A morphological and experimental study of the mesencephalic neural crest cells in the mouse embryo using wheat germ agglutinin-gold conjugate as the cell marker

The distribution of the mesencephalic neural crest cells in the mouse embryo was studied by mapping the colonization pattern of WGA-gold labelled cells following specific labelling of the neuroectoderm and grafting of presumptive neural crest cells to orthotopic and heterotopic sites. The result showed that (1) there were concomitant changes in the morphology of the neural crest epithelium during the formation of neural crest cells, in the 4- to 7-somite-stage embryos, (2) the neural crest cells were initially confined to the lateral subectodermal region of the cranial mesenchyme and there was minimal mixing with the paraxial mesoderm underneath the neural plate, (3) labelled cells from the presumptive crest region colonized the lateral cranio-facial mesenchyme, the developing trigeminal ganglion and the pharyngeal arch, (4) the formation of neural crest cells was facilitated by the focal disruption of the basal lamina and the cell-cell interaction specific to the neural crest site and (5) the trigeminal ganglion was colonized not only by neural crest cells but also by cells from the ectodermal placode.     

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.     

Patterning of the avian intermediate mesoderm by lateral plate and axial tissues

Amniote kidney tissue is derived from the intermediate mesoderm (IM), a strip of mesoderm that lies between the somites and the lateral plate. While much has been learned concerning the later events which regulate the differentiation of IM into tubules and other types of kidney tissue, much less is known concerning the earlier events which regulate formation of the IM itself. In the current study, the chick pronephros was used as a model system to identify tissues that play a role in patterning the IM and the critical time periods during which such patterning events take place. Explant studies revealed that the prospective pronephric IM is already specified to express kidney genes by stage 6, shortly after its gastrulation through the primitive streak, and earlier than previously reported. Transplant and explant experiments revealed that the lateral plate contains an activity that can repress IM formation in tissues that are already specified to express IM genes. In contrast, Hensen's node can promote formation of IM in the lateral plate. Paraxial tissues (presomitic mesoderm plus neural plate and notochord) were found to influence the morphogenesis of the nephric duct, but did not induce IM tissue to an appreciable extent. Combining lateral plate and paraxial tissue in vivo or in vitro led to induction of IM genes in the paraxial mesoderm but not in the lateral plate mesoderm. Based on these results and those of others, we propose a two-step model for the patterning of the IM. While tissue is still in the primitive streak, the prospective IM is relatively uncommitted. By stage 6, shortly after cells leave the primitive streak, a field of cells is generate which is specified to give rise to IM (Step 1). Subsequently, competing signals from the lateral plate and axial tissues modulate the number of cells that commit to an IM fate (Step 2).     

Evidence for the guidance of pronephric duct migration by a craniocaudally traveling adhesion gradient

These experiments were undertaken to identify the general nature of the mechanism that guides the migration of the Ambystoma pronephric duct along the ventral edge of the somite file from its anterior origin to the cloaca. Using scanning electron microscopy in conjunction with microsurgery, we have sought to distinguish among such possibilities as chemotaxis, contact guidance, and gradients of adhesiveness to the substratum. A pronephric duct primordium transplanted to the flank of a host ventral to the primary duct migrates dorsocaudally across the flank to fuse with the primary duct. Removal of potential sources of distant attraction does not alter this behavior, nor do migrating secondary ducts follow any visible structures. A variety of transplantation experiments reveal that the guidance information is not only oriented but also directionally polarized and travels caudad as a wave. These results militate against chemotaxis and contact guidance as guiding influences and indicate that the cells of the pronephric duct tip are directed in their migration by local information which passes caudad over the duct's mesodermal substratum as a wave in register with the advancing wave of somite segmentation. We propose that this duct-guiding information may be a traveling gradient of flank mesoderm cell adhesiveness.     

Temporal and spatial protease nexin 1 expression during chick development

Protease nexin 1 (Pn-1) or glia derived nexin is a secreted protease inhibitor. By screening a chick embryonic cDNA library, we isolated Pn-1 cDNA and analyzed its expression pattern during development by in situ hybridization. Pn-1 was first observed at HH-stage 3 in the primitive pit. At HH-stage 7, expression was observed in the medial part of the neural folds and asymmetrically in the right lateral plate mesoderm and at the left side of Hensen's node. At HH-stage 10-11, Pn-1 was expressed in the closing neural tube, lateral plate mesoderm and paraxial head mesoderm. From HH-stage 12 onwards, expression was observed caudally in the lateral plate mesoderm and cranially in the Wolffian duct. At the level of the compartmentalized somite, expression was seen in the sclerotome. Pn-1 was also expressed in the anterior wall of the pharynx and still in the paraxial head mesoderm. At HH-stage 15, the expression in the Wolffian duct remained caudally while the expression in the sclerotome extended along the whole body axis. A stronger expression was observed in the cranial four somites. From HH-stage 17-18 onwards, expression became visible in the mesenchyme of the developing limb buds. At these stages, expression was no longer observed in the Wolffian duct. At HH-stage 36, Pn-1 was expressed in the vertebral bodies, in the neural tube, and in the metanephros.     

Plasticity in mouse neural crest cells reveals a new patterning role for cranial mesoderm

The anteroposterior identity of cranial neural crest cells is thought to be preprogrammed before these cells emigrate from the neural tube. Here we test this assumption by developing techniques for transposing cells in the hindbrain of mouse embryos, using small numbers of cells in combination with genetic and lineage markers. This technique has uncovered a surprising degree of plasticity with respect to the expression of Hox genes, which can be used as markers of different hindbrain segments and cells, in both hindbrain tissue and cranial neural crest cells. Our analysis shows that the patterning of cranial neural crest cells relies on a balance between permissive and instructive signals, and underscores the importance of cell-community effects. These results reveal a new role for the cranial mesoderm in patterning facial tissues. Furthermore, our findings argue against a permanently fixed prepatterning of the cranial neural crest that is maintained by passive transfer of positional information from the hindbrain to the periphery.     

Embryonic origin of skeletal muscle cells in the iris of the duck and quail

Summary The origin of skeletal muscle cells in avian iris muscle was investigated by quantitative analysis of heterochromatin profiles at the electron-microscopic level in irides of six types of quail-duck chimeras. Each of the following tissues was transplanted into the head region from quail to duck between stages 9 and 10: cranial neural crest; trunk neural crest; midbrain and adjacent mesoderm; forebrain; forebrain without neural crest; and forebrain without neural crest and mesoderm. The average ratio of heterochromatin profile to nucleus profile in iris skeletal muscle cells was high (quail type) in the dorsal iris, but low (duck type) in the ventral iris of the chimeras resulting from isotopic transplantation of cranial neural crest. Heterotopic transplantation of trunk neural crest to cranial position resulted in failure of development of skeletal muscle cells in the dorsal iris, but not in the appearance of skeletal muscle cells in the ventral iris. The average ratio of heterochromatin profile to nucleus profile in iris skeletal muscle cells was high in the chimeras resulting from transplantation of midbrain region and the chimeras resulting from transplantation of forebrain region, intermediate in the chimeras resulting from transplantation of forebrain region without neural crest, and low in the chimeras resulting from transplantation of forebrain region without neural crest and mesoderm. These results indicate that the skeletal muscle cells in the dorsal iris are of cranial neural crest origin while those in the ventral iris are not, and could possibly arise from cranial mesoderm.     

Effect of hyaluronidase treatment on the distribution of cranial neural crest cells in the chick embryo

The cranial paraxial mesoblast is patterned into segmental units termed somitomeres. Recently we demonstrated the morphological relationship between the migratory pathways of cranial neural crest cells and the patterned primary mesenchyme of chick embryos (Anderson and Meier, '81). Since extracellular matrix, particularly hyaluronate, is also distributed in cranial crest pathways, embryos were given sub-blastodisc injections of hyaluronidase just prior to neural tube fusion and neural crest migration to remove matrix. Histological sections of enzyme-treated embryos showed that Alcian blue staining of hyaluronate was significantly reduced. Surface ectoderm appeared collapsed on the subjacent mesoderm as well. Examination of embryos with the scanning electron microscope (SEM) revealed that paraxial mesoderm remained segmentally patterned even though it appeared more condensed because of a reduction in intercellular space between mesenchymal cells. In enzyme-treated embryos, the rostral crest cells spread over the dorsal surfaces of the first four somitomeres, as they would do normally. This distribution of neural crest cells occurs even when enzyme treatment interferes with neural tube fusion at that level. We conclude that 1) neural tube fusion is not a prerequisite for the timely release of cranial crest in the chick embryo and 2) that much of the organized hyaluronate-rich matrix that lies in the path of cranial crest is not essential for crest emigration or patterned distribution.     

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.     

Patterning of the cranial nerves in the chick embryo is dependent on cranial mesoderm and rhombomeric metamerism

The vertebrate peripheral nervous system (PNS) consists of two groups of nerves that have a metamerical series of proximal roots along the body axis: the branchial and spinal nerves. Spinal nerve metamerism is brought about by the presence of somites, while that of the branchial nerves is, in part, intrinsic to rhombomeres, the segmental compartments of the hind-brain. As the distribution pattern of neural crest cells prefigures the morphology of the PNS, we constructed tissue-recombinant chick embryos in order to determine factors that might regulate the crest cell distribution pattern. When the segmental plate was transplanted between the hind-brain and the head mesoderm before crest cell emigration, it developed into ectopic somites that inhibited the dorsolateral migration of crest cells such that formation of the cranial nerve trunks was disturbed. Even so, proximal portions of the nerve roots were intact. An ectopic graft of lateral mesoderm did not inhibit the directional migration of the crest cells, but allowed their ectopic distribution, resulting in the fusion of cranial nerve trunks. When spinal neurectoderm was transplanted into the hind-brain, the graft behaved like an even-numbered rhombomere and caused the fusion of cranial nerve roots. The identity of the spinal neurectoderm was preserved in the ectopic site analyzed by the immunolocalization of Hoxb-5 protein, a spinal cord marker. We conclude that the spatial distribution of cephalic crest cells is regulated by successive processes that act on their proximal and distal distribution. The migratory behavior of crest cells is achieved partly by an embryonic environment that is dependent upon the presence of somitomeres, which do not epithelialize as somites, in the trunk.     

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.     

Cell migration in the formation of the pronephric duct in Xenopus laevis

To determine if cell migration is involved in the formation of the pronephric duct in Xenopus, we used morphometry, ablation, and videomicroscopy of vitally stained cells to study duct formation. In St 23-24 (Nieuwkoop and Faber, 1956) embryos, a ridge of cells forms caudal to the pronephric rudiment. The ridge lengthens at approximately the same rate as the embryonic trunk from St 23 to St 31. Ablation experiments demonstrated that the ridge constitutes the pronephric duct rudiment (PDR); when the ridge was ablated at St 23-24, little or no duct formation occurred, whereas a duct formed when the pronephric rudiment was ablated and the ridge left intact. Vital dye injections showed that the PDR forms from the intermediate mesoderm ventral to myotomes IV-VIII. From St 29/30 to St 33/34, the PDR actively elongates along the ventral edge of the myotomes as far as myotome XIV, where it joins the cloaca as the pronephric duct. Videomicroscopy of vitally stained cells showed that the PDR elongates throughout its length and does not incorporate additional cells from the mesoderm over which it elongates. The results strengthen the case for a common mode of pronephric duct formation among amphibian species.     

A positive correlation between permissiveness of mesoderm to neural crest migration and early DRG growth.

The microenvironment created by grafting rostral somitic halves in place of normal somites leads to the formation of nonsegmented peripheral ganglia (Kalcheim and Teillet, 1989; Goldstein and Kalcheim, 1991) and is mitogenic for neural crest (NC) cells that become dorsal root ganglia (DRG) (Goldstein et al., 1990). We have now extended these studies by using three surgical manipulations to determine how additional mesodermal tissues affected DRG growth in chick embryos. The following experimental manipulations were performed: (1) unilateral deletion of epithelial somites, similar deletions followed by replacing the somites with (2) a three-dimensional collagen matrix, or (3) fragments of quail lateral plate mesoderm. When somites were absent or replaced by collagen matrix, ganglia were unsegmented, and their volumes were decreased by 21% and 12%, respectively, compared to contralateral intact DRG. In contrast, when lateral plate mesoderm was transplanted in place of somitic mesoderm, NC cells migrated into the grafted mesoderm and formed unsegmented DRG whose volumes were increased by 62.6% compared to the contralateral ganglia. These results suggest that although DRG precursors do not require sclerotome to begin migration and condensation processes, DRG size is modulated by the properties of the mesoderm. Permissiveness to migration is positively correlated with an increase in DRG volume. This volume increase observed in grafts of lateral plate mesoderm is likely to result from enhanced proliferation of neural crest progenitors, previously demonstrated for DRG cells in rostral somitic grafts.     

Neural cell adhesion molecule expression in Xenopus embryos

The spatiotemporal pattern of expression of the neural cell adhesion molecule NCAM was mapped immunohistochemically in embryos of the frog Xenopus, from blastula to early swimming stages, using a polyclonal antibody that recognizes Xenopus NCAM. The neural plate stage was the earliest at which NCAM could be detected. The initial sites of NCAM immunoreactivity were neural ectoderm, somitic mesoderm, and chordamesoderm. During formation of the neural tube, NCAM immunoreactivity became restricted to the neuroectoderm and its derivatives. During closure of the neural tube and for 2-4 hr thereafter, NCAM was expressed in a distinctive radial pattern in coronal sections of the neural tube. NCAM was observed in neural crest cells before migration and after formation of cranial and spinal ganglia. During the period of initial neurite outgrowth, NCAM became concentrated in the developing central nerve fiber pathways. NCAM was seen on peripheral nerves from the time of their initial outgrowth and it was strongly expressed at neuromuscular junctions during the period of their formation. These results show that NCAM is expressed after neural induction and functions during morphogenesis of the neural plate and tube, some neural crest derivatives, development of nerve fiber tracts, and formation of neuromuscular connections.     

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.