Cis-regulatory logic driving glial cells missing: self-sustaining circuitry in later embryogenesis

The glial cells missing (gcm) regulatory gene of the sea urchin Strongylocentrotus purpuratus is first expressed in veg2 daughter cells as the genomic target of late cleavage stage Delta-Notch signaling from the skeletogenic mesoderm precursors. Gcm is required in veg2 progeny during late cleavages for the early phase of pigment cell precursor specification. Here we report on a later acting cis-regulatory module that assumes control of gcm expression by the early mesenchyme blastula stage and maintains it through pigment cell differentiation and dispersal. Cis-perturbation analyses reveal that the two critical elements within this late module are consensus matches to Gcm and Six1 binding sites. Significantly, six1 mRNA localizes to gcm+cells from the mesenchyme blastula stage onwards. Trans-perturbations with anti-sense morpholinos reveal a co-dependency between six1 and gcm. Six1 mRNA levels fall sharply after Gcm is depleted, while depleting Six1 leads to significant reductions in output of endogenous gcm or modular-reporters. These results support the conclusion gcm and six1 comprise a positive intergenic feedback loop in the mesodermal GRN. This often employed cross regulatory GRN feature here ensures self-sustaining gcm output in a cohort of fully specified pigment cell precursors at a relatively early developmental stage.     

Signals from primary mesenchyme cells regulate endoderm differentiation in the sea urchin embryo

Primary mesenchyme cells (PMC), the skeletogenic cells derived from the micromeres of the sea urchin embryo, are involved in the differentiation of the gut. When PMC were deleted from the mesenchyme blastula, both formation of the constrictions in the gut and expression of endoderm-specific alkaline phosphatase were significantly delayed. Therefore, the correct timing of gut differentiation depends on the existence of PMC, probably via a type of promotive signal. To date, the only role of PMC in other tissue differentiation has been a suppressive signal for the conversion of secondary mesenchyme cells (SMC) into skeletogenic cells. The present experiments using PMC ablation and transplantation showed that both signaling processes occurred in the same short period during gastrulation, but the embryos kept their competence for gut differentiation until a later stage. Further investigations indicated that conversion of SMC did not cause delay in gut differentiation and that SMC did not mediate the PMC signal to the endoderm. Therefore, the effect of PMC on gut differentiation could be a new role that is independent of the suppressive effect for SMC conversion.     

HpEts, an ets-related transcription factor implicated in primary mesenchyme cell differentiation in the sea urchin embryo

The mechanism of micromere specification is one of the central issues in sea urchin development. In this study we have identified a sea urchin homologue of ets 1 + 2. HpEts, which is maternally expressed ubiquitously during the cleavage stage and which expression becomes restricted to the skeletogenic primary mesenchyme cells (PMC) after the hatching blastula stage. The overexpression of HpEts by mRNA injection into fertilized eggs alters the cell fate of non-PMC to migratory PMC. HpEts induces the expression of a PMC-specific spicule matrix protein, SM50, but suppresses of aboral ectoderm-specific arylsulfatase and endoderm-specific HpEndo16. The overexpression of dominant negative delta HpEts which lacks the N terminal domain, in contrast, specifically represses SM50 expression and development of the spicule. In the upstream region of the SM50 gene there exists an ets binding site that functions as a positive cis-regulatory element. The results suggest that HpEts plays a key role in the differentiation of PMCs in sea urchin embryogenesis.     

Skeletogenic potential of induced secondary mesenchyme cells derived from the presumptive ectoderm in echinoid embryos

 During the normal development of echinoids, an animal cap consisting of 8 mesomeres in a 16-cell stage embryo differentiates exclusively into ectoderm. Micromeres in an embryo at the same stage differentiate into primary mesenchyme cells (PMC) and coelomic pouch constituents. An animal cap and a quartet of micromeres were isolated from a 16-cell stage embryo and recombined to make a chimeric embryo devoid of presumptive endoderm and secondary mesenchyme cells (SMC). The PMC in the chimeric embryo were completely removed at the mesenchyme blastula stage. The PMC-depleted chimeric embryos formed an archenteron derived from the mesomeres. Some secondary mesenchyme-like cells (induced SMC) were released from the archenteron tip. A considerable fraction of the induced SMC formed the typical mesenchyme pattern after migrating into the vegetal region, synthesized skeletogenic mesenchyme cell-surface protein (msp130) and produced the larval skeleton. These findings indicate that induced SMC derived from the presumptive ectoderm have the same nature as natural SMC in both the timing of their release and their skeletogenic potential expressed in the absence of PMC. Received: 14 November 1996 / Accepted: 30 December 1996     

Carbonic anhydrase inhibition blocks skeletogenesis and echinochrome production in Paracentrotus lividus and Heliocidaris tuberculata embryos and larvae

Carbonic anhydrases (CAs) are a family of widely distributed metalloenzymes, involved in diverse physiological processes. These enzymes catalyse the reversible conversion of carbon dioxide to protons and bicarbonate. At least 19 genes encoding for CAs have been identified in the sea urchin genome, with one of these localized to the skeletogenic mesoderm (primary mesenchyme cells, PMCs). We investigated the effects of a specific inhibitor of CA, acetazolamide (AZ), on development of two sea urchin species with contrasting investment in skeleton production, Paracentrotus lividus and Heliocidaris tuberculata, to determine the role of CA on PMC differentiation, skeletogenesis and on non‐skeletogenic mesodermal (NSM) cells. Embryos were cultured in the presence of AZ from the blastula stage prior to skeleton formation and development to the larval stage was monitored. At the dose of 8 mmol/L AZ, 98% and 90% of P. lividus and H. tuberculata embryos lacked skeleton, respectively. Nevertheless, an almost normal PMC differentiation was indicated by the expression of msp130, a PMC‐specific marker. Strikingly, the AZ‐treated embryos also lacked the echinochrome pigment produced by the pigment cells, a subpopulation of NSM cells with immune activities within the larva. Conversely, all ectoderm and endoderm derivatives and other subpopulations of mesoderm developed normally. The inhibitory effects of AZ were completely reversed after removal of the inhibitor from the medium. Our data, together with new information concerning the involvement of CA on skeleton formation, provide evidence for the first time of a possible role of the CAs in larval immune pigment cells.     

Developmental expression of HpNanos, the Hemicentrotus pulcherrimus homologue of nanos

The Hemicentrotus pulcherrimus homologue of nanos (HpNanos), that encodes a protein containing two CCHC zinc finger motifs, was isolated from a gastrula cDNA library. The accumulation of HpNanos mRNA during embryonic development and the spatial expression pattern are reported. Developmental northern blot analysis revealed that HpNanos mRNA markedly accumulated during the blastula stages, and then decreased in abundance at the mesenchyme blastula stage. The second phase of HpNanos mRNA expression occurred during gastrulation, after which the expression returned to a low level. Whole-mount in situ hybridization showed that the HpNanos was exclusively expressed in four to six small micromere-descendant cells at the blastula stage. The expression of HpNanos was restricted to the coelomic pouch, which gives rise to the mesoderm of the ventral surface of the adult rudiment, at the prism stage. These results suggest that HpNanos expression will be instrumental for future analyses of the function of small micromere-descendant cells and of the origin of germ cells during sea urchin development.     

Study of larval and adult skeletogenic cells in developing sea urchin larvae

The larval skeleton of sea urchin embryos is formed by primary mesenchyme cells (PMCs). Thereafter, the larvae start feeding and additional arms develop. An adult rudiment that contains spines, tube feet, tests, and other parts of the adult body is formed in the eight-armed larva. The cellular mechanism of the later skeletogenesis and the lineage of the adult skeletogenic cells are not known. In this study, the morphogenesis of larval and adult skeletons during larval development of the sea urchin Hemicentrotus pulcherrimus was investigated by immunostaining cells with PMC-specific monoclonal antibodies, which are useful markers of skeletogenic cells. All spicules and the associated cells in the later larvae were stained with the antibodies. We could observe the initiation of skeletal morphogenesis at each developmental stage and visualize the cellular basis of skeleton formation in whole-mount embryos that possessed an intact morphology. There were some similarities between PMCs and the later skeletogenic cells. Both had a rounded shape with some filopodia, and the antigen expression started just before overt spicule formation. In the later-stage embryos, cells with filopodia and faint antigen expression were observed migrating in the blastocoel or aggregating in the presumptive location of new skeletogenesis.     

Sequence and expression of FoxB2 (XFD-5) and FoxI1c (XFD-10) in Xenopus embryogenesis

We report on the temporal and spatial expression pattern of two novel genes of the Xenopus fork head/winged helix family, xFoxB2 and xFoxI1c. xFoxB2 is activated at the late blastula stage and first expressed within the dorsolateral ectoderm except for the organiser territory. During gastrulation, xFoxB2 is found in two ectodermal stripes adjacent to the dorsal midline. Expression is completely down-regulated during neurulation. However, two distinct sets of cells expressing xFoxB2 re-appear in the rhombencephalon of swimming tadpoles. xFoxI1c is initially expressed at the early neurula stage in an epidermal ring around the neural field. Subsequent expression is found to be increased, and is exclusively localised to placodal precursor cells. The placodal expression remains until stage 40, when it is restricted to a distinct region in the lateral body wall behind the gills.