Specification of ectoderm restricts the size of the animal plate and patterns neurogenesis in sea urchin embryos

The animal plate of the sea urchin embryo becomes the apical organ, a sensory structure of the larva. In the absence of vegetal signaling, an expanded and unpatterned apical organ forms. To investigate the signaling that restricts the size of the animal plate and patterns neurogenesis, we have expressed molecules that regulate specification of ectoderm in embryos and chimeras. Enhancing oral ectoderm suppresses serotonergic neuron differentiation, whereas enhancing aboral or ciliary band ectoderm increases differentiation of serotonergic neurons. In embryos in which vegetal signaling is blocked, Nodal expression does not reduce the size of the thickened animal plate; however, almost no neurons form. Expression of BMP in the absence of vegetal signaling also does not restrict the size of the animal plate, but abundant serotonergic neurons form. In chimeras in which vegetal signaling is blocked in the entire embryo, and one half of the embryo expresses Nodal, serotonergic neuron formation is suppressed in both halves. In similar chimeras in which vegetal signaling is blocked and one half of the embryo expresses Goosecoid (Gsc), serotonergic neurons form only in the half of the embryo not expressing Gsc. We propose that neurogenesis is specified by a maternal program that is restricted to the animal pole by signaling that is dependent on nuclearization of beta-catenin and specifies ciliary band ectoderm. Subsequently, neurogenesis in the animal plate is patterned by suppression of serotonergic neuron formation by Nodal. Like other metazoans, echinoderms appear to have a phase of neural development during which the specification of ectoderm restricts and patterns neurogenesis.     

Sp-Smad2/3 mediates patterning of neurogenic ectoderm by nodal in the sea urchin embryo

Nodal functions in axis and tissue specification during embryogenesis. In sea urchin embryos, Nodal is crucial for specification of oral ectoderm and is thought to pattern neurogenesis in the animal plate. To determine if Nodal functions directly in suppressing neuron differentiation we have prepared mutant forms of Sp-Smad2/3. Expressing an activated form produces embryos similar to embryos overexpressing Nodal, but with fewer neurons. In chimeras in which Nodal is suppressed, cells expressing activated Sp-Smad2/3 form oral ectoderm, but not neurons. In embryos with vegetal signaling blocked, neurons do not form if activated Smad2/3 is co-expressed. Expression of dominant negative mutants produces embryos identical to those resulting from blocking Nodal expression. In chimeras overexpressing Nodal, cells expressing dominant negative Sp-Smad2/3 form aboral ectoderm and give rise to neurons. In permanent blastula chimeras dominant negative Sp-Smad2/3 is able to suppress the effects of Nodal permitting neuron differentiation. In these chimeras Nodal expression in one half suppresses neural differentiation across the interface. Anti-phospho-Smad3 reveals that the cells adjacent to cells expressing Nodal have nuclear immunoreactivity. We conclude Sp-Smad2/3 is a component of the Nodal signaling pathway in sea urchins and that Nodal diffuses short distances to suppress neural differentiation.     

The homeodomain-containing gene Xdbx inhibits neuronal differentiation in the developing embryo

The development of the vertebrate nervous system depends upon striking a balance between differentiating neurons and neural progenitors in the early embryo. Our findings suggest that the homeodomain-containing gene Xdbx regulates this balance by maintaining neural progenitor populations within specific regions of the neuroectoderm. In posterior regions of the Xenopus embryo, Xdbx is expressed in a bilaterally symmetric stripe that lies at the middle of the mediolateral axis of the neural plate. This stripe of Xdbx expression overlaps the expression domain of the proneural basic/helix-loop-helix-containing gene, Xash3, and is juxtaposed to the expression domains of Xenopus Neurogenin related 1 and N-tubulin, markers of early neurogenesis in the embryo. Xdbx overexpression inhibits neuronal differentiation in the embryo and when co-injected with Xash3, Xdbx inhibits the ability of Xash3 to induce ectopic neurogenesis. One role of Xdbx during normal development may therefore be to restrict spatially neuronal differentiation within the neural plate, possibly by altering the neuronal differentiation function of Xash3.     

T-brain homologue (HpTb) is involved in the archenteron induction signals of micromere descendant cells in the sea urchin embryo

Signals from micromere descendants play a crucial role in sea urchin development. In this study, we demonstrate that these micromere descendants express HpTb, a T-brain homolog of Hemicentrotus pulcherrimus. HpTb is expressed transiently from the hatched blastula stage through the mesenchyme blastula stage to the gastrula stage. By a combination of embryo microsurgery and antisense morpholino experiments, we show that HpTb is involved in the production of archenteron induction signals. However, HpTb is not involved in the production of signals responsible for the specification of secondary mesenchyme cells, the initial specification of primary mesenchyme cells, or the specification of endoderm. HpTb expression is controlled by nuclear localization of beta-catenin, suggesting that HpTb is in a downstream component of the Wnt signaling cascade. We also propose the possibility that HpTb is involved in the cascade responsible for the production of signals required for the spicule formation as well as signals from the vegetal hemisphere required for the differentiation of aboral ectoderm.     

Chordin is required for neural but not axial development in sea urchin embryos

The oral-aboral (OA) axis in the sea urchin is specified by the TGFβ family members Nodal and BMP2/4. Nodal promotes oral specification, whereas BMP2/4, despite being expressed in the oral territory, is required for aboral specification. This study explores the role of Chordin (Chd) during sea urchin embryogenesis. Chd is a secreted BMP inhibitor that plays an important role in axial and neural specification and patterning in Drosophila and vertebrate embryos. In Lytechinus variegatus embryos, Chd and BMP2/4 are functionally antagonistic. Both are expressed in overlapping domains in the oral territory prior to and during gastrulation. Perturbation shows that, surprisingly, Chd is not involved in OA axis specification. Instead, Chd is required both for normal patterning of the ciliary band at the OA boundary and for development of synaptotagmin B-positive (synB) neurons in a manner that is reciprocal with BMP2/4. Chd expression and synB-positive neural development are both downstream from p38 MAPK and Nodal, but not Goosecoid. These data are summarized in a model for synB neural development.     

Very early and transient vegetal-plate expression of SpKrox1, a Krüppel/Krox gene from Strongylocentrotus purpuratus

All endodermal and mesenchymal cells of the sea urchin embryo descend from the vegetal plate, a thickened epithelium of approximately 50 cells arising at the early blastula stage. Cell types that derive from the vegetal plate are specified conditionally by inductive interactions with underlying micromeres, but the molecular details of vegetal-plate specification remain unresolved. In a search for regulatory proteins that have roles in vegetal-plate specification, a screen was performed to clone Krüppel/Krox-related genes from a Strongylocentrotus purpuratus embryo cDNA library. One newly identified clone, named SpKrox1, contained four zinc fingers and a leucine zipper domain. SpKrox1 expression was low in unfertilized eggs, increased severalfold to the early blastula stage and decreased between the early gastrula and pluteus stages. SpKrox1 mRNA was first seen in macromeres of 16-cell stage embryos and was restricted to cells of the developing vegetal plate thereafter. Vegetal-plate expression corresponded to a ring of cells around the blastopore and overlapped the expression patterns of other genes with potential roles in vegetal plate-specification. As the vegetal-plate cells invaginated into the blastopore, SpKrox1 expression was lost, suggesting that its role was not in endoderm differentiation per se but rather in the initial establishment of the vegetal plate.     

Inhibitors of procollagen C-terminal proteinase block gastrulation and spicule elongation in the sea urchin embryo

In the sea urchin embryo, inhibition of collagen processing and deposition affects both gastrulation and embryonic skeleton (spicule) formation. It has been found that cell-free extracts of gastrula-stage embryos of Strongylocentrotus purpuratus contain a procollagen C-terminal proteinase (PCP) activity. A rationally designed non-peptidic organic hydroxamate, which is a potent and specific inhibitor of human recombinant PCP (FG-HL1), inhibited both the sea urchin PCP as well as purified chick embryo tendon PCP. In the sea urchin embryo, FG-HL1 inhibited gastrulation and blocked spicule elongation, but not spicule nucleation. A related compound with a terminal carboxylate rather than a hydroxamate (FG-HL2) did not inhibit either chick PCP or sea urchin PCP activity in a procollagen-cleavage assay. However, FG-HL2 did block spicule elongation without affecting spicule nucleation or gastrulation. Neither compound was toxic, because their effects were reversible on removal. It was shown that the inhibition of gastrulation and spicule elongation were independent of tissue specification events, because both the endoderm specific marker Endo1 and the primary mesenchyme cell specific marker SM50 were expressed in embryos treated with FG-HL1 and FG-HL2. These results suggest that disruption of the fibrillar collagen deposition in the blastocoele blocks the cell movements of gastrulation and may disrupt the positional information contained within the extracellular matrix, which is necessary for spicule formation.     

Expression patterns of HNK-1 carbohydrate and serotonin in sea urchin, amphioxus, and lamprey, with reference to the possible evolutionary origin of the neural crest

We examined deuterostome invertebrates, the sea urchin and amphioxus, and an extant primitive vertebrate, the lamprey, for the presence of structures expressing the HNK-1 carbohydrate and serotonin. In sea urchin embryos and larvae, HNK-1 positive cells were localized in the ciliary bands and in their precursor ectoderm. Serotonergic cells were exclusively observed in the apical organs. In juvenile amphioxus, primary sensory neurons in the dorsal nerve cords were HNK-1 immunoreactive. The juvenile amphioxus nerve cords contained anti-serotonin immunoreactive nerve fibers that seem to be the Rohde axons extending from amphioxus interneurons, the Rohde cells. In lamprey embryos, migrating neural crest cells and primary sensory neurons, including Rohon-Beard cells, expressed the HNK-1 carbohydrate. Lamprey larvae (ammocoetes) contained cell aggregates expressing both the HNK-1 carbohydrate and serotonin in the pronephros and in the regions adjacent to the gut epithelium. Some of these cell aggregates were present in the anti-serotonin positive visceral motor nerve net. Motor neurons and Müller fibers were serotonergic in ammocoetes. Comparison of the expression patterns of HNK-1 carbohydrate among sea urchins, amphioxus and lampreys seem to suggest the possible evolutionary origin of the neural crest, that is, ciliary bands in dipleurula-type ancestors evolved into primary sensory neurons in chordate ancestors, as inferred from Garstang's auricularia hypothesis, and the neural crest originated from the primary sensory neurons.     

Early expression of Tubulin Beta-III in avian cranial neural crest cells

Neural crest cells are a transient stem-like cell population that forms in the dorsal neural tube of vertebrate embryos and then migrates to various locations to differentiate into diverse derivatives such as craniofacial bone, cartilage, and the enteric and peripheral nervous systems. The current dogma of neural crest cell development suggests that there is a specific hierarchical gene regulatory network (GRN) that controls the induction, specification, and differentiation of these cells at specific developmental times. Our lab has identified that a marker of differentiated neurons, Tubulin Beta-III (TUBB3), is expressed in premigratory neural crest cells. TUBB3 has previously been identified as a major constituent of microtubules and is required for the proper guidance and maintenance of axons during development. Using the model organism, Gallus gallus, we have characterized the spatiotemporal localization of TUBB3 in early stages of development. Here we show TUBB3 is expressed in the developing neural plate, is upregulated in the pre-migratory cranial neural crest prior to cell delamination and migration, and it is maintained or upregulated in neurons in later developmental stages. We believe that TUBB3 likely has a role in early neural crest formation and migration separate from its role in neurogenesis.     

Range and stability of cell fate determination in isolated sea urchin blastomeres

We have examined the developmental potential of blastomeres isolated from either the animal (mesomeres) or vegetal (macromeres-micromeres) half of 16-cell embryos of the sea urchin Lytechinus pictus. We have also examined the effects of two known vegetalizing agents on the development of isolated mesomeres; LiCl treatment and combination with micromeres, the small blastomeres found at the vegetal pole of the 16-cell embryo. The markers for differentiation used were both morphological (invaginations, spicules and pigment cells) and molecular (gut-specific alkaline phosphatase activity, and monoclonal antibodies against antigens specific for gut and oral ectoderm). Embryoids derived from isolated mesomeres expressed markers characteristic of vegetal differentiation only at very low levels. They did express an antigen characteristic of animal development, the oral ectoderm antigen, but with an altered pattern. Isolated macromere-micromere pairs expressed all markers characteristic of vegetal development, but did not express the marker characteristic of animal development. Increasing concentrations of LiCl caused isolated mesomeres to give rise to embryoids with an increasing tendency to express vegetal markers of differentiation, and it was found that expression of different vegetal markers begin to appear at different concentrations of LiCl. LiCl also caused the marker for oral ectoderm to be expressed in a more normal pattern. Combining micromeres with mesomeres also induced mesomere derivatives to differentiate in a vegetal manner. Micromeres were not completely effective in inducing a more normal pattern of expression of the marker for oral ectoderm. The treatment of isolated mesomeres with both LiCl and micromeres produces a synergistic effect resulting in embryoids expressing markers not induced by either treatment alone.