Temporospatial study of the migration and distribution of cardiac neural crest in quail-chick chimeras

It has been demonstrated that the septation of the outflow tract of the heart is formed by the cardiac neural crest. Ablation of this region of the neural crest prior to its migration from the neural fold results in anomalies of the outflow and inflow tracts of the heart and the aortic arch arteries. The objective of this study was to examine the migration and distribution of these neural crest cells from the pharyngeal arches into the outflow region of the heart during avian embryonic development. Chimeras were constructed in which each region of the premigratory cardiac neural crest from quail embryos was implanted into the corresponding area in chick embryos. The transplantations were done unilaterally on each side and bilaterally. The quail-chick chimeras were sacrificed between Hamburger-Hamilton stages 18 and 25, and the pharyngeal region and outflow tract were examined in serial paraffin sections to determine the distribution pattern of quail cells at each stage. The neural crest cells derived from the presumptive arch 3 and 4 regions of the neuraxis occupied mainly pharyngeal arches 3 and 4 respectively, although minor populations could be seen in pharyngeal arches 2 and 6. The neural crest cells migrating from the presumptive arch 6 region were seen mainly in pharyngeal arch 6, but they also populated pharyngeal arches 3 and 4. Clusters of quail neural crest cells were found in the distal outflow tract at stage 23.     

The development and evolution of the pharyngeal arches

A muscularised pharynx, with skeletal support, serving the dual functions of feeding and respiration, is a fundamental vertebrate characteristic. Embryologically, the pharyngeal apparatus has its origin in a series of bulges that form on the lateral surface of the embryonic head, the pharyngeal arches, whose development is complex. These structures are composed of a number of disparate embryonic cell types: ectoderm, endoderm, neural crest and mesoderm, whose development must be coordinated to generate the functional adult apparatus. In the past, most studies have emphasised the role played by the neural crest, which generates the skeletal elements of the arches, in directing pharyngeal arch development, but it has also become apparent that the endoderm plays a prominent role in directing arch development. Neural crest cells are not required for arch formation, their regionalisation nor to some extent their sense of identity. Furthermore, the endoderm is the major site of expression of a number of important signalling molecules, and this tissue has been shown to be responsible for promoting the formation of particular components of the arches. Thus vertebrate pharyngeal morphogenesis can now be seen to be a more complex process than was previously believed, and must result from an integration of both neural crest and endodermal patterning mechanisms. Interestingly, this also mirrors the fact that the evolutionary origin of pharyngeal segmentation predates that of the neural crest, which is an exclusively vertebrate characteristic. As such, the evolution of the vertebrate pharynx is also likely to have resulted from an integration between these 2 patterning systems. Alterations in the interplay between neural crest and endodermal patterning are also likely to be responsible for the evolutionary that occurred to the pharyngeal region during subsequent vertebrate evolution.     

The development and evolution of the pharyngeal arches

A muscularised pharynx, with skeletal support, serving the dual functions of feeding and respiration, is a fundamental vertebrate characteristic. Embryologically, the pharyngeal apparatus has its origin in a series of bulges that form on the lateral surface of the embryonic head, the pharyngeal arches, whose development is complex. These structures are composed of a number of disparate embryonic cell types: ectoderm, endoderm, neural crest and mesoderm, whose development must be coordinated to generate the functional adult apparatus. In the past, most studies have emphasised the role played by the neural crest, which generates the skeletal elements of the arches, in directing pharyngeal arch development, but it has also become apparent that the endoderm plays a prominent role in directing arch development. Neural crest cells are not required for arch formation, their regionalisation nor to some extent their sense of identity. Furthermore, the endoderm is the major site of expression of a number of important signalling molecules, and this tissue has been shown to be responsible for promoting the formation of particular components of the arches. Thus vertebrate pharyngeal morphogenesis can now be seen to be a more complex process than was previously believed, and must result from an integration of both neural crest and endodermal patterning mechanisms. Interestingly, this also mirrors the fact that the evolutionary origin of pharyngeal segmentation predates that of the neural crest, which is an exclusively vertebrate characteristic. As such, the evolution of the vertebrate pharynx is also likely to have resulted from an integration between these 2 patterning systems. Alterations in the interplay between neural crest and endodermal patterning are also likely to be responsible for the evolutionary that occurred to the pharyngeal region during subsequent vertebrate evolution.     

Initial migration and distribution of the cardiac neural crest in the avian embryo: An introduction to the concept of the circumpharyngeal crest

The distribution and migration of the cardiac neural crest was studied in chick embryos from stages 11 to 17 that were immunochemically stained in whole-mount and sectioned specimens with a monoclonal antibody, HNK-1. The following results were obtained: (1) The first phase of the migration in the cardiac crest follows the dorsolateral pathway beneath the ectoderm. (2) In the first site of arrest, the cardiac crest forms a longitudinal mass of neural-crest cells, called in the present study, the circumpharyngeal crest; this mass is located dorsolateral to the dorsal edge of the pericardium (pericardial dorsal horn) where splanchic and somatic lateral mesoderm meet. (3) A distinctive strand of neural-crest cells, called the anterior tract, arises from the mid-otic level and ends in the circumpharyngeal crest. (4) By stage 16, after the degeneration of the first somite, another strand of neural-crest cells, called the posterior tract, appears dorsal to the circumpharyngeal crest. It forms an arch-like pathway along the anterior border of the second somite. (5) The seeding of the pharyngeal ectomesenchyme takes place before the formation of pharyngeal arches in the postotic area, i.e., the crest cells are seeded into the lateral body wall ventrally from the circumpharyngeal crest; and, by the ventralward regression of the pericardial dorsal horn, lateral expansion of pharyngeal pouch, and caudal regression of the pericardium, the crest cell population is pushed away by the pharyngeal pouch. Thus the pharyngeal arch ectomesenchyme is segregated. (6) By stage 14, at the occipital somite level, ventrolateral migration of the neural crest is observed within the anterior half of each somite. Some of these crest cells are continuos with the caudal portion of the circumpharyngeal crest. An early contribution to the enteric neuroblasts is apparent in this area.     

Development of the pharyngeal arches

The oro-pharyngeal apparatus has its origin in a series of bulges that is found on the lateral surface of the embryonic head, the pharyngeal arches. The development of the pharyngeal arches is complex involving a number of disparate embryonic cell types: ectoderm, endoderm, neural crest and mesoderm, whose development must be co-ordinated to generate the functional adult apparatus. In the past, most studies have emphasised the role played by the neural crest, which generates the skeletal elements of the arches, in directing pharyngeal arch development, but it has also become apparent that the other tissues of the arches, most notably the endoderm, also plays a prominent role in directing arch development. Thus pharyngeal arch development is more complex, and more consensual, than was previously believed.     

小鼠胚胎细胞视黄酸结合蛋白1阳性神经嵴细胞的时空分布与功能

目的 探讨小鼠胚胎心神经嵴细胞的形成、分布模式及其在心血管系统发育过程中的作用。方法 选用抗细胞视黄酸结合蛋白1（CRABP1）、抗α-平滑肌肌动蛋白（α-SMA）、抗心肌肌球蛋白重链（MHC）、抗胰岛因子1（Isl-1）抗体,对45只胚龄8～12d小鼠胚胎连续切片进行免疫
组织化学染色。结果 胚龄8d,CRABP1在神经褶的外胚层未见阳性表达。胚龄8.5～9d,在心管与鳃弓水平,神经褶开始出现CRABP1阳性细胞,且有部分细胞从神经褶背侧分离进入邻近间充质。胚龄10d,神经管两侧间充质内的CRABP1阳性细胞迁移至鳃弓、弓动脉壁内皮周围以及流出道
心胶质内。胚龄11～12d,弓动脉内皮周围、流出道心内膜垫内CRABP1表达明显下降,但弓动脉管壁α-SMA阳性平滑肌细胞数量增加。主肺动脉隔及其分隔形成的升主动脉和肺动脉干管壁内均可见Isl-1阳性细胞,但未见CRABP1表达。结论 小鼠胚胎CRABP1阳性神经嵴细胞形成的时间窗
限定在胚龄8.5～9d。胚龄10d后,CRABP1阳性神经嵴细胞经过迁移,参与弓动脉中膜平滑肌和流出道心内膜垫的形成。CRABP1不能用于标记迁移后的神经嵴细胞。     

The formation of mesoderm and mesectoderm in 5- to 41-somite rat embryos cultured in vitro, using WGA-Au as a marker

Summary The formation of mesectodermal cells by the neural crest in 5- to 41-somite stage embryos was investigated experimentally in rat embryos cultured in vitro, using lectincoated colloidal gold as a probe. This method labelled all ectodermal cells, among them neural crest, surface ectodermal placodal and epiblastic (primitive streak) cells. The neural crest provides the mesodermal compartment of the entire head region with cells, including the primitive cranial ganglia and the branchial arches. In the head region migration of neural crest cells over a great distance (long-distance migration) was not observed. In the trunk region neural crest derived cells were mainly found to form the primitive spinal ganglia and the sympathetic trunk, once again without long-distance cell migration. Structures and tissues that supposedly were derived from the primitive streak were hardly labelled with colloidal gold. Surface ectodermal placodes were not only found at the expected sites (e.g. epibranchial placodes) but also in the ectoderm covering the transverse septum and lateral abdominal walls.     

An in vitro model for characterizing the post-migratory cranial neural crest cells of the first branchial arch.

The cranial neural crest (CNC) is a transient cell population that originates at the crest of the neural fold and gives rise to multiple cell types during craniofacial development. Traditionally, researchers have used tissue explants, such as the neural tube, to obtain primary neural crest cells for their studies. However, this approach has inevitably resulted in simultaneous isolation of neural and non-neural crest cells as both of these cells migrate away from tissue explants. Using the Wnt1-Cre/R26R mouse model, we have obtained a pure population of neural crest cells and established a primary CNC cell culture system in which the cell culture medium best supports the proliferation of E10.5 first branchial arch CNC cells and maintains these cells in their undifferentiated state. Differentiation of CNC cells can be initiated by switching to a differentiation medium. In this model, cultured CNC cells can give rise to neurons, glial cells, osteoblasts, and other cell types, faithfully mimicking the differentiation process of the post-migratory CNC cells in vivo. Taken together, our study shows that the Wnt1-Cre/R26R mouse first branchial arch provides an excellent model for obtaining post-migratory neural crest cells free of any mesodermal contaminants. The cultured neural crest cells are under sustained proliferative, undifferentiated, or lineage-enhanced conditions, hence, serving as a tool for the investigation of the regulatory mechanism of CNC cell fate determination in normal and abnormal craniofacial development.     

The differentiation and morphogenesis of craniofacial muscles.

Unraveling the complex tissue interactions necessary to generate the structural and functional diversity present among craniofacial muscles is challenging. These muscles initiate their development within a mesenchymal population bounded by the brain, pharyngeal endoderm, surface ectoderm, and neural crest cells. This set of spatial relations, and in particular the segmental properties of these adjacent tissues, are unique to the head. Additionally, the lack of early epithelialization in head mesoderm necessitates strategies for generating discrete myogenic foci that may differ from those operating in the trunk. Molecular data indeed indicate dissimilar methods of regulation, yet transplantation studies suggest that some head and trunk myogenic populations are interchangeable. The first goal of this review is to present key features of these diversities, identifying and comparing tissue and molecular interactions regulating myogenesis in the head and trunk. Our second focus is on the diverse morphogenetic movements exhibited by craniofacial muscles. Precursors of tongue muscles partly mimic migrations of appendicular myoblasts, whereas myoblasts destined to form extraocular muscles condense within paraxial mesoderm, then as large cohorts they cross the mesoderm:neural crest interface en route to periocular regions. Branchial muscle precursors exhibit yet another strategy, establishing contacts with neural crest populations before branchial arch formation and maintaining these relations through subsequent stages of morphogenesis. With many of the prerequisite stepping-stones in our knowledge of craniofacial myogenesis now in place, discovering the cellular and molecular interactions necessary to initiate and sustain the differentiation and morphogenesis of these neglected craniofacial muscles is now an attainable goal.     

Migration and distribution of circumpharyngeal crest cells in the chick embryo. Formation of the circumpharyngeal ridge and E/C8+ crest cells in the vertebrate head region.

The cardiac neural crest is located in a transitional area on the neuraxis between trunk and cephalic regions and gives rise to both the dorsolateral and ventrolateral crest cell populations. Around stage 18 of chick development, a mass of E/C8+ cells surrounds the postotic pharyngeal arches and forms a crescent-shaped arch, termed the circumpharyngeal ridge. Using immunohistochemistry and quail-chick chimeras, it was determined that the E/C8+ cell mass located in the circumpharyngeal ridge derives from the dorsolateral component of the cardiac neural crest. The ventrolateral cell population of the cardiac crest is located more medially and shows long-persistent HNK-1 immunoreactivity dorsolateral to the foregut. The crest cells that populate the gut arise from the caudal portion of the circumpharyngeal crest and are always located caudal to the caudal-most pharyngeal ectomesenchyme. Circumpharyngeal crest cells continuously populate the pharyngeal arch ectomesenchyme and enteric nervous system on the lateral side of the foregut wall, as well as the hypoglossal pathway which develops within the ventral portion of the circumpharyngeal ridge. E/C8 and HNK-1 immunoreactivity are associated with the cells migrating via the dorsolateral (circumpharyngeal) and ventrolateral pathways, respectively, with one exception: there is a population of putative crest cells along the proximal course of the vagal intestinal branch that shows both immunoreactivities around stage 20. DiI labeling of the cells in the circumpharyngeal ridge suggests that the cells are contributed from the circumpharyngeal ridge to this population. Thus, the distribution of the circumpharyngeal crest cells and their derivatives coincides with the peripheral branch distribution of the cranial nerves IX, X, and XII, whose development is selectively affected in the absence of the cardiac neural crest, the source of the circumpharyngeal crest.     

The role of the endoderm in the development and evolution of the pharyngeal arches

The oro-pharyngeal apparatus has its origin in a series of bulges found on the lateral surface of the embryonic head, the pharyngeal arches. Significantly, the development of these structures is extremely complex, involving interactions between a number of disparate embryonic cell types: ectoderm, endoderm, mesoderm and neural crest, each of which generates particular components of the arches, and whose development must be co-ordinated to generate the functional adult oro-pharyngeal apparatus. In the past most studies have emphasized the role played by the neural crest, which generates the skeletal elements of the arches, in directing pharyngeal arch development. However, it is now apparent that the pharyngeal endoderm plays an important role in directing arch development. Here we discuss the role of the pharyngeal endoderm in organizing the development of the pharyngeal arches, and the mechanisms that act to pattern the endoderm itself and those which direct its morphogenesis. Finally, we discuss the importance of modification to the pharyngeal endoderm during vertebrate evolution. In particular, we focus on the emergence of the parathyroid gland, which we have recently shown to be the result of the internalization of the gills.     

Spatiotemporally separated cardiac neural crest subpopulations that target the outflow tract septum and pharyngeal arch arteries

We used lacZ-retrovirus labeling combined with neural crest ablation in chick embryos to determine whether the cardiac neural crest cells constitute one group of multipotent cells, or they emigrate from the neural tube in time-dependent groups with different fates in the developing cardiovascular system. We demonstrated that early-migrating cardiac neural crest cells (HH9-10) massively target the aorticopulmonary septum and pharyngeal arch arteries, while the late-migrating cardiac neural crest cells (HH12) are restricted to the proximal part of the pharyngeal arch arteries. These results suggest a prominent role for early-migrating cells in outflow tract septation, and a function for late-migrating cells in pharyngeal arch artery remodeling. We demonstrated in cultures of neural tube explants an intrinsic difference between the early and late populations. However, by performing heterochronic transplantations we showed that the late-migrating cardiac neural crest cells were not developmentally restricted, and could contribute to the condensed mesenchyme of the aorticopulmonary septum when transplanted to a younger environment. Our findings on the exact timing and migratory behavior of cardiac neural crest cells will help narrow the range of factors and genes that are involved in neural crest-related congenital heart diseases.     

The ultrastructure of early cephalic neural crest cell migration in the mouse

Summary A study of the ultrastructural changes associated with the detachment of the presumptive neural crest cells from the neuroepithelium in the midbrain region in mouse embryos at 9 and 91/2 days of gestation was carried out. The first sign of neural crest cell formation occurred in this region before fusion of the neuroepithelium had occurred. Neural crest cells arose from both the neural plate and the adjoining surface ectoderm. Initially, the cells of the neural plate and the surface ectoderm were attached to each other by zonula occludens and zonula adherans at their apical surfaces however, these junctions disappeared just prior to the beginning of the migration of the crest cells. The first sign of migration of the crest cells was the disappearance of the basal lamina in the region of the presumptive crest cells. Once the basal lamina was lost, cell junctions were formed between the epithelial cells and the underlying mesenchymal cells. Once the crest cells had migrated into the underlying mesenchyme, they tended to form clumps of closely related, irregularly shaped cells. Phagosomes and accumulations of glycogen particles were found within some crest cells when they were still within 50 to 100 microns of the epithelium.     

Migration and distribution of circumpharyngeal crest cells in the chick embryo

The cardiac neural crest is located in a transitional area on the neuraxis between trunk and cephalic regions and gives rise to both the dorsolateral and ventrolateral crest cell populations. Around stage 18 of chick development, a mass of E/C8⁺ cells surrounds the postotic pharyngeal arches and forms a crescent-shaped arch, termed the circumpharyngeal ridge. Using immunohistochemistry and quail-chick chimeras, it was determined that the E/C8⁺ cell mass located in the circumpharyngeal ridge derives from the dorsolateral component of the cardiac neural crest. The ventrolateral cell population of the cardiac crest is located more medially and shows long-persistent HNK-1 immunoreactivity dorsolateral to the foregut. The crest cells that populate the gut arise from the caudal portion of the circumpharyngeal crest and are always located caudal to the caudalmost pharyngeal ectomesenchyme. Circumpharyngeal crest cells continuously populate the pharyngeal arch ectomesenchyme and enteric nervous system on the lateral side of the foregut wall, as well as the hypoglossal pathway which develops within the ventral portion of the circumpharyngeal ridge. E/C8 and HNK-1 immunoreactivity are associated with the cells migrating via the dorsolateral (circumpharyngeal) and ventrolateral pathways, respectively, with one exception: there is a population of putative crest cells along the proximal course of the vagal intestinal branch that shows both immunoreactivities around stage 20. Dil labeling of the cells in the circumpharyngeal ridge suggests that the cells are contributed from the circumpharyngeal ridge to this population. Thus, the distribution of the circumpharyngeal crest cells and their derivatives coincides with the peripheral branch distribution of the cranial nerves IX, X, and XII, whose development is selectively affected in the absence of the cardiac neural crest, the source of the circumpharyngeal crest.© Willey-Liss, Inc.     

Multilineage differentiation of ectomesenchymal cells isolated from the first branchial arch

Cranial neural crest-derived ectomesenchymal cells may be pluripotent stem cells that are capable of generating a range of phenotypes. The fate of these cells appears to be determined in part by intrinsic genetic programs and also by the influence of extracellular signals in the local environment. The extent of lineage determination once neural crest cells have migrated to the first branchial arch is not clear, although branchial arch pattern is not thought to be the result of crest predetermination. The aim of the present study was to test the hypothesis that ectomesenchymal cells of the first branchial arch show properties of pluripotent stem cells, the lineage of which may be directed by specific molecular signaling. Ectomesenchymal cells were enzymatically isolated from the mandibular processes of BALB/c mice and maintained in an undifferentiated state while cultured with leukemia inhibitory factor or induced to differentiate by lineage-specific induction factors or growth conditions, including transforming growth factor beta, forskolin, and a mineralization-promoting medium. Morphological observations and immunocytochemistry demonstrated that cells could be induced to differentiate into smooth muscle cells, glial cells, and osteoblasts, respectively. In the presence of the mineralization-promoting medium, alkaline phosphatase activity increased significantly and mineralization nodules formed. The data reported support the concept that many, although not all, first branchial arch-derived ectomesenchymal cells show properties of multipotent stem cells, the subsequent fate of which can be influenced by induction factors and growth conditions. Some cells, however, showed a degree of commitment with respect to their fate. The possible application of first branchial arch-derived stem cells to tissue engineering of the orofacial tissues should involve consideration of the developmental stage of cell harvesting and the desired cell fate.     

Neural crest and placodal contributions in the development of the glossopharyngeal-vagal complex in the chick

By using the method of quail-to-chick transplantation of neural crest in one series (VNG) and placodal ectoderm in a second series (VPG) we were able to determine the relative contribution of cranial neural crest and placodal ectoderm to the formation of the Glossopharyngeal-vagal complex. In chimeric embryos, quail cells originating from cranial neural crest grafts of postotic levels end up in the root ganglia, while quail cells originating from placodal ectoderm of postotic levels end up in the trunk ganglia. The results clearly indicate that the caudal levels of the medulla and rostral cervical segments represent the site, and the neural crest the source, for the neurons of the root ganglia. The neurons form a homogenous population of the small-cell type. This clearly rules out any contribution to the root ganglia from placodal ectoderm. On the basis of our experiments, it is also concluded that the neurons of the trunk ganglia are purely placodal in origin and are composed of a population of cells of the large-cell type. Our experiments also provide convincing evidence for a neural crest origin for Schwann cell and ganglionic Satellite cells.     

Neuropilin‐1 interacts with the second branchial arch microenvironment to mediate chick neural crest cell dynamics

Cranial neural crest cells (NCCs) require neuropilin signaling to reach and invade the branchial arches. Here, we use an in vivo chick model to investigate whether the neuropilin‐1 knockdown phenotype is specific to the second branchial arch (ba2), changes in NCC behaviors and phenotypic consequences, and whether neuropilins work together to facilitate entry into and invasion of ba2. We find that cranial NCCs with reduced neuropilin‐1 expression displayed shorter protrusions and decreased cell body and nuclear length‐to‐width ratios characteristic of a loss in polarity and motility, after specific interaction with ba2. Directed NCC migration was rescued by transplantation of transfected NCCs into rhombomere 4 of younger hosts. Lastly, reduction of neuropilin‐2 expression by shRNA either solely or with reduction of neuropilin‐1 expression did not lead to a stronger head phenotype. Thus, NCCs, independent of rhombomere origin, require neuropilin‐1, but not neuropilin‐2 to maintain polarity and directed migration into ba2. Developmental Dynamics 239:1664–1673, 2010. © 2010 Wiley‐Liss, Inc.     

Tbx1 mutation causes multiple cardiovascular defects and disrupts neural crest and cranial nerve migratory pathways

TBX1 is the major candidate gene for DiGeorge syndrome (DGS). Mouse studies have shown that the Tbx1 gene is haploinsufficient, as expected for a DGS candidate gene, and that it is required for the development of pharyngeal arches and pouches, as predicted by the DGS clinical phenotype. However, a detailed analysis of the cardiovascular phenotype associated with Tbx1 mutations has not been reported. Here we show that Tbx1 deficiency causes a number of distinct vascular and heart defects, suggesting multiple roles in cardiovascular development - specifically formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Comparison of phenotype and gene expression using a Tbx1-lacZ reporter allele supports a cell-autonomous function in the growth of the pharyngeal apparatus, and a cell non-autonomous function in the growth and early remodeling of the pharyngeal arch arteries. Our data do not support a direct role of neural crest cells in the pathogenesis of the Tbx1 mutant phenotype; however, these cells, and the cranial nerves, are misdirected. We hypothesize that this is due to the lack of a guidance role from the pouch endoderm, which is missing in these mutants.     

Expression of Hox 2.1 protein in restricted populations of neural crest cells and pharyngeal ectoderm.

A polyclonal antibody, alpha Hox 2.1a, was used to localize Hox 2.1 protein in presumptive neural crest cells and nodose ganglion of 8.5-10.0 day p.c. mouse embryos. The following results were obtained: (1) The nodose placode, in its epithelial state, first expresses Hox 2.1 protein at 9.0 d.p.c. By 9.5 d.p.c. presumptive migrating neuroblasts between the nodose placode and ganglion primordium also express Hox 2.1 protein. (2) At 9.5 d.p.c., presumptive crest cells lateral to the cephalic cardinal vein and within pharyngeal arches 4 and 6 are immunoreactive for alpha Hox 2.1a. In the arch 6 region, positive cells extend medially to a mesenchymal cell population on the lateral aspect of the foregut wall. (3) At 10.0 d.p.c., Hox 2.1 protein expression in putative crest cells is restricted to the arch 6 cell population. A similar staining pattern is seen using alpha Hox 2.1a with chick embryos. Comparison with the chicken embryo suggests that the Hox 2.1 positive cells in the pharyngeal arch and those on the lateral aspect of the foregut in the mouse embryo correspond to the caudalmost subpopulation of the circumpharyngeal crest (Kuratani and Kirby: Am. J. Anat. 191:215-227, 1991; Anat. Rec. 234:263-280, 1992). These results are consistent with a role for Hox 2.1 in pattern formation in the caudalmost region of the vertebrate head.