Regulation of programmed cell death during neural induction in the chick embryo

To study early responses to neural inducing signals from the organizer (Hensen's node), a differential screen was performed in primitive streak stage chick embryos, comparing cells that had or had not been exposed to a node graft for 5 hours. Three of the genes isolated have been implicated in Programmed Cell Death (PCD): Defender Against Cell Death (Dad1), Polyubiquitin II (UbII) and Ferritin Heavy chain (fth1). We therefore explored the potential involvement of PCD in neural induction. Dad1, UbII and fth1 are expressed in partly overlapping domains during early neural plate development, along with the pro-apoptotic gene Cas9 and the death effector Cas3. Dad1 and UbII are induced by a node graft within 3 hours. TUNEL staining revealed that PCD is initially random, but both during normal development and following neural induction by a grafted node, it becomes concentrated at the border of the forming neural plate and anterior non-neural ectoderm and downregulated from the neural plate itself. PCD was observed in regions of Caspase expression that are free from Dad1, consistent with the known anti-apoptotic role of Dad1. However, gain- and loss-of-function of any of these genes had no detectable effect on cell identity or on neural plate development. This study reveals that early development of the neural plate is accompanied by induction of putative pro- and anti-apoptotic genes in distinct domains. We suggest that the neural plate is protected against apoptosis, confining cell death to its border and adjacent non-neural ectoderm.     

The L5 epitope: an early marker for neural induction in the chick embryo and its involvement in inductive interactions.

The pattern of expression of the carbohydrate epitope L5 was studied during early development of the chick neuroepithelium. Immunoreactivity first appears during gastrulation, at mid-primitive streak stage, and persists until at least 3.5 days of development. The epitope is expressed on all the components of the developing nervous system, both central and peripheral. In immunoblots, the antibody recognises a major component of about Mr 500,000 and several more minor components of lower molecular mass. If a Hensen's node from a donor embryo is transplanted into the area opaca of a host embryo, L5 immunoreactivity appears in the epiblast surrounding the graft. If hybridoma cells secreting the antibody are grafted together with Hensen's node into a host chick embryo, the induction of a supernumerary nervous system is inhibited. We suggest that the L5 epitope is an early and general marker for neural induction and that it may be involved directly in inductive interactions.     

Tissue interactions affecting the migration and differentiation of neural crest cells in the chick embryo.

A series of microsurgical operations was performed in chick embryos to study the factors that control the polarity, position and differentiation of the sympathetic and dorsal root ganglion cells developing from the neural crest. The neural tube, with or without the notochord, was rotated by 180 degrees dorsoventrally to cause the neural crest cells to emerge ventrally. In some embryos, the notochord was ablated, and in others a second notochord was implanted. Sympathetic differentiation was assessed by catecholamine fluorescence after aldehyde fixation. Neural crest cells emerging from an inverted neural tube migrate in a ventral-to-dorsal direction through the sclerotome, where they become segmented by being restricted to the rostral half of each sclerotome. Both motor axons and neural crest cells avoid the notochord and the extracellular matrix that surrounds it, but motor axons appear also to be attracted to the notochord until they reach its immediate vicinity. The dorsal root ganglia always form adjacent to the neural tube and their dorsoventral orientation follows the direction of migration of the neural crest cells. Differentiation of catecholaminergic cells only occurs near the aorta/mesonephros and in addition requires the proximity of either the ventral neural tube (floor plate/ventral root region) or the notochord. Prior migration of presumptive catecholaminergic cells through the sclerotome, however, is neither required nor sufficient for their adrenergic differentiation.     

Neural induction and regionalisation in the chick embryo.

Induction and regionalisation of the chick nervous system were investigated by transplanting Hensen's node into the extra-embryonic region (area opaca margin) of a host embryo. Chick/quail chimaeras were used to determine the contributions of host and donor tissue to the supernumerary axis, and three molecular markers, Engrailed, neurofilaments (antibody 3A10) and XlHbox1/Hox3.3 were used to aid the identification of particular regions of the ectopic axis. We find that the age of the node determines the regions of the nervous system that form: young nodes (stages 2-4) induced both anterior and posterior nervous system, while older nodes (stages 5-6) have reduced inducing ability and generate only posterior nervous system. By varying the age of the host embryo, we show that the competence of the epiblast to respond to neural induction declines after stage 4. We conclude that during normal development, the initial steps of neural induction take place before stage 4 and that anteroposterior regionalisation of the nervous system may be a later process, perhaps associated with the differentiating notochord. We also speculate that the mechanisms responsible for induction of head CNS differ from those that generate the spinal cord: the trunk CNS could arise by homeogenetic induction by anterior CNS or by elongation of neural primordia that are induced very early.     

Notochordal induction of cell wedging in the chick neural plate and its role in neural tube formation

Cells in the median hinge point (MHP) of the bending chick neural plate are tightly apposed to the underlying notochord. These cells differ from those in adjacent lateral neuroepithelial areas (L) in that MHP cells are short and mainly wedge-shaped and line a furrow, whereas L cells are tall and mainly spindle-shaped and do not line a furrow. Cell generation time also differs in these regions. These consistent differences are detectable only after the notochord has formed and established contact with the neural plate; it is unclear whether they result from self-differentiation or induction. Two experiments were performed to evaluate the hypothesis that MHP characteristics develop owing to inductive interactions between the notochord and overlying neuroepithelial cells. First, notochordless chick embryos were generated to determine whether midline neuroepithelial cells still developed typical MHP characteristics. In the absence of the notochord, such characteristics did not develop. Second, isolated segments of quail notochord were transplanted subjacent to L of chick hosts to ascertain whether the notochord is capable of inducing MHP characteristics in L cells. When transplanted notochordal segments established apposition with host L cells, the apposing L cells usually developed typical MHP characteristics. Collectively, these results provide strong evidence that the notochord plays an inductive role in the formation of MHP characteristics. This investigation further revealed that bending can occur in the absence of MHP characteristics, forming a neural tube with an abnormal morphology. Thus, the formation of such characteristics, particularly cell wedging, is not required for bending but plays a major role in generating the normal cross-sectional morphology of the neural tube.     

Glutamine synthetase induction in chick embryo retina monolayers

Induction of glutamine synthetase (GS) by cortisol has been shown to occur in monolayer cultures of cells obtained by enzymatic dissociation of retinas from 8- and 12-day-old chick embryos with papain (0.1%) or trypsin (0.25%). Although essentially single cells when plated, monolayers obtained by enzymatic dissociation show significant aggregation by 4--6 h. Monolayers prepared by mechanical dispersion (cells forced through successively smaller gage needles) are minimally inducible, perhaps owing to poor viability in such cultures. Storage at 4 degrees C for 24 h prior to treatment with cortisol significantly elevated both basal GS activity and inducibility in whole (but not in monolayer) retina cultures.     

Glutamine synthetase induction in chick embryo retina monolayers

Induction of glutamine synthetase (GS) by cortisol has been shown to occur in monolayer cultures of cells obtained by enzymatic dissociation of retinas from 8- and 12-day-old chick embryos with papain (0.1%) or trypsin (0.25%). Although essentially sigle cells when plated, monolayers obtained by enzymatic dissociation show significant aggregation by 4–6 h. Monolayers prepared by mechanical dispersion (cells forced through successively smaller gage needles) are minimally inducible, perhaps owing to poor viability in such cultures. Storage at 4°C for 24 h prior to treatment with cortisol significantly elevated both basal GS activity and inducibility in whole (but not in monolayer) retina cultures.     

Distinct modes of floor plate induction in the chick embryo

To begin to reconcile models of floor plate formation in the vertebrate neural tube, we have performed experiments aimed at understanding the development of the early floor plate in the chick embryo. Using real-time analyses of cell behaviour, we provide evidence that the principal contributor to the early neural midline, the future anterior floor plate, exists as a separate population of floor plate precursor cells in the epiblast of the gastrula stage embryo, and does not share a lineage with axial mesoderm. Analysis of the tissue interactions associated with differentiation of these cells to a floor plate fate reveals a role for the nascent prechordal mesoderm, indicating that more than one inductive event is associated with floor plate formation along the length of the neuraxis. We show that Nr1, a chick nodal homologue, is expressed in the nascent prechordal mesoderm and we provide evidence that Nodal signalling can cooperate with Shh to induce the epiblast precursors to a floor-plate fate. These results indicate that a shared lineage with axial mesoderm cells is not a pre-requisite for floor plate differentiation and suggest parallels between the development of the floor plate in amniote and anamniote embryos.     

An early requirement for FGF signalling in the acquisition of neural cell fate in the chick embryo

BACKGROUND: In Xenopus embryos, fibroblast growth factors (FGFs) and secreted inhibitors of bone morphogenetic protein (BMP)-mediated signalling have been implicated in neural induction. The precise roles, if any, that these factors play in neural induction in amniotes remains to be established. RESULTS: To monitor the initial steps of neural induction in the chick embryo, we developed an in vitro assay of neural differentiation in epiblast cells. Using this assay, we found evidence that neural cell fate is specified in utero, before the generation of the primitive streak or Hensen's node. Early epiblast cells expressed both Bmp4 and Bmp7, but the expression of both genes was downregulated as cells acquired neural fate. During prestreak and gastrula stages, exposure of epiblast cells to BMP4 activity in vitro was sufficient to block the acquisition of neural fate and to promote the generation of epidermal cells. Fgf3 was also found to be expressed in the early epiblast, and ongoing FGF signalling in epiblast cells was required for acquisition of neural fate and for the suppression of Bmp4 and Bmp7 expression. CONCLUSIONS: The onset of neural differentiation in the chick embryo occurs in utero, before the generation of Hensen's node. Fgf3, Bmp4 and Bmp7 are each expressed in prospective neural cells, and FGF signalling appears to be required for the repression of Bmp expression and for the acquisition of neural fate. Subsequent exposure of epiblast cells to BMPs, however, can prevent the generation of neural tissue and induce cells of epidermal character.     

Fibroblast growth factor during mesoderm induction in the early chick embryo

A chick genomic clone that reveals a high degree of homology to the mammalian and Xenopus bFGF gene has been isolated. The pattern of expression of bFGF has been examined during early chick embryogenesis. RNA blot analysis revealed that chick bFGF is already transcribed at pregastrula stages. Immunolabeling analysis indicated that bFGF protein is present at these early developmental stages and is distributed evenly in the epiblast, hypoblast and marginal zone of the chick blastula. Substances that can inhibit FGF action were applied to early chick blastoderms grown in vitro under defined culture conditions (DCM). Both heparin and suramin were capable of blocking the formation of mesodermal structures in a dose-dependent manner. Our results indicate that FGF-like substances may need to be present for axial structures to develop although they may be acting earlier during the induction of non-axial mesoderm.     

Acetylcholinesterase activity in neural crest cells of the early chick embryo

The appearance and distribution of AChE activity in the neural crest cells of the chick embryo were histochemically investigated. Prior to closure of the neural tube, neural crests were not demonstrated and most of the cells constituting the neural plate and the more lateral ectoderm were AChE-negative. With the closure of the neural tube, the neural crests assumed the form of a cell mass in its mid-dorsal portion and AChE activity was demonstrated in some elements of both tube and crests. The neural crest cells beginning to migrate ventrally or laterally were AChE-positive, and some showed intense enzymatic activity. Electron microscopically, the neural crest cells and the cells migrating from the neural crest displayed AChE activity in the cisternae of the nuclear envelope and in a few r-ER profiles, but were morphologically undifferentiated. As assessed by 3H-thymidine autoradiography, these cells possessed the potential to proliferate. These findings indicate that with the formation of the neural tube and neural crest, cells constituting these structures begin to differentiate with respect to AChE activity and that the enzyme appears in the neural crest cells before the onset of neuronal differentiation.     

Extracellular matrix fibrils and cell contacts in the chick embryo

Summary The migration of neural crest and sclerotome cells and the extension of ventral root axons in chick embryos at stages 16–20 were studied by light microscopy as well as scanning and transmission electron microscopy at the leg bud level of fixed specimens. Extensive cellular movements take place in association with an extracellular matrix consisting of microfibrils. The neural crest and sclerotome cells migrate into the large matrix-filled extracellular space surrounding the neural tube and notochord, apparently using microfibril bundles as substratum. The cells exhibit pseudopodia which are closely associated with the matrix fibrils. The fibrils around the notochord show a spatial arrangement indicating that the sclerotome cells are contact-guided to their subsequent positions. Mutual cell contacts, including those established by cell processes, frequently show cytoplasmic electron dense plaques at adjacent membranes. These small plaque contacts might be correlated to contact inhibition of locomotion between the cells and participate in the guidance of cells. The growth cones of extending axons exhibit filopodia contacting both surrounding mesenchyme cells and extracellular fibrils. The orientation of the axons might thus be affected by contacts with cell surfaces as well as with extracellular material.Technical assistance was given by Mrs. Kerstin Ahlfors, Mrs. Charlotte Fällström, Mrs. Annika Kylberg and Mrs. Stine SöderströmSupported by grants from The Swedish Natural Science Research Council