A Spatio-Temporal-Dependent Requirement of Sonic Hedgehog in the Early Development of Sclerotome-Derived Vertebrae and Ribs.

Derived from axial structures, Sonic Hedgehog (Shh) is secreted into the paraxial mesoderm, where it plays crucial roles in sclerotome induction and myotome differentiation. Through conditional loss-of-function in quail embryos, we investigate the timing and impact of Shh activity during early formation of sclerotome-derived vertebrae and ribs, and of lateral mesoderm-derived sternum. To this end, Hedgehog interacting protein (Hhip) was electroporated at various times between days 2 and 5. While the vertebral body and rib primordium showed consistent size reduction, rib expansion into the somatopleura remained unaffected, and the sternal bud developed normally. Additionally, we compared these effects with those of locally inhibiting BMP activity. Transfection of Noggin in the lateral mesoderm hindered sternal bud formation. Unlike Hhip, BMP inhibition via Noggin or Smad6 induced myogenic differentiation of the lateral dermomyotome lip, while impeding the growth of the myotome/rib complex into the somatic mesoderm, thus affirming the role of the lateral dermomyotome epithelium in rib guidance. Overall, these findings underscore the continuous requirement for opposing gradients of Shh and BMP activity in the morphogenesis of proximal and distal flank skeletal structures, respectively. Future research should address the implications of these early interactions to the later morphogenesis and function of the musculo-skeletal system and of possible associated malformations.

From neural tube to spinal cord: The dynamic journey of the dorsal neuroepithelium.

In a developing embryo, formation of tissues and organs is remarkably precise in both time and space. Through cell-cell interactions, neighboring progenitors coordinate their activities, sequentially generating distinct types of cells. At present, we only have limited knowledge, rather than a systematic understanding, of the underlying logic and mechanisms responsible for cell fate transitions. The formation of the dorsal aspect of the spinal cord is an outstanding model to tackle these dynamics, as it first generates the peripheral nervous system and is later responsible for transmitting sensory information from the periphery to the brain and for coordinating local reflexes. This is reflected first by the ontogeny of neural crest cells, progenitors of the peripheral nervous system, followed by formation of the definitive roof plate of the central nervous system and specification of adjacent interneurons, then a transformation of roof plate into dorsal radial glia and ependyma lining the forming central canal. How do these peripheral and central neural branches segregate from common progenitors? How are dorsal radial glia established concomitant with transformation of the neural tube lumen into a central canal? How do the dorsal radial glia influence neighboring cells? This is only a partial list of questions whose clarification requires the implementation of experimental paradigms in which precise control of timing is crucial. Here, we outline some available answers and still open issues, while highlighting the contributions of avian models and their potential to address mechanisms of neural patterning and function.

Recent advances in understanding cell type transitions during dorsal neural tube development.

The vertebrate neural tube is a representative example of a morphogen-patterned tissue that generates different cell types with spatial and temporal precision. More specifically, the development of the dorsal region of the neural tube is of particular interest because of its highly dynamic behavior. First, early premigratory neural crest progenitors undergo an epithelial-to-mesenchymal transition, exit the neural primordium, and generate, among many derivatives, most of the peripheral nervous system. Subsequently, the dorsal neural tube becomes populated by definitive roof plate cells that constitute an organizing center for dorsal interneurons and guide axonal patterning. In turn, roof plate cells transform into dorsal radial glia that contributes to and shapes the formation of the dorsal ependyma of the central nervous system. To form a normal functional spinal cord, these extraordinary transitions should be tightly regulated in time and space. Thus far, the underlying cellular changes and molecular mechanisms are only beginning to be uncovered. In this review, we discuss recent results that shed light on the end of neural crest production and delamination, the early formation of the definitive roof plate, and its further maturation into radial glia. The last of these processes culminate in the formation of the dorsal ependyma, a component of the stem cell niche of the central nervous system. We highlight how similar mechanisms operate throughout these transitions, which may serve to reveal common design principles applicable to the ontogeny of epithelial tissues.

Completion of neural crest cell production and emigration is regulated by retinoic-acid-dependent inhibition of BMP signaling.

Production and emigration of neural crest cells is a transient process followed by the emergence of the definitive roof plate. The mechanisms regulating the end of neural crest ontogeny are poorly understood. Whereas early crest development is stimulated by mesoderm-derived retinoic acid, we report that the end of the neural crest period is regulated by retinoic acid synthesized in the dorsal neural tube. Inhibition of retinoic acid signaling in the neural tube prevents the normal upregulation of BMP inhibitors in the nascent roof plate and prolongs the period of BMP responsiveness which otherwise ceases close to roof plate establishment. Consequently, neural crest production and emigration are extended well into the roof plate stage. In turn, extending the activity of neural crest-specific genes inhibits the onset of retinoic acid synthesis in roof plate suggesting a mutual repressive interaction between neural crest and roof plate traits. Although several roof plate-specific genes are normally expressed in the absence of retinoic acid signaling, roof plate and crest markers are co-expressed in single cells and this domain also contains dorsal interneurons. Hence, the cellular and molecular architecture of the roof plate is compromised. Collectively, our results demonstrate that neural tube-derived retinoic acid, via inhibition of BMP signaling, is an essential factor responsible for the end of neural crest generation and the proper segregation of dorsal neural lineages.

The division between the central nervous system ­ formed by the brain and spinal cord ­ and the peripheral nervous system ­ which consists of the neurons that sense and relay information to and from the body ­ takes place early during embryonic development. Initially, the nervous system consists of a tube of cells called the neural tube. From the top region of this tube, some cells change their shape, exit the tube and migrate to different places in the developing body. These cells are called the 'neural crest', and they form many different structures, including the peripheral nervous system. Neural crest cells keep leaving the neural tube for a period of time, but after that, the neural tube stops producing them. At this point, the region of the neural tube that had been producing neural crest cells becomes the 'roof plate' of the central nervous system, a structure that is essential for the development of specific groups of neurons in the brain and spinal cord. In bird embryos, a protein called bone morphogenetic protein (BMP) is essential for neural crest production because it triggers the migration of these cells away from the neural tube. Before the roof plate is formed, the activity of BMP is blocked by proteins known as BMP inhibitors, which stop more cells from leaving the neural tube. Around the time when neural crest formation stops, another molecule called retinoic acid begins to be synthesized in the top region of the neural tube. Rekler and Kalcheim asked whether retinoic acid is involved in the transition from neural crest to roof plate. To test this hypothesis, Rekler and Kalcheim blocked the activity of retinoic acid in the neural tube of quail embryos at the time when they should stop producing neural crest cells. This resulted in embryos in which the neural tube keeps producing neural crest cells after the roof plate has formed. In these embryos, individual cells in the resulting 'roof plate' produced both proteins that are normally only found in neural crest cells, and proteins typically exclusive to the roof plate. This suggests that, in the absence of retinoic acid activity, the segregation of neural crest identity from roof plate identity is compromised. Rekler and Kalcheim also found that, in the embryos where retinoic acid activity had been blocked, the cells in the area where the roof plate should be produced virtually no BMP inhibitors, and exhibited extended BMP activity. This allowed neural crest cells to continue forming and migrating away from the neural tube well after the period when they would stop in a normal embryo. These results indicate that retinoic acid stops the production of neural crest cells by repressing BMP activity in the roof plate of the neural tube. Rekler and Kalcheim's experiments shed light on the mechanisms that allow the central and peripheral nervous systems to become segregated. This could increase our understanding of the origin of several neurodevelopmental disorders, potentially providing insights into their treatment or prevention. Additionally, the process of neural crest production and exit from the neural tube is highly similar to the process of metastasis in many invasive cancers. Thus, by understanding how the production of neural crest cells is terminated, it may be possible to learn how to prevent malignant cancer cells from spreading through the body.

From Bipotent Neuromesodermal Progenitors to Neural-Mesodermal Interactions during Embryonic Development.

To ensure the formation of a properly patterned embryo, multiple processes must operate harmoniously at sequential phases of development. This is implemented by mutual interactions between cells and tissues that together regulate the segregation and specification of cells, their growth and morphogenesis. The formation of the spinal cord and paraxial mesoderm derivatives exquisitely illustrate these processes. Following early gastrulation, while the vertebrate body elongates, a population of bipotent neuromesodermal progenitors resident in the posterior region of the embryo generate both neural and mesodermal lineages. At later stages, the somitic mesoderm regulates aspects of neural patterning and differentiation of both central and peripheral neural progenitors. Reciprocally, neural precursors influence the paraxial mesoderm to regulate somite-derived myogenesis and additional processes by distinct mechanisms. Central to this crosstalk is the activity of the axial notochord, which, via sonic hedgehog signaling, plays pivotal roles in neural, skeletal muscle and cartilage ontogeny. Here, we discuss the cellular and molecular basis underlying this complex developmental plan, with a focus on the logic of sonic hedgehog activities in the coordination of the neural-mesodermal axis.

From Neural Crest to Definitive Roof Plate: The Dynamic Behavior of the Dorsal Neural Tube.

Research on the development of the dorsal neural tube is particularly challenging. In this highly dynamic domain, a temporal transition occurs between early neural crest progenitors that undergo an epithelial-to-mesenchymal transition and exit the neural primordium, and the subsequent roof plate, a resident epithelial group of cells that constitutes the dorsal midline of the central nervous system. Among other functions, the roof plate behaves as an organizing center for the generation of dorsal interneurons. Despite extensive knowledge of the formation, emigration and migration of neural crest progenitors, little is known about the mechanisms leading to the end of neural crest production and the transition into a roof plate stage. Are these two mutually dependent or autonomously regulated processes? Is the generation of roof plate and dorsal interneurons induced by neural tube-derived factors throughout both crest and roof plate stages, respectively, or are there differences in signaling properties and responsiveness as a function of time? In this review, we discuss distinctive characteristics of each population and possible mechanisms leading to the shift between the above cell types.

Notch signaling is a critical initiator of roof plate formation as revealed by the use of RNA profiling of the dorsal neural tube.

BACKGROUND: The dorsal domain of the neural tube is an excellent model to investigate the generation of complexity during embryonic development. It is a highly dynamic and multifaceted region being first transiently populated by prospective neural crest (NC) cells that sequentially emigrate to generate most of the peripheral nervous system. Subsequently, it becomes the definitive roof plate (RP) of the central nervous system. The RP, in turn, constitutes a patterning center for dorsal interneuron development. The factors underlying establishment of the definitive RP and its segregation from NC and dorsal interneurons are currently unknown. RESULTS: We performed a transcriptome analysis at trunk levels of quail embryos comparing the dorsal neural tube at premigratory NC and RP stages. This unraveled molecular heterogeneity between NC and RP stages, and within the RP itself. By implementing these genes, we asked whether Notch signaling is involved in RP development. First, we observed that Notch is active at the RP-interneuron interface. Furthermore, gain and loss of Notch function in quail and mouse embryos, respectively, revealed no effect on early NC behavior. Constitutive Notch activation caused a local downregulation of RP markers with a concomitant development of dI1 interneurons, as well as an ectopic upregulation of RP markers in the interneuron domain. Reciprocally, in mice lacking Notch activity, both the RP and dI1 interneurons failed to form and this was associated with expansion of the dI2 population. CONCLUSIONS: Collectively, our results offer a new resource for defining specific cell types, and provide evidence that Notch is required to establish the definitive RP, and to determine the choice between RP and interneuron fates, but not the segregation of RP from NC.

Guidelines and definitions for research on epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by 'the EMT International Association' (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data generated in various experimental models and to promote cross-disciplinary collaboration to identify and address key open questions in this research field. While recognizing the importance of maintaining diversity in experimental approaches and conceptual frameworks, we emphasize that lasting contributions of EMT research to increasing our understanding of developmental processes and combatting cancer and other diseases depend on the adoption of a unified terminology to describe EMT.

Neural tube development depends on notochord-derived sonic hedgehog released into the sclerotome.

Sonic hedgehog (Shh), produced in the notochord and floor plate, is necessary for both neural and mesodermal development. To reach the myotome, Shh has to traverse the sclerotome and a reduction of sclerotomal Shh affects myotome differentiation. By investigating loss and gain of Shh function, and floor-plate deletions, we report that sclerotomal Shh is also necessary for neural tube development. Reducing the amount of Shh in the sclerotome using a membrane-tethered hedgehog-interacting protein or Patched1, but not dominant active Patched, decreased the number of Olig2+ motoneuron progenitors and Hb9+ motoneurons without a significant effect on cell survival or proliferation. These effects were a specific and direct consequence of Shh reduction in the mesoderm. In addition, grafting notochords in a basal but not apical location, vis-à-vis the tube, profoundly affected motoneuron development, suggesting that initial ligand presentation occurs at the basal side of epithelia corresponding to the sclerotome-neural tube interface. Collectively, our results reveal that the sclerotome is a potential site of a Shh gradient that coordinates the development of mesodermal and neural progenitors.

YAP promotes neural crest emigration through interactions with BMP and Wnt activities.

BACKGROUND: Premigratory neural crest progenitors undergo an epithelial-to-mesenchymal transition and leave the neural tube as motile cells. Previously, we showed that BMP generates trunk neural crest emigration through canonical Wnt signaling which in turn stimulates G1/S transition. The molecular network underlying this process is, however, not yet completely deciphered. Yes-associated-protein (YAP), an effector of the Hippo pathway, controls various aspects of development including cell proliferation, migration, survival and differentiation. In this study, we examined the possible involvement of YAP in neural crest emigration and its relationship with BMP and Wnt. METHODS: We implemented avian embryos in which levels of YAP gene activity were either reduced or upregulated by in ovo plasmid electroporation, and monitored effects on neural crest emigration, survival and proliferation. Neural crest-derived sensory neuron and melanocyte development were assessed upon gain of YAP function. Imunohistochemistry was used to assess YAP expression. In addition, the activity of specific signaling pathways including YAP, BMP and Wnt was monitored with specific reporters. RESULTS: We find that the Hippo pathway transcriptional co-activator YAP is expressed and is active in premigratory crest of avian embryos. Gain of YAP function stimulates neural crest emigration in vivo, and attenuating YAP inhibits cell exit. This is associated with an accumulation of FoxD3-expressing cells in the dorsal neural tube, with reduced proliferation, and enhanced apoptosis. Furthermore, gain of YAP function inhibits differentiation of Islet-1-positive sensory neurons and augments the number of EdnrB2-positive melanocytes. Using specific in vivo reporters, we show that loss of YAP function in the dorsal neural tube inhibits BMP and Wnt activities whereas gain of YAP function stimulates these pathways. Reciprocally, inhibition of BMP and Wnt signaling by noggin or Xdd1, respectively, downregulates YAP activity. In addition, YAP-dependent stimulation of neural crest emigration is compromised upon inhibition of either BMP or Wnt activities. Together, our results suggest a positive bidirectional cross talk between these pathways. CONCLUSIONS: Our data show that YAP is necessary for emigration of neural crest progenitors. In addition, they incorporate YAP signaling into a BMP/Wnt-dependent molecular network responsible for emigration of trunk-level neural crest.

The Neural Crest: A Remarkable Model System for Studying Development and Disease.

Neural crest cells are the embryonic precursors of most neurons and all glia of the peripheral nervous system, pigment cells, some endocrine components, and connective tissue of the head, face, neck, and heart. Following induction, crest cells undergo an epithelial to mesenchymal transition that enables them to migrate along specific pathways culminating in their phenotypic differentiation. Researching this unique embryonic population has revealed important understandings of basic biological and developmental principles. These principles are likely to assist in clarifying the etiology and help in finding strategies for the treatment of neural crest diseases, collectively termed neurocristopathies. The progress achieved in neural crest research is made feasible thanks to the continuous development of species-specific in vivo and in vitro paradigms and more recently the possibility to produce neural crest cells and specific derivatives from embryonic or induced pluripotent stem cells. All of the above assist us in elucidating mechanisms that regulate neural crest development using state-of-the art cellular, molecular, and imaging approaches.

Neural crest emigration: From start to stop.

Within the dynamic context of a developing embryo, the multicellular patterns formed are extraordinarily precise. Through cell-cell communication, neighboring progenitors coordinate their activities, sequentially generating distinct tissues. The development of the dorsal neural tube remarkably illustrates this principle. It first generates neural crest (NC) cells, precursors of most of the peripheral nervous system, and then becomes the roof plate (RP) of the central nervous system. While the molecular network regulating emigration of NC progenitors has been extensively studied, the mechanisms by which dorsal neural tube precursors transit from an initial NC fate to a definitive RP identity remain widely open to investigation. Critical differences exist between premigratory NC and RP cells. Whereas the former extensively proliferate and undergo an epithelial-to-mesenchymal transition that generates cellular migrations, the latter progressively exit the cell cycle and regain epithelial traits including apico-basal polarity and regeneration of a laminin-containing basement membrane. To understand this transition, the nature of the cross-talk between these two sequentially forming progenitor subsets should be unraveled, including the identity and mode of action of signals that, on the one hand, induce the arrest of NC emigration, and, on the other hand, promote formation of a definitive RP.

Cell fate decisions during neural crest ontogeny.

The neural crest (NC) originates in the central nervous system (CNS) primordium. Born as an epithelium, NC progenitors undergo an epithelial-to-mesenchymal transition that generates cellular movement away from the CNS. Mesenchymal NC progenitors then migrate through stereotypic pathways characteristic of various axial levels until homing to distinct primordia where phenotypic differentiation takes place. Being the source of most of the peripheral nervous system, pigment cells and ectomesenchyme, the embryonic NC is considered to be a multipotent population of precursors. In spite of numerous recent studies, an essential and still unsolved question is when during ontogeny do the different lineages segregate from putative homogeneous and multipotent progenitors. Evidence suggests that the premigratory NC still resident in the dorsal neural tube epithelium is composed both of multipotent as well as of fate-restricted precursors, supporting the notion of an early appearance of cellular heterogeneity. Understanding these changing states of commitment is a prerequisite for deciphering molecular mechanisms that regulate fate segregation of the embryonic NC. In this review, we present data illustrating the existence of progenitors harboring different states of specification and their emergence as a function of time and space.

Dynamics of BMP and Hes1/Hairy1 signaling in the dorsal neural tube underlies the transition from neural crest to definitive roof plate.

BACKGROUND: The dorsal midline region of the neural tube that results from closure of the neural folds is generally termed the roof plate (RP). However, this domain is highly dynamic and complex, and is first transiently inhabited by prospective neural crest (NC) cells that sequentially emigrate from the neuroepithelium. It only later becomes the definitive RP, the dorsal midline cells of the spinal cord. We previously showed that at the trunk level of the axis, prospective RP progenitors originate ventral to the premigratory NC and progressively reach the dorsal midline following NC emigration. However, the molecular mechanisms underlying the end of NC production and formation of the definitive RP remain virtually unknown. RESULTS: Based on distinctive cellular and molecular traits, we have defined an initial NC and a subsequent RP stage, allowing us to investigate the mechanisms responsible for the transition between the two phases. We demonstrate that in spite of the constant production of BMP4 in the dorsal tube at both stages, RP progenitors only transiently respond to the ligand and lose competence shortly before they arrive at their final location. In addition, exposure of dorsal tube cells at the NC stage to high levels of BMP signaling induces premature RP traits, such as Hes1/Hairy1, while concomitantly inhibiting NC production. Reciprocally, early inhibition of BMP signaling prevents Hairy1 mRNA expression at the RP stage altogether, suggesting that BMP is both necessary and sufficient for the development of this RP-specific trait. Furthermore, when Hes1/Hairy1 is misexpressed at the NC stage, it inhibits BMP signaling and downregulates BMPR1A/Alk3 mRNA expression, transcription of BMP targets such as Foxd3, cell-cycle progression, and NC emigration. Reciprocally, Foxd3 inhibits Hairy1, suggesting that repressive cross-interactions at the level of, and downstream from, BMP ensure the temporal separation between both lineages. CONCLUSIONS: Together, our data suggest that BMP signaling is important both for NC and RP formation. Given that these two structures develop sequentially, we speculate that the longer exposure of RP progenitors to BMP compared with that of premigratory NC cells may be translated into a higher signaling level in the former. This induces changes in responsiveness to BMP, most likely by downregulating the expression of Alk3 receptors and, consequently, of BMP-dependent downstream transcription factors, which exhibit spatial complementary expression patterns and mutually repress each other to generate alternative fates. This molecular dynamic is likely to account for the transition between the NC and definitive RP stages and thus be responsible for the segregation between central and peripheral lineages during neural development.

Epithelial-Mesenchymal Transitions during Neural Crest and Somite Development.

Epithelial-to-mesenchymal transition (EMT) is a central process during embryonic development that affects selected progenitor cells of all three germ layers. In addition to driving the onset of cellular migrations and subsequent tissue morphogenesis, the dynamic conversions of epithelium into mesenchyme and vice-versa are intimately associated with the segregation of homogeneous precursors into distinct fates. The neural crest and somites, progenitors of the peripheral nervous system and of skeletal tissues, respectively, beautifully illustrate the significance of EMT to the above processes. Ongoing studies progressively elucidate the gene networks underlying EMT in each system, highlighting the similarities and differences between them. Knowledge of the mechanistic logic of this normal ontogenetic process should provide important insights to the understanding of pathological conditions such as cancer metastasis, which shares some common molecular themes.

A Novel Role for VICKZ Proteins in Maintaining Epithelial Integrity during Embryogenesis.

BACKGROUND: VICKZ (IGF2BP1,2,3/ZBP1/Vg1RBP/IMP1,2,3) proteins bind RNA and help regulate many RNA-mediated processes. In the midbrain region of early chick embryos, VICKZ is expressed in the neural folds and along the basal surface of the neural epithelium, but, upon neural tube closure, is down-regulated in prospective cranial neural crest (CNC) cells, concomitant with their emigration and epithelial-to-mesenchymal transition (EMT). Electroporation of constructs that modulate cVICKZ expression demonstrates that this down-regulation is both necessary and sufficient for CNC EMT. These results suggest that VICKZ down-regulation in CNC cell-autonomously promotes EMT and migration. Reduction of VICKZ throughout the embryo, however, inhibits CNC migration non-cell-autonomously, as judged by transplantation experiments in Xenopus embryos. RESULTS AND CONCLUSIONS: Given the positive role reported for VICKZ proteins in promoting cell migration of chick embryo fibroblasts and many types of cancer cells, we have begun to look for specific mRNAs that could mediate context-specific differences. We report here that the laminin receptor, integrin alpha 6, is down-regulated in the dorsal neural tube when CNC cells emigrate, this process is mediated by cVICKZ, and integrin alpha 6 mRNA is found in VICKZ ribonucleoprotein complexes. Significantly, prolonged inhibition of cVICKZ in either the neural tube or the nascent dermomyotome sheet, which also dynamically expresses cVICKZ, induces disruption of these epithelia. These data point to a previously unreported role for VICKZ in maintaining epithelial integrity.

Mechanisms of myogenic specification and patterning.

Mesodermal somites are initially composed of columnar cells arranged as a pseudostratified epithelium that undergoes sequential and spatially restricted changes to generate the sclerotome and dermomyotome, intermediate structures that develop into vertebrae, striated muscles of the body and limbs, dermis, smooth muscle, and endothelial cells. Regional cues were elucidated that impart differential traits upon the originally multipotent progenitors. How do somite cells and their intermediate progenitors interpret these extrinsic cues and translate them into various levels and/or modalities of intracellular signaling that lead to differential gene expression profiles remains a significant challenge. So is the understanding of how differential fate specification relates to complex cellular migrations prefiguring the formation of body muscles and vertebrae. Research in the past years has largely transited from a descriptive phase in which the lineages of distinct somite-derived progenitors and their cellular movements were traced to a more mechanistic understanding of the local function of genes and regulatory networks underlying lineage segregation and tissue organization. In this chapter, we focus on some major advances addressing the segregation of lineages from the dermomyotome, while discussing both cellular as well as molecular mechanisms, where possible.

Segregation of striated and smooth muscle lineages by a Notch-dependent regulatory network.

BACKGROUND: Lineage segregation from multipotent epithelia is a central theme in development and in adult stem cell plasticity. Previously, we demonstrated that striated and smooth muscle cells share a common progenitor within their epithelium of origin, the lateral domain of the somite-derived dermomyotome. However, what controls the segregation of these muscle subtypes remains unknown. We use this in vivo bifurcation of fates as an experimental model to uncover the underlying mechanisms of lineage diversification from bipotent progenitors. RESULTS: Using the strength of spatio-temporally controlled gene missexpression in avian embryos, we report that Notch harbors distinct pro-smooth muscle activities depending on the duration of the signal; short periods prevent striated muscle development and extended periods, through Snail1, promote cell emigration from the dermomyotome towards a smooth muscle fate. Furthermore, we define a Muscle Regulatory Network, consisting of Id2, Id3, FoxC2 and Snail1, which acts in concert to promote smooth muscle by antagonizing the pro-myogenic activities of Myf5 and Pax7, which induce striated muscle fate. Notch and BMP closely regulate the network and reciprocally reinforce each other¿s signal. In turn, components of the network strengthen Notch signaling, while Pax7 silences this signaling. These feedbacks augment the robustness and flexibility of the network regulating muscle subtype segregation. CONCLUSIONS: Our results demarcate the details of the Muscle Regulatory Network, underlying the segregation of muscle sublineages from the lateral dermomyotome, and exhibit how factors within the network promote the smooth muscle at the expense of the striated muscle fate. This network acts as an exemplar demonstrating how lineage segregation occurs within epithelial primordia by integrating inputs from competing factors.