P16 is an essential regulator of skeletogenesis in the sea urchin embryo

The primary mesenchyme cells (PMCs) of the sea urchin embryo undergo a dramatic sequence of morphogenetic behaviors that culminates in the formation of the larval endoskeleton. Recent studies have identified components of a gene regulatory network that underlies PMC specification and differentiation. In previous work, we identified novel gene products expressed specifically by PMCs (Illies, M.R., Peeler, M.T., Dechtiaruk, A.M., Ettensohn, C.A., 2002. Identification and developmental expression of new biomineralization proteins in the sea urchin, Strongylocentrotus purpuratus. Dev. Genes Evol. 212, 419-431). Here, we show that one of these gene products, P16, plays an essential role in skeletogenesis. P16 is not required for PMC specification, ingression, migration, or fusion, but is essential for skeletal rod elongation. We have compared the predicted sequences of P16 from two species and show that this small, acidic protein is highly conserved in both structure and function. The predicted amino acid sequence of P16 and the subcellular localization of a GFP-tagged form of the protein suggest that P16 is enriched in the plasma membrane. It may function to receive signals required for skeletogenesis or may play a more direct role in the deposition of biomineral. Finally, we place P16 downstream of Alx1 in the PMC gene network, thereby linking the network to a specific “effector” protein involved in biomineralization.     

P58-A and P58-B: novel proteins that mediate skeletogenesis in the sea urchin embryo

During sea urchin embryogenesis, the skeleton is produced by primary mesenchyme cells (PMCs). PMCs undergo a sequence of morphogenetic behaviors that includes ingression, directed migration, and cell–cell fusion. Ultimately, PMCs deposit the calcite-containing biomineral that forms the endoskeleton of the late embryo and early larva. The endoskeleton has a stereotypical structure and is the major determinant of the distinctive, angular shape of the larva. Although many candidate biomineralization proteins have been identified, functional data concerning these proteins are scant. Here, we identify and characterize two new biomineralization genes, p58-a and p58-b. We show that these two genes are highly conserved in Strongylocentrotus purpuratus and Lytechinus variegatus, two sea urchin species whose ancestors diverged approximately 100 mya. The p58-a and p58-b genes lie in tandem on the chromosome, suggesting that one of the two genes arose via a gene duplication event. The two genes encode closely related, type I transmembrane proteins. We have established by whole mount in situ hybridization that p58-a and p58-b are expressed specifically in the PMCs in both species. Knockdown of either gene by morpholino antisense oligonucleotides leads to profound defects in skeletogenesis, although skeletal elements are not completely eliminated. The P58-A and P58-B proteins do not appear to play a role in the specification, directed migration or differentiation of the PMCs, but most likely are directly involved in biomineralization during sea urchin embryonic development.     

Opposing Nodal/Vg1 and BMP signals mediate axial patterning in embryos of the basal chordate amphioxus

The basal chordate amphioxus resembles vertebrates in having a dorsal, hollow nerve cord, a notochord and somites. However, it lacks extensive gene duplications, and its embryos are small and gastrulate by simple invagination. Here we demonstrate that Nodal/Vg1 signaling acts from early cleavage through the gastrula stage to specify and maintain dorsal/anterior development while, starting at the early gastrula stage, BMP signaling promotes ventral/posterior identity. Knockdown and gain-of-function experiments show that these pathways act in opposition to one another. Signaling by these pathways is modulated by dorsally and/or anteriorly expressed genes including Chordin, Cerberus, and Blimp1. Overexpression and/or reporter assays in Xenopus demonstrate that the functions of these proteins are conserved between amphioxus and vertebrates. Thus, a fundamental genetic mechanism for axial patterning involving opposing Nodal and BMP signaling is present in amphioxus and probably also in the common ancestor of amphioxus and vertebrates or even earlier in deuterostome evolution.     

Involvement of Delta and Nodal signals in the specification process of five types of secondary mesenchyme cells in embryo of the sea urchin, Hemicentrotus pulcherrimus

Secondary mesenchyme cells (SMCs) of the sea urchin embryo are composed of pigment cells, blastocoelar cells, spicule tip cells, coelomic pouch cells and muscle cells. To learn how and when these five types of SMCs are specified in the veg₂ descendants, Notch or Nodal signaling was blocked with γ‐secretase inhibitor or Nodal receptor inhibitor, respectively. All types of SMCs were decreased with DAPT, while sensitivity to this inhibitor varied among them. Pulse‐treatment revealed that five types of SMCs are divided into “early” (pigment cells and blastocoelar cells) and “late” (spicule tip cells, coelomic pouch cells and muscle cells) groups; the “early” group was sensitive to DAPT up to the hatching, and the “late” group was sensitive until the mesenchyme blastula stage. Judging from timing of the shift of Delta‐expressing regions, it was suggested that the “early” group and “late” groups are derived from the lower and the middle tier of veg₂ descendants, respectively. Interestingly, numbers of SMCs were also altered with SB431542; blastocoelar cells, coelomic pouch cells and circum‐esophageal muscles decreased, whereas pigment cells and spicule tip cells increased in number. Pulse‐treatment showed that the “early” group was sensitive up to the mesenchyme blastula stage, while the “late” group up to the onset of gastrulation. Thus, it became clear that precursor cells of the “early” and “late” groups, which are located in different regions in the vegetal plate, receive Delta and Nodal signals at different timings, resulting in the diversification of SMCs. Based on the obtained results, the specification processes of five types of SMCs are diagrammatically presented.     

Identification of a new isoform of eEF2 whose phosphorylation is required for completion of cell division in sea urchin embryos

Elongation factor 2 (eEF2) is the main regulator of peptide chain elongation in eukaryotic cells. Using sea urchin eggs and early embryos, two isoforms of eEF2 of respectively 80 and 83 kDa apparent molecular weight have been discovered. Both isoforms were identified by immunological analysis as well as mass spectrometry, and appeared to originate from a unique post-translationally modified protein. Accompanying the net increase in protein synthesis that occurs in early development, both eEF2 isoforms underwent dephosphorylation in the 15 min period following fertilization, in accordance with the active role of dephosphorylated eEF2 in regulation of protein synthesis. After initial dephosphorylation, the major 83 kDa isoform remained dephosphorylated while the 80 kDa isoform was progressively re-phosphorylated in a cell-cycle dependent fashion. In vivo inhibition of phosphorylation of the 80 kDa isoform impaired the completion of the first cell cycle of early development implicating the involvement of eEF2 phosphorylation in the exit from mitosis.     

Oral-aboral axis specification in the sea urchin embryo II. Mitochondrial distribution and redox state contribute to establishing polarity in Strongylocentrotus purpuratus

The initial asymmetry that specifies the oral-aboral (OA) axis of the sea urchin embryo has long been a mystery. It was shown previously that OA polarity can be entrained in embryos by imposing a respiratory asymmetry, with the most oxidizing side of the embryo tending to develop as the oral pole. This suggests that one of the earliest observable asymmetries along the incipient OA axis, a redox gradient established by a higher density and/or activity of mitochondria on the prospective oral side of the embryo, might play a causal role in establishing the axis. Here, we examine the origin and functional significance of this early redox gradient. Using MitoTracker Green, we show that mitochondria are asymmetrically distributed in the unfertilized egg of Strongylocentrotus purpuratus, and that the polarity of the maternal asymmetry is maintained in the zygote. Vital staining indicates that the side of the embryo that inherits the highest density of mitochondria tends to develop into the oral pole. This correlation holds when mitochondria are redistributed by centrifugation of eggs or by transfer of purified mitochondria into zygotes, indicating that an asymmetric mitochondrial distribution can entrain OA polarity, possibly through effects on intracellular redox state. In support of this possibility, we find that specification of oral ectoderm is suppressed when embryos are cultured under hypoxic conditions that enforce a relatively reducing redox state. This effect is reversed by overexpression of nodal, an early zygotic marker of oral specification whose localized expression suffices to organize the entire OA axis, indicating that redox state is upstream of nodal expression. We therefore propose that a threshold level of intracellular oxidation is required to effectively activate nodal, and that precocious attainment of this threshold within the blastomeres containing the highest density of mitochondria results in asymmetric nodal activity and consequent specification of the OA axis.     

R11: a cis-regulatory node of the sea urchin embryo gene network that controls early expression of SpDelta in micromeres

A gene regulatory network (GRN) controls the process by which the endomesoderm of the sea urchin embryo is specified. In this GRN, the program of gene expression unique to the skeletogenic micromere lineage is set in train by activation of the pmar1 gene. Through a double repression system, this gene is responsible for localization of expression of downstream regulatory and signaling genes to cells of this lineage. One of these genes, delta, encodes a Notch ligand, and its expression in the right place and time is crucial to the specification of the endomesoderm. Here we report a cis-regulatory element R11 that is responsible for localizing the expression of delta by means of its response to the pmar1 repression system. R11 was identified as an evolutionarily conserved genomic sequence located about 13 kb downstream of the last exon of the delta gene. We demonstrate here that this cis-regulatory element is able to drive the expression of a reporter gene in the same cells and at the same time that the endogenous delta gene is expressed, and that temporally, spatially, and quantitatively it responds to the pmar1 repression system just as predicted for the delta gene in the endomesoderm GRN. This work illustrates the application of cis-regulatory analysis to the validation of predictions of the GRN model. In addition, we introduce new methodological tools for quantitative measurement of the output of expression constructs that promise to be of general value for cis-regulatory analysis in sea urchin embryos.     

An increase in surface area is not required for cell division in early sea urchin development

Cell division requires an increase in surface area to volume ratio. During early development, surface area can increase, volume can decrease, or surface topography can be optimized to allow for division. While exocytosis is thought to be essential for division [Mol. Biol. Cell 10 (1999), 2735; Proc. Natl. Acad. Sci. USA 99 (2002), 3633], exocytosis doesn't always yield an increase in surface area [Proc. Natl. Acad. Sci. USA 79 (1982), 6712]. We used multiphoton laser scanning microscopy, fluorescence spectroscopy, and electron microscopy to monitor membrane trafficking, surface area, volume, and surface topography during early sea urchin development. Despite extensive membrane trafficking monitored by FM 1-43 fluorescence, we find that the net surface area of the embryo does not change prior to the eight-cell stage. During this period, embryo volume decreases by 15%, and microvilli disappear from interior facing membrane segments. Thus, the first three cell divisions utilize residual membrane liberated by decreasing cytoplasmic volume, and reducing microvilli density on interior facing membranes. Only after the eight-cell stage was a net increase in FM 1-43 fluorescence from the embryo surface detected. Our data suggest that compensatory endocytosis is downregulated after this developmental stage to yield an increase in surface area for cell division.     

cDNA cloning, expression, and characterization of methyl-CpG-binding domain type 2/3 proteins from starfish and sea urchin

Two kinds of cDNAs that are highly homologous to mammalian MBD2 and MBD3 cDNAs were cloned from ovary of the starfish Asterina pectinifera. They are splicing variants and designated sMBD2/3a and sMBD2/3b cDNAs. sMBD2/3a cDNA spans 1378 bp and consists of a 48-bp upstream untranslated region, a 807-bp open reading frame encoding sMBD2/3a, and a 523-bp downstream untranslated region. sMBD2/3a and sMBD2/3b cDNAs encode proteins with predicted molecular weights of 30,724 and 29,635 consisting of 268 and 260 amino acid residues, respectively. The deduced amino acid sequences of these two are identical from residues 1 to 255, but different from residues 256 to the C-terminal ends. sMBD2/3a is expressed in all the tissues of starfish, whereas sMBD2/3b is highly expressed in ovary and oocytes, slightly in testis, but not in somatic cells. As suggested from the whole-genome sequence of Strongylocentrotus purpuratus, a sea urchin MBD2/3 cDNA was cloned from eggs of Hemicentrotus pulcherrimus and designated suMBD2/3 cDNA. It encodes a protein with predicted molecular weight of 30,778 consisting of 274 amino acid residues. All the three echinodermal MBD2/3 proteins consist of a methy-CpG-binding domain (MBD) and a coiled-coil domain, and only sMBD2/3a contains a glutamate-rich C-terminal region, a key mark in vertebrate MBD3. The three MBD2/3 proteins expressed in Escherichia coli and purified to homogeneity were capable to bind specifically to methylated DNA. It was shown that sMBD2/3a exists as dimer or in the monomer-dimer equilibrium, whereas sMBD2/3b and suMBD2/3 exist as monomer and dimer, respectively.     

Expression of univin, a TGF-beta growth factor, requires ectoderm-ECM interaction and promotes skeletal growth in the sea urchin embryo

Pl-nectin is an ECM protein located on the apical surface of ectoderm cells of Paracentrotus lividus sea urchin embryo. Inhibition of ECM-ectoderm cell interaction by the addition of McAb to Pl-nectin to the culture causes a dramatic impairment of skeletogenesis, offering a good model for the study of factor(s) involved in skeleton elongation and patterning. We showed that skeleton deficiency was not due to a reduction in the number of PMCs ingressing the blastocoel, but it was correlated with a reduction in the number of Pl-SM30-expressing PMCs. Here, we provide evidence on the involvement of growth factor(s) in skeleton morphogenesis. Skeleton-defective embryos showed a strong reduction in the levels of expression of Pl-univin, a growth factor of the TGF-beta superfamily, which was correlated with an equivalent strong reduction in the levels of Pl-SM30. In contrast, expression levels of Pl-BMP5-7 remained low and constant in both skeleton-defective and normal embryos. Microinjection of horse serum in the blastocoelic cavity of embryos cultured in the presence of the antibody rescued skeleton development. Finally, we found that misexpression of univin is also sufficient to rescue defects in skeleton elongation and SM30 expression caused by McAb to Pl-nectin, suggesting a key role for univin or closely related factor in sea urchin skeleton morphogenesis.     

ankAT-1 is a novel gene mediating the apical tuft formation in the sea urchin embryo

In sea urchin embryos, the apical tuft forms within the neurogenic animal plate. When FoxQ2, one of the earliest factors expressed specifically in the animal plate by early blastula stage, is knocked down, the structure of the apical tuft is altered. To determine the basis of this phenotype, we identified FoxQ2-dependent genes using microarray analysis. The most strongly down-regulated gene in FoxQ2 morphants encodes a protein with ankyrin repeats region in its N-terminal domain. We named this gene ankAT-1, Ankyrin-containing gene specific for Apical Tuft. Initially its expression in the animal pole region of very early blastula stage embryos is FoxQ2-independent but becomes FoxQ2-dependent beginning at mesenchyme blastula stage and continuing in the animal plate of 3-day larvae. Furthermore, like FoxQ2, this gene is expressed throughout the expanded apical tuft region that forms in embryos lacking nuclear β-catenin. When AnkAT-1 is knocked-down by injecting a morpholino, the cilia at the animal plate in the resulting embryos are much shorter and their motility is less than that of motile cilia in other ectoderm cells, and remains similar to that of long apical tuft cilia. We conclude that AnkAT-1 is involved in regulating the length of apical tuft cilia.     

A spatially dynamic cohort of regulatory genes in the endomesodermal gene network of the sea urchin embryo

A gene regulatory network subcircuit comprising the otx, wnt8, and blimp1 genes accounts for a moving torus of gene expression that sweeps concentrically across the vegetal domain of the sea urchin embryo. Here we confirm by mutation the inputs into the blimp1cis-regulatory module predicted by network analysis. Its essential design feature is that it includes both activation and autorepression sites. The wnt8 gene is functionally linked into the subcircuit in that cells receiving this ligand generate a β-catenin/Tcf input required for blimp1 expression, while the wnt8 gene in turn requires a Blimp1 input. Their torus-like spatial expression patterns and gene regulatory analysis indicate that the genes even-skipped and hox11/13b are also entrained by this subcircuit. We verify the cis-regulatory inputs of even-skipped predicted by network analysis. These include activation by β-catenin/Tcf and Blimp1, repression within the torus by Hox11/13b, and repression outside the torus by Tcf in the absence of Wnt8 signal input. Thus even-skipped and hox11/13b, along with blimp1 and wnt8, are members of a cohort of torus genes with similar regulatory inputs and similar, though slightly out-of-phase, expression patterns, which reflect differences in cis-regulatory design.