Atsuko Yamazaki

Structure, regulation, and function of micro1 in the sea urchin Hemicentrotus pulcherrimus

The animal-vegetal axis of sea urchin embryos is morphologically apparent at the 16-cell stage, when the mesomeres, macromeres, and micromeres align along it. At this stage, the micromere is the only autonomously specified blastomere that functions as a signaling center. We used a subtraction PCR survey to identify the homeobox gene micro1 as a micromere-specific gene. The micro1 gene is a representative of a novel family of paired-like class homeobox genes, along with PlHbox12 from Paracentrotus lividus and pmar1 from Strongylocentrotus purpuratus. In the present study, we showed that micro1 is a multicopy gene with six or more polymorphic loci, at least three of which are clustered in a 30-kb region of the genome. The micro1 gene is transiently expressed during early cleavage stages in the micromere. Recently, nuclear β-catenin was shown to be essential for the specification of vegetal cell fates, including micromeres, and the temporal and spatial coincidence of micro1 expression with the nuclear entry of β-catenin is highly suggestive. We demonstrated that micro1 is a direct target of β-catenin. In addition, we showed that micro1 is necessary and sufficient for micromere specification. These observations on the structure, regulation, and function of micro1 lead to the conclusion that micro1 and pmar1 (and potentially PlHbox12) are orthologous.

The micro1 gene is necessary and sufficient for micromere differentiation and mid/hindgut-inducing activity in the sea urchin embryo

In the sea urchin embryo, micromeres have two distinct functions: they differentiate cell autonomously into the skeletogenic mesenchyme cells and act as an organizing center that induces endomesoderm formation. We demonstrated that micro1 controls micromere specification as a transcriptional repressor. Because micro1 is a multicopy gene with at least six polymorphic loci, it has been difficult to consistently block micro1 function by morpholino-mediated knockdown. Here, to block micro1 function, we used an active activator of micro1 consisting of a fusion protein of the VP16 activation domain and the micro1 homeodomain. Embryos injected with mRNA encoding the fusion protein exhibited a phenotype similar to that of micromere-less embryos. To evaluate micro1 function in the micromere, we constructed chimeric embryos composed of animal cap mesomeres and a micromere quartet from embryos injected with the fusion protein mRNA. The chimeras developed into dauerblastulae with no vegetal structures, in which the micromere progeny constituted the blastula wall. We also analyzed the phenotype of chimeras composed of an animal cap and a mesomere expressing micro1. These chimeras developed into pluteus larvae, in which the mesomere descendants ingressed as primary mesenchyme cells and formed a complete set of skeletal rods. The hindgut and a part of the midgut were also generated from host mesomeres. However, the foregut and nonskeletogenic mesoderm were not formed in the larvae. From these observations, we conclude that micro1 is necessary and sufficient for both micromere differentiation and mid/hindgut-inducing activity, and we also suggest that micro1 may not fulfill all micromere functions.

Larval mesenchyme cell specification in the primitive echinoid occurs independently of the double-negative gate

Echinoids (sea urchins) are divided into two major groups – cidaroids (a ‘primitive’ group) and euechinoids (a ‘derived’ group). The cidaroids are a promising model species for understanding the ancestral developmental mechanisms in echinoids, but little is known about the molecular mechanisms of cidaroid development. In euechinoids, skeletogenic mesenchyme cell specification is regulated by the double-negative gate (DNG), in which hesC represses the transcription of the downstream mesenchyme specification genes (alx1, tbr and ets1), thereby defining the prospective mesenchyme region. To estimate the ancestral mechanism of larval mesenchyme cell specification in echinoids, the expression patterns and roles of mesenchyme specification genes in the cidaroid Prionocidaris baculosa were examined. The present study reveals that the expression pattern and function of hesC in P. baculosa were inconsistent with the DNG model, suggesting that the euechinoid-type DNG is not utilized during cidaroid mesenchyme specification. In contrast with hesC, the expression patterns and functions of alx1, tbr and ets1 were similar between P. baculosa and euechinoids. Based on these results, we propose that the roles of alx1, tbr and ets1 in mesenchyme specification were established in the common ancestor of echinoids, and that the DNG system was acquired in the euechinoid lineage after divergence from the cidaroid ancestor. The evolutionary timing of the establishment of the DNG suggests that the DNG was originally related to micromere and/or primary mesenchyme cell formation but not to skeletogenic cell differentiation.

Conserved early expression patterns of micromere specification genes in two echinoid species belonging to the orders clypeasteroida and echinoida.

The micromere gene regulatory network (GRN) has been extensively examined using sea urchins belonging to the order Echinoida. To examine whether the network of Echinoida species is conserved in Scaphechinus mirabilis, an irregular echinoid of the order Clypeasteroida, the genes micro1, hesC, alx1, ets1, and delta were isolated from S. mirabilis and their expression patterns were compared with those from Hemicentrotus pulcherrimus, a species belonging to the order Echinoida. Data from this study suggest that the early GRN architecture had been largely established in a common ancestor of these two species. On the other hand, we found vegetal shifts in expression domains of some GRN members in H. pulcherrimus embryos compared to S. mirabilis embryos. Developmental Dynamics 239:3391–3403, 2010.

Micromere formation and expression of endomesoderm regulatory genes during embryogenesis of the primitive echinoid Prionocidaris baculosa.

Blastomere composition and expression profiles of wnt8 and hox11/13b orthologues were examined in the primitive indirect‐developing echinoid Prionocidaris baculosa. We found that blastomere composition in the 16‐cell‐stage Prionocidaris embryos was different from that of the indirect‐developing echinoids belonging to Euechinoidea, a derived group of the echinoids. The sizes of the blastomeres in the 16‐cell‐stage embryo varied, and no embryos formed a “micromere quartet,” a group of four equal‐sized micromeres. The smallest blastomere was usually located around the vegetal pole. We also found significant differences in early expression profiles of wnt8 orthologues of the Prionocidaris and euechinoids. Unlike euechinoids, the expression of wnt8 orthologue of Prionocidaris was not detected at the 16‐cell stage; it began at the 32‐cell stage in the broad area containing the vegetal pole. However, in later stages, the expression profiles of hox11/13b and wnt8 orthologues of Prionocidaris were similar to that of euechinoid orthologues. The present study suggests that there are considerable differences between Prionocidaris and euechinoids in early developmental mechanisms in the vicinity of the vegetal pole.

Structure–function correlation of micro1 for micromere specification in sea urchin embryos

The micromeres of sea urchin embryos have two functions: to promote the autonomous differentiation of skeletogenic cells and to induce endomesodermal tissues. Micromere specification is controlled by a double-repression gate consisting of two repressors, Pmar1 and HesC. Micro1/pmar1 encodes a transcriptional repressor with a paired-type N-terminal homeodomain and two C-terminal serine-rich repeats, each of which includes a sequence similar to engrailed homology region 1, which interacts with the co-repressor Groucho. To understand the molecular mechanisms of the double-repression gate, we examined the correlation between the structure and function of micro1. Phenotypic and gene expression pattern analyses of embryos injected with mutated micro1 mRNA revealed that micro1 consists of five functional domain and motifs; namely, a DNA-binding homeodomain, a nuclear localization signal in the C-terminal flanking region of the homeodomain, and two eh1-like motifs plus a short C-terminal stretch that together mediate transcriptional repression. Our data suggest that micro1 represses target genes, including hesC, via two redundant means: its eh1-like and C-terminal motifs. The C-terminal motif requires unidentified sequences for micro1 function; a micro1 mutant with the motif but lacking the unidentified sequences failed to trigger the double-repression gate for early micromere regulatory genes, except for delta, though it did repress hesC. Our results suggest that the spatial regulation of primary mesenchyme cell specification genes, including tbr, alx1, and ets1, may be different from that of delta.

Expession patterns of mesenchyme specification genes in two distantly related echinoids, Glyptocidaris crenularis and Echinocardium cordatum

The molecular mechanism of the larval mesenchyme cell specification in echinoids has been well analyzed. However, most of the data have been provided by studies of a single group of echinoids, the order Camarodonta. Little is known about this mechanism in other echinoid orders. We examined the expression patterns of mesenchyme specification genes, micro1, hesC, alx1, tbr, ets1, cyp1, and gcm, in the two non-Camarodonta echinoids, Glyptocidaris crenularis and Echinocardium cordatum. We found that the expression patterns of some genes contained characteristics that were unique to one of the species; others were shared by the two species. Some of the shared characteristics of G. crenularis and E. cordatum are not found in the species belonging to Camarodonta, suggesting the derived status of this order. The expression of ets1 in E. cordatum aboral ectoderm is one of the molecular level modifications possibly related to an evolutionarily novel larval structure, the posterior process. Our results suggest that a considerable number of modifications in the mesenchyme specification mechanisms have been introduced during the echinoid evolution.

Krüppel-like is required for nonskeletogenic mesoderm specification in the sea urchin embryo

The canonical Wnt pathway plays a central role in specifying vegetal cell fate in sea urchin embryos. SpKrl has been cloned as a direct target of nuclear beta-catenin. Using Hemicentrotus pulcherrimus embryos, here we show that HpKrl controls the specification of secondary mesenchyme cells (SMCs) through both cell-autonomous and non-autonomous means. Like SpKrl, HpKrl was activated in both micromere and macromere progenies. To examine the functions of HpKrl in each blastomere, we constructed chimeric embryos composed of blastomeres from control and morpholino-mediated HpKrl-knockdown embryos and analyzed the phenotypes of the chimeras. Micromere-swapping experiments showed that HpKrl is not involved in micromere specification, while micromere-deprivation assays indicated that macromeres require HpKrl for cell-autonomous specification. Transplantation of normal micromeres into a micromere-less host with morpholino revealed that macromeres are able to receive at least some micromere signals regardless of HpKrl function. From these observations, we propose that two distinct pathways of endomesoderm formation exist in macromeres, a Krl-dependent pathway and a Krl-independent pathway. The Krl-independent pathway may correspond to the Delta/Notch signaling pathway via GataE and Gcm. We suggest that Krl may be a downstream component of nuclear beta-catenin required by macromeres for formation of more vegetal tissues, not as a member of the Delta/Notch pathway, but as a parallel effector of the signaling (Krl-dependent pathway).

Par6 regulates skeletogenesis and gut differentiation in sea urchin larvae

Partitioning-defective (par) genes were originally identified as genes that are essential for the asymmetric division of the Caenorhabditis elegans zygote. Studies have since revealed that the gene products are part of an evolutionarily conserved PAR–atypical protein kinase C system involved in cell polarity in various biological contexts. In this study, we analyzed the function of par6 during sea urchin morphogenesis by morpholino-mediated knockdown and by manipulation swapping of the primary mesenchyme cells (PMCs). Loss of Par6 resulted in defects in skeletogenesis and gut differentiation in larvae. Phenotypic analyses of chimeras constructed by PMC swapping showed that Par6 in non-PMCs is required for differentiation of archenteron into functional gut. In contrast, Par6 in both PMCs and ectodermal cells cooperatively regulates skeletogenesis. We suggest that Par6 in PMCs plays an immediate role in the deposition of biomineral in the syncytial cable, whereas Par6 in ectoderm may stabilize skeletal rods via an unknown signal(s).

Roles of hesC and gcm in echinoid larval mesenchyme cell development

To understand the roles of hesC and gcm during larval mesenchyme specification and differentiation in echinoids, we performed perturbation experiments for these genes in two distantly related euechinoids, Hemicentrotus pulcherrimus and Scaphechinus mirabilis. The number of larval mesenchyme cells increased when the translation of hesC was inhibited, thereby suggesting that hesC has a general role in larval mesenchyme development. We confirmed previous results by demonstrating that gcm is involved in pigment cell differentiation. Simultaneous inhibition of the translation of hesC and gcm induced a significant increase in the number of skeletogenic cells, which suggests that gcm functions in skeletogenic fate repression. Based on these observations, we suggest that: (i) hesC participates in some general aspects of mesenchymal cell development; and (ii) gcm is involved in the mechanism responsible for the binary specification of skeletogenic and pigment cell fates.