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.     

TGFβ signaling positions the ciliary band and patterns neurons in the sea urchin embryo

The ciliary band is a distinct region of embryonic ectoderm that is specified between oral and aboral ectoderm. Flask-shaped ciliary cells and neurons differentiate in this region and they are patterned to form an integrated tissue that functions as the principal swimming and feeding organ of the larva. TGFβ signaling, which is known to mediate oral and aboral patterning of the ectoderm, has been implicated in ciliary band formation. We have used morpholino knockdown and ectopic expression of RNA to alter TGFβ signaling at the level of ligands, receptors, and signal transduction components and assessed the differentiation and patterning of the ciliary band cells and associated neurons. We propose that the primary effects of these signals are to position the ciliary cells, which in turn support neural differentiation. We show that Nodal signaling, which is known to be localized by Lefty, positions the oral margin of the ciliary band. Signaling from BMP through Alk3/6, affects the position of the oral and aboral margins of the ciliary band. Since both Nodal and BMP signaling produce ectoderm that does not support neurogenesis, we propose that formation of a ciliary band requires protection from these signals. Expression of BMP2/4 and Nodal suppress neural differentiation. However, the response to receptor knockdown or dominant-negative forms of signal transduction components indicate signaling is not acting directly on unspecified ectoderm cells to prevent their differentiation as neurons. Instead, it produces a restricted field of ciliary band cells that supports neurogenesis. We propose a model that incorporates spatially regulated control of Nodal and BMP signaling to determine the position and differentiation of the ciliary band, and subsequent neural patterning.     

Novel population of embryonic secondary mesenchyme cells in the keyhole sand dollar Astriclypeus manni

We have found a novel embryonic cell population in the keyhole sand dollar Astriclypeus manni, which we refer to as lucent fluorescent cells (LFCs). Live LFCs are transparent, but emit autofluorescence after formaldehyde fixation. LFCs become noticeable in the vegetal plate of early gastrulae immediately after the appearance of pigment cells. As development progresses, LFCs increase in number and migrate from the vegetal plate toward the animal pole in a manner similar to pigment cells. Notably, LFCs also migrate into the oral ectoderm, while pigment cells do not. In addition, we determined that there were nearly 300 LFCs per embryo, which greatly exceeds the number of pigment cells. Treatment with the Notch signaling inhibitor N‐[(3,5‐Difluorophenyl)acetyl]‐l ‐alanyl‐2‐phenyl]glycine‐1,1‐dimethylethyl ester (DAPT) resulted in a marked decrease in pigment cell number, but only a modest decrease in LFCs. In DAPT‐treated embryos, LFCs had a distribution pattern similar to pigment cells and were excluded from the oral ectoderm. Unlike other sea urchins, Nodal signaling was not involved in the specification of pigment cells and LFCs in these embryos. Pulse treatment and measurement of cell diameters revealed that LFCs underwent 13–15 cycles of cell division and were specified during the 11th cleavage, one cell cycle later than observed for pigment cells. At the pluteus stage, a cluster of LFCs was observed in the animal plate in addition to two rows of LFCs running along the ciliary band. In addition, dozens of LFCs aligned at the uppermost level of the stomodaeum. Therefore, though the two cell populations share some features, LFCs are considerably different from pigment cells.     

The pitx2 homeobox protein is required early for endoderm formation and nodal signaling.

Nodal and Nodal-related factors play fundamental roles in a number of developmental processes, including mesoderm and endoderm formation, patterning of the anterior neural plate, and determination of bilateral asymmetry in vertebrates. pitx2, a paired-like homeobox gene, has been proposed to act downstream of Nodal in the gene cascade providing left-right cues to the developing organs. Here, we report that pitx2 is required early in the Nodal signaling pathway for specification of the endodermal and mesodermal germ layers. We found that pitx2 is expressed very early during Xenopus and zebrafish development and in many regions where Nodal signaling is required, including the presumptive mesoderm and endoderm at the blastula and gastrula stages and the prechordal mesoderm at later stages. In Xenopus embryos, overexpression of pitx2 caused ectopic expression of goosecoid and sox-17 and interfered with mesoderm formation. Overexpression of pitx2 in Xenopus animal cap explants partially mimics the effects of Nodal overexpression, suggesting that pitx2 is a mediator of Nodal signaling during specification of the endoderm and prechordal plate, but not during mesoderm induction. We further demonstrate that pitx2 is induced by Nodal signaling in Xenopus animal caps and that the early expression of zebrafish pitx2 is absent when the Nodal signaling pathway is inactive. Inhibition of pitx2 function using a chimeric EnR-pitx2 blocked specification of the mesoderm and endoderm and caused severe embryonic defects resembling those seen when Nodal signaling is inhibited. Following inhibition of pitx2 function, the fate of ventral vegetal blastomeres was shifted from an endodermal to a more mesodermal fate, an effect that was reversed by wild-type pitx2. Finally, we show that inhibition of pitx2 function interferes with the response of cells to Nodal signaling. Our results provide direct evidence that pitx2 function is required for normal specification of the endodermal and mesodermal germ layers.     

Direct and indirect control of oral ectoderm regulatory gene expression by Nodal signaling in the sea urchin embryo

The Nodal signaling pathway is known from earlier work to be an essential mediator of oral ectoderm specification in the sea urchin embryo, and indirectly, of aboral ectoderm specification as well. Following expression of the Nodal ligand in the future oral ectoderm during cleavage, a sequence of regulatory gene activations occur within this territory which depend directly or indirectly on nodal gene expression. Here we describe additional regulatory genes that contribute to the oral ectoderm regulatory state during specification in Strongylocentrotus purpuratus, and show how their spatial expression changes dynamically during development. By means of system wide perturbation analyses we have significantly improved current knowledge of the epistatic relations among the regulatory genes of the oral ectoderm. From these studies there emerge diverse circuitries relating downstream regulatory genes directly and indirectly to Nodal signaling. A key intermediary regulator, the role of which had not previously been discerned, is the not gene. In addition to activating several genes earlier described as targets of Nodal signaling, the not gene product acts to repress other oral ectoderm genes, contributing crucially to the bilateral spatial organization of the embryonic oral ectoderm.     

Axial patterning interactions in the sea urchin embryo: suppression of nodal by Wnt1 signaling

Wnt and Nodal signaling pathways are required for initial patterning of cell fates along anterior-posterior (AP) and dorsal-ventral (DV) axes, respectively, of sea urchin embryos during cleavage and early blastula stages. These mechanisms are connected because expression of nodal depends on early Wnt/β-catenin signaling. Here, we show that an important subsequent function of Wnt signaling is to control the shape of the nodal expression domain and maintain correct specification of different cell types along the axes of the embryo. In the absence of Wnt1, the posterior-ventral region of the embryo is severely altered during early gastrulation. Strikingly, at this time, nodal and its downstream target genes gsc and bra are expressed ectopically, extending posteriorly to the blastopore. They override the initial specification of posterior-ventral ectoderm and endoderm fates, eliminating the ventral contribution to the gut and displacing the ciliary band dorsally towards, and occasionally beyond, the blastopore. Consequently, in Wnt1 morphants, the blastopore is located at the border of the re-specified posterior-ventral oral ectoderm and by larval stages it is in the same plane near the stomodeum on the ventral side. In normal embryos, a Nodal-dependent process downregulates wnt1 expression in dorsal posterior cells during early gastrulation, focusing Wnt1 signaling to the posterior-ventral region where it suppresses nodal expression. These subsequent interactions between Wnt and Nodal signaling are thus mutually antagonistic, each limiting the range of the other's activity, in order to maintain and stabilize the body plan initially established by those same signaling pathways in the early embryo.     

Absence of Nodal signaling promotes precocious neural differentiation in the mouse embryo

After implantation, mouse embryos deficient for the activity of the transforming growth factor-beta member Nodal fail to form both the mesoderm and the definitive endoderm. They also fail to specify the anterior visceral endoderm, a specialized signaling center which has been shown to be required for the establishment of anterior identity in the epiblast. Our study reveals that Nodal-/- epiblast cells nevertheless express prematurely and ectopically molecular markers specific of anterior fate. Our analysis shows that neural specification occurs and regional identities characteristic of the forebrain are established precociously in the Nodal-/- mutant with a sequential progression equivalent to that of wild-type embryo. When explanted and cultured in vitro, Nodal-/- epiblast cells readily differentiate into neurons. Genes normally transcribed in organizer-derived tissues, such as Gsc and Foxa2, are also expressed in Nodal-/- epiblast. The analysis of Nodal-/-;Gsc-/- compound mutant embryos shows that Gsc activity plays no critical role in the acquisition of forebrain characters by Nodal-deficient cells. This study suggests that the initial steps of neural specification and forebrain development may take place well before gastrulation in the mouse and highlights a possible role for Nodal, at pregastrula stages, in the inhibition of anterior and neural fate determination.     

p38 MAPK is essential for secondary axis specification and patterning in sea urchin embryos

Most eggs in the animal kingdom establish a primary, animal-vegetal axis maternally, and specify the remaining two axes during development. In sea urchin embryos, the expression of Nodal on the oral (ventral) side of the embryo is the first known molecular determinant of the oral-aboral axis (the embryonic dorsoventral axis), and is crucial for specification of the oral territory. We show that p38 MAPK acts upstream of Nodal and is required for Nodal expression in the oral territory. p38 is uniformly activated early in development, but, for a short interval at late blastula stage, is asymmetrically inactivated in future aboral nuclei. Experiments show that this transient asymmetry of p38 activation corresponds temporally to both oral specification and the onset of oral Nodal expression. Uniform inhibition of p38 prevents Nodal expression and axis specification, resulting in aboralized embryos. Nodal and its target Gsc each rescue oral-aboral specification and patterning when expressed asymmetrically in p38-inhibited embryos. Thus, our results indicate that p38 is required for oral specification through its promotion of Nodal expression in the oral territory.     

β-catenin specifies the endomesoderm and defines the posterior organizer of the hemichordate Saccoglossus kowalevskii

The canonical Wnt/β-catenin pathway is a key regulator of body plan organization and axis formation in metazoans, being involved in germ layer specification, posterior growth and patterning of the anteroposterior axis. Results from animals spanning a wide phylogenetic range suggest that a unifying function of β-catenin in metazoans is to define the posterior/vegetal part of the embryo. Although the specification of vegetal territories (endoderm) by β-catenin has been demonstrated in distantly related animals (cnidarians, a protostome, echinoderms and ascidians), the definition of the posterior part of the embryo is well supported only for vertebrates and planarians. To gain insights into β-catenin functions during deuterostome evolution, we have studied the early development of the direct developing hemichordate Saccoglossus kowalevskii. We show that the zygote is polarized after fertilization along the animal-vegetal axis by cytoplasmic rearrangements resembling the ascidian vegetal contraction. This early asymmetry is translated into nuclear accumulation of β-catenin at the vegetal pole, which is necessary and sufficient to specify endomesoderm. We show that endomesoderm specification is crucial for anteroposterior axis establishment in the ectoderm. The endomesoderm secretes as yet unidentified signals that posteriorize the ectoderm, which would otherwise adopt an anterior fate. Our results point to a conserved function at the base of deuterostomes for β-catenin in germ layer specification and to a causal link in the definition of the posterior part of the embryonic ectoderm by way of activating posteriorizing endomesodermal factors. Consequently, the definition of the vegetal and the posterior regions of the embryo by β-catenin should be distinguished and carefully re-examined.     

Fez function is required to maintain the size of the animal plate in the sea urchin embryo

Partitioning ectoderm precisely into neurogenic and non-neurogenic regions is an essential step for neurogenesis of almost all bilaterian embryos. Although it is widely accepted that antagonism between BMP and its inhibitors primarily sets up the border between these two types of ectoderm, it is unclear how such extracellular, diffusible molecules create a sharp and precise border at the single-cell level. Here, we show that Fez, a zinc finger protein, functions as an intracellular factor attenuating BMP signaling specifically within the neurogenic region at the anterior end of sea urchin embryos, termed the animal plate. When Fez function is blocked, the size of this neurogenic ectoderm becomes smaller than normal. However, this reduction is rescued in Fez morphants simply by blocking BMP2/4 translation, indicating that Fez maintains the size of the animal plate by attenuating BMP2/4 function. Consistent with this, the gradient of BMP activity along the aboral side of the animal plate, as measured by pSmad1/5/8 levels, drops significantly in cells expressing Fez and this steep decline requires Fez function. Our data reveal that this neurogenic ectoderm produces an intrinsic system that attenuates BMP signaling to ensure the establishment of a stable, well-defined neural territory, the animal plate.     

Regulatory expression of Brachyury and Goosecoid in P19 embryonal carcinoma cells

Mouse P19 embryonal carcinoma cells can differentiate into various cell types depending on culture conditions. Here we show that the expression of the mesodermal genes Brachyury (Bra) and Goosecoid (Gsc) are under regulatory control in P19 cells. When P19 cells were cultured in a tissue culture dish in the presence of serum, Bra and Gsc were unexpectedly expressed. Expression of Bra and Gsc was greatly reduced with culture time, and expression levels at 144 h of culture were below 25% those at 48 h of culture. Members of the Tgf-beta family such as Activin and Nodal have been known to up-regulate expression of mesodermal genes. Treatment with SB431542, an Alk4/5/7 inhibitor, decreased Bra and Gsc in a dose-dependent manner, whereas it induced the expression of the neuroectodermal genes Mash-1 and Pax-6. Quantitative RT-PCR and dsRNAi transfection indicated Nodal as a possible ligand responsible for the regulation of Bra and Gsc. In addition, exogenous Nodal increased expression of Bra and Gsc in a dose-dependent manner. Serum concentration in culture medium positively related to expression of Nodal, Bra, Gsc, and Cripto, which encodes a membrane-tethered protein required for Nodal signaling. Addition of the culture supernatant of P19 cells at 144 h of culture to medium decreased expression of these genes. The present study reveals that stimulation and inhibition of the Nodal pathway increases mesodermal genes and neuroectodermal genes, respectively, indicating the importance of control of Nodal and Cripto expression for mesodermal formation and neurogenesis.     

A conserved role for the nodal signaling pathway in the establishment of dorso-ventral and left-right axes in deuterostomes

Nodal factors play crucial roles during embryogenesis of chordates. They have been implicated in a number of developmental processes, including mesoderm and endoderm formation and patterning of the embryo along the anterior-posterior and left-right axes. We have analyzed the function of the Nodal signaling pathway during the embryogenesis of the sea urchin, a non-chordate organism. We found that Nodal signaling plays a central role in axis specification in the sea urchin, but surprisingly, its first main role appears to be in ectoderm patterning and not in specification of the endoderm and mesoderm germ layers as in vertebrates. Starting at the early blastula stage, sea urchin nodal is expressed in the presumptive oral ectoderm where it controls the formation of the oral-aboral axis. A second conserved role for nodal signaling during vertebrate evolution is its involvement in the establishment of left-right asymmetries. Sea urchin larvae exhibit profound left-right asymmetry with the formation of the adult rudiment occurring only on the left side. We found that a nodal/lefty/pitx2 gene cassette regulates left-right asymmetry in the sea urchin but that intriguingly, the expression of these genes is reversed compared to vertebrates. We have shown that Nodal signals emitted from the right ectoderm of the larva regulate the asymmetrical morphogenesis of the coelomic pouches by inhibiting rudiment formation on the right side of the larva. This result shows that the mechanisms responsible for patterning the left-right axis are conserved in echinoderms and that this role for nodal is conserved among the deuterostomes. We will discuss the implications regarding the reference axes of the sea urchin and the ancestral function of the nodal gene in the last section of this review.