Patterning of avian craniofacial muscles

Vertebrate voluntary muscles are composed of myotubes and connective tissue cells. These two cell types have different embryonic origins: myogenic cells arise from paraxial mesoderm, while in the head many of the connective tissues are formed by neural crest cells. The objective of this research was to study interactions between heterotopically transplanted trunk myotomal cells and presumptive connective tissue-forming cephalic neural crest mesenchyme. Presumptive or newly formed cervical somites from quail embryos were implanted lateral to the midbrain of chick hosts prior to the onset of neural crest emigration. Hosts were sacrificed between 7 and 12 days of incubation, and sections examined for the presence of quail cells. Some grafted tissues differentiated in situ, forming ectopic skeletal, connective, and muscle tissues. However, many myotomal cells broke away from the implant, became integrated into adjacent neural crest mesenchyme, and subsequently formed normal extrinsic ocular or jaw muscles. In these muscles it was evident that only the myogenic populations were derived from grafted trunk cells. Ancillary findings were that grafted trunk paraxial mesoderm frequently interfered with the movement of neural crest cells which form the corneal posterior epithelial and stromal tissues, and that some grafted cells formed ectopic intramembranous bones adjacent to the eye. These results verify that presumptive connective tissue-forming mesenchyme derived from the neural crest imparts spatial patterning information upon myogenic cells that invade it. Moreover, interactions between myotomal cells and both lateral plate somatic mesoderm in the trunk and neural crest mesenchyme in the head appear to operate according to similar mechanisms.     

The embryonic origins of avian cephalic and cervical muscles and associated connective tissues

The objective of these experiments was to determine the embryonic origins of craniofacial and cervical voluntary muscles and associated connective tissues in the chick. To accomplish this, suspected primordia, including somitomeres 3-7, somites 1-7, and cephalic neural crest primordia have been transplanted from quail into chick embryos. Quail cells can be detected by the presence of a species-specific nuclear marker. The results are summarized as follows: (table; see text) These results indicate that muscles associated with branchial arch skeletal structures are derived from paraxial mesoderm, as are all other voluntary muscles in the vertebrate embryo. Thus, theories of vertebrate ontogeny and phylogeny based in part on proposed unique features of branchiomeric muscles must be critically reappraised. In addition, many of these cephalic muscles are composites of two separate primordia: the myogenic stem cells of mesodermal origin and the supporting and connective tissues derived from the neural crest or lateral plate mesoderm. Defining these embryonic origins is a necessary prerequisite to understanding how the mesenchymal primordia of cephalic muscles and connective tissues interact to form patterned, species-unique musculoskeletal systems.     

Cell lineage in mammalian craniofacial mesenchyme

We have analysed the contributions of neural crest and mesoderm to mammalian craniofacial mesenchyme and its derivatives by cell lineage tracing experiments in mouse embryos, using the permanent genetic markers Wnt1-cre for neural crest and Mesp1-cre for mesoderm, combined with the Rosa26 reporter. At the end of neural crest cell migration (E9.5) the two patterns are reciprocal, with a mutual boundary just posterior to the eye. Mesodermal cells expressing endothelial markers (angioblasts) are found not to respect this boundary; they are associated with the migrating neural crest from the 5-somite stage, and by E9.5 they form a pre-endothelial meshwork throughout the cranial mesenchyme. Mesodermal cells of the myogenic lineage also migrate with neural crest cells, as the branchial arches form. By E17.5 the neural crest-mesoderm boundary in the subectodermal mesenchyme becomes out of register with that of the underlying skeletogenic layer, which is between the frontal and parietal bones. At E13.5 the primordia of these bones lie basolateral to the brain, extending towards the vertex of the skull during the following 4-5 days. We used DiI labelling of the bone primordia in ex-utero E13.5 embryos to distinguish between two possibilities for the origin of the frontal and parietal bones: (1) recruitment from adjacent connective tissue or (2) proliferation of the original primordia. The results clearly demonstrated that the bone primordia extend vertically by intrinsic growth, without detectable recruitment of adjacent mesenchymal cells.     

Cell movements and control of patterned tissue assembly during craniofacial development.

Craniofacial mesenchyme is heterogeneous with respect to origins (e.g., paraxial mesoderm, lateral mesoderm, prechordal mesoderm, neural crest, placodes) and fates. The many disparate cell migratory behaviors exhibited by these mesenchymal populations have only recently been revealed, necessitating a reappraisal of how these different populations come together to form specific tissues and organs. The objectives of this review are to characterize the diverse migratory behaviors of craniofacial mesenchymal subpopulations, to define the interactions necessary for their assembly into tissues, and to discuss these data in the context of recent discoveries concerning the molecular basis of craniofacial development. The application of antibodies that recognize features unique to migrating neural crest cells has verified the results of previous transplantation experiments in birds and shown the migratory pathways in murine embryos to be similar. Within paraxial or prechordal mesoderm arise myoblasts that are precursors of craniofacial voluntary muscles. These cells migrate, usually en masse, to the sites where overt muscle differentiation occurs. Whereas the initial alignment of primary myotubes presages the fiber orientation seen in the adult, the time at which individual myotubes appear relative to the formation of discrete, individual muscle bundles and attachments with connective tissues varies with each muscle. The pattern of primary myotube alignment is determined by local connective tissue-forming mesenchyme and is independent of the source of myoblasts. Also found within paraxial and lateral mesodermal tissues are endothelial precursors (angioblasts). Some of these aggregate in situ, forming vesicles that coalesce with ingrowing endothelial cords. Others are highly invasive, moving in all directions and infiltrating tissues such as the neural crest, which lacks endogenous angioblasts. The patterns of initial blood vessel formation in the head are also determined by local connective tissue-forming mesenchyme and are independent of the origin of endothelial cells. Neural crest cells, which constitute the predominant connective tissue-forming mesenchyme in the facial, oral, and branchial regions of the head, acquire a regional identity while still part of the neural epithelium, and carry this with them as they move into the mandibular, hyoid, and branchial arches. Some of these regionally unique propensities correspond spatially to genetic and cellular patterns unique to rhombomeres, although the links between gene expression and crest population phenotypes are not yet known. In contrast, the inherent spatial programming of those crest cells that populate the maxillary and frontonasal regions is altered by their proximity to the prosencephalon.     

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.     

The concerted action of Meox homeobox genes is required upstream of genetic pathways essential for the formation,patterning and differentiation of somites

The paraxial mesoderm of the somites of the vertebrate embryo contains the precursors of the axial skeleton, skeletal muscles and dermis. The Meox1 and Meox2 homeobox genes are expressed in the somites and their derivatives during embryogenesis. Mice homozygous for a null mutation in Meox1 display relatively mild defects in sclerotome derived vertebral and rib bones, whereas absence of Meox2 function leads to defective differentiation and morphogenesis of the limb muscles. By contrast, mice carrying null mutations for both Meox genes display a dramatic and wide-ranging synthetic phenotype associated with extremely disrupted somite morphogenesis, patterning and differentiation. Mutant animals lack an axial skeleton and skeletal muscles are severely deficient. Our results demonstrate that Meox1 and Meox2 genes function together and upstream of several genetic hierarchies that are required for the development of somites. In particular, our studies place Meox gene function upstream of Pax genes in the regulation of chondrogenic and myogenic differentiation of paraxial mesoderm.     

The neural crest in presomite to 40-somite murine embryos

The production of cells by the neural crest is studied light-microscopically in 10 microns and 1 micron serially sectioned mouse and rat embryos, ranging in age from presomite to 40-somite stages. In the head region, mesectoderm formation starts in a pre-neural plate stage. It continues to the 20-somite stage. This implies that the contribution of the neural crest to the head mesoderm must be considerable. In the trunk, the neural crest only produces cells after adhesion of the neural walls. Mesectoderm formation continues for a long time, slowly retreating in a caudal direction. At the 40-somite stage, mesectoderm formation still occurs in the most caudal part of the trunk. Compared to the head, the contribution of the neural crest in the trunk seems to be less important than that of the primitive streak.     

Neural crest can form cartilages normally derived from mesoderm during development of the avian head skeleton

The lateral wall of the avian braincase, which is indicative of the primitive amniote condition, is formed from mesoderm. In contrast, mammals have replaced this portion of their head skeleton with a nonhomologous bone of neural crest origin. Features that characterize the local developmental environment may have enabled a neural crest-derived skeletal element to be integrated into a mesodermal region of the braincase during the course of evolution. The lateral wall of the braincase lies along a boundary in the head that separates neural crest from mesoderm, and also, neural crest cells migrate through this region on their way to the first visceral arch. Differences in the availability of one skeletogenic population versus the other may determine the final composition of the lateral wall of the braincase. Using the quail-chick chimeric system, this investigation tests if populations of neural crest, when augmented and expanded within populations of mesoderm, will give rise to the lateral wall of the braincase. Results demonstrate that neural crest can produce cartilages that are morphologically indistinguishable from elements normally generated by mesoderm. These findings (1) indicate that neural crest can respond to the same cues that both promote skeletogenesis and enable proper patterning in mesoderm, (2) challenge hypotheses on the nature of the boundary between neural crest and mesoderm in the head, and (3) suggest that changes in the allocation of migrating cells could have enabled a neural crest-derived skeletal element to replace a mesodermal portion of the braincase during evolution.     

Vertebrate somitogenesis: a novel paradigm for animal segmentation?

In vertebrates, the primary segmented tissue of the body axis is the paraxial mesoderm, which lies bilaterally to the axial organs, neural tube and notochord. The segmental pattern of the paraxial mesoderm is established during embryogenesis through the production of the somites which are transient embryonic segments giving rise to the vertebrae, the skeletal muscles and the dorsal dermis. Somitogenesis can be subdivided into three major phases (see Fig. 1). First a growth phase during which new paraxial mesoderm cells are produced by a growth zone (epiblast and blastopore margin or primitive streak and later on tail bud) and become organized as two rods of mesenchymal tissue,forming the presomitic mesoderm. Second a patterning phase occuring in the PSM, during which the segmental pattern is established at the molecular level. Third, the somitic boundaries are formed during the morphological segmentation phase. In all vertebrates, all cells of the paraxial mesoderm, during their maturation in the PSM, go successively through these three phases, which are tightly regulated at the spatio-temporal level. The first phase of paraxial mesoderm production falls out of the scope of this review, as it essentially pertains to the gastrulation process. Here, I essentially discuss the segmental patterning phase in vertebrates. Recent data suggest that establishment of the segmental pattern relies on a clock and wavefront mechanism which has been conserved in vertebrates. Furthermore, conservation of this system could extend to invertebrates, suggesting that the clock and wavefront is an ancestral mechanism.     

Rostral paraxial mesoderm regulates refinement of the eye field through the bone morphogenetic protein (BMP) pathway

The eye field is initially a large single domain at the anterior end of the neural plate and is the first indication of optic potential in the vertebrate embryo. During the course of development, this domain is subject to interactions that shape and refine the organogenic field. The action of the prechordal mesoderm in bisecting this single region into two bilateral domains has been well described, however the role of signalling interactions in the further restriction and refinement of this domain has not been previously characterised. Here we describe a role for the rostral cephalic paraxial mesoderm in limiting the extent of the eye field. The anterior transposition of this mesoderm or its ablation disrupted normal development of the eye. Importantly, perturbation of optic vesicle development occurred in the absence of any detectable changes in the pattern of neighbouring regions of the neural tube. Furthermore, negative regulation of eye development is a property unique to the rostral paraxial mesoderm. The rostral paraxial mesoderm expresses members of the bone morphogenetic protein (BMP) family of signalling molecules and manipulation of endogenous BMP signalling resulted in abnormalities of the early optic primordia.     

Temporal and spatial requirements for Nodal‐induced anterior mesendoderm and mesoderm in anterior neurulation

Zebrafish with defective Nodal signaling have a phenotype analogous to the fatal human birth defect anencephaly, which is caused by an open anterior neural tube. Previous work in our laboratory found that anterior open neural tube phenotypes in Nodal signaling mutants were caused by lack of mesendodermal/mesodermal tissues. Defects in these mutants are already apparent at neural plate stage, before the neuroepithelium starts to fold into a tube. Consistent with this, we found that the requirement for Nodal signaling maps to mid‐late blastula stages. This timing correlates with the timing of prechordal plate mesendoderm and anterior mesoderm induction, suggesting these tissues act to promote neurulation. To further identify tissues important for neurulation, we took advantage of the variable phenotypes in Nodal signaling‐deficient sqt mutant and Lefty1‐overexpressing embryos. Statistical analysis indicated a strong, positive correlation between a closed neural tube and presence of several mesendoderm/mesoderm‐derived tissues (hatching glands, cephalic paraxial mesoderm, notochord, and head muscles). However, the neural tube was closed in a subset of embryos that lacked any one of these tissues. This suggests that several types of Nodal‐induced mesendodermal/mesodermal precursors are competent to promote neurulation. genesis 54:3–18, 2016. © 2016 Wiley Periodicals, Inc.     

Combined intrinsic and extrinsic influences pattern cranial neural crest migration and pharyngeal arch morphogenesis in axolotl

Cranial neural crest cells migrate in a precisely segmented manner to form cranial ganglia, facial skeleton and other derivatives. Here, we investigate the mechanisms underlying this patterning in the axolotl embryo using a combination of tissue culture, molecular markers, scanning electron microscopy and vital dye analysis. In vitro experiments reveal an intrinsic component to segmental migration; neural crest cells from the hindbrain segregate into distinct streams even in the absence of neighboring tissue. In vivo, separation between neural crest streams is further reinforced by tight juxtapositions that arise during early migration between epidermis and neural tube, mesoderm and endoderm. The neural crest streams are dense and compact, with the cells migrating under the epidermis and outside the paraxial and branchial arch mesoderm with which they do not mix. After entering the branchial arches, neural crest cells conduct an "outside-in" movement, which subsequently brings them medially around the arch core such that they gradually ensheath the arch mesoderm in a manner that has been hypothesized but not proven in zebrafish. This study, which represents the most comprehensive analysis of cranial neural crest migratory pathways in any vertebrate, suggests a dual process for patterning the cranial neural crest. Together with an intrinsic tendency to form separate streams, neural crest cells are further constrained into channels by close tissue apposition and sorting out from neighboring tissues.     

The endoderm plays an important role in patterning the segmented pharyngeal region in zebrafish (Danio rerio)

The development of the vertebrate head is a highly complex process involving tissues derived from all three germ layers. The endoderm forms pharyngeal pouches, the paraxial mesoderm gives rise to endothelia and muscles, and the neural crest cells, which originate from the embryonic midbrain and hindbrain, migrate ventrally to form cartilage, connective tissue, sensory neurons, and pigment cells. All three tissues form segmental structures: the hindbrain compartmentalizes into rhombomeres, the mesoderm into somitomeres, and the endoderm into serial gill slits. It is not known whether the different segmented tissues in the head develop by the same molecular mechanism or whether different pathways are employed. It is also possible that one tissue imposes segmentation on the others. Most recent studies have emphasized the importance of neural crest cells in patterning the head. Neural crest cells colonize the segmentally arranged arches according to their original position in the brain and convey positional information from the hindbrain into the periphery. During the screen for mutations that affect embryonic development of zebrafish, one mutant, called van gogh (vgo), in which segmentation of the pharyngeal region is absent, was isolated. In vgo, even though hindbrain segmentation is unaffected, the pharyngeal endoderm does not form reiterated pouches and surrounding mesoderm is not patterned correctly. Accordingly, migrating neural crest cells initially form distinct streams but fuse when they reach the arches. This failure to populate distinct pharyngeal arches is likely due to the lack of pharyngeal pouches. The results of our analysis suggest that the segmentation of the endoderm occurs without signaling from neural crest cells but that tissue interactions between the mesendoderm and the neural crest cells are required for the segmental appearance of the neural crest-derived cartilages in the pharyngeal arches. The lack of distinct patches of neural crest cells in the pharyngeal region is also seen in mutants of one-eyed pinhead and casanova, which are characterized by a lack of endoderm, as well as defects in mesodermal structures, providing evidence for the important role of the endoderm and mesoderm in governing head segmentation.     

The role of the neural crest in patterning of avian cranial skeletal, connective, and muscle tissues

The morphology of skeletal tissues formed in each of the branchial arches of higher vertebrates is unique. In addition to these structures, which are derived from the neural crest, the crest-derived connective tissues and mesodermal muscles also form different patterns in each of the branchial arches. The objective of this study was to examine how these patterns arise during avian embryonic development. Presumptive second or third arch neural crest cells were excised from chick hosts and replaced with presumptive first arch crest cells. Both quail and chick embryos were used as donors; orthotopic crest grafts were performed as controls. Following heterotopic transplantation, the hosts developed several unexpected anomalies. Externally they were characterized by the appearance of ectopic, beak-like projections from the ventrolateral surface of the neck and also by the formation of supernumerary external auditory depressions located immediately caudal to the normal external ear. Internally, the grafted cells migrated in accordance with normal, second arch pathways but then formed a complete, duplicate first arch skeletal system in their new location. Squamosal, quadrate, pterygoid, Meckel's, and angular elements were present in most cases. In addition, anomalous first arch-type muscles were found associated with the ectopic skeletal tissues in the second arch. These results indicate that the basis for patterning of branchial arch skeletal and connective tissues resides within the neural crest population prior to its emigration from the neural epithelium, and not within the pharynx or pharyngeal pouches as had previously been suggested. Furthermore, the patterns of myogenesis by mesenchymal populations derived from paraxial mesoderm is dependent upon properties inherent to the neural crest.     

The Hectd1 ubiquitin ligase is required for development of the head mesenchyme and neural tube closure

Closure of the cranial neural tube depends on normal development of the head mesenchyme. Homozygous-mutant embryos for the ENU-induced open mind (opm) mutation exhibit exencephaly associated with defects in head mesenchyme development and dorsal-lateral hinge point formation. The head mesenchyme in opm mutant embryos is denser than in wildtype embryos and displays an abnormal cellular organization. Since cells that originate from both the cephalic paraxial mesoderm and the neural crest populate the head mesenchyme, we explored the origin of the abnormal head mesenchyme. opm mutant embryos show apparently normal development of neural crest-derived structures. Furthermore, the abnormal head mesenchyme in opm mutant embryos is not derived from the neural crest, but instead expresses molecular markers of cephalic mesoderm. We also report the identification of the opm mutation in the ubiquitously expressed Hectd1 E3 ubiquitin ligase. Two different Hectd1 alleles cause incompletely penetrant neural tube defects in heterozygous animals, indicating that Hectd1 function is required at a critical threshold for neural tube closure. This low penetrance of neural tube defects in embryos heterozygous for Hectd1 mutations suggests that Hectd1 should be considered as candidate susceptibility gene in human neural tube defects.     

Anterior cephalic neural crest is required for forebrain viability.

The prosencephalon, or embryonic forebrain, grows within a mesenchymal matrix of local paraxial mesoderm and of neural crest cells (NCC) derived from the posterior diencephalon and mesencephalon. Part of this NCC population forms the outer wall of capillaries within the prosencephalic leptomeninges and neuroepithelium itself. The surgical removal of NCC from the anterior head of chick embryos leads to massive cell death within the forebrain neuroepithelium during an interval that precedes its vascularization by at least 36 hours. During this critical period, a mesenchymal layer made up of intermingled mesodermal cells and NCC surround the neuroepithelium. This layer is not formed after anterior cephalic NCC ablation. The neuroepithelium then undergoes massive apoptosis. Cyclopia ensues after forebrain deterioration and absence of intervening frontonasal bud derivatives. The deleterious effect of ablation of the anterior NC cannot be interpreted as a deficit in vascularization because it takes place well before the time when blood vessels start to invade the neuroepithelium. Thus the mesenchymal layer itself exerts a trophic effect on the prosencephalic neuroepithelium. In an assay to rescue the operated phenotype, we found that the rhombencephalic but not the truncal NC can successfully replace the diencephalic and mesencephalic NC. Moreover, any region of the paraxial cephalic mesoderm can replace NCC in their dual function: in their early trophic effect and in providing pericytes to the forebrain meningeal blood vessels. The assumption of these roles by the cephalic neural crest may have been instrumental in the rostral expansion of the vertebrate forebrain over the course of evolution.     

Respective contribution of the cephalic neural crest and mesoderm to SIX1-expressing head territories in the avian embryo

Background

Vertebrate head development depends on a series of interactions between many cell populations of distinct embryological origins. Cranial mesenchymal tissues have a dual embryonic source: - the neural crest (NC), which generates most of craniofacial skeleton, dermis, pericytes, fat cells, and tenocytes; and - the mesoderm, which yields muscles, blood vessel endothelia and some posterior cranial bones. The molecular players that orchestrate co-development of cephalic NC and mesodermal cells to properly construct the head of vertebrates remain poorly understood. In this regard, Six1 gene, a vertebrate homolog of Drosophila Sine Oculis, is known to be required for development of ear, nose, tongue and cranial skeleton. However, the embryonic origin and fate of Six1-expressing cells have remained unclear. In this work, we addressed these issues in the avian embryo model by using quail-chick chimeras, cephalic NC cultures and immunostaining for SIX1.

Results

Our data show that, at early NC migration stages, SIX1 is expressed by mesodermal cells but excluded from the NC cells (NCC). Then, SIX1 becomes widely expressed in NCC that colonize the pre-otic mesenchyme. In contrast, in the branchial arches (BAs), SIX1 is present only in mesodermal cells that give rise to jaw muscles. At later developmental stages, the distribution of SIX1-expressing cells in mesoderm-derived tissues is consistent with a possible role of this factor in the myogenic program of all types of head muscles, including pharyngeal, extraocular and tongue muscles. In NC derivatives, SIX1 is notably expressed in perichondrium and chondrocytes of the nasal septum and in the sclera, although other facial cartilages such as Meckel’s were negative at the stages considered. Moreover, in cephalic NC cultures, chondrocytes and myofibroblasts, not the neural and melanocytic cells express SIX1.

Conclusion

The present results point to a dynamic tissue-specific expression of SIX1 in a variety of cephalic NC- and mesoderm-derived cell types and tissues, opening the way for further analysis of Six1 function in the coordinated development of these two cellular populations during vertebrate head formation.

     

Head morphogenesis in embryonic avian chimeras: evidence for a segmental pattern in the ectoderm corresponding to the neuromeres

Areas of the superficial cephalic ectoderm, including or excluding the neural fold at the same level, were surgically removed from 3-somite chick embryos and replaced by their counterparts excised from a quail embryo at the same developmental stage. Strips of ectoderm corresponding to the presumptive branchial arches were delineated, thus defining anteroposterior 'segments' (designated here as 'ectomeres') that coincided with the spatial distribution of neural crest cells arising from the adjacent levels of the neural fold. This discrete ectodermal metamerisation parallels the segmentation of the hindbrain into rhombomeres. It seems, therefore, that not only is the neural crest patterned according to its rhombomeric origin but that the superficial ectoderm covering the branchial arches may be part of a larger developmental unit that includes the entire neurectoderm, i.e., the neural tube and the neural crest.     

Vertical versus planar induction in amphibian early development

In the Urodeles, the archenteron roof invaginates as a single continuous sheet of cells, vertically inducing the neural anlage in the overlying ectoderm during invagination. The induction comprises first the activation process, leading, to forebrain differentiation tendencies, and then the superimposed transformation process, which changes presumptive forebrain development into that of hindbrain and spinal cord acting with a caudally increasing intensity. The activating action, being maximal anteriorly, decreases caudally to nearly zero. In the double-layered Xenopus embryo, the internal mesodermal marginal zone shows much more independent and earlier regional segregation and involution than the external marginal zone in the Urodeles; its prechordal mesoderm already initiating vertical neural induction in overlying ectoderm at stages 10 to 10⁺ before any visible archenteron invagination. In Xenopus incomplete exogastrulae the prechordal mesoderm involutes normally prior to evagination of the endoderm and mesodem. Artificially produced Xenopus total exogastrulae, made at stage 9 before mesoderm involution, behave just like axolotl total exogastrulae, showing no neural differentiation. The notion of planar neural induction in Xenopus can only be applied in exogastrulae and Keller explants for the transforming action, which is maximal in the caudal archenteron roof. In normal Xenopus development, the formation of the entire nervous system is essentially due to vertical induction by the successively involuting prechordal and notochordal mesoderm. The different behavior of Xenopus embryos in comparison with Urodele embryos can essentially be explained by the double-layered character of the animal moiety of the Xenopus embryo.