New Perspectives in Neural Crest Development
Séminaire FSER organisé par Chaya Kalcheim (The Hebrew University of Jerusalem) et Marianne Bronner-Fraser (University of Irvine, California) du 2 au 7 mai 2006
Yves-Alain Barde, James Briscoe, Marianne Bronner-Fraser, Alan Burns, Sophie Creuzet, José De Brito, Elisabeth Dupin, Carol Erickson, Scott Fraser, Mike Gershon, Chaya Kalcheim, Paul Kulesa, Carole LaBonne, Nicole Le Douarin, Roberto Mayor, Anne-Helene Monsoro-Burq, Angela Nieto, Jean-Pierre Saint-Jeannet, Lukas Sommer, Yoshiko Takahashi, Paul Trainor, Klaus Unsicker, Yoshio Wakamatsu, David Wilkinson.
The colloquium, a timely initiative of Prof. Nicole Le Douarin, addressed state of the art questions on the ontogeny of the neural crest (NC). This transient embryonic structure segregates early from the neuroepithelium and superficial ectoderm; its component cells undergo a process of epithelial-to-mesenchymal transition, separate from the neural primordium and migrate extensively to yield a rich variety of neural and non-neural derivatives. Several major questions were discussed. A classical and still puzzling problem is elucidating the state of commitment of NC cells at different stages of their ontogeny. Although much has been done to address the determination of specific sublineages, the molecular definition of a generic concept of “cell specification” is still vague and requires continuous elaboration. In this regard, useful insights on how cells establish and maintain their fates came from researchers working in the stem cell field.
An intriguing conclusion of many studies is that a limited number of molecules act reiteratively to induce distinct outcomes. Examples are the activities of BMPs and Wnts which participate in early NC induction, then in cell delamination and later in specification of particular sublineages. The molecular cassettes activated downstream of these genes at each stage were discussed with the aim of understanding the rules that operate at different spatial and temporal contexts.
Next, the participants dealt with fascinating problems of morphogenesis, sharing their findings on possible molecular mechanisms that regulate NC cell migration and their further organization into discrete tissues. Finally, some attempts were made to integrate present knowledge on genes operating on NC development in an evolutionary context.
Below, we summarize the main concepts and findings discussed by each participant in sequential order of presentation:
Nicole Le Douarin started her talk with an interesting presentation of the history and mission of Treilles. She then discussed the work that led to her interest in the NC. Her initial experiments examined development of the liver, but she was surprised to find that cocultures of endoderm with mesenchyme led to the production of catecholamine-containing cells. She suspected these were of NC origin and developed the quail/chick chimera system to test this. Her suspicions were correct and this raised her interest in the NC, a field to which she made pivotal contributions to understanding the pathways of NC migration and fates. She finished her talk discussing new data on the mechanisms underlying facial patterning. She has found that there is an interesting segregation in the ability of Hox-positive versus Hox-negative NC in their ability to form elements of the face. Ablation of the midbrain NC can be rescued by Hox-negative but not by Hox-positive NC. Furthermore, her new data suggest that Shh from the endoderm may be critical for the survival of cranial NC cells.
Marianne Bronner-Fraser discussed regulatory interactions that lead to NC formation at the neural plate border. First, inductive interactions lead to establishment of neural plate border genes. These regulate NC specifier genes that in turn regulate downstream targets involved in generating migratory cells. She showed that NC cells were specified during gastrulation and that the neuropilin-2 receptor was responsible for patterning migration in the trunk. Finally, this regulatory network is present at the base of vertebrates, but NC specifiers are absent from the neural plate border in non-vertebrate chordates.
Jean-Pierre Saint-Jeannet presented data on the roles of Pax3 and Zic1 as neural plate border specifiers and SoxE genes as NC specifiers in Xenopus. He showed that Zic1 synergizes with Pax3 to promote NC fate. A balance of Pax3 activity controls whether these embryonic regions form NC versus hatching gland, whereas a balance of Zic activities determines NC versus placode fate. Finally, he showed that SoxE genes have some overlapping and some unique functions in the early embryo. Knock-down of Sox8 causes severe depletion of multiple NC lineages and can be rescued by Sox8 or Sox9 but not Sox10.
Anne-Helene Monsoro-Burq discussed the molecular mechanisms of NC induction that are involved in building a NC gene regulatory network using Xenopus as a model. She showed that NC induction involves FGF8, Msx1 and Pax3. Induction by Msx1 requires Pax3 activity but Pax3 by itself is not sufficient. She found that there are parallel FGF8 and Wnt pathways that act at neural plate border and that each is necessary for induction of Snail2 and FoxD3. Both pathways converge on Pax3 activity at the neural plate border.
Yoshio Wakamatsu discussed the modulation of neural induction and epithelial/mesenchymal transitions in the cranial NC by PKA signaling. He analyzed transcriptional regulation of Snail2 using an 800 base pair regulatory element of the Snail2 promoter. He presented data showing that Snail2 can act as a positive regulator of itself. Furthermore, it can bind to and cooperate with Sox9, causing these cells to undergo an epithelial/mesenchymal transition and emigrate. Finally, he showed that PKA was important for Snail2 expression in vivo.
Carole LaBonne discussed the regulation of proteins by post-translational modifications involving ubiquitin and SUMOylation. She found that the E3 ligase Ppa is expressed in the NC forming region and is a physiological regulator of Snail2 such that Snail2 is stabilized once it is down-regulated. Other NC markers, the SoxE genes, are regulated by SUMOylation such that SoxE mutants in SUMOylation are more active in making NC precursors whereas constitutively SUMOylated forms block formation of precursors.
Chaya Kalcheim presented data on the molecular basis of NC delamination from the neural tube. She showed that there are reciprocal gradients of BMP and noggin in the neural tube and only when BMP reached appropriate levels are NC cells able to emigrate. The dermomyotome of the somite produces an inhibitor of noggin that releases BMP from its inhibition thereby controlling the onset of delamination. BMP in turn upregulates Wnt1 which stimulates cyclin D1 and synchronous delamination of NC in the S-phase of the cell cycle. Another important inhibitor of delamination is N-cadherin which acts by antagonizing canonical Wnt signaling. N-cadherin is cleaved by a BMP-dependent ADAM10 metalloprotease to a soluble cytoplasmic product that, contrary to the full-length protein, stimulates cyclin D1 transcription and subsequently promotes delamination.
Roberto Mayor discussed the role of Wnt signaling in NC induction and migration in Xenopus and zebrafish. He showed that canonical Wnt8 signaling from the dorsolateral mesoderm is required for NC induction; later, the intermediate mesoderm derived from this dorsolateral mesoderm, is required for maintenance of NC specification. After emigration, Wnt signaling via the non-canonical PCP pathway is also required for NC migration. Finally, he showed that syndecan4 is expressed in the NC and necessary for migration, also functioning in the PCP pathway.
James Briscoe’s data suggest, using coelectroporation of several genes simultaneously, that there is a combinatorial transcriptional code involved in making neuroepithelial cells undergo specification into NC-like cells and then epithelial-mesenchymal transitions in aves. He found that Sox9 was important for survival and induces many features of this EMT whereas FoxD3 induces Sox10 and also affects cell-cell adhesion. Triple electroporation of FoxD3, Sox9 and Slug causes virtually all neural tube cells to emigrate. Sox induces a set of neural genes, promotes survival, provides competence to undergo EMT whereas Rho induces EMT in NC progenitors but induces apoptosis in absence of Sox9.
Angela Nieto discussed the role of Snail genes in development and cancer. The Snail superfamily consists of Scratch and Snail, with vertebrates having three Snail genes (1, 2 and 3). Snail genes have two main functions: as inducers of EMT and repressors of E-cadherin. In health and disease, Snail controls acquisition of migratory properties, decreases proliferation, and confers resistance to cell death. In zebrafish, morpholino-mediated knock-down of Snail1b down-regulates the NC markers FoxD3, crestin and AP-2. The results suggest that Snail genes act as inducers of survival or of EMT depending upon the cellular context.
David Wilkinson talked about the regulation of cell repulsion and invasion by cross-talk to Eph receptors and FGF-receptors. He found that co-expression of activated FGF receptor in cells expressing Eph receptors alters their interactions with ephrin expressing cells from a repulsive to an attractive response. He presented evidence showing that the receptor tyrosine phosphatase LAR and Eph have partly overlapping roles in axon guidance, consistent with the possibility that LAR may dephosphorylate Eph. Finally, he showed that the vertebrate homolog of cross-veinless2 is a modulator of BMP signaling expressed in the neural tube which can either synergize with or antagonize BMP depending upon the level of activity.
Paul Kulesa described new techniques for imaging NC cells. He studied the cellular dynamics within rhombomere 4 stream of NC cells. He showed that NC cells fill their derivatives in a distal to proximal order and tend to maintain neighbor relationships. They migrate in chain-like arrays. He went on to show that modulation of Cdc42 activity disrupts normal chain formation, decreases velocity and directionality of cell movement. Finally, he showed that melanoma cells, NC-derived tumor cells, could migrate along NC pathways when introduced into the embryo.
Scott Fraser presented novel techniques for imaging vertebrate embryos which will allow detailed analysis of cell movements, cell lineages and cell interactions. These tools are intended to follow a large number of cells, permitting clonal histories to be generated in vivo. These reveal that cells in the embryo can take a variety of paths to reach their final positions and fates in the embryo, and argue that cell interactions may take place in positions and stages previously unanticipated. In addition, the results show that NC and other cells respond to mechanical as well as molecular signals.
Yves Barde discussed ways in which one can generate homogeneous progenitor populations of cells from mouse embryonic stem cells by selecting for rapidly dividing cells. These can be generated from both wild type and mutant lines and can be assayed for differentiation by transplantation into embryos. By adding retinoic acid, ES cells generate a Pax6+ population of progenitors similar to radial glia. In vitro, these form glutaminergic neurons. When transplanted in vivo, these cells form neurons that normally are derived from a Pax6 positive population like motor neurons and glutamatergic neurons in the brain but fail to make dorsal root ganglion cells. Mutant cells lacking Pax6 fail to form these same cell types, but rather form GABAergic neurons with high levels of p75 and eventually die. Finally, he showed a proteomic analysis whereby they were able to identify Galectin, a lectin made by p75 expressing cells that promoted atrophy of the axons and led to cell death.
Elizabeth Dupin described the developmental potential and stem cell properties of NC cells derived from the mesencephalic and trunk neural tube. By placing these cells in culture with a feeder cell layer and medium that allowed differentiation of all derivatives, she showed that various combinations of phenotypes arose from a single cell. Some progenitors formed one, two, three, four or as many as five different cell types, suggesting multipotency. Endothelin3 promotes survival and proliferation of glial-melanocyte precursors, significantly increasing the number of melanocytes, and promoting self-renewal. Finally, she showed plasticity in NC derived-melanocytes. Single pigment cells in culture could give rise to clones containing multiple NC derivatives and could be serially recloned.
Lukas Sommer discussed molecular and cellular mechanisms governing lineage decisions in NC progenitor cells. NC cells have some stem cell properties: they can form multiple derivatives and self-renew at least transiently. NC stem cells persist in the numerous tissues including the skin, where they seem to arise from glial and melanocyte lineages. TGF-b signaling via the TbRII receptor is important in some NC lineage decisions. Receptor mutant embryos die prenatally due to multiple developmental defects including craniofacial anomalies that resemble DiGeorge’s syndrome as well as Axenfeld-Rieger’s anomaly in the eye. The data suggest that TGF-b is essential for differentiation into multiple lineages ranging from smooth muscle, to chondrocytes, and keratinocytes. In the absence of TGF-b signaling, these cells fail to differentiate properly. In general, TGF-b may prime cells to stay in a progenitor state. In trunk NC stem cells, TGF-b allows clonal populations to respond to FGF by turning on Sox9 and forming chondrocytes.
Paul Trainor talked about understanding the role of NC cells in congenital craniofacial birth defects such as Treacher-Collin’s syndrome, which is caused by a mutation in the Tcof1 gene that encodes a putative nucleolar phosphoprotein called treacle. Mutant mice exhibit severe facial hypoplasia and microopthalmia. The defect appears to be due to striking cell death of NC precursors in the dorsal neural plate. In addition, the proliferative ability of the remaining migrating NC cells is significantly compromised. Many of the genes affected were involved in p53 function; consequently, lowering the dosage of p53 in treacle mutant embryos leads to at least partial rescue of the phenotype. Finally, he discussed ongoing experiments to generate NC stem cells lines from cortex-derived neurospheres that express Sox2; these appear to be multipotent and able to self-renew. However, they fail to make NC when transplanted to embryos whereas neurospheres lacking Sox2 can form NC, migrate and contribute to multiple derivatives.
Sophie Creuzet described the role of NC in coordinating head and brain development. Ablation of midbrain neural folds results in severe loss of skeleton and brain abnormalities. These defects are rescued by small portions of midbrain to rhombomere1/2 but not more caudal neural folds that express Hox genes. r3 NC was found to be able to regenerate parts of the facial skeleton when more caudal NC was grafted anteriorly after facial crest ablation next to the FGF8 domain. This demonstrates a high degree of regulative ability in this population. Electroporation of Hoxa2 into the midbrain leads to similar deficits as ablation whereas FGF8 beads partially rescue both skeletal and brain defects. The NC plays an important role in maintaining FGF8 expression in the anterior neural ridge, apparently indirectly via gremlin-mediated inhibition of BMP signaling.
Michael Gershon discussed formation of the enteric nervous system from the NC and the role of Hand 2. Defects in enteric development lead to debilitating diseases like aganglionic megacolon and irritable bowel syndrome. Hand2 is expressed in NC-derived precursors to both neurons and glia in the gut. These precursors express a variety of transcription factors and receptors including Phox2b, Mash-1, Sox10 and ret. Hand2 is developmentally regulated and is expressed intranuclearly at early stages but later has cytoplasmic localization. In mutants for Hand2, the precursors fail to differentiate into neurons. Finally, he discussed molecules involved in synapse formation in the enteric nervous system. He has found that neurexins and neuroligins, previously found to function in synapse formation in the central nervous system, can mediate formation of synapses by enteric neurons as well.
Alan Burns described the colonization of the gut and lungs by vagal and sacral NC cells. By performing quail/chick chimera, he was able to follow the colonization of these tissues over time in the bird. He found that cells colonize the entire length of the gut by E8.5. Similar results were seen in human. In addition to the gut, vagal NC migrate from foregut to lung buds and form intrinsic ganglia but the mechanism of migration unknown. Sacral NC cells also contribute to the gut and initially colonize the nerve of Remak, then sending projections into the gut wall that the NC derived cells follow. Grafts of sacral neural tube to vagal levels leads to many NC cells entering the gut. In the reciprocal graft, vagal NC cells transplanted to sacral levels migrate caudorostrally and express normal patterns of enteric genes, but the two populations exhibit some differences in developmental potential. These differences can be mimicked by lowering ret levels in vagal cells or increasing them in sacral cells.
Carol Erickson discussed pathfinding by the NC with focus on the melanoblast pathway. She showed that melanoblasts are specified before entering the dorsolateral pathway and that this specification confers the ability to take a particular path. Ephrins are expressed at early stages along the dorsolateral pathway and is inhibitory for early migrating NC but appears to promote migration of melanoblasts. The balance of promotion versus inhibition of migratory behavior in NC-derived cells may be mediated by the Rho family of GTPases. A model for specification in the pigment lineage is that FoxD3 is expressed early in neural tube where it represses transcription of Mitf. Then FoxD3 is down-regulated, allowing transcription of genes controlling pigmentation. At vagal levels as well, there may be a developmental bias correlated with timing of emigration, especially of cardiac crest, and ephrins may be responsible for pathway choice.
Klaus Unsicker presented data that revisits the question of how the chromaffin cell lineage develops from the NC. The classic view is that glucocorticoids from the adrenal cortex cause chromaffin cells to develop from a common sympathoadrenal precursor. When analyzing mutant mice that lack the adrenal cortex, however, he found that chromaffin cells formed normally, expressing dense core vesicles and having normal morphology. The only noted differences were fewer numbers of cells and failure to down-regulate c-ret or up-regulate PNMT. In contrast, Mash1 null mice have very few chromaffin cells, with only a small subpopulation persisting. The phenotype of Phox2b null mice is even more severe, though chromaffin precursors maintain Mash1 for some time. However the adrenal cortex does appear to regulate the numbers of chromaffin cells via expression of BMP4. The results suggest that sympathetic precursors and chromaffin cells may arise from separate precursor populations.
Yoshiko Takahashi discussed the migratory behaviors of somitic cells as well as of adrenomedullary cells. In the case of somites, she finds that Notch signaling is activated in posterior somitic cells and these subsequently migrate ventrally to the dorsal aorta. The dorsal aorta appears to be a source of signals that attract these cells since either grafting the somite cells dorsally or an ectopic dorsal aorta ventrally leads to the Notch-activated cells migrating toward the dorsal aorta. EphrinB2 on the somite cells appears essential for the migration of this population. To understand how the presumptive adrenomedullary and adrenal cortical cells interact, she over-expressed the cortical marker SF1 into the coelomic epithelium to create ectopic cortical cells. The results suggest that NC-derived cells are attracted to the ectopic cortical cells and that BMP4 expression in the cortex may be important for attractive activity.