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In vertebrates, the embryonic dorsal midline is a crucial signalling centre that patterns the surrounding tissues during development. Members of the FoxA subfamily of transcription factors are expressed in the structures that compose this centre. Foxa2 is essential for dorsal midline development in mammals, since knock-out mouse embryos lack a definitive node, notochord and floor plate. The related gene foxA4 is only present in amphibians. Expression begins in the blastula -chordin and -noggin expressing centre (BCNE) and is later restricted to the dorsal midline derivatives of the Spemann's organiser. It was suggested that the early functions of mammalian foxa2 are carried out by foxA4 in frogs, but functional experiments were needed to test this hypothesis. Here, we show that some important dorsal midline functions of mammalian foxa2 are exerted by foxA4 in Xenopus. We provide new evidence that the latter prevents the respecification of dorsal midline precursors towards contiguous fates, inhibiting prechordal and paraxial mesoderm development in favour of the notochord. In addition, we show that foxA4 is required for the correct regionalisation and maintenance of the central nervous system. FoxA4 participates in constraining the prospective rostral forebrain territory during neural specification and is necessary for the correct segregation of the most anterior ectodermal derivatives, such as the cement gland and the pituitary anlagen. Moreover, the early expression of foxA4 in the BCNE (which contains precursors of the whole forebrain and most of the midbrain and hindbrain) is directly required to restrict anterior neural development.

The midline organ the notochord and its overlying dorsal neural tube are the most prominent features of the chordate body plan. Although the molecular mechanisms involved in the formation of the central nervous system (CNS) have been studied extensively in vertebrate embryos, none of the genes that are expressed exclusively in notochord cells has been shown to function in this process. Here, we report a gene in the urochordate Ciona intestinalis encoding a fibrinogen-like protein that plays a pivotal role in the notochord-dependent positioning of neuronal cells. While this gene (Ci-fibrn) is expressed exclusively in notochord cells, its protein product is not confined to these cells but is distributed underneath the CNS as fibril-like protrusions. We demonstrated that Ci-fibrn interacts physically and functionally with Ci-Notch that is expressed in the central nervous system, and that the correct distribution of Ci-fibrn protein is dependent on Notch signaling. Disturbance of the Ci-fibrn distribution caused an abnormal positioning of neuronal cells and an abnormal track of axon extension. Therefore, it is highly likely that the interaction between the notochord-based fibrinogen-like protein and the neural tube-based Notch signaling plays an essential role in the proper patterning of CNS.

The formation of the secondary neural tube was traced in serial sections of human embryos of developmental stages 13 to 17 (32-41 days after fertilisation). It was found that the secondary neural tube formation begins with cavitation of the neural cord. The minute cavities are seen in embryos at stages 13 and 15. At stages 16 and 17 the numerous cavities coalesce to form a single central canal.

Using degenerated PCR-primers to identify known and novel BMPs that are expressed in the developing chicken heart, we identified not only BMP2, -4, and -7 mRNA, but also the TGFbeta superfamily member cVg1. The expression pattern of cVg1 mRNA was determined during chicken development from HH4 to HH44. In early developmental stages, cVg1 mRNA is expressed in the primitive streak, paraxial mesoderm, developing somites, and developing neural tube. Subsequently, cVg1 mRNA is expressed in the developing central and peripheral nervous system, retina, auditory vesicle, notochord, lung alveoli, and olfactory mucosa. In the heart, cVg1 is initially expressed through the linear heart tube, but becomes restricted to the forming chamber myocardium, in an expression domain similar to that of atrial natriuretic factor (ANF) mRNA., (Copyright 2003 Wiley-Liss, Inc.)

In an attempt to understand the events in the secondary neurulation in embryonic stage, we investigated morphological changes in the tail bud of normal developing chick embryos. Hamburger and Hamilton stage 16-45 embryos were harvested and processed for light microscopic studies. The secondary neural tube is formed by aggregation of the caudal cell mass. Cells are arranged into a cord-like mass (medullary cord), which is continuous with the primary neural tube. Multiple small cavities develop in the medullary cord, and these cavities coalesce into one single lumen. The process of coalescence is completed by stage 35, and the whole neural tube is transformed into one tube with a single continuous lumen. At this stage, the terminal portion of the neural tube is bulged dorsally. Thereafter, the caudal portion of the neural tube regresses, and the proximal portion develops into normal spinal cord. Transient occlusion of the central canal was observed at stage 40 in one sample. The sequence of events elucidated in this study can be used as base-line data for experiments concerning congenital malformations involving secondary neurulation.

Primary neurulation is a form-shaping event during the early development of the vertebrate embryo in which the neural plate is rolled up into the neural tube, the rudiment of the central nervous system. In an effort to identify genes specifically expressed in tissues lateral to the chick neural plate--tissues known to generate extrinsic forces for primary neurulation--we designed a subtractive scheme and identified a positive clone as the gene encoding chick cartilage linking protein 1 (CRTL1). CRTL1 (also known as link protein) is a small glycoprotein of the extracellular matrix that was originally identified for its role in stabilizing aggregates of aggrecan and hyaluronan in cartilage. In addition to being expressed in cartilage, CRTL1 is also immunolocalized in several noncartilaginous tissues as assessed with the 4B6 monoclonal antibody. Using the 4B6 antibody and the G9 riboprobe derived from our subtraction, we report the detailed distribution of CRTL1 protein and crtl1 transcripts during primary neurulation in chick embryos. This report emphasizes and briefly discusses important differences between the RNA expression pattern and the domains of accumulation of the protein. CRTL1 prominently accumulates in the basal lamina of the epidermal ectoderm just lateral to the neural plate. Based on the crucial role of the interface between this tissue and the neuroepithelium in the formation of the neural folds, and because of the biophysical role of hyaluronan in tissue morphogenesis, we propose that crtl1 represents is an excellent candidate neurulation gene, worthy of further study., (Copyright 2003 Elsevier Science B.V.)

It has been known for some time that ablation of the neural tube and/or the notochord in the chick embryo leads to a massive wave of cell death in the adjacent somites. It is postulated that in the normal embryo, survival signals emanate from the neural tube and/or notochord that suppress apoptosis in the cells of the somites, except for a small population of sclerotome cells that are programmed to die naturally. In this study we show that axial ablation results in the death of sclerotome and not somitic neural crest cells, and we have examined the apoptotic response of these cells to the ablation. We show that several elements of the apoptotic cascade become detectable in somite cells in response to the withdrawal of survival signals. We demonstrate the down-regulation of bcl-2 protein in the somites adjacent to, and caudal to, the site of ablation, corresponding to the region that displays an elevated level of cell death. Although caspase-9 appeared to be activated in somites at all levels of the trunk, caspase-2 showed a clear response to the ablation of the axial structures. Removal of the neural tube and notochord produced an up-regulation of caspase-2 activity in somites in the region of the operation. Cleavage of two down-stream substrates of these caspases was examined. The cleavage of poly (ADP-ribose) polymerase (PARP) was apparent in somites at all levels of the trunk, and showed only a modest up-regulation after ablation. By contrast, the cleavage of DNA fragmentation factor (DFF45) showed a marked up-regulation in response to ablation, suggesting that this is a primary substrate for a caspase-dependent apoptotic mechanism. Evidence was also found for a caspase-independent mechanism, since the expression of apoptosis-inducing factor (AIF) was found to be very sensitive to, and up-regulated in somites by, axial ablation. Because the wave of apoptosis that is precipitated in somites by removal of the axial structures may be mediated by BMP-4, we examined the levels of BMP-4 in somites in response to axial ablation. BMP-4 expression was clearly up-regulated in somites adjacent to, or close to, the site of operation.

In the ascidian embryo, the nerve cord and notochord of the tail of tadpole larvae originate from the precursor blastomeres for both tissues in the 32-cell-stage embryo. Each fate is separated into two daughter blastomeres at the next cleavage. We have examined mechanisms that are responsible for nerve cord and notochord specification through experiments involving blastomere isolation, cell dissociation, and treatment with basic fibroblast growth factor (bFGF) and inhibitors for the mitogen-activated protein kinase (MAPK) cascade. It has been shown that inductive cell interaction at the 32-cell stage is required for notochord formation. Our results show that the nerve cord fate is determined autonomously without any cell interaction. Presumptive notochord blastomeres also assume a nerve cord fate when they are isolated before induction is completed. By contrast, not only presumptive notochord blastomeres but also presumptive nerve cord blastomeres forsake their default nerve cord fate and choose the notochord fate when they are treated with bFGF. When the FGF-Ras-MAPK signaling cascade is inhibited, both blastomeres choose the default nerve cord pathway, supporting the results of blastomere isolation. Thus, binary choice of alternative fates and asymmetric division are involved in this nerve cord/notochord fate determination system, mediated by FGF signaling.

Members of the netrin gene family have been identified in vertebrates, Drosophila and Caenorhabditis elegans and found to encode secreted molecules involved in axon guidance. Here I use the conserved function of netrins in triploblasts, coupled with the phylogenetic position of amphioxus (the closest living relative of the vertebrates), to investigate the evolution of an axon guidance cue in chordates. A single amphioxus netrin gene was isolated by PCR and cDNA library screening and named AmphiNetrin. The predicted AmphiNetrin protein showed high identity to other netrin family members but differed in that the third of three EGF repeats found in other netrins was absent. Molecular phylogene-tic analysis showed that despite the absent EGF repeat AmphiNetrin is most closely related to the vertebrate netrins. AmphiNetrin expression was identified in embryonic notochord and floor plate, a pattern similar to that of vertebrate netrin-1 expression. AmphiNetrin expression was also identified more widely in the posterior larval brain, and in the anterior extension of the notochord that underlies the anterior of the amphioxus brain. All of these areas of expression are correlated with developing axon trajectories: The floor plate with ventrally projecting somatic motor neurons and Rohde cell projections, the posterior brain with the ventral commissure and primary motor centre and the anterior extension of the notochord with ventrally projecting neurons associated with the median eye. Amphioxus is naturally cyclopaedic and also lacks the ventral brain cells that the induction of which results in the splitting of the vertebrate eye field and, when missing, result in cyclopaedia. These cells normally express netrins required for developing axon tracts in the brain, and the expression of AmphiNetrin in the anterior extension of the notochord underlying the brain may explain how amphioxus is able to maintain ventral guidance cues while lacking these cells.

Neural tube defects (NTDs), a set of structural abnormalities affecting the brain, spinal cord, and the skeletal and connective tissues that protect them, are common malformations among humans and laboratory animals. The embryogenesis of the neural tube is presented to convey the complexity of the phenomenon, the multiplicity of requisite cellular and subcellular processes, and the precise timing of events that must occur for successful neural tube development. Interruption, even transitory, of any of these intricate processes or disruption of an embryo's developmental schedule can lead to an NTD. The population distribution of human NTDs demonstrates that genetic predisposition functions in susceptibility to NTDs. Data from animal studies support these concepts. NTDs are common outcomes in developmental toxicity safety assessments, occurring among control and treated groups. Numerous agents have caused increased levels of NTDs in laboratory animals, and species with shorter gestational periods appear more prone to toxicant-induced NTDs than those with longer gestations. Data from post-implantation whole embryo culture, although not predictive of human risk, are useful in studying neurulation mechanisms and in demonstrating the importance of maintaining embryonic schedules of development. We conclude that the concept that NTDs are produced by only a few toxicants that selectively target the developing nervous system is untenable. Rather, the combination of the time in gestation that an agent is applied, its dose, and its ability to disrupt critical processes in neurulation leads to NTDs. We further conclude that, because of both the relatively high prevalence and the multifactorial nature of NTDs, the mere occurrence of an NTD is insufficient for inferring that the defect was caused by an exogenous agent., (Copyright 1999 Wiley-Liss, Inc.)

There has long been controversy concerning the role of the axial mesoderm in the induction and rostrocaudal patterning of the vertebrate nervous system. Here we investigate the neural inducing and regionalising properties of defined rostrocaudal regions of head process/prospective notochord in the chick embryo by juxtaposing these tissues with extraembryonic epiblast or neural plate explants. We localise neural inducing signals to the emerging head process and using a large panel of region-specific neural markers, show that different rostrocaudal levels of the head process derived from headfold stage embryos can induce discrete regions of the central nervous system. However, we also find that rostral and caudal head process do not induce expression of any of these molecular markers in explants of the neural plate. During normal development the head process emerges beneath previously induced neural plate, which we show has already acquired some rostrocaudal character. Our findings therefore indicate that discrete regions of axial mesendoderm at headfold stages are not normally responsible for the establishment of rostrocaudal pattern in the neural plate. Strikingly however, we do find that caudal head process inhibits expression of rostral genes in neural plate explants. These findings indicate that despite the ability to induce specific rostrocaudal regions of the CNS de novo, signals provided by the discrete regions of axial mesendoderm do not appear to establish regional differences, but rather refine the rostrocaudal character of overlying neuroepithelium.

Induction of the floor plate at the ventral midline of the neural tube is one of the earliest events in the establishment of dorsoventral (d/v) polarity in the vertebrate central nervous system (CNS). The secreted molecule, Sonic hedgehog, has been shown to be both necessary and sufficient for this induction. In vertebrates, several downstream components of this signalling pathway have been identified, including members of the Gli transcription factor family. In this study, we have examined d/v patterning of the CNS in Gli2 mouse mutants. We have found that the floor plate throughout the midbrain, hindbrain and spinal cord does not form in Gli2 homozygotes. Despite this, motoneurons and ventral interneurons form in their normal d/v positions at 9.5 to 12.5 days postcoitum (dpc). However, cells that are generated in the region flanking the floor plate, including dopaminergic and serotonergic neurons, were greatly reduced in number or absent in Gli2 homozygous embryos. These results suggest that early signals derived from the notochord can be sufficient for establishing the basic d/v domains of cell differentiation in the ventral spinal cord and hindbrain. Interestingly, the notochord in Gli2 mutants does not regress ventrally after 10.5 dpc, as in normal embryos. Finally, the spinal cord of Gli1/Gli2 zinc-finger-deletion double homozygous mutants appeared similar to Gli2 homozygotes, indicating that neither gene is required downstream of Shh for the early development of ventral cell fates outside the ventral midline.

The notochord and dorsal ectoderm induce dorsoventral compartmentalization of the vertebrate neural tube through the differential regulation of genes such as HNF-3beta, Pax3, Pax6 and snail. Here we analyze the expression of HNF-3beta and snail homologues in the ascidian, Ciona intestinalis, a member of the subphylum Urochordata, the earliest branch in the chordate phylum. A combination of in situ hybridization and promoter fusion analyses was used to demonstrate that the Ciona HNF-3beta homologue is expressed in the ventralmost ependymal cells of the neural tube, while the Ciona snail homologue is expressed at the junction between the invaginating neuroepithelium and dorsal ectoderm, similar to the patterns seen in vertebrates. These findings provide evidence that dorsoventral compartmentalization of the chordate neural tube is not an innovation of the vertebrates. We propose that precursors of the floor plate and neural crest were present in a common ancestor of both vertebrates and ascidians.

The fork head/HNF-3 genes form a subclass of a family of transcription factors united by the possession of a conserved DNA binding domain known as the fork head domain. Most vertebrate HNF-3 class genes show several conserved sites of expression during development, including the dorsal lip/Hensen's node, notochord and floor plate, all structures known to organise adjacent tissues. In this paper I report the characterisation of HNF-3 class genes from the cephalochordate amphioxus. I show that amphioxus has two HNF-3 class genes, named AmHNF-3-1 and AmHNF-3-2; molecular phylogenetic analysis reveals that these derive from an independent duplication in the cephalochordate lineage. The expression of both genes in early development appears identical and shows striking similarities to that of vertebrates. In neurulae, transcripts of both genes were detected in the presumed organiser, endoderm, and notochord, supporting morphological and embryological evidence that these are homologous between vertebrates and amphioxus. This expression pattern overlaps considerably with that of amphioxus brachyury, suggesting that the functional relationship between these genes in vertebrates is conserved with amphioxus. Expression of both genes was maintained in the endoderm and notochord up to the 6 somite stage. After the 6 somite stage no expression of AmHNF-3-2 was detected and expression of AmHNF-3-1 began to decrease in the notochord, such that by the 10 somite stage transcripts were only detected in the terminal regions. At this stage, however, a column of AmHNF-3-1-expressing cells was detected at the ventral midline of the neural tube, a position occupied by the floor plate in vertebrates. This is the first evidence that amphioxus has a floor plate which is specified by a mechanism conserved with vertebrates. Taken together these data support two conclusions: Firstly, that the role of the dorsal lip/Hensen's node, notochord, and floor plate as organisers of the vertebrate body plan evolved prior to the separation of the vertebrate and cephalochordate lineages, at least 520 million years ago. Secondly, that the role of HNF-3 genes in these structures predates the origin of the multiple HNF-3 genes found in vertebrates.

Midline structures, such as the notochord and floor plate, are crucial to the developing central nervous system (CNS). Previously, we demonstrated that annexin IV is an excellent marker of midline structures. In the present study, we explore the possible role of annexin IV in development of the CNS midline. Using immunocytochemistry with an antibody to annexin IV, we have elucidated the temporal and spatial expression of this molecule. Annexin IV is present in the notochord at embryonic day (E) 8.5, prior to its expression in any structures within the neural tube. Subsequently, annexin IV is expressed by floor plate cells at E9.5. Annexin IV is also expressed in the roof plate, but not until E10.5. To determine if normal morphogenesis of these midline structures is essential for annexin IV expression, we analyzed two strains of mutant mice that have defective formation of either the floor or the roof plate. In Danforth's short-tail mice, the floor plate is absent from the caudal spinal cord, and annexin IV immunopositivity disappears at the level where the floor plate is missing. In curly tail mutant mice, there can be a failure of the neural tube to close, and in these regions there is no annexin IV expression in presumptive roof plate cells. Finally, annexin IV immunolabeling is present from the caudal spinal cord, through the brainstem up to the diencephalon and lamina terminalis. Thus, annexin IV is an excellent marker for differentiated midline cells, is temporally and spatially correlated with development of the floor and roof plates, and is expressed in a rostral-caudal manner that supports the hypothesis that the floor plate extends the full length of the original neural tube.

Patterning of the ventral half of the neural tube results from the inductive influence of the notochord and of the floor plate. We have studied here the effect of an ectopically grafted notochord on the development of the dorsalmost part of the neural tube i.e. roof plate and alar plates. We show that at their early stages, dorsal genes are repressed by the dorsal graft of a notochord, as shown previously in other studies. We found also that when the notochord is implanted in a mediodorsal position on top of the roof plate (and not laterally as previously performed in other studies) the genes specifics of the floor plate are not induced, and motoneurons do not differentiate. The notochord prevents the formation of the medial septum from roof plate cells and induces their active proliferation between E5 and E7. Roof and dorsal alar plates derived cells start to die from E7 onward leaving a dorsally truncated spinal cord. If the notochord is grafted at 20 degrees-30 degrees from the sagittal plane ventral genes and structures are induced and the roof plate differentiates normally. We conclude that roof plate cells exhibit a specific response to notochord signals, the short range effect of which is thus strikingly demonstrated.

The identity and patterning of ventral cell types in the vertebrate central nervous system depends on cell interactions. For example, induction of a specialized population of ventral midline cells, the floor plate, appears to require contact-mediated signalling by the underlying notochord, whereas diffusible signals from the notochord and floor plate can induce ventrolaterally positioned motor neurons. Sonic hedgehog (Shh), a vertebrate hedgehog-family member, is processed to generate two peptides (M(r) 19K and 26/27K) which are secreted by both of these organizing centres. Moreover, experiments in a variety of vertebrate embryos, and in neural explants in vitro, indicate that Shh can mediate floor-plate induction. Here we have applied recombinant Shh peptides to neural explants in serum-free conditions. High concentrations of Shh bound to a matrix induce floor plate and motor neurons, and addition of Shh to the medium leads to dose-dependent induction of motor neurons. All inducing activity resides in a highly conserved amino-terminal peptide (M(r) 19K). Moreover, antibodies that specifically recognize this peptide block induction of motor neurons by the notochord. We propose that Shh acts as a morphogen to induce distinct ventral cell types in the vertebrate central nervous system.

HNF-3 beta, a transcription factor of the winged-helix family, is expressed in embryonic and adult endoderm and also in midline cells of the node, notochord, and floor plate in mouse embryos. To define the function of HNF-3 beta, a targeted mutation in the HNF-3 beta locus was generated by homologous recombination in embryonic stem cells. Mice lacking HNF-3 beta die by embryonic day (E) 10-11. Mutant embryos examined from E6.5 to E9.5 do not form a distinct node and lack a notochord. In addition, mutant embryos show marked defects in the organization of somites and neural tube that may result from the absence of the notochord. The neural tube of mutant embryos exhibits overt anteroposterior polarity but lacks a floor plate and motor neurons. Endodermal cells are present but fail to form a gut tube in mutant embryos. These studies indicate that HNF-3 beta has an essential role in the development of axial mesoderm in mouse embryos.

HNF-3 beta, a member of the HNF-3/fork head family of transcription factors, is expressed in the node, notochord, floor plate, and gut in mouse embryos. A null mutation of this gene leads to embryonic lethality. The primary defect of HNF-3 beta -/- embryos is an absence of organized node and notochord formation, which leads to secondary defects in dorsal-ventral patterning of the neural tube. In contrast, patterning along the anterior-posterior axis was surprisingly little affected. Although HNF-3 beta is required for node and notochord formation, some organizer activity persists in the absence of these structures. HNF-3 beta is not required for the development of definitive endoderm cells, but foregut morphogenesis is severely affected in HNF-3 beta -/- embryos.

The embryonic zebrafish provides a relatively simple and accessible experimental system for understanding the underlying Bauplan of a vertebrate central nervous system (CNS) and for uncovering interactions critical for patterning. We show that features of CNS organization in normal, mutant, and developmentally ventralized zebrafish embryos can be explained by a three-axis hypothesis: Specifications occur according to position relative to coordinates along the anterior-posterior, dorsal-ventral, and luminal-pial axes of the neural tube. Midline tissues may be responsible for directly establishing the dorsal-ventral coordinates, but may only be indirectly involved in patterning along the anterior-posterior axis.

The floor plate is located at the ventral midline of the neural tube and has been implicated in neural cell patterning and axon guidance. To address the cellular mechanisms involved in floor plate differentiation, we have used an assay that monitors the expression of floor-plate-specific antigens in neural plate explants cultured in the presence of inducing tissues. Contact-mediated signals from both the notochord and the floor plate act directly on neural plate cells to induce floor plate differentiation. Floor plate induction is initiated medially by a signal from the notochord, but appears to be propagated to more lateral cells by homeogenetic signals that derive from medial floor plate cells. The response of neural plate cells to inductive signals declines with embryonic age, suggesting that the mediolateral extent of the floor plate is limited by a loss of competence of neural cells. The rostral boundary of the floor plate at the midbrain-forebrain junction appears to result from the lack of inducing activity in prechordal mesoderm and the inability of rostral neural plate cells to respond to inductive signals.

The aim of this work was to investigate the role played by the axial organs, neural tube and notochord, on the differentiation of muscle cells from the somites in the avian embryo. Two of us have previously shown that neuralectomy and notochordectomy is followed by necrosis of the somites and consecutive absence of vertebrae and of most muscle cells derived from the myotomes while the limbs develop normally with muscles. Here we have focused our attention on muscle cell differentiation by using the 13F4 mAb that recognizes a cytoplasmic antigen specific of all types of muscle cells. We show that differentiation of muscle cells of myotomes can occur in the absence of notochord and neural tube provided that the somites from which they are derived have been in contact with the axial organs for a defined period of time, about 10 hours for the first somites formed at the cervical level, a duration that progressively reduces caudalward (i.e. for thoracic and lumbar somites). Either one or the other of the two axial organs, the neural tube or the notochord can prevent somitic cell death and fulfill the requirements for myotomal muscle cell differentiation. Separation of the neural tube/notochord complex from the somites by a surgical slit on one side of the embryo gave the same results as extirpation of these organs and provided a perfect control on the non-operated side. A striking finding was that limb and body wall muscles, although derived from the somites, differentiated in the absence of the axial organs. However, limb muscles that develop after excision of the neural tube started to degenerate from E10 onward due to lack of innervation. In vitro explantation of somites from different axial levels confirmed and defined precisely the chronology of muscle cell commitment in the myotomes as revealed by the in vivo experiments.

Notochord has been implicated in previous studies in both the dorsoventral and rostrocaudal patterning of the developing neural tube. This possibility has been further explored by analyzing the expression of Engrailed-2 in chick embryos developing with cranial notochord defects. Control embryos containing intact notochords expressed Engrailed-2 protein within the neural tube and in a subset of the neural crest and overlying surface ectoderm at the future mesencephalon and cranial metencephalon levels. Within the neural tube, expression was confined to cell nuclei in the roof plate and lateral walls; floor plate nuclei directly overlying the notochord typically failed to show expression. After surgical removal of Hensen's node, the source of notochord precursor cells, embryos were cultured through neurulation and assayed for expression of Engrailed-2 protein. All embryos that partially or completely lacked cranial notochord expressed Engrailed-2 in a pattern similar to that of control embryos containing intact notochords, except that when notochord and floor plate were absent, Engrailed-2 was also expressed in the most ventral part of the neural tube. These results indicate that 1) Engrailed-2 expression is suppressed in the most ventral neural tube owing to induction of the floor plate by the notochord, and 2) that the presence of an underlying notochord is not required for correct rostrocaudal expression, suggesting that multiple pathways act in the patterning of the rudiment of the central nervous system.

Cell contact and adhesion between somites and the axial extracellular matrix (ECM) is likely to play a fundamental role in vertebrate development. In a preliminary report we showed that injection of the monoclonal antibody CSAT, which recognizes the avian beta 1 integrins, causes a lateral separation of both somites and segmental plate tissue from the embryonic axis (Drake and Little, 1991). In this study we addressed the cell biological response to CSAT injection, particularly the cell-ECM interactions involved in maintaining normal somite-axial relationships. A total of 150 stage 7-10 quail embryos have been injected with CSAT and then cultured for varying periods (1-30 hr). CSAT caused somitic cells to behave abnormally. Changes include, rounding-up, extensive blebbing, and formation of retraction fibers. A majority of separated somites were able to assume normal axial position with further time culture. Whether a somite subsequently aligned at the axis was dependent on the amount of CSAT injected and the postinjection culture period. Embryos in which somites remained separated from the axis after relatively long culture intervals (18-24 hr) displayed abnormal sclerotomal cell migrations. In no case did control injected embryos exhibit cellular alterations. Similarly, the injection of RGD-containing peptides had no detectable effect on somitogenesis or somite/segmental plate adhesion to the axis. On the basis of these data, we conclude that beta 1 integrins are necessary for normal somitic cell adhesions to the axis, but not somite segmentation and differentiation.

In the epiblast of elongating primitive-streak-stage avian embryos, MHP cells--short wedge-shaped neurepithelial cells contained within the median hinge point of the bending neural plate--arise from the midline prenodal and nodal area, whereas L cells--tall spindle-shaped neurepithelial cells constituting the lateral neural plate--arise from paired areas flanking the cranial primitive streak. These characteristic differences in neurepithelial cell shape are acquired as a result of inductive interactions with the notochord. Both MHP and L cells undergo extensive rearrangement (intercalation) during shaping and bending of the neural plate, but their pattern of rearrangement differs. MHP cells intercalate with other MHP cells and the population always spans the midline, whereas L cells intercalate with other L cells, remaining in bulk lateral to the midline. The following experiment was performed to establish whether these distinctive rearrangement patterns are determined prior to notochordal inductive interactions. Quail prospective MHP and L cells were transplanted isochronically and heterotopically to chick host blastoderms at stages prior to formation of the notochord (to wit, prospective MHP cells were transplanted into prospective L cell territory and vice versa) and the distribution, fate, and morphological characteristics of grafted cells were determined in chimeras collected 24 hr later. Our results demonstrate that heterotopic MHP and L cells do not adopt the rearrangement pattern characteristic of their new site; rather, they change their position so that grafted MHP cells intermix with MHP cells of the host and grafted L cells intermix with L cells of the host. Thus, patterns of neurepithelial cell rearrangement are determined prior to notochordal inductive interactions. When and how this determination occurs are topics for further studies.