Neural tube closure requires the endocytic receptor Lrp2 and its functional interaction with intracellular scaffolds.

Pathogenic mutations in the endocytic receptor LRP2 in humans are associated with severe neural tube closure defects (NTDs) such as anencephaly and spina bifida. Here, we have combined analysis of neural tube closure in mouse and in the African Clawed Frog Xenopus laevis to elucidate the etiology of Lrp2-related NTDs. Lrp2 loss of function impaired neuroepithelial morphogenesis, culminating in NTDs that impeded anterior neural plate folding and neural tube closure in both model organisms. Loss of Lrp2 severely affected apical constriction as well as proper localization of the core planar cell polarity (PCP) protein Vangl2, demonstrating a highly conserved role of the receptor in these processes, which are essential for neural tube formation. In addition, we identified a novel functional interaction of Lrp2 with the intracellular adaptor proteins Shroom3 and Gipc1 in the developing forebrain. Our data suggest that, during neurulation, motifs within the intracellular domain of Lrp2 function as a hub that orchestrates endocytic membrane removal for efficient apical constriction, as well as PCP component trafficking in a temporospatial manner.

Loss of Grhl3 is correlated with altered cellular protrusions in the non-neural ectoderm during neural tube closure.

BACKGROUND: The transcription factor Grainyhead-like 3 (GRHL3) has multiple roles in a variety of tissues during development including epithelial patterning and actin cytoskeletal regulation. During neural tube closure (NTC) in the mouse embryo, GRHL3 is expressed and functions in the non-neural ectoderm (NNE). Two important functions of GRHL3 are regulating the actin cytoskeleton during NTC and regulating the boundary between the NNE and neural ectoderm. However, an open question that remains is whether these functions explain the caudally restricted neural tube defect (NTD) of spina bifida observed in Grhl3 mutants. RESULTS: Using scanning electron microscopy and immunofluorescence based imaging on Grhl3 mutants and wildtype controls, we show that GRHL3 is dispensable for NNE identity or epithelial maintenance in the caudal NNE but is needed for regulation of cellular protrusions during NTC. Grhl3 mutants have decreased lamellipodia relative to wildtype embryos during caudal NTC, first observed at the onset of delays when lamellipodia become prominent in wildtype embryos. At the axial level of NTD, half of the mutants show increased and disorganized filopodia and half lack cellular protrusions. CONCLUSION: These data suggest that altered cellular protrusions during NTC contribute to the etiology of NTD in Grhl3 mutants.

Live Imaging of the Neural Crest Cell Epithelial-to-Mesenchymal Transition in the Chick Embryo.

Live embryo imaging may provide a wealth of information on intact cell and tissue dynamics, but can be technically challenging to sustain embryo orientation and health for long periods under a microscope. In this protocol, we describe an in vivo method to mount and image cell movements during the epithelial-to-mesenchymal transition (EMT) of neural crest cells within the chick dorsal neural tube. We focus on describing the collection of images and data preparation for image analysis throughout the developmental stages HH15-21 in the chick trunk. Trunk neural crest cell EMT is crucial to development of the peripheral nervous system and pigment cell patterning. The methods we describe may also be applied to other cell and tissue phenomena at various chick developmental stages with some modifications.

Expression of Connexins 37, 43 and 45 in Developing Human Spinal Cord and Ganglia.

Direct intercellular communication via gap junctions has an important role in the development of the nervous system, ranging from cell migration and neuronal differentiation to the formation of neuronal activity patterns. This study characterized and compared the specific spatio-temporal expression patterns of connexins (Cxs) 37, 43 and 45 during early human developmental stages (since the 5th until the 10th developmental week) in the spinal cord (SC) and dorsal root ganglia (DRG) using double immunofluorescence and transmission electron microscopy. We found the expression of all three investigated Cxs during early human development in all the areas of interest, in the SC, DRG, developing paravertebral ganglia of the sympathetic trunk, notochord and all three meningeal layers, with predominant expression of Cx37. Comparing the expression of different Cxs between distinct developmental periods, we did not find significant differences. Specific spatio-temporal pattern of Cxs expression might reflect their relevance in the development of all areas of interest via cellular interconnectivity and synchronization during the late embryonic and early fetal period of human development.

Non-neural surface ectodermal rosette formation and F-actin dynamics drive mammalian neural tube closure.

The mechanisms underlying mammalian neural tube closure remain poorly understood. We report a unique cellular process involving multicellular rosette formation, convergent cellular protrusions, and F-actin cable network of the non-neural surface ectodermal cells encircling the closure site of the posterior neuropore, which are demonstrated by scanning electron microscopy and genetic fate mapping analyses during mouse spinal neurulation. These unique cellular structures are severely disrupted in the surface ectodermal transcription factor Grhl3 mutants that exhibit fully penetrant spina bifida. We propose a novel model of mammalian neural tube closure driven by surface ectodermal dynamics, which is computationally visualized.

Cell biological mechanisms regulating chick neurogenesis.

Signalling pathways that regulate neural progenitor proliferation and neuronal differentiation have been identified. However, we know much less about how transduction of such signals is regulated within neuroepithelial cells to direct cell fate choice during mitosis and subsequent neuronal differentiation. Here we review recent advances in the experimentally amenable chick embryo, which reveal that this involves association of signalling pathway components with cell biological entities, including mitotic centrosomes and ciliary structures. This includes changing centrosomal localization of protein kinase A, which regulates Sonic hedgehog signalling and so neural progenitor status, and Mindbomb1, a mediator of Notch ligand activation, which promotes Notch signalling in neighbouring cells, and so is active in presumptive neurons. We further review cell biological events that underlie the later step of neuronal delamination, during which a newborn neuron detaches from its neighbouring cells and undergoes a process known as apical abscission. This involves inter-dependent actin and microtubule dynamics and includes dissociation of the centrosome from the ciliary membrane, which potentially alters the signalling repertoire of this now post-mitotic cell. Open questions and future directions are discussed along with technological advances which improve accuracy of gene manipulation, monitoring of protein dynamics and quantification of cell biological processes in living tissues.

Atg7-Mediated Autophagy Is Involved in the Neural Crest Cell Generation in Chick Embryo.

Autophagy plays a very important role in numerous physiological and pathological events. However, it still remains unclear whether Atg7-induced autophagy is involved in the regulation of neural crest cell production. In this study, we found the co-location of Atg7 and Pax7+ neural crest cells in early chick embryo development. Upregulation of Atg7 with unilateral transfection of full-length Atg7 increased Pax7+ and HNK-1+ cephalic and trunk neural crest cell numbers compared to either Control-GFP transfection or opposite neural tubes, suggesting that Atg7 over-expression in neural tubes could enhance the production of neural crest cells. BMP4 in situ hybridization and p-Smad1/5/8 immunofluorescent staining demonstrated that upregulation of Atg7 in neural tubes suppressed the BMP4/Smad signaling, which is considered to promote the delamination of neural crest cells. Interestingly, upregulation of Atg7 in neural tubes could significantly accelerate cell progression into the S phase, implying that Atg7 modulates cell cycle progression. However, ß-catenin expression was not significantly altered. Finally, we demonstrated that upregulation of the Atg7 gene could activate autophagy as did Atg8. We have also observed that similar phenotypes, such as more HNK-1+ neural crest cells in the unilateral Atg8 transfection side of neural tubes, and the transfection with full-length Atg8-GFP certainly promote the numbers of BrdU+ neural crest cells in comparison to the GFP control. Taken together, we reveal that Atg7-induced autophagy is involved in regulating the production of neural crest cells in early chick embryos through the modification of the cell cycle.

hmmr mediates anterior neural tube closure and morphogenesis in the frog Xenopus.

Development of the central nervous system requires orchestration of morphogenetic processes which drive elevation and apposition of the neural folds and their fusion into a neural tube. The newly formed tube gives rise to the brain in anterior regions and continues to develop into the spinal cord posteriorly. Conspicuous differences between the anterior and posterior neural tube become visible already during neural tube closure (NTC). Planar cell polarity (PCP)-mediated convergent extension (CE) movements are restricted to the posterior neural plate, i.e. hindbrain and spinal cord, where they propagate neural fold apposition. The lack of CE in the anterior neural plate correlates with a much slower mode of neural fold apposition anteriorly. The morphogenetic processes driving anterior NTC have not been addressed in detail. Here, we report a novel role for the breast cancer susceptibility gene and microtubule (MT) binding protein Hmmr (Hyaluronan-mediated motility receptor, RHAMM) in anterior neurulation and forebrain development in Xenopus laevis. Loss of hmmr function resulted in a lack of telencephalic hemisphere separation, arising from defective roof plate formation, which in turn was caused by impaired neural tissue narrowing. hmmr regulated polarization of neural cells, a function which was dependent on the MT binding domains. hmmr cooperated with the core PCP component vangl2 in regulating cell polarity and neural morphogenesis. Disrupted cell polarization and elongation in hmmr and vangl2 morphants prevented radial intercalation (RI), a cell behavior essential for neural morphogenesis. Our results pinpoint a novel role of hmmr in anterior neural development and support the notion that RI is a major driving force for anterior neurulation and forebrain morphogenesis.

[Primary cilia control different steps of brain development]. / Le cil primaire, orchestrateur de la morphogenèse cérébrale.

The role of primary cilia in adult neurons remains elusive, however their developmental functions during brain morphogenesis have been recently highlighted thanks to mouse models. Unmistakably, they are needed for Hedgehog (Hh)-dependent patterning in the forebrain. Not only for Hh reception itself, but most importantly for a downstream event in the Hh transduction pathway, independent of Hh ligand: the Gli3 processing. Indeed, phenotypes due to cilia disruption in the developing brain, such as early patterning, olfactory bulb or corpus callosum formation, can be rescued by reintroducing Gli3-R (the short truncated form of Gli3 working as a transcriptional repressor of Hh target gene). In addition, primary cilia control the proliferation rate in different neural progenitors in the cortex, the hippocampus and the cerebellum; they are required for proper migration of interneurons. And cilia dysfunction is correlated with hydrocephaly, synaptogenesis defects and aberrant axonal tract projections. Most of these neurodevelopmental defects can be related to the various neurological features frequently observed across the ciliopathy spectrum. And thus, understanding the underlying mechanisms of these diverse functions of primary cilia in the brain is a new fundamental challenge.

Transgenic increase of Wnt7b in neural progenitor cells decreases expression of T-domain transcription factors and impairs neuronal differentiation.

Wnt/beta-catenin signaling plays an important role in neural development, instructing both progenitor cell division and differentiation. During early corticogenesis, Wnt7b is expressed in a restricted expression pattern in the ventricular zone progenitor cells. However, its influence on progenitor cell behavior has not been fully studied. We report that transgenic overexpression of Wnt7b in neural progenitor cells impairs neuronal differentiation and the development of forebrain structures at embryonic day 10.5 (E10.5). This was accompanied by a decreased expression of T-domain transcription factors Tbr1 and Tbr2, in both progenitor cells and post-mitotic neurons. However, proliferation, apoptosis and the overall proportion of pax6(+) neural progenitor cells were similar to wild-type litter mates. These results suggest that Wnt signaling may affect early neural progenitor differentiation by regulating the expression of pro-neural transcription factors.

Forward genetics uncovers Transmembrane protein 107 as a novel factor required for ciliogenesis and Sonic hedgehog signaling.

Cilia are dynamic organelles that are essential for a vast array of developmental patterning events, including left-right specification, skeletal formation, neural development, and organogenesis. Despite recent advances in understanding cilia form and function, many key ciliogenesis components have yet to be identified. By using a forward genetics approach, we isolated a novel mutant allele (schlei) of the mouse Transmembrane protein 107 (Tmem107) gene, which we show here is critical for cilia formation and embryonic patterning. Tmem107 is required for normal Sonic hedgehog (Shh) signaling in the neural tube and acts in combination with Gli2 and Gli3 to pattern ventral and intermediate neuronal cell types. schlei mutants also form extra digits, and we demonstrate that Tmem107 acts in the Shh pathway to determine digit number, but not identity, by regulating a subset of Shh target genes. Phenotypically, schlei mutants share several features with other cilia mutants; however, spatial restriction of mutant phenotypes and lack of left-right patterning defects in schlei animals suggest differential requirements for Tmem107 in cilia formation in distinct tissues. Also, in contrast to mutants with complete loss of cilia, schlei mutants retain some function of both Gli activator and repressor forms. Together, these studies identify a previously unknown regulator of ciliogenesis and provide insight into how ciliary factors affect Shh signaling and cilia biogenesis in distinct tissues.

Trophic and proliferative perturbations of in vivo/in vitro cephalic neural crest cells after ethanol exposure are prevented by neurotrophin 3.

Neural crest cells (NCCs), a transient population that migrates from the developing neural tube, distributes through the embryo and differentiates into many derivatives, are clearly involved in the damage induced by prenatal exposure to ethanol. The aim of this work was to evaluate alterations of trophic parameters of in vivo (in ovo) and in vitro NCCs exposed to teratogenic ethanol doses, and their possible prevention by trophic factor treatment. Chick embryos of 24-30h of incubation were treated during 10h with 100mM ethanol, or 40 ng/ml Neurotrophin 3 (NT3), or 10 ng/ml Ciliary Neurotrophic Factor (CNTF), or ethanol plus NT3 or CNTF, or defined medium; then the topographic distribution of NCC apoptosis was assessed using a whole-mount acridine orange supravital method. Cultures of cephalic NCCs were exposed to the same ethanol or NT3, or CNTF treatments, or ethanol plus one of both trophic factors, or N2 medium. A viability assay was performed using the calcein-ethidium test, apoptosis was evaluated with the TUNEL test, and proliferative capacity after BrdU labeling. After direct exposure of embryos to 100mM ethanol for 10h, a high level of NCC apoptosis was coincident with the abnormal closure of the neural tube. These anomalies were prevented in embryos exposed to ethanol plus NT3 but not with CNTF. In NCC cultures, high cell mortality and a diminution of proliferative activity were observed after 3h of ethanol treatment. Incubation with ethanol plus NT3 (but not with CNTF) prevented NCC mortality as well as a fall in NCC proliferation. The consequences of direct exposure to ethanol expand data from our and other laboratories, supporting current opinion on the potential risk of alcohol ingestion (even at low doses and/or during a short time), in any period of pregnancy or lactation. Our in vivo/in vitro model encourages us to examine the pathogenic mechanism(s) of the ethanol-exposed embryo as well as the use of trophic factors for the treatment and/or prevention of anomalies induced by prenatal alcohol.

Disruption of Mks1 localization to the mother centriole causes cilia defects and developmental malformations in Meckel-Gruber syndrome.

Meckel-Gruber syndrome (MKS) is a recessive disorder resulting in multiple birth defects that are associated with mutations affecting ciliogenesis. We recovered a mouse mutant with a mutation in the Mks1 gene (Mks1(del64-323)) that caused a 260-amino-acid deletion spanning nine amino acids in the B9 domain, a protein motif with unknown function conserved in two other basal body proteins. We showed that, in wild-type cells, Mks1 was localized to the mother centriole from which the cilium was generated. However, in mutant Mks1(del64-323) cells, Mks1 was not localized to the centriole, even though it maintained a punctate distribution. Resembling MKS patients, Mks1 mutants had craniofacial defects, polydactyly, congenital heart defects, polycystic kidneys and randomized left-right patterning. These defects reflected disturbance of functions subserved by motile and non-motile cilia. In the kidney, glomerular and tubule cysts were observed along with short cilia, and cilia were reduced in number to a near-complete loss. Underlying the left-right patterning defects were fewer and shorter nodal cilia, and analysis with fluorescent beads showed no directional flow at the embryonic node. In the cochlea, the stereocilia were mal-patterned, with the kinocilia being abnormally positioned. Together, these defects suggested disruption of planar cell polarity, which is known to regulate node, kidney and cochlea development. In addition, we also showed that Shh signaling was disrupted. Thus, in the neural tube, the floor plate was not specified posteriorly even as expression of the Shh mediator Gli2 increased. By contrast, the Shh signaling domain was expanded in the anterior neural tube and anterior limb bud, consistent with reduced Gli3-repressor (Gli3R) function. The latter probably accounted for the preaxial digit duplication exhibited by the Mks1(del64-323) mutants. Overall, these findings indicate that centriole localization of Mks1 is required for ciliogenesis of motile and non-motile cilia, but not for centriole assembly. On the basis of these results, we hypothesize a role for the B9 domain in mother centriole targeting, a possibility that warrants further future investigations.

Foxj1 regulates floor plate cilia architecture and modifies the response of cells to sonic hedgehog signalling.

Sonic hedgehog signalling is essential for the embryonic development of many tissues including the central nervous system, where it controls the pattern of cellular differentiation. A genome-wide screen of neural progenitor cells to evaluate the Shh signalling-regulated transcriptome identified the forkhead transcription factor Foxj1. In both chick and mouse Foxj1 is expressed in the ventral midline of the neural tube in cells that make up the floor plate. Consistent with the role of Foxj1 in the formation of long motile cilia, floor plate cells produce cilia that are longer than the primary cilia found elsewhere in the neural tube, and forced expression of Foxj1 in neuroepithelial cells is sufficient to increase cilia length. In addition, the expression of Foxj1 in the neural tube and in an Shh-responsive cell line attenuates intracellular signalling by decreasing the activity of Gli proteins, the transcriptional mediators of Shh signalling. We show that this function of Foxj1 depends on cilia. Nevertheless, floor plate identity and ciliogenesis are unaffected in mouse embryos lacking Foxj1 and we provide evidence that additional transcription factors expressed in the floor plate share overlapping functions with Foxj1. Together, these findings identify a novel mechanism that modifies the cellular response to Shh signalling and reveal morphological and functional features of the amniote floor plate that distinguish these cells from the rest of the neuroepithelium.

Distribution and structural diversity of cilia in tadpole larvae of the ascidian Ciona intestinalis.

Accumulating evidence demonstrates that cilia play important roles in a variety of processes in embryogenesis. For functional survey of larval cilia at the cellular level, we exploited the simple cell organization of tadpole larvae in the ascidian Ciona intestinalis. Immunofluorescent microscopy showed distribution of cilia not only in previously described tissues but also in a subpopulation of ependymal cells in the sensory vesicle, gut primordium, papillae, apical trunk epidermal neurons, and the endodermal strand. Transmission electron microscopy revealed a variety of axonemal structures, including a 9+0 structure similar to vertebrate primary cilia, a 9+0 structure with electron-dense materials in the center, a 9+2 structure with no dynein arms, and an axoneme with a disorganized structure at the distal end. Extensive description of cilia in the present study gives important insights into the evolution of the ciliary structure and provides a basis for analysis of ciliary functions in establishment of chordate body plan.

Apical accumulation of Rho in the neural plate is important for neural plate cell shape change and neural tube formation.

Although Rho-GTPases are well-known regulators of cytoskeletal reorganization, their in vivo distribution and physiological functions have remained elusive. In this study, we found marked apical accumulation of Rho in developing chick embryos undergoing folding of the neural plate during neural tube formation, with similar accumulation of activated myosin II. The timing of accumulation and biochemical activation of both Rho and myosin II was coincident with the dynamics of neural tube formation. Inhibition of Rho disrupted its apical accumulation and led to defects in neural tube formation, with abnormal morphology of the neural plate. Continuous activation of Rho also altered neural tube formation. These results indicate that correct spatiotemporal regulation of Rho is essential for neural tube morphogenesis. Furthermore, we found that a key morphogenetic signaling pathway, the Wnt/PCP pathway, was implicated in the apical accumulation of Rho and regulation of cell shape in the neural plate, suggesting that this signal may be the spatiotemporal regulator of Rho in neural tube formation.

Initial motor axon outgrowth from the developing central nervous system.

Rat and chick studies show that the earliest motor rootlet axon bundles emerge from all levels of the neural tube between radial glial end feet which comprise the presumptive glia limitans. The loose arrangement of the end feet at the time of emergence facilitates this passage. The points of emergence are regularly spaced in relation to the long axis of the neural tube and are not defined by any cell contact with its surface. Each rootlet carries a covering of basal lamina from the neural tube surface, which forms a sleeve around it. It is only after bundles of ventral rootlet axons have emerged that cells associate with them, forming clusters on the rootlet surface at a distance peripheral to the CNS surface of both species. A tight collar of glial end feet develops around the axon bundle at the neural tube surface shortly after initial emergence. These arrangements are in sharp contrast to those seen in the sensory rootlets, where clusters of boundary cap cells prefigure the sensory entry zones at the attachments of the prospective dorsal spinal and cranial sensory rootlets. Boundary cap cells resemble cluster cells and a neural crest origin seems the most likely for them. The study clearly demonstrates that no features resembling boundary caps are found in relation to the developing motor exit points.