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).     

Localized VEGF signaling from ectoderm to mesenchyme cells controls morphogenesis of the sea urchin embryo skeleton

During development, cell migration plays an important role in morphogenetic processes. The construction of the skeleton of the sea urchin embryo by a small number of cells, the primary mesenchyme cells (PMCs), offers a remarkable model to study cell migration and its involvement in morphogenesis. During gastrulation, PMCs migrate and become positioned along the ectodermal wall following a stereotypical pattern that determines skeleton morphology. Previous studies have shown that interactions between ectoderm and PMCs regulate several aspects of skeletal morphogenesis, but little is known at the molecular level. Here we show that VEGF signaling between ectoderm and PMCs is crucial in this process. The VEGF receptor (VEGFR) is expressed exclusively in PMCs, whereas VEGF expression is restricted to two small areas of the ectoderm, in front of the positions where the ventrolateral PMC clusters that initiate skeletogenesis will form. Overexpression of VEGF leads to skeletal abnormalities, whereas inhibition of VEGF/VEGFR signaling results in incorrect positioning of the PMCs, downregulation of PMC-specific genes and loss of skeleton. We present evidence that localized VEGF acts as both a guidance cue and a differentiation signal, providing a crucial link between the positioning and differentiation of the migrating PMCs and leading to morphogenesis of the embryonic skeleton.     

Gene expression patterns in a novel animal appendage: the sea urchin pluteus arm

The larval arms of echinoid plutei are used for locomotion and feeding. They are composed of internal calcite skeletal rods covered by an ectoderm layer bearing a ciliary band. Skeletogenesis includes an autonomous molecular differentiation program in primary mesenchyme cells (PMCs), initiated when PMCs leave the vegetal plate for the blastocoel, and a patterning of the differentiated skeletal units that requires molecular cues from the overlaying ectoderm. The arms represent a larval feature that arose in the echinoid lineage during the Paleozoic and offers a subject for the study of gene co-option in the evolution of novel larval features. We isolated new molecular markers in two closely related but differently developing species, Heliocidaris tuberculata and Heliocidaris erythrogramma. We report the expression of a larval arm-associated ectoderm gene tetraspanin, as well as two new PMC markers, advillin and carbonic anhydrase. Tetraspanin localizes to the animal half of blastula stage H. tuberculata and then undergoes a restriction into the putative oral ectoderm and future location of the postoral arms, where it continues to be expressed at the leading edge of both the postoral and anterolateral arms. In H. erythrogramma, its expression initiates in the animal half of blastulae and expands over the entire ectoderm from gastrulation onward. Advillin and carbonic anhydrase are upregulated in the PMCs postgastrulation and localized to the leading edge of the growing larval arms of H. tuberculata but do not exhibit coordinated expression in H. erythrogramma larvae. The tight spatiotemporal regulation of these genes in H. tuberculata along with other ontogenetic and phylogenetic evidence suggest that pluteus arms are novel larval organs, distinguishable from the processes of skeletogenesis per se. The dissociation of expression control in H. erythrogramma suggest that coordinate gene expression in H. tuberculata evolved as part of the evolution of pluteus arms, and is not required for larval or adult development.     

Skeletogenesis by transfated secondary mesenchyme cells is dependent on extracellular matrix-ectoderm interactions in Paracentrotus lividus sea urchin embryos

In the sea urchin embryo, primary mesenchyme cells (PMCs) are committed early in development to direct skeletogenesis, provided that a permissive signal is conveyed from adjacent ectoderm cells. We showed that inhibition of extracellular matrix (ECM)-ectoderm cells interaction, by monoclonal antibodies (mAb) to Pl-nectin, causes an impairment of skeletogenesis and reduced expression of Pl-SM30, a spicule-specific matrix protein. When PMCs are experimentally removed, some secondary mesenchyme cells (SMCs) switch to skeletogenic fate. Here, for the first time we studied SMC transfating in PMC-less embryos of Paracentrotus lividus. We observed the appearance of skeletogenic cells within 10 h of PMCs removal, as shown by binding of wheat germ agglutinin (WGA) to cell surface molecules unique to PMCs. Interestingly, the number of WGA-positive cells, expressing also msp130, another PMC-specific marker, doubled with respect to that of PMCs present in normal embryos, though the number of SM30-expressing cells remained constant. In addition, we investigated the ability of SMCs to direct skeletogenesis in embryos exposed to mAbs to Pl-nectin after removal of PMCs. We found that, although phenotypic SMC transfating occurred, spicule development, as well as Pl-SM30-expression was strongly inhibited. These results demonstrate that ectoderm inductive signals are necessary for transfated SMCs to express genes needed for skeletogenesis.     

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.     

A switch in the cellular basis of skeletogenesis in late-stage sea urchin larvae

Primary mesenchyme cells (PMCs) are solely responsible for the skeletogenesis during early larval development of the sea urchin, but the cells responsible for late larval and adult skeletal formation are not clear. To investigate the origin of larval and adult skeletogenic cells, I first performed transplantation experiments in Pseudocentrotus depressus and Hemicentrotus pulcherrimus, which have different skeletal phenotypes. When P. depressus PMCs were transplanted into H. pulcherrimus embryos, the donor phenotype was observed only in the early larval stage, whereas when secondary mesenchyme cells (SMCs) were transplanted, the donor phenotype was observed in late and metamorphic larvae. Second, a reporter construct driven by the spicule matrix protein 50 (SM50) promoter was introduced into fertilized eggs and their PMCs/SMCs were transplanted. In the resultant 6-armed pluteus, green fluorescent protein (GFP) expression was observed in both PMC and SMC transplantations, suggesting SMC participation in late skeletogenesis. Third, transplanted PMCs or SMCs tagged with GFP were analyzed by PCR in the transgenic chimeras. As a result, SMCs were detected in both larval and adult stages, but GFP from PMCs was undetectable after metamorphosis. Thus, it appears that SMCs participate in skeletogenesis in late development and that PMCs disappear in the adult sea urchin, suggesting that the skeletogenesis may pass from PMCs to SMCs during the late larval stage.     

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.     

The Wnt antagonist Frzb-1 regulates chondrocyte maturation and long bone development during limb skeletogenesis

The Wnt antagonist Frzb-1 is expressed during limb skeletogenesis, but its roles in this complex multistep process are not fully understood. To address this issue, we determined Frzb-1 gene expression patterns during chick long bone development and carried out gain- and loss-of-function studies by misexpression of Frzb-1, Wnt-8 (a known Frzb-1 target), or different forms of the intracellular Wnt mediator LEF-1 in developing limbs and cultured chondrocytes. Frzb-1 expression was quite strong in mesenchymal prechondrogenic condensations and then characterized epiphyseal articular chondrocytes and prehypertrophic chondrocytes in growth plates. Virally driven Frzb-1 misexpression caused shortening of skeletal elements, joint fusion, and delayed chondrocyte maturation, with consequent inhibition of matrix mineralization, metalloprotease expression, and marrow/bone formation. In good agreement, misexpression of Frzb-1 or a dominant-negative form of LEF-1 in cultured chondrocytes maintained the cells at an immature stage. Instead, misexpression of Wnt-8 or a constitutively active LEF-1 strongly promoted chondrocyte maturation, hypertrophy, and calcification. Immunostaining revealed that the distribution of endogenous Wnt mediator beta-catenin changes dramatically in vivo and in vitro, from largely cytoplasmic in immature proliferating and prehypertrophic chondrocytes to nuclear in hypertrophic mineralizing chondrocytes. Misexpression of Frzb-1 prevented beta-catenin nuclear relocalization in chondrocytes in vivo or in vitro. The data demonstrate that Frzb-1 exerts a strong influence on limb skeletogenesis and is a powerful and direct modulator of chondrocyte maturation, phenotype, and function. Phases of skeletogenesis, such as terminal chondrocyte maturation and joint formation, appear to be particularly dependent on Wnt signaling and thus very sensitive to Frzb-1 antagonistic action.     

Identification and developmental expression of new biomineralization proteins in the sea urchin Strongylocentrotus purpuratus

The endoskeleton of the sea urchin larva is a network of calcareous rods secreted by primary mesenchyme cells (PMCs). In this study, we identified seven new biomineralization-related proteins through an analysis of a large database of gene products expressed by PMCs. The proteins include three new spicule matrix proteins (SpSM29, SpSM32, and SpC-lectin), two proteins related to the PMC-specific cell surface glycoprotein MSP130 (MSP130-related-1 and -2), and two novel proteins (SpP16 and SpP19). The genes encoding these proteins are expressed specifically by cells of the large micromere-PMC lineage and are activated zygotically beginning at the blastula stage, prior to PMC ingression. Several of the mRNAs show regulated patterns of expression within the PMC syncytium that correlate with the pattern of skeletal rod growth. This work identifies new proteins that may regulate the process of biomineralization in this tractable model system.     

Cell lineage conversion in the sea urchin embryo

The mesoderm of the sea urchin embryo conventionally is divided into two populations of cells; the primary mesenchyme cells (PMCs), which produce the larval skeleton, and the secondary mesenchyme cells (SMCs), which differentiate into a variety of cell types but do not participate in skeletogenesis. In this study we examine the morphogenesis of embryos from which the PMCs have been removed microsurgically. We confirm the observation of Fukushi (1962) that embryos lacking PMCs form a complete skeleton, although in a delayed fashion. We demonstrate by microsurgical and cell marking experiments that the appearance of skeletogenic cells in such PMC-deficient embryos is due exclusively to the conversion of other cells to the PMC phenotype. Time-lapse video recordings of PMC-deficient embryos indicate that the converting cells are a subpopulation of late-ingressing SMCs. The conversion of these cells to the skeletogenic phenotype is accompanied by their de novo expression of cell surface determinants normally unique to PMCs, as shown by binding of wheat germ agglutinin and a PMC-specific monoclonal antibody. Cell transplantation and cell marking experiments have been carried out to determine the number of SMCs that convert when intermediate numbers of PMCs are present in the embryo. These experiments indicate that the number of converting SMCs is inversely proportional to the number of PMCs in the blastocoel. In addition, they show that PMCs and converted SMCs cooperate to produce a skeleton that is correct in both size and configuration. This regulatory system should shed light on the nature of cell-cell interactions that control cell differentiation and on the way in which evolutionary processes modify developmental programs.     

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.     

Signal-dependent regulation of the sea urchin skeletogenic gene regulatory network

The endoskeleton of the sea urchin embryo is produced by primary mesenchyme cells (PMCs). Maternal inputs activate a complex gene regulatory network (GRN) in the PMC lineage in a cell-autonomous fashion during early development, initially creating a uniform population of prospective skeleton-forming cells. Previous studies showed that at post-blastula stages of development, several effector genes in the network exhibit non-uniform patterns of expression, suggesting that their regulation becomes subject to local, extrinsic cues. Other studies have identified the VEGF and MAPK pathways as regulators of PMC migration, gene expression, and biomineralization. In this study, we used whole mount in situ hybridization (WMISH) to examine the spatial expression patterns of 39 PMC-specific/enriched mRNAs in Strongylocentrotus purpuratus embryos at the late gastrula, early prism and pluteus stages. We found that all 39 mRNAs (including several regulatory genes) showed non-uniform patterns of expression within the PMC syncytium, revealing a global shift in the regulation of the skeletogenic GRN from a cell-autonomous to a signal-dependent mode. In general, localized regions of elevated gene expression corresponded to sites of rapid biomineral deposition. We used a VEGFR inhibitor (axitinib) and a MEK inhibitor (U0126) to show that VEGF signaling and the MAPK pathway are essential for maintaining high levels of gene expression in PMCs at the tips of rods that extend from the ventral region of the embryo. These inhibitors affected gene expression in the PMCs in similar ways, suggesting that VEGF acts via the MAPK pathway. In contrast, axitinib and U0126 did not affect the localized expression of genes in PMCs at the tips of the body rods, which form on the dorsal side of the embryo. Our results therefore indicate that multiple signaling pathways regulate the skeletogenic GRN during late stages of embryogenesis-VEGF/MAPK signaling on the ventral side and a separate, unidentified pathway on the dorsal side. These two signaling pathways appear to be activated sequentially (ventral followed by dorsal) and many effector genes are subject to regulation by both pathways.