Mice lacking ZFHX1B,the gene that codes for Smad-interacting protein-1, reveal a role for multiple neural crest cell defects in the etiology of Hirschsprung disease-mental retardation syndrome

Recently, mutations in ZFHX1B, the gene that encodes Smad-interacting protein-1 (SIP1), were found to be implicated in the etiology of a dominant form of Hirschsprung disease-mental retardation syndrome in humans. To clarify the molecular mechanisms underlying the clinical features of SIP1 deficiency, we generated mice that bear a mutation comparable to those found in several human patients. Here, we show that Zfhx1b-knockout mice do not develop postotic vagal neural crest cells, the precursors of the enteric nervous system that is affected in patients with Hirschsprung disease, and they display a delamination arrest of cranial neural crest cells, which form the skeletomuscular elements of the vertebrate head. This suggests that Sip1 is essential for the development of vagal neural crest precursors and the migratory behavior of cranial neural crest in the mouse. Furthermore, we show that Sip1 is involved in the specification of neuroepithelium.     

Overlapping origins of pharyngeal arch crest cells on the postotic hind-brain

The developing hind-brain of vertebrates consists of segmental units called rhombomeres. Although crest cells emigrate from the hind-brain, they are subsequently subdivided into several cell populations that are attached to restricted regions of the hind-brain. At the preotic level, only even-numbered rhombomeres are accompanied by crest cells, while the odd-numbered ones are not. At the postotic level, such the birhombomeric repetition becomes obscure. In order to map the origins and distributions of postotic crest cells, focal injections of Dil were made into various axial levels of the postotic neural tube. Cephalic crest cells at the postotic level first form a single cell population deposited by cells along the dorsolateral pathway. They are called the circumpharyngeal crest cells (CP cells) and are secondarily subdivided into each pharyngeal arch ectomesenchyme. The neural tube extending from r5 to the somite 3/4 boundary gave rise to CP cells. The neuraxial origins of each pharyngeal ectomesenchyme extended for more than three somite lengths, most of which overlapped with the other. Unlike in the preotic region, there is no segmental registration between neuraxial levels and pharyngeal arches. Caudal portions of the CP cell population show a characteristic distribution pattern that circumscribes the postotic pharyngeal arches caudally. Heterotopic transplantation of the Dil-labeled neural crest into the somite 3 level resulted in a distribution of labeled cells similar to that of CP cells, suggesting that the pattern of distribution depends upon dynamic modification of the body wall associated with pharyngeal arch formation.     

Development of cephalic neural crest cells in embryos of Lampetra japonica, with special reference to the evolution of the jaw

Neural crest cells contribute extensively to vertebrate head morphogenesis and their origin is an important question to address in understanding the evolution of the craniate head. The distribution pattern of cephalic crest cells was examined in embryos of one of the living agnathan vertebrates, Lampetra japonica. The initial appearance of putative crest cells was observed on the dorsal aspect of the neural rod at stage 20.5 and ventral expansion of these cells was first seen at the level of rostral somites. As in gnathostomes, cephalic crest cells migrate beneath the surface ectoderm and form three major cell populations, each being separated at the levels of rhombomeres (r) 3 and r5. The neural crest seems initially to be produced at all neuraxial levels except for the rostral-most area, and cephalic crest cells are secondarily excluded from levels r3 and r5. Such a pattern of crest cell distribution prefigures the morphology of the cranial nerve anlage. The second or middle crest cell population passes medial to the otocyst, implying that the otocyst does not serve as a barrier to separate the crest cell populations. The three cephalic crest cell populations fill the pharyngeal arch ventrally, covering the pharyngeal mesoderm laterally with the rostral-most population covering the premandibular region and mandibular arch. The third cell population is equivalent to the circumpharyngeal crest cells in the chick, and its influx into the pharyngeal region precedes the formation of postotic pharyngeal arches. Focal injection of DiI revealed the existence of an anteroposterior organization in the neural crest at the neurular stage, destined for each pharyngeal region. The crest cells derived from the posterior midbrain that express the LjOtxA gene, the Otx2 cognate, were shown to migrate into the mandibular arch, a pattern which is identical to gnathostome embryos. It was concluded that the head region of the lamprey embryo shares a common set of morphological characters with gnathostome embryos and that the morphological deviation of the mandibular arch between the gnathostomes and the lamprey is not based on the early embryonic patterning.     

The fate of cranial neural crest cells in the Australian lungfish, Neoceratodus forsteri

The cranial neural crest has been shown to give rise to a diversity of cells and tissues, including cartilage, bone and connective tissue, in a variety of tetrapods and in the zebrafish. It has been claimed, however, that in the Australian lungfish these tissues are not derived from the cranial neural crest, and even that no migrating cranial neural crest cells exist in this species. We have earlier documented that cranial neural crest cells do migrate, although they emerge late, in the Australian lungfish. Here, we have used the lipophilic fluorescent dye, DiI, to label premigratory cranial neural crest cells and follow their fate until stage 43, when several cranial skeletal elements have started to differentiate. The timing and extent of their migration was investigated, and formation of mandibular, hyoid and branchial streams documented. Cranial neural crest was shown to contribute cells to several parts of the head skeleton, including the trabecula cranii and derivatives of the mandibular arch (e.g., Meckel's cartilage, quadrate), the hyoid arch (e.g., the ceratohyal) and the branchial arches (ceratobranchials I-IV), as well as to the connective tissue surrounding the myofibers in cranial muscles. We conclude that cranial neural crest migration and fate in the Australian lungfish follow the stereotyped pattern documented in other vertebrates.     

Combined deficiencies of Msx1 and Msx2 cause impaired patterning and survival of the cranial neural crest

The neural crest is a multipotent, migratory cell population that contributes to a variety of tissues and organs during vertebrate embryogenesis. Here, we focus on the function of Msx1 and Msx2, homeobox genes implicated in several disorders affecting craniofacial development in humans. We show that Msx1/2 mutants exhibit profound deficiencies in the development of structures derived from the cranial and cardiac neural crest. These include hypoplastic and mispatterned cranial ganglia, dysmorphogenesis of pharyngeal arch derivatives and abnormal organization of conotruncal structures in the developing heart. The expression of the neural crest markers Ap-2alpha, Sox10 and cadherin 6 (cdh6) in Msx1/2 mutants revealed an apparent retardation in the migration of subpopulations of preotic and postotic neural crest cells, and a disorganization of neural crest cells paralleling patterning defects in cranial nerves. In addition, normally distinct subpopulations of migrating crest underwent mixing. The expression of the hindbrain markers Krox20 and Epha4 was altered in Msx1/2 mutants, suggesting that defects in neural crest populations may result, in part, from defects in rhombomere identity. Msx1/2 mutants also exhibited increased Bmp4 expression in migratory cranial neural crest and pharyngeal arches. Finally, proliferation of neural crest-derived mesenchyme was unchanged, but the number of apoptotic cells was increased substantially in neural crest-derived cells that contribute to the cranial ganglia and the first pharyngeal arch. This increase in apoptosis may contribute to the mispatterning of the cranial ganglia and the hypoplasia of the first arch.     

Expression of zebrafish fkd6 in neural crest-derived glia

The zebrafish fkd6 gene is a marker for premigratory neural crest. In this study, we analyze later expression in putative glia of the peripheral nervous system. Prior to neural crest migration, fkd6 expression is downregulated in crest cells. Subsequently, expression appears initially in loose clusters of cells in positions corresponding to cranial ganglia. Double labelling with a neuronal marker shows that fkd6-expressing cells are not differentiated neurones and generally lie peripheral to neurones in ganglia. Later, expression appears associated with the posterior lateral line and other cranial nerves. For the posterior lateral line nerve, we show that fkd6-labeling extends caudally along this nerve in tight correlation with lateral line primordium migration and axon elongation. Expression in colourless mutant embryos is consistent with these cells being satellite glia and Schwann cells.     

Neuropilin 2/semaphorin 3F signaling is essential for cranial neural crest migration and trigeminal ganglion condensation

In the head of vertebrate embryos, neural crest cells migrate from the neural tube into the presumptive facial region and condense to form cranial ganglia and skeletal elements in the branchial arches. We show that newly formed neural folds and migrating neural crest cells express the neuropilin 2 (npn2) receptor in a manner that is highly conserved in amniotes. The repulsive npn2 ligand semaphorin (sema) 3F is expressed in a complementary pattern in the mouse. Furthermore, mice carrying null mutations for either npn2 or sema3F have abnormal cranial neural crest migration. Most notably, "bridges" of migrating cells are observed crossing between neural crest streams entering branchial arches 1 and 2. In addition, trigeminal ganglia fail to form correctly in the mutants and are improperly condensed and loosely organized. These data show that npn2/sema3F signaling is required for proper cranial neural crest development in the head.     

Neural crest survival and differentiation in zebrafish depends on mont blanc/tfap2a gene function

Neural crest progenitor cells are the main contributors to craniofacial cartilage and connective tissue of the vertebrate head. These progenitor cells also give rise to the pigment, neuronal and glial cell lineages. To study the molecular basis of neural crest differentiation, we have cloned the gene disrupted in the mont blanc (mob(m610)) mutation, which affects all neural crest derivatives. Using a positional candidate cloning approach we identified an A to G transition within the 3' splice site of the sixth intron of the tfap2a gene that abolishes the last exon encoding the crucial protein dimerization and DNA-binding domains. Neural crest induction and specification are not hindered in mob(m610) mutant embryos, as revealed by normal expression of early neural crest specific genes such as snail2, foxd3 and sox10. In addition, the initial stages of cranial neural crest migration appear undisturbed, while at a later phase the craniofacial primordia in pharyngeal arches two to seven fail to express their typical set of genes (sox9a, wnt5a, dlx2, hoxa2/b2). In mob(m610) mutant embryos, the cell number of neuronal and glial derivatives of neural crest is greatly reduced, suggesting that tfap2a is required for their normal development. By tracing the fate of neural crest progenitors in live mont blanc (mob(m610)) embryos, we found that at 24 hpf neural crest cells migrate normally in the first pharyngeal arch while the preotic and postotic neural crest cells begin migration but fail to descend to the pharyngeal region of the head. TUNEL assay and Acridine Orange staining revealed that in the absence of tfap2a a subset of neural crest cells are unable to undergo terminal differentiation and die by apoptosis. Furthermore, surviving neural crest cells in tfap2a/mob(m610) mutant embryos proliferate normally and later differentiate to individual derivatives. Our results indicate that tfap2a is essential to turn on the normal developmental program in arches 2-7 and in trunk neural crest. Thus, tfap2a does not appear to be involved in early specification and cell proliferation of neural crest, but it is a key regulator of an early differentiation phase and is required for cell survival in neural crest derived cell lineages.     

Morphogenesis of the cranial segments and distribution of neural crest in the embryos of the snapping turtle,Chelydra serpentina

Recent studies of the heads of vertebrates have shown a primitive pattern of segmentation in the mesoderm and neural plate not previously recognized. The role of this pattern in the subsequent distribution of cranial crest and the development of branchial arches and cranial nerves, may resolve century-old arguments about the evolution of vertebrate segmentation. In this study, we examine the early embryonic development of the cranium of a primitive amniote, the snapping turtle, with the SEM. We show that the paraxial mesoderm cranial to the first-formed somites is segmented and that this pattern is based on somitomeres, similar to those described in the embryos of chick and mouse. Seven contiguous pairs of somitomeres comprise the “head mesoderm”; the first pair of somites actually arise from the eighth pair of somitomeres added to the axis. Cranial somitomeres are associated with specific brain regions, in that the first pair lie adjacent to prosencephalon, the second and third pair are adjacent to the mesencephalon, and the fourth, fifth, sixth, and seventh pair of somitomeres lie adjacent to individual neuromeres of the rhombencephalon. Prior to the closure of the anterior neuropore, cranial neural crest cells first emerge from the mesencephalon and migrate onto the second and third somitomeres. Shortly thereafter, neural crest cells emerge at more caudal levels of the rhombencephalon, beginning at the juncture of the fifth and sixth somitomeres. Eventually, neural crest originating from the mesencephalon spreads caudally as far as the fourth somitomere, leaving a gap in crest emigration adjacent to the fifth somitomere. The otic placode develops from the surface ectoderm covering the sixth and seventh somitomeres, and the adjacent rhombencephalic neural crest moves around the cranial and caudal edge of the placode. At more caudal levels, rhombencephalic crest cells merge with cervical crest populations to form a continuous sheet over the somites. By the time the anterior neuropore closes, some of the mesencephalic crest cells return from the paraxial mesoderm to spread onto the rostral wall of the optic vesicle and future telencephalon. The segmentation of the mesoderm and patterned distribution of cranial neural crest seen in snapping turtle embryos, further strengthens the argument that the heads of amniotes are derived from a common metameric pattern established early during gastrulation.     

Neural crest and placode roles in formation and patterning of cranial sensory ganglia in lamprey

Vertebrates possess paired cranial sensory ganglia derived from two embryonic cell populations, neural crest and placodes. Cranial sensory ganglia arose prior to the divergence of jawed and jawless vertebrates, but the developmental mechanisms that facilitated their evolution are unknown. Using gene expression and cell lineage tracing experiments in embryos of the sea lamprey, Petromyzon marinus, we find that in the cranial ganglia we targeted, development consists of placode‐derived neuron clusters in the core of ganglia, with neural crest cells mostly surrounding these neuronal clusters. To dissect functional roles of neural crest and placode cell associations in these developing cranial ganglia, we used CRISPR/Cas9 gene editing experiments to target genes critical for the development of each population. Genetic ablation of SoxE2 and FoxD‐A in neural crest cells resulted in differentiated cranial sensory neurons with abnormal morphologies, whereas deletion of DlxB in cranial placodes resulted in near‐total loss of cranial sensory neurons. Taken together, our cell‐lineage, gene expression, and gene editing results suggest that cranial neural crest cells may not be required for cranial ganglia specification but are essential for shaping the morphology of these sensory structures. We propose that the association of neural crest and placodes in the head of early vertebrates was a key step in the organization of neurons and glia into paired sensory ganglia.     

Cadherin‐7 mediates proper neural crest cell–placodal neuron interactions during trigeminal ganglion assembly

The cranial trigeminal ganglia play a vital role in the peripheral nervous system through their relay of sensory information from the vertebrate head to the brain. These ganglia are generated from the intermixing and coalescence of two distinct cell populations: cranial neural crest cells and placodal neurons. Trigeminal ganglion assembly requires the formation of cadherin‐based adherens junctions within the neural crest cell and placodal neuron populations; however, the molecular composition of these adherens junctions is still unknown. Herein, we aimed to define the spatio‐temporal expression pattern and function of Cadherin‐7 during early chick trigeminal ganglion formation. Our data reveal that Cadherin‐7 is expressed exclusively in migratory cranial neural crest cells and is absent from trigeminal neurons. Using molecular perturbation experiments, we demonstrate that modulation of Cadherin‐7 in neural crest cells influences trigeminal ganglion assembly, including the organization of neural crest cells and placodal neurons within the ganglionic anlage. Moreover, alterations in Cadherin‐7 levels lead to changes in the morphology of trigeminal neurons. Taken together, these findings provide additional insight into the role of cadherin‐based adhesion in trigeminal ganglion formation, and, more broadly, the molecular mechanisms that orchestrate the cellular interactions essential for cranial gangliogenesis.     

Regional differences in neural crest morphogenesis

Neural crest cells are pluripotent cells that emerge from the neural epithelium, migrate extensively, and differentiate into numerous derivatives, including neurons, glial cells, pigment cells and connective tissue. Major questions concerning their morphogenesis include: 1) what establishes the pathways of migration and 2) what controls the final destination and differentiation of various neural crest subpopulations. These questions will be addressed in this review. Neural crest cells from the trunk level have been explored most extensively. Studies show that melanoblasts are specified shortly after they depart from the neural tube, and this specification directs their migration into the dorsolateral pathway. We also consider other reports that present strong evidence for ventrally migrating neural crest cells being similarly fate restricted. Cranial neural crest cells have been less analyzed in this regard but the preponderance of evidence indicates that either the cranial neural crest cells are not fate-restricted, or are extremely plastic in their developmental capability and that specification does not control pathfinding. Thus, the guidance mechanisms that control cranial neural crest migration and their behavior vary significantly from the trunk. The vagal neural crest arises at the axial level between the cranial and trunk neural crest and represents a transitional cell population between the head and trunk neural crest. We summarize new data to support this claim. In particular, we show that: 1) the vagal-level neural crest cells exhibit modest developmental bias; 2) there are differences in the migratory behavior between the anterior and the posterior vagal neural crest cells reminiscent of the cranial and the trunk neural crest, respectively; 3) the vagal neural crest cells take the dorsolateral pathway to the pharyngeal arches and the heart, but the ventral pathway to the peripheral nervous system and the gut. However, these pathways are not rigidly specified because of prior fate restriction. Understanding the molecular, cellular and behavioral differences between these three populations of neural crest cells will be of enormous assistance when trying to understand the evolution of the neck.     

Neuropilin 1 and 2 control cranial gangliogenesis and axon guidance through neural crest cells

Neuropilin (NRP) receptors and their class 3 semaphorin (SEMA3) ligands play well-established roles in axon guidance, with loss of NRP1, NRP2, SEMA3A or SEMA3F causing defasciculation and errors in growth cone guidance of peripherally projecting nerves. Here we report that loss of NRP1 or NRP2 also impairs sensory neuron positioning in the mouse head, and that this defect is a consequence of inappropriate cranial neural crest cell migration. Specifically, neural crest cells move into the normally crest-free territory between the trigeminal and hyoid neural crest streams and recruit sensory neurons from the otic placode; these ectopic neurons then extend axons between the trigeminal and facioacoustic ganglia. Moreover, we found that NRP1 and NRP2 cooperate to guide cranial neural crest cells and position sensory neurons; thus, in the absence of SEMA3/NRP signalling, the segmentation of the cranial nervous system is lost. We conclude that neuropilins play multiple roles in the sensory nervous system by directing cranial neural crest cells, positioning sensory neurons and organising their axonal projections.     

Relationship between Neural Crest Cells and Cranial Mesoderm during Head Muscle Development

Background

In vertebrates, the skeletal elements of the jaw, together with the connective tissues and tendons, originate from neural crest cells, while the associated muscles derive mainly from cranial mesoderm. Previous studies have shown that neural crest cells migrate in close association with cranial mesoderm and then circumscribe but do not penetrate the core of muscle precursor cells of the branchial arches at early stages of development, thus defining a sharp boundary between neural crest cells and mesodermal muscle progenitor cells. Tendons constitute one of the neural crest derivatives likely to interact with muscle formation. However, head tendon formation has not been studied, nor have tendon and muscle interactions in the head.

Methodology/Principal Findings

Reinvestigation of the relationship between cranial neural crest cells and muscle precursor cells during development of the first branchial arch, using quail/chick chimeras and molecular markers revealed several novel features concerning the interface between neural crest cells and mesoderm. We observed that neural crest cells migrate into the cephalic mesoderm containing myogenic precursor cells, leading to the presence of neural crest cells inside the mesodermal core of the first branchial arch. We have also established that all the forming tendons associated with branchiomeric and eye muscles are of neural crest origin and express the Scleraxis marker in chick and mouse embryos. Moreover, analysis of Scleraxis expression in the absence of branchiomeric muscles in Tbx1^−/− mutant mice, showed that muscles are not necessary for the initiation of tendon formation but are required for further tendon development.

Conclusions/Significance

This results show that neural crest cells and muscle progenitor cells are more extensively mixed than previously believed during arch development. In addition, our results show that interactions between muscles and tendons during craniofacial development are similar to those observed in the limb, despite the distinct embryological origin of these cell types in the head.     

Trunk neural crest has skeletogenic potential

During early vertebrate development, neural crest cells emerge from the dorsal neural tube, migrate into the periphery, and form a wide range of derivatives. There is, however, a significant difference between the cranial and trunk neural crest with respect to the diversity of cell types that each normally produces. Thus, while crest cells from all axial levels form neurons, glia, and melanocytes, the cranial crest additionally generates skeletal derivatives such as bone and cartilage; trunk crest cells are generally thought to lack skeletogenic potential. Here, we show, however, that if avian trunk neural crest cells are cultured in appropriate media, they form both bone and cartilage cells, and if placed into the developing head, they contribute to cranial skeletal components. Thus, the neural crest from all axial levels can generate the full repertoire of crest derivatives. The skeletogenic potential of the trunk neural crest is significant, as it was likely realized in early vertebrates, which had extensive postcranial exoskeletal coverings.     

Angiopoietin 2 signaling plays a critical role in neural crest cell migration

Background

Collective neural crest cell migration is critical to the form and function of the vertebrate face and neck, distributing bone, cartilage, and nerve cells into peripheral targets that are intimately linked with head vasculature. The vasculature and neural crest structures are ultimately linked, but when and how these patterns develop in the early embryo are not well understood.

Results

Using in vivo imaging and sophisticated cell behavior analyses, we show that quail cranial neural crest and endothelial cells share common migratory paths, sort out in a dynamic multistep process, and display multiple types of motion. To better understand the underlying molecular signals, we examined the role of angiopoietin 2 (Ang2), which we found expressed in migrating cranial neural crest cells. Overexpression of Ang2 causes neural crest cells to be more exploratory as displayed by invasion of off-target locations, the widening of migratory streams into prohibitive zones, and differences in cell motility type. The enhanced exploratory phenotype correlates with increased phosphorylated focal adhesion kinase activity in migrating neural crest cells. In contrast, loss of Ang2 function reduces neural crest cell exploration. In both gain and loss of function of Ang2, we found disruptions to the timing and interplay between cranial neural crest and endothelial cells.

Conclusions

Together, these data demonstrate a role for Ang2 in maintaining collective cranial neural crest cell migration and suggest interdependence with endothelial cell migration during vertebrate head patterning.

     

Cranial neural crest cell migration in the Australian lungfish, Neoceratodus forsteri

SUMMARY A crucial role for the cranial neural crest in head development has been established for both actinopterygian fishes and tetrapods. It has been claimed, however, that the neural crest is unimportant for head development in the Australian lungfish ( Neoceratodus forsteri   ), a member of the group (Dipnoi) which is commonly considered to be the living sister group of the tetrapods. In the present study, we used scanning electron microscopy to study cranial neural crest development in the Australian lungfish. Our results, contrary to those of Kemp, show that cranial neural crest cells do emerge and migrate in the Australian lungfish in the same way as in other vertebrates, forming mandibular, hyoid, and branchial streams. The major difference is in the timing of the onset of cranial neural crest migration. It is delayed in the Australian lungfish in comparison with their living sister group the Lissamphibia. Furthermore, the delay in timing between the emergence of the hyoid and branchial crest streams is very long, indicating a steeper anterior-posterior gradient than in amphibians. We are now extending our work on lungfish head development to include experimental studies (ablation of selected streams of neural crest cells) and fate mapping (using fluoresent tracer dyes such as DiI) to document the normal fate as well as the role in head patterning of the cranial neural crest in the Australian lungfish.     

Regional differences in neural crest morphogenesis

Neural crest cells are pluripotent cells that emerge from the neural epithelium, migrate extensively and differentiate into numerous derivatives, including neurons, glial cells, pigment cells and connective tissue. Major questions concerning their morphogenesis include: (1) what establishes the pathways of migration? And (2), what controls the final destination and differentiation of various neural crest subpopulations? These questions will be addressed in this Review. Neural crest cells from the trunk level have been explored most extensively. Studies show that melanoblasts are specified shortly after they depart from the neural tube and this specification directs their migration into the dorsolateral pathway. We also consider other reports that present strong evidence for ventrally migrating neural crest cells being similarly fate restricted. Cranial neural crest cells have been less analyzed in this regard but the preponderance of evidence indicates that either the cranial neural crest cells are not fate-restricted or are extremely plastic in their developmental capability and that specification does not control pathfinding. Thus, the guidance mechanisms that control cranial neural crest migration and their behavior vary significantly from the trunk.The vagal neural crest arises at the axial level between the cranial and trunk neural crest and represents a transitional cell population between the head and trunk neural crest. We summarize new data to support this claim. In particular, we show that: (1) the vagal-level neural crest cells exhibit modest developmental bias; (2) there are differences in the migratory behavior between the anterior and the posterior vagal neural crest cells reminiscent of the cranial and the trunk neural crest, respectively and (3) the vagal neural crest cells take the dorsolateral pathway to the pharyngeal arches and the heart, but take the ventral pathway to the peripheral nervous system and the gut. However, these pathways are not rigidly specified because of prior fate restriction. Understanding the molecular, cellular and behavioral differences between these three populations of neural crest cells will be of enormous assistance when trying to understand the evolution of the neck.Key words: neural crest, morphogenesis, cell migration, chicken embryo, fate restriction, vagal neural crest, pathways     

Inhibition of sonic hedgehog signaling in vivo results in craniofacial neural crest cell death

BACKGROUND: Sonic hedgehog (Shh) is well known for its role in patterning tissues, including structures of the head. Haploinsufficiency for SHH in humans results in holoprosencephaly, a syndrome characterized by facial and forebrain abnormalities. Shh null mice have cyclopia and loss of branchial arch structures. It is unclear, however, whether these phenotypes arise solely from the early function of Shh in patterning midline structures, or whether Shh plays other roles in head development. RESULTS: To address the role of Shh after floorplate induction, we inhibited Shh signaling by injecting hybridoma cells that secrete a function-blocking anti-Shh antibody into the chick cranial mesenchyme. The antibody subsequently bound to Shh in the floorplate, notochord, and the pharyngeal endoderm. Perturbation of Shh signaling at this stage resulted in a significant reduction in head size after 1 day, loss of branchial arch structures after 2 days, and embryos with smaller heads after 7 days. Cell death was significantly increased in the neural tube and neural crest after 1 day, and neural crest cell death was not secondary to the loss of neural tube cells. CONCLUSIONS: Reduction of Shh signaling after neural tube closure resulted in a transient decrease in neural tube cell proliferation and an extensive increase in cell death in the neural tube and neural crest, which in turn resulted in decreased head size. The phenotypes observed after reduction of Shh are similar to those observed after cranial neural crest ablation. Thus, our results demonstrate a role for Shh in coordinating the proliferation and survival of cells of the neural tube and cranial neural crest.