A molecular switch for the orientation of epithelial cell polarization[END_REF][START_REF] Martin-Belmonte | Cell-polarity dynamics controls the mechanism of lumen formation in epithelial morphogenesis[END_REF], and in the early mouse embryo [START_REF] Bedzhov | Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon[END_REF].

To characterize the subcellular events accompanying NMPs epithelialisation, we analysed cell shape, centrosome positioning, Golgi distribution, and polarity protein localisation at early MET stages (Fig. 3E-L, see also Supp.Fig. S4A-D). During MET in the SNT, cells change their shape from polygonal to elongated (median±IQR circularity central cells=0.8±0.1 vs peripheral cells=0.3±0.2), concomitantly to basal nuclear positioning (Fig. 3E-I). The perinuclear centrosome becomes apically localised, as determined by the distance from FOP+ labelled centrosomes [START_REF] Yan | A complex of two centrosomal proteins, CAP350 and FOP, cooperates with EB1 in microtubule anchoring[END_REF] to the nucleus (median±IQR distance central cells=0.9±0.5m vs peripheral cells=9.7±10.3m: Fig. 3E,J). The high variability found in peripheral cell centrosome-to-nucleus distance associates to the onset of interkinetic nuclear migration, which separates or brings together the centrosome and the nucleus depending on the phase of the cell cycle. Additionally, the Golgi elongates and it is confined to the apical cellular process, as determined by labelling with the cis-Golgi matrix protein GM130 [START_REF] Nakamura | Characterization of a cis-Golgi matrix protein, GM130[END_REF] (median±IQR distance central cells=3.3±1.6m vs peripheral cells=9.9±6.8m: Fig. 3F,K), similar to that observed for NPCs in the developing cerebral cortex [START_REF] Taverna | Non-canonical features of the Golgi apparatus in bipolar epithelial neural stem cells[END_REF]. Finally, cell epithelialization includes the organization of the apical membrane in discrete micro-domains where N-cadherin and the ZO-1/occludin complex occupy internal positions, while aPKC concentrates at the most apical domain [START_REF] Aaku-Saraste | Loss of occludin and functional tight junctions, but not ZO-1, during neural tube closure--remodeling of the neuroepithelium prior to neurogenesis[END_REF][START_REF] Afonso | PAR3 acts as a molecular organizer to define the apical domain of chick neuroepithelial cells[END_REF][START_REF] Chenn | Intrinsic polarity of mammalian neuroepithelial cells[END_REF][START_REF] Marthiens | Adherens junction domains are split by asymmetric division of embryonic neural stem cells[END_REF]. Thus, apical proteins such as N-cadherin or ZO-1 are progressively accumulated in the apical pole of the cell (Fig. 3G,H,L).

To establish the sequence of apical polarisation and the possible correlation with lumen initiation, we analysed the subcellular localisation of FOP, N-cadherin and aPKC at the initial stages of MET (Supp.Fig. S4E-M). While all the analysed cells already showed an apically localised centrosome, several also presented apical N-cadherin, and only a few apical aPKC. Neither N-cadherin nor aPKC were found before apical centrosome positioning and, furthermore, apical aPKC was never found before N-cadherin apical accumulation (mean±sem % of peripheral cells with apical FOP=73.7±5.1 vs apical FOP and N-cadherin=26.3±5.1; apical FOP=73.8±8.0 vs apical FOP and N-cadherin=6.6±1.3 vs apical FOP, N-cadherin and aPKC=19.6±8.9: Supp.Fig. S 4E-I). Moreover, co-staining of FOP, N-cadherin and ZO-1 revealed N-cadherin and ZO-1 co-localisation, being always apically found at the same time and, again, never before FOP (mean±sem % of peripheral cells with apical FOP=72.6±9.9 vs apical FOP, N-cadherin and ZO-1=27.4±9.9: Supp.Fig. S4E-I). Finally, peripheral cells with apical FOP already showed basal 1 Integrin, and no cells were found with only one of these components apicobasally polarised, which suggests that apical and basal polarities are organised concomitantly (mean±sem % of peripheral cells with apical FOP and 1 integrin=64.5±6.6 vs apical FOP, 1 integrin and N-cadherin=35.5±6.6: Supp.Fig. S4H,J). In summary, we found the subcellular events of apico-basal polarisation to be sequential in polarising NPCs, as such apical centrosome positioning and basal 1 integrin localisation is followed by N-cadherin/ZO-1 apical membrane accumulation, and last, aPKC apical-most membrane localization (Fig 3L).

As NMPs transformed into fully epithelialized NPCs, we observed multiple small lumens emerging at the interface between the peripheral epithelial and central mesenchymal cell populations (Fig 3L ,M). These small lumen foci are always formed at one-cell distance from the basement membrane, as the distance from lumen foci to the basement membrane was comparable to that of cell length (median±IQR cell length=26.5±9.2m vs LF to BM distance=26.8±6.4m: Fig. 3M-P). We next tested for a possible role of canonical TGF- signaling in triggering MET and lumen initiation. At 24hpe, sh-SMAD3 cells properly locate N-cadherin and aPKC to their apical pole and multiple small lumens still open up at one-cell distance from the BM (median±IQR LF to BM distance control=26.8±6.4m vs sh-SMAD3=28.0±5.5m: (Fig. 3P). Altogether, results show that SMAD3 activity is dispensable for the subcellular processes involved in MET triggering and lumen foci initiation, which prompted us to search for defects in late stages of MET and in SNT lumen resolution. We next tested for a possible role of canonical TGF- signalling in final NPCs epithelialization. To that end, we analyzed cell shape, centrosome positioning, Golgi elongation and polarity protein localization in sh-SMAD3 electroporated cells of the SNT, at cranio-caudal levels where the morphogenesis of the neighbouring somites and underlying notochord was completed. 24hpe, sh-SMAD3 cells of the SNT properly elongate (median±IQR circularity control=0.1±0.1 vs sh-SMAD3=0.2±0.1: Fig. 3Q,R), and apically localize their centrosomes, as control electroporated cells (median±IQR FOP to nucleus distance control=16.6±10.6m vs sh-SMAD3=19.3±13.0m: Fig. 3S,T). Although sh-SMAD3 electroporated cells fail to elongate their Golgi (median±IQR GM130 length control=13.3±6.9m vs sh-SMAD3=6.3±3.7m: Fig. 3U,V), these cells organize apical membrane micro-domains containing N-cadherin, ZO-1, and aPKC (Fig. 3W), similar to control electroporated cells. Although the apico-basal polarity is properly organized in NPCs, sh-SMAD3 electroporated embryos present a multiple lumen phenotype of the SNT in which small lumens are correctly initiated but fail to coalesce into a single central cavity (Fig. 3X). Altogether, results indicate that SMAD3 activity is dispensable for the subcellular processes involved in cell epithelialization; however, it appeared to be required for the resolution of a centrally positioned single lumen during SNT formation.

SMAD3 activity is required for cell intercalation and the resolution of a single central lumen in the SNT

Formation of a single and continuous lumen in the SNT requires distinct luminal rearrangements and cellular events of 3D tissue remodelling. We used Imaris reconstruction to generate 3D images of the lumen size and shape from ZO-1 in toto immunostained HH15 chick embryos (Fig. 4A-F). Analysis revealed that the caudal small lumen foci tend to coalesce first into three enlarged lumens (one dorsal-central and two ventral-lateral) which will finally fuse into a single central cavity in cranial regions (Fig. 4A-F, Sup Movie S1 and S2). We also found a population of cells that remain in between the resolving lumens until the end of lumen coalescence. Lumen resolution therefore requires the clearance of this central cell population to generate the SNT composed of NPCs arranged around a single central cavity (Supp.Fig. S5A,I). Analysis of central cells revealed that even though they have already lost T/Bra expression (Supp.Fig. S5A,B), that is why we call them here central NPCs, they retain mesenchymal characteristics. Central NPCs maintain a circular cell shape, while peripheral NPCs at the stage of lumen resolution have already lost circularity, heading towards the fully elongation of NPCs in the formed SNT (median±IQR circularity central NPCs (LR)=0.8±0.1, peripheral NPCs (LR)=0.2±0.1, NPCs (SNT)=0.1±0.03: Supp.Fig. S5C,F). Central NPCs also present the centrosome close to their nucleus (median±IQR FOP to nucleus distance central NPCs (LR)=1.1±0.6m, peripheral NPCs (LR)=14.3±11.0m, NPCs(SNT)=27.7±21.2m: Supp.Fig. S5D,E,G), a pericentrosomal Golgi (median±IQR FOP to distal GM130 distance central NPCs =4.8±2.8m, peripheral NPCs =16.8±10.3m, NPCs (SNT)=22.2±20.3m: Supp.Fig. S5D,E,H) and disorganised apical polarity (Supp.Fig. S5E).

Resolution into a single continuous lumen in the developing SNT requires the clearing of central mesenchymal NPCs. To test whether cavitation participates in lumen resolution, we examined sections of stage HH15 chick embryos at various cranio-caudal levels for cleaved caspase-3-positive or TUNEL-positive cells. Whereas there were variable numbers of apoptotic cells in the dorsal NT and dorsal non-neural ectoderm, such cells were almost absent from the forming SNT (mean±sem %c-Caspase3 NPCs =0.6±0.1), or even from the inner cell mass surrounded by forming lumens (mean±sem %c-Caspase3 central NPCs =0.3±0.2), indicating that cavitation does not contribute to lumen resolution in the SNT (Supp.Fig. S5M-O). However, the population of centrally located NPCs appeared to intercalate among epithelialized NPCs of the forming SNT, as observed in transverse sections stained for actin cytoskeleton (Phalloidin) (Fig. 4G). Interestingly, this process of lumen resolution by cell intercalation appeared to require SMAD3 activity, since 24hpe of sh-SMAD3 electroporation showed the presence of centrally located NPCs at lumen resolution stages which do not die by apoptosis either (Supp.Fig. S2M,N,P).

To directly test this hypothesis, we set the electroporation of stage HH9 chick embryos for the in vivo analysis of the process, which were later cultured and imaged under an upright wide-field microscope. This system allows chick embryos to elongate and to develop at the approximate same rate as they do in ovo (Gonzalez-Gobartt et al., 2020 (chapter) [START_REF] Benazeraf | A random cell motility gradient downstream of FGF controls elongation of an amniote embryo[END_REF];[START_REF] Rupp | Culturing of avian embryos for time-lapse imaging[END_REF]. It also permits the tracking over time of electroporated fluorescently labelled cells of the elongating SNT. These cells exhibit high motility, high rate of cell division, and important cell mixing. Analysis of membrane-GFP electroporated cells along time revealed circular mesenchymal cells centrally localized in the elongating SNT (green arrows in Fig. 5Ai and first timepoint in D-F). We consistently observed central cell intercalation into the lateral walls of the neuroepithelium, perceived as a lateral cell movement away from the centre of the tissue accompanied with a cell shape change from circular to elongated (Fig. 5A-C; Supp. Movies S3). Notably, intercalating cells presented high protrusive activity (Fig. 5A-E) and central cell intercalation always occurred just after cell division (dotted circles in Fig. 5B-F). Indeed, quantification of dividing cells in fixed sections of stage HH15 embryos showed the proportion of mitotic cells to be significantly higher in the area where the single lumen is resolving, as compared to the formed SNT or to the caudal tail bud (mean±SD %pH3 cells SNT=4.4±1.0m vs LR=7.0±2.5m vs Tailbud=3.3±1.1m: Fig. 5G).

We next quantified cell distance from the centre, circularity and cranio-caudal location along time in spotted membrane-GFP+ central mitosis and generated daughter cells. Cranio-caudal location is assessed as the distance to the last pair of formed somites, Results showed that the outcome of central cell division varies along the cranio-caudal axis (Fig. 5D-F). At the anterior SNT (median±IQR distance to the last pair of somites=494.8±464.0m), central cells divide symmetrically before both daughter cells elongate and migrate laterally to intercalate among epithelialized NPCs (Fig. 5D,H,I; Supp. Movie S4). We termed this mode of division as II, as it produces two intercalating daughter cells (IDCs) (Fig. 5D'). At intermediate regions (median±IQR distance to the last pair of somites = 803.2±163.2m), central cells divide asymmetrically, such that one daughter cell remains mesenchymal and centrally located, while the other daughter cell elongates and migrates laterally to intercalate among epithelialized NPCs forming the SNT (Fig. 5E,H,I; Supp. Movie S3). We termed this mode of division as IC, as it gives rise to one intercalating (IDC) and one central daughter cell (CDC) (Fig. 5E'). At the caudal tail bud (median±IQR distance to the last pair of somites=1040.0±193.1m) mesenchymal central cells divide symmetrically, such that both daughter cells remain rounded and centrally located (Fig. 5F,H,I; Supp. Movie S5). We termed this mode of division as CC, as it generates two central daughter cells (CDCs) (Fig. 5F').

In order to test for a possible role of canonical TGF- signaling in cell intercalation and single lumen resolution during SNT formation, stage HH9 chick embryos were co electroporated with either sh-SMAD3 and sh-control and Sox2p:GFP vector [START_REF] Saade | Sonic hedgehog signaling switches the mode of division in the developing nervous system[END_REF][START_REF] Uchikawa | Functional analysis of chicken Sox2 enhancers highlights an array of diverse regulatory elements that are conserved in mammals[END_REF] and imaged for tracking of fluorescently labelled cells over time (Fig. 6A-D). Control embryos presented the three modes of division at the corresponding cranio-caudal levels (median±IQR distance to the last pair of somites control II=732±378.8m, IC=843±123m, CC=1008±163m: Fig. 6E) and daughter cells intercalated normally (Supp.Fig. S6J,K; Supp.Movie S6). Even though sh-SMAD3 electroporated central cells were able to divide in a similar ratio to control electroporated cells (Supp.Fig. S2M,O,Q), most of sh-SMAD3 electroporated mitoses generated two daughter cells that remained centrally located. They failed to intercalate into the lateral epithelialized SNT (Fig. 6F-I; Supp.Movies S7, S8), regardless of the mitotic craniocaudal location (median±IQR distance to the last pair of somites sh-SMAD3 IC=718.8±503.5m, CC=831.9±445.8m: Fig. 6E). Faulty cell intercalation in sh-SMAD3 electroporated embryos resulted in an abnormal persistence of central cells at cranial levels (Fig. 6C,D), that eventually elongate at central positions (last time points in Fig. 6G-I). This disturbed the resolution of the developing lumen and ultimately led to the NTDs with a multilumen phenotype.

DISCUSSION

In this study, we provide an exhaustive description of the morphogenetic events that built the SNT in the chick embryo, crucial for unravelling the causes of caudal NTDs, which rank among the most common categories of birth defects. Importantly, we have found that the final resolution of the central lumen involves cell intercalation into the lateral walls of the neuroepithelium, leading to a revision of the pre-existent model for secondary neurulation in the chick embryo [START_REF] Schoenwolf | Ultrastructure of secondary neurulation in the chick embryo[END_REF]. We also show that defective TGF-/SMAD3 activity leads to caudal NTDs characterised by the presence of multiple small lumens. This phenotype is not due to changes in cell viability, cell identity, lumen initiation or apico-basal polarity disruption, but arises from a failure in central cell intercalation during lumen resolution.

We studied the process of secondary neurulation starting at the mesenchymal NMPs driving caudal body axis elongation, until the complete formation of the caudal secondary neural tube (SNT), composed of epithelial neural progenitor cells (NPCs) enclosing a single central lumen. Based on our findings, we divided the morphogenesis of the SNT into three fundamental steps: (i) cell confinement of NMPs and neural lineage restriction, (ii) mesenchymal-to-epithelial (MET) and de novo multiple lumen formation, (iii) lumen resolution into a single central cavity. Our model proposes that SNT formation involves two related cell events: first, a change in cell identity from NMPs to NPCs, and second, a switch in cell polarity from mesenchymal (front-rear) to epithelial (apico-basal). Both transformations highly associate to the growing basement membrane, which suggests an important role for the basement membrane/integrin signalling in this process. Concomitant to these cellular changes, the SNT lumen is formed de novo in between cells. Small lumens open up at one-cell distance from the basement membrane and isolate a central mesenchymal cell population. The accepted model for SN does not explain how this population of central cells is cleared from the lumen, although it considers the possibility of central cell intercalation into the lateral walls of the neuroepithelium [START_REF] Schoenwolf | Ultrastructure of secondary neurulation in the chick embryo[END_REF]. However, even though this hypothesis was posed decades ago, it was never tested. By performing in vivo time-lapse imaging in electroporated chick embryos, we were able to follow central NPCs along time. Results show that central cells indeed intercalate into the lateral walls of the neuroepithelium as lumens coalesce. The result of this process is that a hollow SNT composed of epithelial NPCs around a single central lumen arises. Altogether, we have merged studies on cell identity, cell polarity and cell dynamics to extend and complete the pre-existing model of normal SNT development, setting the basis to understand the pathology of caudal NTDs.

The molecular signals driving SNT formation remain largely unknown. WNT and FGF signalling play important roles in the maintenance and expansion of NMPs [START_REF] Garriock | Lineage tracing of neuromesodermal progenitors reveals novel Wnt-dependent roles in trunk progenitor cell maintenance and differentiation[END_REF][START_REF] Takemoto | Convergence of Wnt and FGF signals in the genesis of posterior neural plate through activation of the Sox2 enhancer N-1[END_REF][START_REF] Wymeersch | Position-dependent plasticity of distinct progenitor types in the primitive streak[END_REF][START_REF] Yamaguchi | A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo[END_REF] and in the induction of the mesodermal or neural lineage in NMPs derivatives [START_REF] Del Corral | Onset of neuronal differentiation is regulated by paraxial mesoderm and requires attenuation of FGF signalling[END_REF][START_REF] Gouti | A Gene Regulatory Network Balances Neural and Mesoderm Specification during Vertebrate Trunk Development[END_REF][START_REF] Martin | Canonical Wnt signaling dynamically controls multiple stem cell fate decisions during vertebrate body formation[END_REF][START_REF] Nowotschin | Interaction of Wnt3a, Msgn1 and Tbx6 in neural versus paraxial mesoderm lineage commitment and paraxial mesoderm differentiation in the mouse embryo[END_REF][START_REF] Yoshikawa | Evidence That Absence ofWnt-3aSignaling Promotes Neuralization Instead of Paraxial Mesoderm Development in the Mouse[END_REF]. Here we show that both TGF- and BMP signalling pathways are active during the process of SN, supporting the results obtained from our analysis of gene expression. While the inhibition of BMP SMADs results in normal SNTs, the depletion of TGF- SMAD3, but not SMAD2, generates a NTD characterised by a multilumen phenotype. We cannot completely rule out a possible role for BMP in the process, as the negative results obtained with BMP sh-SMADs electroporation could still be explained if the inhibition of the BMP pathway is normally required for SNT formation. The different outcome of the two TGF- SMADs inhibition might reside in the fact that SMAD3 and SMAD2 can either cooperate or antagonize to regulate their transcriptional targets [START_REF] Miguez | Smad2 and Smad3 cooperate and antagonize simultaneously in vertebrate neurogenesis[END_REF]. Phosphorylated R-SMAD proteins form heterotrimeric complexes with SMAD4 that enter the nuclei, recruit various co-factors and bind to DNA in order to regulate target gene expression (A [START_REF] Moustakas | Smad regulation in TGFbeta signal transduction[END_REF][START_REF] Moustakas | From mono-to oligo-Smads: the heart of the matter in TGF-beta signal transduction[END_REF]. These complexes can contain of either SMAD2or SMAD3-homodimers, or SMAD2/SMAD3-heterodimers [START_REF] Shi | Mechanisms of TGF-beta signaling from cell membrane to the nucleus[END_REF]. As SMAD2 and SMAD3 recruit different co-factors and target different regulatory sequences [START_REF] Brown | A tale of two proteins: differential roles and regulation of Smad2 and Smad3 in TGF-beta signaling[END_REF], the balance between the heterotrimeric complexes formed upon ligand stimulation dictates the set of genes that ultimately will be activated. Our results therefore indicate that the activation of SMAD3 targets, but not of SMAD2 targets, is crucial for SNT morphogenesis.

A role of TGF- signalling in the disruption of epithelial organisation and EMT is well established [START_REF] Yang | Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis[END_REF]. However, we show here that SMAD3 depletion does not affect the capability of cells to apico-basally polarise and initiate multiple lumen foci in the developing SNT. Indeed, sh-SMAD3 cells normally elongate and correctly localise their centrosomes to the apical pole. The Golgi apparatus is the only organelle that we have found altered in sh-SMAD3 electroporated NPCs, as it fails to extend as control cells. Although the Golgi is abnormally short, it is still confined to the apical process of NPCs, between the nucleus and the apical surface. It is important to denote that we only studied the cis-Golgi, through the staining of GM-130, and left the trans-Golgi unexplored. Nevertheless, the Golgi apparatus in NPCs of the developing neocortex is oriented with their cis-to-trans axis perpendicular to the apico-basal axis of the cell [START_REF] Taverna | Non-canonical features of the Golgi apparatus in bipolar epithelial neural stem cells[END_REF]. If this is conserved in spinal cord NPCs, which is highly probable, then the extension of the Golgi will not affect apico-basal trafficking to a large extent but other Golgi cisternae functionalities. Indeed, junctional and apical markers such as Ncadherin, ZO-1 and aPKC present a normal subcellular distribution and completely line the multiple luminal surfaces, suggesting that apical intracellular trafficking occurs normally. The fact that TGF- activity depletion results in a multilumen phenotype with proper apico-basal polarity, rather than in the absence of polarisation and the complete inexistence of a lumen, points to a possible role of the pathway in the last steps of SNT lumen formation.

We found that the TGF- pathway is active and SMAD3 highly expressed in central cells at the lumen resolution stage. We hypothesize that TGF- signalling, through SMAD3, could be replacing the lost T/Bra activity in central NPCs and ensure that they retain the mesenchymal features. The maintained mesenchymal capabilities confer high cell motility and invasive protrusions to central cells and permit their intercalation into the lateral walls of the developing neuroepithelium. Indeed, TGF- activity controls actin polymerisation and actomyosin contractility during the lumen expansion of the Ciona intestinallis notochord [START_REF] Denker | Regulation by a TGFbeta-ROCK-actomyosin axis secures a non-linear lumen expansion that is essential for tubulogenesis[END_REF]. Both actin polymerisation and actomyosin contractility are essential for mesenchymal cell migration [START_REF] Chi | Rear actomyosin contractility-driven directional cell migration in threedimensional matrices: a mechano-chemical coupling mechanism[END_REF][START_REF] Ridley | Cell migration: integrating signals from front to back[END_REF]. Moreover, TGF- activity also controls stress fibres formation, reorganization of cytoskeletal structures and mesenchymal cell characteristics through the activation of RhoA-dependent signalling (64-66). However, the precise mechanisms by which TGF-SMAD3 regulates motility of central cells and promotes cell intercalation remains to be determined. Altogether, here we propose a model in which, in the absence of SMAD3 activity, central cells loose motility and protrusive activity so they become unable to intercalate and to be cleared from central areas. These prevents central lumen resolution ultimately generating neural tube defects (NTDs). (A-E (A-E (H) Selected images of transverse sections 24hpe of sh-SMAD3 (green) at the lumen resolution stage, stained for ZO-1 (white). A higher magnification of the boxed region is shown in E'. Scale bars = 20 m. (I) Selected images of transverse sections 24hpe of sh-SMAD3 (green) stained for the actin cytoskeleton (white) and cell nuclei (DAPI, blue). A higher magnification of the boxed region is shown in F'. Scale bars = 20 m.

FIGURE LEGENDS (max 350 words)

FIGURE1: SNT formation in the chick embryos requires TGFb mediated SMAD3 activity

FIGURE2: Cell confinement of NMPs and lineage restriction into NPCs are independent of SMAD3 activity

FIGURE5: Three modes of division coexist in the developing SNT but localise at different cranio-caudal levels. (A)

  Fig S2K,L), it did not compromise tissue or NMPs viability, as assessed by the rate of apoptosis and proliferation of total cells (mean±SD %c-Casp3+ cells control=1.9±1.0 vs sh-SMAD3=3.0±1.8: Supp.Fig S2N; mean±SD %pH3+ cells control=4.7±1.5 vs sh-SMAD3=3.8±1.1: Supp.Fig S2O) and electroporated cells (mean±SD %c-Casp3 H2B-RFP+ cells control=3.1±3.3 vs sh-SMAD3=4.0±1.9: Supp.Fig S2P; mean±SD %pH3+ H2B-RFP+ cells control=3.4±2.5 vs sh-SMAD3=3.4±2.1: Supp.Fig S2Q), respectively.

)

  FIGURE1: SNT formation in the chick embryos requires TGFb mediated SMAD3 activity(A-E) Scheme showing a stage HH15 chick embryo, and the cellular processes occurring along the posterior to anterior axis during secondary neural tube (SNT) formation. SNT cells are shown in light blue, the surrounding mesoderm is in brown and the notochord appears in dark blue. (A'-E') Transverse sections at different craniocaudal levels showing the distribution of actin (white). DAPI (blue) stains cell nuclei. Scale bars = 40 m. (F,G) Scheme summarizing the BMP and TGF- signalling components. (H) Scheme showing the method and timing of the co-electroporation in chick embryos and the electroporated DNAs (EP, electroporation; hpe, hours post-electroporation). (I) The BMP reporter is active in NMPs, 24hpe of BRE:GFP (green) and control H2B-RFP (red). DAPI (blue) stains nuclei. Scale bar = 40 m. (J) The TGF- reporter is active in NMPs, 24hpe of CAGA12:GFP (green) and control H2B-RFP (red). DAPI (blue) stains nuclei. Scale bar = 40 m. (K,L) Dorsal views of control H2B-GFP (green, F) or sh-SMAD3 (green, G) electroporated NTs. N-cadherin (white) lines the NT lumen and laminin (pink) stains the basement membrane and somites. Scale bars = 40 m. (M-Q) Selected images of transverse sections 24hpe of the indicated DNAs (green), ZO-1 staining (white) lines the NT lumen. sh-SMAD3 electroporation results in multiple lumens (L). Scale bar = 20 m.

)FIGURE3:

  FIGURE2: Cell confinement of NMPs and lineage restriction into NPCs are independent of SMAD3 activity(A-E) Selected images of transverse sections at the indicated cranio-caudal levels, stained for T/Bra (green) and Sox2 (red). Scale bars = 20 m. (F) Plots fluorescence nuclear intensity of T/Bra and Sox2 at the indicated tissue remodeling events (horizontal bold lines show the median; n=62, 109, 122, 122, 40 cells from 10 embryos; *p<0.05, **p<0.01, ***p<0.001 Kruskal-Wallis test). (G) Scheme showing SNT developing section and the lineage restriction of NMPs into PSNT cells. Cells contacting the BM (purple) are the first to convert to PSNT cells, this transition being characterised by T/Bra downregulation and Sox2 upregulation. (H) Plots ratio of Sox2 fluorescence intensity in control and sh-SMAD3 electroporated cells (horizontal bold lines show the median; n=100, 100 cells from 10 embryos/condition; p>0.05 Mann-Whitney test).

  FIGURE5: Three modes of division coexist in the developing SNT but localise at different cranio-caudal levels.(A) Selected frames of membrane-GFP electroporated embryos time-lapse movies showing a representative IC mitosis. A central mesenchymal cell (i, green arrow), divides (ii, orange dotted circle) and generates one intercalating daughter cell (IDCs) (iii-viii, red arrow) and one central daughter cell (CDC) (iii-viii, green arrow). Scale bars = 100m, 20m. (B) Plots cell distance from the centre of the cells in D along the time-lapse movie (10h). One daughter cell remains in the centre (green) while the other moves away and intercalates into the SNT lateral wall (red). (C) Plots circularity of the cells in D along the time-lapse movie (10h). One daughter cell maintains the high circularity of the mother cell (green) while the other elongates (red).

  FIGURE6: sh-SMAD3 electroporated cells fail to intercalate into the lateral walls of the forming SNT.(A) Drawing of an electroporated HH15 chick embryo showing the cranio-caudal levels of B-E. (B) Selected frame of Sox2p:GFP electroporated control embryos time-lapse movies at the cranio-caudal level indicated in A. Red dotted lines delineate the apicobasal surfaces of the SNT. Scale bar = 100m. (C-E) Selected frames of sh-SMAD3 Sox2p:GFP electroporated control embryos time-lapse movies at the cranio-caudal levels indicated in A. Frames are the last time points of G,H,I mitotic sequences, as indicated by the boxed regions. Red dotted lines delineate the apico-basal surfaces of the SNT. Note the abnormal accumulation of cells in the centre of the SNT in C and D. Scale bars = 100m. (G-H) Selected frames of sh-SMAD3 Sox2p:GFP movies showing two different cell divisions at the indicated cranio-caudal positions in C,D. The dotted circles indicate the mitosis (CC divisions, green), the green arrows point to central daughter cells and the red arrow to an intercalating daughter cell. Scale bars = 10m. (F) Plots the cranio-caudal location for each of the three modes of division as the distance from each measured division to the last formed pair of somites in both Sox2p:GFP control and sh-SMAD3 electroporated embryos. Mostly all sh-SMAD3 divisions generate two central daughter cells (CC divisions), regardless of the craniocaudal position (horizontal bold lines show the median; n=9,10,12 divisions from 5 Sox2p:GFP control and n=0,3,37 divisions from 6 sh-SMAD3 time-lapse movies; *p<0.05 Kruskal-Wallis test). (J) Selected frame of Sox2p:GFP control time-lapse movies. The boxed region refers to the last time point (v) of the sequence in B. Scale bar = 100m. (K) Selected frames of HH15 Sox2p:GFP time-lapse movies showing a representative IC mitosis. A mesenchymal cell (i, green arrow), divides (ii, orange dotted circle) and generates a daughter cell which remains in the centre (iii-v, green arrow) and another which intercalates into the SNT lateral wall (iii-v, red arrow). Scale bars = 10m.

( C )

 C Plots T/Bra fluorescence nuclear intensity in central and peripheral NPCs, at the indicated tissue remodeling events (horizontal bold lines show the median; n=54,54; 61, 61; 60,62 cells from 10 embryos; ***p<0.001 Kruskal-Wallis test). (D) Plots Sox2 fluorescence nuclear intensity in central and peripheral NPCs, at the indicated tissue remodeling events (horizontal bold lines show the median; n=54,54; 61, 61; 60,62 cells from 10 embryos; *p<0.05, ***p<0.001 Kruskal-Wallis test). (E) Selected images of transverse sections of control and sh-SMAD3 electroporated embryos (white), showing the developing SNT stained for Sox2(red). Scale bars = 20 m.SUPPLEMENTARY FIGURE S4: Mesenchymal-to-epithelial transition occurs in association to the basement membrane(A) Selected images of transverse sections showing fibronectin deposition (green) and centrosome positioning (purple). Higher magnifications of the boxed regions are shown in A'. Scale bars = 20 m. (B) Selected images of transverse sections showing Laminin deposition (red) and Golgi polarization (green). Higher magnifications of the boxed regions are shown in B'. Scale bars = 20 m. (C) Selected images of transverse sections showing Fibronectin deposition (purple) and N-Cadherin polarization (green). Higher magnifications of the boxed regions are shown in C'. Scale bars = 20 m. (D) Selected images of transverse sections showing Laminin deposition (red) and ZO-1 polarization (green). Higher magnifications of the boxed regions are shown in C'. Scale bars = 20 m. (E) Selected image of transverse sections stained for the centrosomes (purple), Ncadherin (red) and aPKC (green) at the first steps of peripheral cell polarisation. The yellow arrow points to apically localized centrosomes with the absence of apical protein accumulation. Scale bars = 10 m. (F) Selected image of transverse sections stained for the centrosomes (purple), Ncadherin (red) and aPKC (green) at the first steps of peripheral cell polarisation. Yellow arrows point to apically localized centrosomes with apical N-cadherin either with or without apical aPKC. Scale bars = 10 m. (G) Selected image of transverse sections stained for the centrosomes (purple), Ncadherin (red) and aPKC (green) at the first steps of peripheral cell polarisation. The yellow arrow points to apically localized centrosomes with N-cadherin and aPKC apical protein accumulation. Scale bars = 10 m. (H) Selected images of transverse sections stained for the centrosomes (purple), the basal protein 1 integrin (green) and the apical protein N-cadherin (red). 1 integrin is basally localised in all cells with apically localised centrosomes. The yellow arrow points to apically localized centrosomes with apical N-cadherin accumulation and without aPKC. Scale bars = 10m. (I) Plots the percentage of polarizing peripheral chord cells presenting only apical centrosome (FOP); apical centrosome and N-cadherin; apical centrosome, N-cadherin and aPKC; or apical centrosome, N-cadherin and ZO-1 (plot shows the mean±sem; n=100, 60, 87 cells from 10 embryos). (J) Plots the percentage of polarizing peripheral chord cells presenting apical centrosome (FOP) and basal 1 integrin or apical centrosome, basal 1 integrin and Ncadherin (plot shows the mean±sem; n=84 cells from 10 embryos). (K-M) Schemes showing the measurements performed in I,J. Cell nuclei are depicted in blue, centrosomes in purple (FOP) and the cis-Golgi in green (GM130). SUPPLEMENTARY FIGURE S5: Central NPCs at the stage of lumen resolution maintain mesenchymal characteristics (A) Scheme of the cellular events occurring in secondary lumen resolution (LR). The basement membrane appears in green. The two populations of peripheral and central NPCs are shown in A'. (B) Selected image of transverse sections at the lumen resolution phase stained for Sox2 (red) and T/Bra (green). Both central and peripheral NPCs are Sox2+ and T/Bra-at this stage. Dotted white line delineates central NPCs and a higher magnification is shown in B', B''. Scale bars = 20 m. (C) Selected image of transverse sections stained for the actin cytoskeleton (white). A higher magnification of the boxed region is shown in C', where blue dotted lines delineate cell shape of peripheral and central NPCs. Scale bars = 20 m. (D) Selected image of transverse sections stained for the centrosomes (purple) and the cis-Golgi (green). DAPI (blue) stains cell nuclei. A higher magnification of the boxed region is shown in D'. Scale bars = 20 m. (E) Selected image of transverse sections stained for the centrosomes (purple) and the polarity protein aPKC (green). DAPI (blue) stains cell nuclei. A higher magnification of the boxed region is shown E'. Scale bars = 20 m. (F) Plots cells shape/circularity of central and peripheral NPCs at the stage of lumen resolution (LR) and of NPCs once the secondary neural tube (SNT) is fully formed (horizontal bold lines show the median; n=100, 100, 100 cells from 10 embryos; ***p<0.001 Kruskal-Wallis test). (G) Plots the distance from apical FOP to the nucleus in central and peripheral NPCs at the stage of LR and in NPCs of the formed SNT (horizontal bold lines show the median; n=100, 100, 100 cells from 10 embryos; ***p<0.001 Kruskal-Wallis test). (H) Plots the distance from FOP to the basal-most Golgi (distal GM130) in central and peripheral NPCs at the stage of LR and in NPCs of the formed SNT (horizontal bold lines show the median; n=100, 100, 110 cells from 10 embryos; ***p<0.001 Kruskal-Wallis test).(I) Scheme of the formed secondary neural tube (SNT). SN results in a SNT with a single lumen in its centre surrounded by dividing epithelial NPCs as shown in I'. Apical and basal surfaces are indicated. The basal membrane appears in green. (J) Selected image of transverse sections of the formed SNT stained for the actin cytoskeleton (white). A higher magnification is shown in J'. Dotted lines delineate cell shape of central and peripheral NPCs. Scale bars = 20 m, 5m. (K) Selected image of transverse sections of the formed SNT stained for the actin cytoskeleton (red) and the cis-Golgi (green). DAPI (blue) stains cell nuclei. A higher magnification is shown in K'. Scale bars = 20 m, 5 m. (L) Selected image of transverse sections stained for the centrosomes (purple), the polarity protein aPKC (green) and the junctional protein N-cadherin (red). DAPI (blue) stains cell nuclei. A higher magnification is shown in L', where the yellow arrows point to the two apical complexes shown at the bottom left. Scale bars = 20m, 5 m.SUPPLEMENTARY FIGURE S6: The majority of sh-SMAD3 divisions generate two daughter central cells that fail to intercalate into the lateral walls of the forming SNT. (A)Drawing of an electroporated HH15 chick embryo showing the cranio-caudal levels of B-E. (B) Selected frame of Sox2p:GFP electroporated control embryos time-lapse movies at the cranio-caudal level indicated in A. Red dotted lines delineate the apicobasal surfaces of the SNT. Scale bar = 100m. (C,D) Selected frames of sh-SMAD3 Sox2p:GFP electroporated control embryos time-lapse movies at the cranio-caudal levels indicated in A. Frames are the last time points of G,H mitotic sequences, as indicated by the boxed regions. Red dotted lines delineate the apico-basal surfaces of the SNT. Note the abnormal accumulation of cells in the centre of the SNT in C and D. Scale bars = 100m. (E,F) Selected frames of sh-SMAD3 Sox2p:GFP movies showing three different cell divisions at the indicated cranio-caudal positions in C,D. The dotted circles indicate the mitosis (CC divisions, green), the green arrows point to central. (G,H) Plots cell distance from the centre along the time-lapse movie of shSMAD3 Sox2p:GFP cells. All daughter cells remain close to the centre (both dark and light green). CDC, central daughter cell; IDC, intercalating daughter cell.

SUPPLEMENTARY MOVIE LEGENDS

Movie S1. Transversal z-stack of the stage HH15 chick embryo in Figure 25E, advancing from caudal to cranial sections. shows the emerging lumen foci that coalesce into a single central cavity, laminin (purple) stains the growing basal lamina and DAPI (blue) stains cell nuclei. Related to Figure 4E.