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In the sea urchin embryo, the animal-vegetal axis is established during oogenesis and the oral-aboral axis is specified sometime after fertilization. The mechanisms by which either of these axes are specified and patterned during embryogenesis are poorly understood. Here, we investigated the role of cellular interactions in the specification of the ectoderm territories and polarization of the ectoderm along the oral-aboral axis. Isolated animal halves (mesomeres), which are fated to give rise to oral and aboral ectoderm, developed into polarized embryoids that expressed an oral ectoderm-specific marker uniformly. These embryoids also produced neuron-like cells and serotonergic neurons, suggesting that mesomeres are autonomously specified as oral ectoderm. Mesomere-derived embryoids did not express any aboral ectoderm-specific markers, although we previously showed that aboral ectoderm-specific genes can be induced by 25 mM lithium chloride, which also induced endoderm formation (Wikramanayake, A. H., Brandhorst, B. P. and Klein, W. H.(1995). Development 121, 1497-1505). To ascertain if endoderm formation is a prerequisite for induction of aboral ectoderm by lithium and for normal ectoderm patterning in animal halves, we modulated the lithium treatment to ensure that no endoderm formed. Remarkably, treating animal halves with 10 mM LiCl at approximately 7 hours postfertilization resulted in embryoids that displayed oral-aboral axis patterning in the absence of endoderm. Application of 25 mM LiCl to animal halves at approximately 16 hours postfertilization, which also did not induce endoderm, resulted in polarized expression of the aboral ectoderm-specific LpS1 protein, but global expression of the Ecto V antigen and no induction of the stomodeum or ciliary band. These results suggest that at least two signals, a positive inductive signal to specify the aboral ectoderm and a negative suppressive signal to inactivate oral ectoderm-specific genes in the prospective aboral ectoderm territory, are needed for correct spatial expression of oral and aboral ectoderm-specific genes. Transmission of both these signals may be prerequisite for induction of secondary ectodermal structures such as the ciliary band and stomodeum. Thus, differentiation of ectoderm and polarization of the oral-aboral axis in Lytechinus pictus depends on cellular interactions with vegetal blastomeres as well as interactions along the oral-aboral axis.
The vertebrate nervous system is initially induced from a section of dorsal ectoderm by signal(s) from the underlying dorsal mesoderm during gastrulation. In an effort to identify the neural inducing factor(s) emanating from the dorsal mesoderm, we have examined the inductive action of various growth factors by applying them to ectoderm cells from Xenopus gastrulae (8- to 12.5-hour age; embryonic stage 9+ to 11 1/2) in a microculture system. Monoclonal antibodies that specifically recognize cellular differentiation antigens from three distinct ectoderm lineages (N1 for CNS neurons from neural tube, Me1 for melanophores from neural crest and E3 for skin epidermal cells from epidermal lineages, respectively) and a mesoderm lineage (Mu1 for muscle cells) were used as markers to monitor the differentiation of cultured ectoderm cells. We found that basic fibroblast growth factor (bFGF) was capable of specifically and reproducibly inducing gastrula ectoderm cells to produce CNS neurons and melanophores at concentrations as low as 5 pM, a value about 50-fold lower than that required to induce the formation of muscle cells from blastula animal cap cells (6-hour age; stage 8+). The induction of neural lineages by bFGF was correlated with a suppression of epidermal differentiation in a dose-dependent manner. bFGF never induced the formation of muscle cells from gastrula ectoderm cells even at concentrations as high as 5 nM. The response of ectoderm cells to bFGF changed dramatically during gastrulation. Ectoderm cells from early (8- to 9-hour age; stage 9+ to 10) gastrula gave rise to CNS neurons, but yielded few melanophores. As ectoderm cells were prepared from gastrulae of increasing age, their competence to form neurons was gradually lost, whereas the production of melanophores was enhanced and peaked in 11-hour gastrula (stage 10 1/2). The ability to form both neurons and melanophores was substantially reduced in 12.5-hour gastrula (stage 11 1/2). By examining ectoderm cells from the ventral and dorsal sides independently, it was also shown that during gastrulation the change in response to bFGF of the ventral ectoderm preceded that of the dorsal ectoderm. The state of competence of the ectoderm changed primarily due to intrinsic factors rather than by instruction from other parts of the gastrula embryo. This was shown by adding bFGF to cultures of ectoderm cells that were isolated at 9-hour (stage 10) and cultured for increasing periods to allow their autonomous development. The time course of both loss of neuronal competence and gain and loss of melanophore competence closely paralleled that observed in vivo during gastrulation.(ABSTRACT TRUNCATED AT 400 WORDS)
Reports on the stages when chick flank ectoderm can respond to ridge induction are contradictory. Different results have been obtained using presumptive wing or leg bud mesoderm as the inducing tissue with flank ectoderm as the responding tissue. In addition, although incomplete outgrowths have been obtained from recombinants with stage-19 flank ectoderm in a small percentage of cases, no complete outgrowths have been obtained from recombinants with ectoderm older than stage 18. We reinvestigated when chick flank ectoderm can respond to ridge induction and promote outgrowth of complete limbs. To do this, we combined flank ectoderm with in situ chick presumptive wing bud mesoderm using a pre-limb bud recombinant technique. When presumptive wing bud ectoderm was removed from the host and not replaced, wing development was suppressed. When host ectoderm was replaced with stage-15 through -18 chick flank ectoderm, limbs grew out in all cases; 86.4% of these recombinant limbs were distally complete. Stage-19 flank ectoderm formed a ridge and promoted limb outgrowth in 80.9% of recombinants; 52.9% of these were distally complete limbs. Recombinants made by grafting early stage-20 (40-somite donor) flank ectoderm to stage-15 hosts resulted in outgrowths in 60% of the cases and 33.3% of these were distally complete. Graft ectoderm from older donors did not respond to inductive mesoderm. Our results demonstrate that chick flank ectoderm from stage-15 through early stage-20 donors can respond to inductive signals from presumptive wing bud mesoderm to form an apical ridge. This ridge can promote outgrowth of distally complete wings in a substantial proportion of recombinants. This is two stages beyond when the ability to promote outgrowth of distally complete wings appeared to be lost using other methods.
The distribution of lens self-differentiation capacity in the head ectoderm of chick embryos during stages 4-13 (defined by Hamburger and Hamilton) was investigated. Different fragments of head ectoderm were cultivated in organ culture in vitro. Lens cells and lentoids formed in explants were identified by the indirect immunofluorescence technique with monospecific rabbit antisera to chick lens proteins: alpha-, beta-, and delta-crystallins. It was established that besides presumptive lens ectoderm, the lateral head ectoderm and ectoderm of stomodeal area of stages 10-11 chick embryos differentiated into lens cells when separated and cultured alone. The cultivation of presumptive lens ectoderm resulted in lens fiber differentiation in most explants. In the explants of lateral head ectoderm and those of the ectoderm of the stomodeal area, the lentoids and groups of cells containing crystallins were also found in most cases. In some, all cells of the explants taken from these areas differentiated into lens tissue. These results show that in the chick embryos at stages 10-11, capacity for lens self-differentiation is distributed over a large continuous area of the head ectoderm. The potential seems to arise during the process of gastrulation. The head ectoblast of stage 4-5 chick embryos can achieve at least three differentiation in vitro--lens, epidermal-like, and neutral. In the chick head ectoderm, at the beginning of neurulation two areas can be distinguished according to their capacity for self-differentiation. The area situated in front of the head fold develops into lens and epidermal-like tissues in vitro, whereas the area between the head fold and Hensen's node develops into neural tissue. In the course of further development, lens potential was restricted to the presumptive lens ectoderm. Most of the head ectoderm develops in normogenesis in several directions without any traits of lens differentiation. The formation of Rathke's pouch (adanohypaphysis anlage) by cells which have synthesized beta-crystallins is the only known case (besides the lens anlage) in which an expression of lens potential of head ectoderm occurs.
Cell determination in vertebrates requires integration of many events, although the mechanisms controlling the different stages in this process are poorly understood. While studies of lens determination have helped define some of these stages, we know very little about the intermediate steps involved in the commitment of ectoderm to lens formation. Lens determination begins during gastrulation when ectoderm is briefly competent to respond to lens-inducing signals and progresses to a point, at the neural tube stage, when the presumptive lens ectoderm is specified. Between these two stages important regulatory genes are activated in the presumptive lens ectoderm, including the transcription factor Pax-6, and transplantation experiments presented here suggest that the presumptive lens ectoderm is becoming "biased" toward lens formation. We show that competent ectoderm from Xenopus laevis embryos forms well-differentiated lenses in most cases when transplanted to the presumptive lens area of neural plate stage hosts, where the lens-inductive environment is strong. When placed into later, neural tube stage hosts, optimally competent ectoderm does form small lenses in about half the cases, but the overall response is much weaker. Even in this weakly inducing environment, however, lens ectoderm that is part way through the inductive process (at the neural plate stage) is shown to have a lens-forming bias, since it forms well differentiated lenses in nearly all cases. While we find that ectoderm surrounding the lens-forming area at neural plate stages does not have a lens-forming bias, non-lens head ectoderm at the neural tube stage does, suggesting that a large region of head ectoderm is biased during neurulation. Using Rana palustris embryos, a species used in the earliest lens induction studies, we were also able to show that the optic vesicle can induce lenses in non-lens head ectoderm at neural tube stages. These results lead us to refine the definition of lens cell determination and provide a context that should allow clarification of determination mechanisms.
The ectoderm of the sea urchin embryo has been a useful system for understanding how regions of a simple epithelium are specified during early development, as well as how pattern formation leads to the correct localization of mesenchyme cells during morphogenesis. This study examines cell-cell signals that regulate precise patterning of ectoderm within the oral region of embryos of the sea urchin, Lytechinus variegatus. The oral ectoderm contains at least two types of patterned tissues: (1) the ectoderm that forms the stomodeum and (2) ectoderm expressing pattern information required for formation of parallel oral skeletal rods by primary mesenchyme cells (PMCs). Using microsurgical isolations and cell transplantation, we show (1) that cell-cell signaling is capable of producing new oral ectodermal structures until immediately prior to the gastrula stage, (2) that the presumptive oral ectoderm is not committed to produce oral structures until the early gastrula stage, (3) that oral ectodermal patterning cues for PMCs are highly local in character, and (4) that interactions between the tip of the archenteron and the presumptive oral ectoderm are not required for the differentiation of cells within either tissue. These studies suggest that short-range cell-cell signals within the ectoderm are involved in specifying regionalized oral ectodermal tissues immediately prior to gastrulation, and that this patterned ectoderm then influences the localization of skeletogenic mesenchyme cells in the oral region.
The Spdeadringer (Spdri) gene encodes an ARID-class transcription factor not previously known in sea urchin embryos. We show that Spdri is a key player in two separate developmental gene regulatory networks (GRNs). Spdri is expressed in a biphasic manner, first, after 12 h and until ingression in the skeletogenic descendants of the large micromeres; second, after about 20 h in the oral ectoderm, where its transcripts remain present at 30-50 mRNA molecules/cell far into development. In both territories, the periods of Spdri expression follow prior territorial specification events. The functional significance of each phase of expression was assessed by determining the effect of an alphaSpdri morpholino antisense oligonucleotide (MASO) on expression of 17 different mesodermal genes, 8 different oral ectoderm genes, and 18 other genes expressed specifically during endomesoderm specification. These effects were measured by quantitative PCR, supplemented by whole-mount in situ hybridization and morphological observations. Spdri is shown to act in the micromere descendants in the pathways that result in the expression of batteries of terminal skeletogenic genes. But, in the oral ectoderm, the same gene participates in the central GRN controlling oral ectoderm identity. Spdri is linked in the oral ectoderm GRN with several other genes encoding transcriptional regulators that are expressed specifically in various regions of the oral ectoderm. If its expression is blocked by treatment with alphaSpdri MASO, oral-specific features disappear and expression of the aboral ectoderm marker spec1 encompasses the whole of the ectoderm. In addition to disappearance of the oral ectoderm, morphological consequences of alphaSpdri MASO treatment include failure of spiculogenesis and of correct primary mesenchyme cell (pmc) patterning in the postgastrular embryo, and also failure of gastrulation. To further analyze these phenotypes, chimeric embryos were constructed consisting of two labeled micromeres combined with micromereless 4th cleavage host embryos; either the micromeres or the hosts contained alphaSpdri MASO. These experiments showed that, while Spdri expression is required autonomously for expression of skeletogenic genes prior to ingression, complete skeletogenesis also requires the expression of oral ectoderm patterning information. Presentation of this information on the oral side of the blastocoel in turn depends on Spdri expression in the oral ectoderm. Failure of gastrulation is not due to indirect interference with endomesodermal specification per se, since all endomesodermal genes tested function normally in alphaSpdri MASO embryos. Part of its cause is interference by alphaSpdri MASO with a late signaling function on the part of the micromere descendants that is needed to complete clearance of the Soxb1 repressor of gastrulation from the prospective endoderm, but in addition there is a nonautonomous oral ectoderm effect.
The ability of a tissue to respond to induction, termed its competence, is often critical in determining both the timing of inductive interactions and the extent of induced tissue. We have examined the lens-forming competence of Xenopus embryonic ectoderm by transplanting it into the presumptive lens region of open neural plate stage embryos. We find that early gastrula ectoderm has little lens-forming competence, but instead forms neural tissue, despite its location outside the neural plate; we believe that the transplants are being neuralized by a signal originating in the host neural plate. This neural competence is not localized to a particular region within the ectoderm since both dorsal and ventral portions of early gastrula ectoderm show the same response. As ectoderm is taken from gastrulae of increasing age, its neural competence is gradually lost, while lens competence appears and then rapidly disappears during later gastrula stages. To determine whether these developmental changes in competence result from tissue interactions during gastrulation, or are due to autonomous changes within the ectoderm itself, ectoderm was removed from early gastrulae and cultured for various periods of time before transplantation. The loss of neural competence, and the gain and loss of lens competence, all occur in ectoderm cultured in vitro with approximately the same time course as seen in ectoderm in vitro. Thus, at least from the beginning of gastrulation onwards, changes in competence occur autonomously within ectoderm. We propose that there is a developmental timing mechanism in embryonic ectoderm that specifies a sequence of competences solely on the basis of the age of the ectoderm.
Previous studies have indicated possible dual effects of the limb ectoderm in cartilage differentiation. On one hand, explants from early (stage 15) wing buds are dependent on contact with the limb ectoderm for cartilage differentiation (Gumpel-Pinot, J. Embryol. Exp. Morph. 59:157-173, 1980). On the other hand, limb ectoderm from stage 23/24 wing buds inhibits cartilage differentiation by cultured limb mesenchyme cells even without direct contact (Solursh et al., Dev. Biol. 86:471-482, 1981). In the present study, ectoderms from both stage 15/16 and stage 23/24 wings are cultured under the same conditions, and ectoderms from each source are shown to have two effects. Each stimulates chondrogenesis in stage 15 wing bud mesenchyme, and each inhibits chondrogenesis in older wing mesenchyme. The results suggest that the limb ectoderm has at least dual effects on cartilage differentiation, depending on the stage of the mesenchyme. One effect involves an early mesenchymal dependence on the ectoderm. This effect requires contact between the ectoderm and mesoderm (Gumpel-Pinot, J. Embryol. Exp. Morphol. 59:157-173, 1980) but also can be observed at a distance from the ectoderm. Later, the ectoderm can act without direct contact between the ectoderm and mesoderm to inhibit chondrogenesis over some distance.
When presumptive ectoderm is treated with high concentrations of activin A, it mainly differentiates into axial mesoderm (notochord, muscle) in Xenopus and into yolk-rich endodermal cells in newt (Cynops pyrrhogaster). Xenopus ectoderm consists of multiple layers, different from the single layer of Cynops ectoderm. This multilayer structure of Xenopus ectoderm may prevent complete treatment of activin A and subsequent whole differentiation into endoderm. In the present study, therefore, Xenopus ectoderm was separated into an outer layer and an inner layer, which were individually treated with a high concentration of activin A (100 ng/mL). Then the differentiation and inductive activity of these ectodermal cells were examined in explantation and transplantation experiments. In isolation culture, ectoderm treated with activin A formed endoderm. Ectodermal and mesodermal tissues were seldom found in these explants. The activin-treated ectoderm induced axial mesoderm and neural tissues, and differentiated into endoderm when it was sandwiched between two sheets of ectoderm or was transplanted into the ventral marginal zone of other blastulae. These findings suggest that Xenopus ectoderm treated with a high concentration of activin A forms endoderm and mimics the properties of the organizer as in Cynops.
During early vertebrate development, a series of inductive tissue interactions appear to be involved in establishing regional specializations that are eventually elaborated in the basic body plan of the embryo. These early inductive interactions are particularly difficult to study because they often occur in the absence of any associated morphological changes. In the chick embryo, the regional subdivision of the early ectoderm is evidenced by a marked lens-forming bias in the head ectoderm, which is absent from the presumptive dorsal epidermis of the trunk region. This striking divergence in developmental state is present long before any differentiation into lens or epidermal phenotypes can be detected. As a strategy for isolating genes whose differential expression might be a reflection of this regional subdivision, a cDNA library was prepared from early embryos and screened for differential hybridization to radiolabelled probes prepared from head ectoderm and trunk ectoderm. Two related cDNA clones were isolated that hybridize to transcripts present at much higher levels in trunk ectoderm than in head ectoderm. Sequence analysis of one of these clones revealed a high degree of similarity to members of the type II subfamily of intermediate filament cytokeratins. This clone (pCKse1) was used to examine cytokeratin gene expression in ectodermal tissues. A large increase in the level of CKse1 transcripts was found to take place in trunk ectoderm, approximately coordinate with neurulation, contrasting sharply with the much lower levels detected in head ectoderm and neural ectoderm at all stages tested. These results indicate that differential cytokeratin gene expression can occur within a contiguous layer of simple embryonic epithelia, and that this expression pattern coincides closely to the subdivision of the early ectoderm into regions with distinct developmental potencies. This type of regulation has not been described previously for members of the cytokeratin gene family.
The shape of the embryonic ectoderm of early post-implantation mouse embryos changes greatly in the period of 6.2-7.3 days post coitum. The subcellular morphology of the embryonic ectoderm remains unchanged, except in the primitive-streak region. Cell kinetics differ between ectodermal regions. These differences may be related to the changes in the shape of the ectoderm. The increase in cell number in the lateral ectoderm (the prospective surface ectoderm) exceeds that in the frontal ectoderm (the future neurectoderm). This is not due to differences in the duration of the cell cycle. It can be explained, however, by the occurence of different relative numbers of dividing and non-dividing cells. These numbers vary between the two regions. The percentage of non-dividing cells in the frontal ectoderm may reach 45, whereas in the lateral ectoderm this percentage is not higher than 15. Autoradiography in tritiated thymidine-treated embryos combined with the mitotic indices gave us all of the parameters necessary to present a model capable of clarifying the growth of the ectoderm during gastrulation, as well as the changes in the shape of the ectoderm.
Interaction of ectoderm and underlying mesoderm is essential for normal vertebrate limb morphogenesis. One of the functions of limb bud ectoderm is its influence on the composition of extracellular matrix in subectodermal mesoderm, which in turn participates in morphogenesis of this region of the limb. This matrix is highly enriched in hyaluronan, even at the time when the level of hyaluronan in the chondrogenic and myogenic regions of the limb decreases, due to secretion of a stimulatory factor by the ectoderm. In this study we show that limb bud ectoderm not only stimulates hyaluronan synthesis but induces formation of large hyaluronan-dependent, pericellular matrices around cultured limb bud mesodermal cells. The ectodermal activity is mimicked in great part by fibroblast growth factor-2 and transforming growth factor-beta, and antibodies to these proteins inhibit induction of mesodermal pericellular matrix by the ectodermal factor. It has been shown by other investigators that fibroblast growth factor-2 is produced by limb ectoderm whereas transforming growth factor-2 is produced by limb ectoderm whereas transforming growth factor-beta is present in limb mesodermal tissues. Thus we conclude that the unique properties of mesodermally produced matrix underlying limb bud ectoderm are regulated, at least in part, by ectodermal fibroblast growth factor-2, probably in concert with mesodermal transforming growth factor-beta.
Ectodermal dysplasias are a large group of heritable conditions characterized by congenital defects of one or more ectodermal structures and their appendages: hair (hypotrichosis, partial, or total alopecia), nails (dystrophic, hypertrophic, abnormally keratinized), teeth (enamel defect or absent), and sweat glands (hypoplastic or aplastic). The ectodermal dysplasias, as a rule, are not pure "one-layer diseases." Mesodermal and, rarely, endodermal dysplasias coexist. Embryogenesis exhibits distinct tissue organizational fields and specific interactions among the germ layers that may lead to a wide range of ectodermal dysplasias when genes important for development are mutated or otherwise altered in expression. Of the approximately 200 different ectodermal dysplasias, about 30 have been studied at the molecular level with identification of the causative gene. Freire-Maia and Pinheiro used the clinical aspects for their classification, and Priolo integrated molecular genetic and clinical aspects for her scheme. Those two more historical classification schemes have the difficulty that, when applied strictly, several additional groups of diseases should be integrated within the term "ectodermal dysplasias," e.g. keratodermas with skin or hair alterations or the ichthyoses with associated features. Such consequent classification would lead to an endless list of diseases and would be useless for the practical work. Recent evidence implicates a genetic defect in different pathways orchestrating ectodermal organogenesis. Modern molecular genetics will increasingly elucidate the basic defects of the different syndromes and yield more insight into the regulatory mechanisms of embryology. In this way, a reclassification of ectodermal dysplasias will be possible according to the function of their involved mutated genes. Lamartine recently proposed a helpful classification according to the functions of the genes discovered in different types of ectodermal dysplasias. Accordingly, the present overview categorizes the various ectodermal dysplasias into four major functional subgroups: cell-cell communication and signaling, adhesion, transcription regulation, and development.