The small GTPase Cdc42 regulates shell field morphogenesis in a gastropod mollusk.

In most mollusks (conchiferans), the early tissue responsible for shell development, namely, the shell field, shows a common process of invagination during morphogenesis. Moreover, lines of evidence indicated that shell field invagination is not an independent event, but an integrated output reflecting the overall state of shell field morphogenesis. Nevertheless, the underlying mechanisms of this conserved process remain largely unknown. We previously found that actomyosin networks (regularly organized filamentous actin (F-actin) and myosin) may play essential roles in this process by revealing the evident aggregation of F-actin in the invaginated region and demonstrating that nonmuscle myosin II (NM II) is required for invagination in the gastropod Lottia peitaihoensis (= Lottia goshimai). Here, we investigated the roles of the Rho family of small GTPases (RhoA, Rac1, and Cdc42) to explore the upstream regulators of actomyosin networks. Functional assays using small molecule inhibitors suggested that Cdc42 modulates key events of shell field morphogenesis, including invagination and cell rearrangements, while the roles of RhoA and Rac1 may be nonspecific or negligible. Further investigations revealed that the Cdc42 protein was concentrated on the apical side of shell field cells and colocalized with F-actin aggregation. The aggregation of these two molecules could be prevented by treatment with Cdc42 inhibitors. These findings suggest a possible regulatory cascade of shell field morphogenesis in which Cdc42 recruits F-actin (actomyosin networks) on the apical side of shell field cells, which then generates resultant mechanical forces that mediate correct shell field morphogenesis (cell shape changes, invagination and cell rearrangement). Our results emphasize the roles of the cytoskeleton in early shell development and provide new insights into molluscan shell evolution.

The combination of high temperature and Vibrio infection worsens summer mortality in the clam Meretrix petechialis by increasing apoptosis and oxidative stress.

The interaction between environmental factors and Vibrio in bivalves is not well understood, despite the widely held belief that pathogen infection and seawater temperature significantly impact summer mortality. In the present study, we conducted simulated experiments to explore the effects of high temperature and Vibrio infection on the clam Meretrix petechialis. The survival curve analysis revealed that the combined challenge of high temperature and Vibrio infection (31°C-vibrio) led to significantly higher clam mortality compared to the groups exposed solely to Vibrio (27°C-vibrio), high temperature (31°C-control), and the control condition (27°C-control). Furthermore, PCoA analysis of 11 immune genes indicated that Vibrio infection predominated during the incubation period, with a gradual equilibrium between these factors emerging during the course of the infection. Additionally, our investigations into apoptosis and autophagy processes exhibited significant induction of mTOR and Bcl2 of the 31°C-vibrio group in the early challenge stage, followed by inhibition in the later stage. Oxidative stress analysis demonstrated a substantial additive effect on malondialdehyde (MDA) and glutathione (GSH) content in the combined challenge group compared to the control group. Comparative transcriptome analysis revealed a significant increase in differentially expressed genes related to immunity, such as complement C1q-like protein, C-type lectin, big defensin, and lysozyme, in the 31°C-vibrio group, suggesting that the synergistic effect of high temperature and Vibrio infection triggers more robust antibacterial immune responses. These findings provide critical insights for understanding the infection process and uncovering the causes of summer mortality.

Molluskan Dorsal-Ventral Patterning Relying on BMP2/4 and Chordin Provides Insights into Spiralian Development and Evolution.

Although a conserved mechanism relying on BMP2/4 and Chordin is suggested for animal dorsal-ventral (DV) patterning, this mechanism has not been reported in spiralians, one of the three major clades of bilaterians. Studies on limited spiralian representatives have suggested markedly diverse DV patterning mechanisms, a considerable number of which no longer deploy BMP signaling. Here, we showed that BMP2/4 and Chordin regulate DV patterning in the mollusk Lottia goshimai, which was predicted in spiralians but not previously reported. In the context of the diverse reports in spiralians, it conversely represents a relatively unusual case. We showed that BMP2/4 and Chordin coordinate to mediate signaling from the D-quadrant organizer to induce the DV axis, and Chordin relays the symmetry-breaking information from the organizer. Further investigations on L. goshimai embryos with impaired DV patterning suggested roles of BMP signaling in regulating the behavior of the blastopore and the organization of the nervous system. These findings provide insights into the evolution of animal DV patterning and the unique development mode of spiralians driven by the D-quadrant organizer.

The gastropod Lottia peitaihoensis as a model to study the body patterning of trochophore larvae.

The body patterning of trochophore larvae is important for understanding spiralian evolution and the origin of the bilateral body plan. However, considerable variations are observed among spiralian lineages, which have adopted varied strategies to develop trochophore larvae or even omit a trochophore stage. Some spiralians, such as patellogastropod mollusks, are suggested to exhibit ancestral traits by producing equal-cleaving fertilized eggs and possessing "typical" trochophore larvae. In recent years, we developed a potential model system using the patellogastropod Lottia peitaihoensis (= Lottia goshimai). Here, we introduce how the species were selected and establish sources and techniques, including gene knockdown, ectopic gene expression, and genome editing. Investigations on this species reveal essential aspects of trochophore body patterning, including organizer signaling, molecular and cellular processes connecting the various developmental functions of the organizer, the specification and behaviors of the endomesoderm and ectomesoderm, and the characteristic dorsoventral decoupling of Hox expression. These findings enrich the knowledge of trochophore body patterning and have important implications regarding the evolution of spiralians as well as bilateral body plans.

Dorsoventral decoupling of Hox gene expression underpins the diversification of molluscs.

In contrast to the Hox genes in arthropods and vertebrates, those in molluscs show diverse expression patterns with differences reported among lineages. Here, we investigate 2 phylogenetically distant molluscs, a gastropod and a polyplacophoran, and show that the Hox expression in both species can be divided into 2 categories. The Hox expression in the ventral ectoderm generally shows a canonical staggered pattern comparable to the patterns of other bilaterians and likely contributes to ventral patterning, such as neurogenesis. The other category of Hox expression on the dorsal side is strongly correlated with shell formation and exhibits lineage-specific characteristics in each class of mollusc. This generalized model of decoupled dorsoventral Hox expression is compatible with known Hox expression data from other molluscan lineages and may represent a key characteristic of molluscan Hox expression. These results support the concept of widespread staggered Hox expression in Mollusca and reveal aspects that may be related to the evolutionary diversification of molluscs. We propose that dorsoventral decoupling of Hox expression allowed lineage-specific dorsal and ventral patterning, which may have facilitated the evolution of diverse body plans in different molluscan lineages.

Early shell field morphogenesis of a patellogastropod mollusk predominantly relies on cell movement and F-actin dynamics.

BACKGROUND: The morphogenesis of the shell field is an essential step of molluscan shell formation, which exhibits both conserved features and interlineage variations. As one major gastropod lineage, the patellogastropods show different characters in its shell field morphogenesis compared to other gastropods (e.g., the pulmonate gastropod Lymnaea stagnalis), likely related to its epibolic gastrulation. The investigation on the shell field morphogenesis of patellogastropods would be useful to reveal the lineage-specific characters in the process and explore the deep conservation among different molluscan lineages. RESULTS: We investigated the early shell field morphogenesis in the patellogastropod Lottia goshimai using multiple techniques. Electron microscopy revealed distinct morphological characters for the central and peripheral cells of the characteristic rosette-like shell field. Gene expression analysis and F-actin staining suggested that the shell field morphogenesis in this species predominantly relied on cell movement and F-actin dynamics, while BrdU assay revealed that cell proliferation contributed little to the process. We found constant contacts between ectodermal and meso/endodermal tissues during the early stages of shell field morphogenesis, which did not support the induction of shell field by endodermal tissues in general, but a potential stage-specific induction was indicated. CONCLUSIONS: Our results emphasize the roles of cell movement and F-actin dynamics during the morphogenesis of the shell field in Lo. goshimai, and suggest potential regulators such as diffusible factors and F-actin modulators. These findings reflect the differences in shell field morphogenesis of different gastropods, and add to the knowledge of molluscan larval shell formation.

Identification of three cell populations from the shell gland of a bivalve mollusc.

The molluscan larval shell formation is a complicated process. There is evidence that the mantle of the primary larva (trochophore) contains functionally different cell populations with distinct gene expression profiles. However, it remains unclear how these cells are specified. In the present study, we identified three cell populations from the shell gland in earlier stages (gastrula) from the bivalve mollusc Crassostrea gigas. These cell populations were determined by analyzing the co-expression relationships among six potential shell formation (pSF) genes using two-color hybridization. The three cell populations, which we designated as SGCPs (shell gland cell populations), formed a concentric-circle pattern from outside to inside of the shell gland. SGCP I was located in the outer edge of the shell gland and the cells expressed pax2/5/8, gata2/3, and bmp2/4. SGCP II was located more internally and the cells expressed two engrailed genes. The last population, SGCP III, was located in the central region of the shell gland and the cells expressed lox4. Determination of the gene expression profiles of SGCPs would help trace their origins and fates and elucidate how these cell populations are specified. Moreover, potential roles of the SGCPs, e.g., development of sensory cells and shell biogenesis, are suggested. Our results reveal the internal organization of the embryonic shell gland at the molecular level and add to the knowledge of larval shell formation.

The sea cucumber genome provides insights into morphological evolution and visceral regeneration.

Apart from sharing common ancestry with chordates, sea cucumbers exhibit a unique morphology and exceptional regenerative capacity. Here we present the complete genome sequence of an economically important sea cucumber, A. japonicus, generated using Illumina and PacBio platforms, to achieve an assembly of approximately 805 Mb (contig N50 of 190 Kb and scaffold N50 of 486 Kb), with 30,350 protein-coding genes and high continuity. We used this resource to explore key genetic mechanisms behind the unique biological characters of sea cucumbers. Phylogenetic and comparative genomic analyses revealed the presence of marker genes associated with notochord and gill slits, suggesting that these chordate features were present in ancestral echinoderms. The unique shape and weak mineralization of the sea cucumber adult body were also preliminarily explained by the contraction of biomineralization genes. Genome, transcriptome, and proteome analyses of organ regrowth after induced evisceration provided insight into the molecular underpinnings of visceral regeneration, including a specific tandem-duplicated prostatic secretory protein of 94 amino acids (PSP94)-like gene family and a significantly expanded fibrinogen-related protein (FREP) gene family. This high-quality genome resource will provide a useful framework for future research into biological processes and evolution in deuterostomes, including remarkable regenerative abilities that could have medical applications. Moreover, the multiomics data will be of prime value for commercial sea cucumber breeding programs.

A comparative proteomic analysis reveals important proteins for the fertilization and early embryonic development of the oyster Crassostrea gigas.

Molluscan development involves important features that are important to understanding not only molluscan ontogeny but also animal evolution. To gain insight into the gamete proteome and protein function in fertilization and early development, we analyzed the proteomes of unfertilized oocytes and early embryos (2/4-cell stage) of the Pacific oyster, Crassostrea gigas. An oocyte reference map containing 116 protein spots, of which 69 were identified, revealed a high abundance of vitellogenin-derived protein spots. The differentially regulated protein spots during fertilization were screened using comparative proteomic approaches. In total, 18 differentially regulated protein spots were screened, and 15 of these were identified and divided into three groups. The proteins belonging to the first group function in energy supply and antioxidation and are proposed to ensure successful fertilization by regulating the levels of adenosine triphosphate, resisting oxidative stress, and preventing polyspermy. The proteins of the second group are associated with protein synthesis and modification, reflecting active protein synthesis after fertilization. The three proteins belonging to the final group are hypothesized to function in the regulation of embryonic development through the establishment of cell polarity and modulation of methylation reactions in nuclei. These results will enhance our knowledge of molluscan fertilization and development.

Expression patterns indicate that BMP2/4 and Chordin, not BMP5-8 and Gremlin, mediate dorsal-ventral patterning in the mollusk Crassostrea gigas.

Though several bilaterian animals use a conserved BMP2/4-Chordin antagonism to pattern the dorsal-ventral (DV) axis, the only lophotrochozoan species in which early DV patterning has been studied to date, the leech Helobdella robusta, appears to employ BMP5-8 and Gremlin. These findings call into question the conservation of a common DV patterning mechanism among bilaterian animals. To explore whether the unusual DV patterning mechanism in H. robusta is also used in other lophotrochozoan species, we investigated the expression of orthologous genes in the early embryo of a bivalve mollusk, Crassostrea gigas. Searching of the genome and phylogenetic analysis revealed that C. gigas possesses single orthologs of BMP2/4, Chordin, and BMP5-8 and no Gremlin homolog. Whole mount in situ hybridization revealed mRNA localization of BMP2/4 and Chordin on the opposite sides of embryos, suggesting the potential involvement of a BMP2/4-Chordin antagonism in DV patterning in this species. Furthermore, universal BMP5-8 expression and the absence of a Gremlin homolog in the C. gigas genome called into question any major contribution by BMP5-8 and Gremlin to early DV patterning in this species. Additionally, we identified seven genes showing asymmetric expression along the DV axis, providing further insight into DV patterning in C. gigas. We present the first report of a Chordin gene in a lophotrochozoan species and of the opposite expression of BMP2/4 (dorsal) and Chordin (ventral) along the D/V axis of a lophotrochozoan embryo. The findings of this study further the knowledge of axis formation in lophotrochozoan species and provide insight into the evolution of the animal DV patterning mechanism.

A SoxC gene related to larval shell development and co-expression analysis of different shell formation genes in early larvae of oyster.

Among the potential larval shell formation genes in mollusks, most are expressed in cells surrounding the shell field during the early phase of shell formation. The only exception (cgi-tyr1) is expressed in the whole larval mantle and thus represents a novel type of expression pattern. This study reports another gene with such an expression pattern. The gene encoded a SoxC homolog of the Pacific oyster Crassostrea gigas and was named cgi-soxc. Whole-mount in situ hybridization revealed that the gene was highly expressed in the whole larval mantle of early larvae. Based on its spatiotemporal expression, cgi-soxc is hypothesized to be involved in periostracum biogenesis, biomineralization, and regulation of cell proliferation. Furthermore, we investigated the interrelationship between cgi-soxc expression and two additional potential shell formation genes, cgi-tyr1 and cgi-gata2/3. The results confirmed co-expression of the three genes in the larval mantle of early D-veliger. Nevertheless, cgi-gata2/3 was only expressed in the mantle edge, and the other two genes were expressed in all mantle cells. Based on the spatial expression patterns of the three genes, two cell groups were identified from the larval mantle (tyr1 +/soxc +/gata2/3 + cells and tyr1 +/soxc +/gata2/3 - cells) and are important to study the differentiation and function of this tissue. The results of this study enrich our knowledge on the structure and function of larval mantle and provide important information to understand the molecular mechanisms of larval shell formation.

A GATA2/3 gene potentially involved in larval shell formation of the Pacific oyster Crassostrea gigas.

Shells are one of the most notable features of the majority of mollusks. In addition, the shell is also considered a key characteristic during molluscan evolution and development. However, although the morphological changes during larval shell formation have been well described, the underlying molecular mechanisms remain poorly understood. In this study, we focused on the potential involvement of a GATA gene in shell formation because GATA genes are often downstream genes of BMP (bone morphogenetic protein) signaling pathways, which have been suggested to participate in molluscan shell formation. In the Pacific oyster Crassostrea gigas, we observed that the expression of a GATA2/3 homolog (cgi-gata2/3) was clearly restricted to the edge of the shell field in early larval stages (trochophore and D-veliger). This expression pattern supports the notion that cgi-gata2/3 gene plays conserved roles in bilaterian ectodermal development. It is possible that cgi-gata2/3 is one shell-formation gene under the regulation of BMP signaling pathways. In addition, cgi-gata2/3 was also detected in the ventral side of embryos. The expression of cgi-gata2/3 away from the shell field may be involved in hematopoiesis. Our results provide fundamental support for studies into the molecular mechanisms of larval shell formation and the functions of molluscan GATA genes.

A Calaxin Gene in the Pacific Oyster Crassostrea gigasand Its Potential Roles in Cilia.

calaxin is a newly identified calcium sensor gene that modulates the movement of flagella (and possibly cilia). It was first identified from the ascidian Ciona intestinalis, and its orthologs have been observed in a wide range of animals and choanoflagellates. However, no calaxin-ortholog in a Lophotrochozoa species has been reported so far. This leaves open the question of whether the modulation of ciliary motility by calaxin is conserved among animals. We report a calaxin gene from the Pacific oyster Crassostrea gigas. This gene, termed cgi-calaxin, possesses three conserved EF-hand motifs as its orthologs from chordates. A phylogenetic analysis confirmed its orthology. Expression analysis revealed high expression in typical ciliated tissues such as gill and hepatopancreas. The spatiotemporal expression of the gene during early development was investigated using whole mount in situ hybridization. The results revealed that cgi-calaxin mRNA was aggregated in ciliated tissues of early larvae such as prototroch and velum. Immunofluorescence experiment further certified the ciliary localization of Cgi-Calaxin protein in D-veligers. We prepared the recombinant protein of cgi-calaxin and proved it had the capacity to bind to calcium. These results support the conserved roles of cgi-calaxin as a calcium sensor in ciliated cells and enrich the current knowledge on regulatory mechanisms of molluscan cilia. Moreover, we found the gene to be expressed in poorly ciliated tissues, such as adductor muscle, indicating possible roles in non-ciliated cells, which merits further investigation.

An EGFR gene of the Pacific oyster Crassostrea gigas functions in wound healing and promotes cell proliferation.

The epidermal growth factor receptor (EGFR) is an important receptor tyrosine kinase member in animals, which plays versatile functions in development, growth, tissue regeneration etc. Current knowledge on EGFR is poor in bivalve mollusks. In this study, we cloned and analyzed an EGFR gene from the Pacific oyster Crassostrea gigas (cgegfr). A 5,731 bp full-length cDNA of cgegfr was obtained, encoding a peptide with 1,494 amino acids which exhibited a typical EGFR structure, including an extracellular region, a single transmembrane region and an intracellular region. A conserved tyrosine kinase domain was predicted in the intracellular region, while the extracellular region responsible for ligand binding showed comparatively poor conservation. Expression analysis revealed that cgefgr was expressed widely in C. gigas tissues and a highest expression level was observed in adductor tissue. Expression of cgegfr was revealed to be up-regulated during wound healing of mantle, indicating that EGFR might function in the cell proliferation and migration during wound healing. Further functional analysis of cgegfr was conducted in mouse myoblast cell line C2C12, in which different parts of cgegfr were expressed and their effects were measured. The results revealed that cgegfr was able to accelerate cell proliferation of C2C12 cells and the transmembrane region was necessary for self-activation of truncated cgegfr. Our results would provide supports for further studies on the roles of cgegfr in development and growth in C. gigas.

Comprehensive Multi-omics Approaches Provide Insights to Summer Mortality in the Clam Meretrix petechialis.

Bivalve mass mortalities have been reported worldwide, which not only can be explained as a result of pathogen infection, but may reflect changes in environments. Although these episodes were often reported, there was limited information concerning the molecular responses to various stressors leading to summer mortality. In the present work, RNA sequencing (RNA-seq), tandem mass tagging (TMT)-based quantitative proteomics, and 16S rRNA sequencing were used to explore the natural outbreak of summer mortality in the clam Meretrix petechialis. We identified a total of 172 differentially expressed genes (DEGs) and 222 differentially expressed proteins (DEPs) in the diseased group compared to the normal group. The inconsistent expression profiles of immune DEGs/DEPs may be due to the immune dysregulation of the diseased clams. Notably, 11 solute carrier family genes were found among the top 20 down-regulated genes in the diseased group, indicating that weakened transmembrane transport ability might occur in the diseased clams. Integration analysis of transcriptomic and proteomic results showed that many metabolic processes such as "arginine and proline metabolism" and "tyrosine metabolism" were inhibited in the diseased group, suggesting metabolic inhibition. Moreover, 16S rRNA sequencing revealed that the microbial composition of clam hepatopancreas was disordered in the diseased group. The comparison of DEGs expression between the natural summer mortality event and an artificial challenge experiment involving both Vibrio infection and heat stress revealed 9/15 genes showing similar expression trends between the two conditions, suggesting that the summer mortality might be caused by a combination of high temperature and Vibrio infection. These results would deepen our understanding of summer mortality and provide candidate resistance markers for clam resistance breeding.

Identification of a tyrosinase gene potentially involved in early larval shell biogenesis of the Pacific oyster Crassostrea gigas.

The larval shell emerges early in embryogenesis of mollusks, but the detailed mechanisms of its biogenesis remain to be determined. In this study, we cloned a tyrosinase gene (cgi-tyr1) that potentially functioned in larval shell biogenesis from the Pacific oyster Crassostrea gigas, a worldwide bivalve species. Sequence analysis of cgi-tyr1 revealed that it had typical copper-binding domains and a signal peptide. Through whole mount in situ hybridization and an electron scanning microscopic observation, we detected the expression of cgi-tyr1 firstly in the saddle-shaped shell field in trochophores, indicating that cgi-tyr1 might participate in the biogenesis of the initial non-calcified shell of trochophores. In the following development to early D-veliger, cells in the central region of shell field exhibited no detectable cgi-tyr1 expression, and cgi-tyr1 expression was sustained only in the edge of the shell field and the hinge region, indicating that cgi-tyr1 might function fundamentally in shell growth from trochophore to early D-veliger. Unexpectedly, cgi-tyr1 expression was not detected after the D-veliger stage. This indicated that other molecules might function in later shell development. Our results suggested a role for a tyrosinase gene that specifically functioned in the initial phase of the larval shell biogenesis of C. gigas. This work would shed light on future studies on larval shell development and might be helpful to understand how the molluscan shell emerged during evolution.

Functional analysis of the promoter of the heat shock cognate 70 gene of the Pacific white shrimp, Litopenaeus vannamei.

Current knowledge on cis-regulatory elements of immune genes of the Pacific white shrimp (Litopenaeus vannamei) is poor. In this study, we identified the promoter of the heat shock cognate protein 70 (HSC70) gene of L. vannamei (lvhsc70). The promoter activity of lvhsc70 promoter was analyzed in insect sf9 cell lines. First, the putative promoter sequence was proved to be able to drive the expression of reporter EGFP gene successfully. Then serial deletion experiments were conducted to investigate functional transcription elements in the promoter region. The results revealed that both positive and negative transcription elements existed in this region. These results are quite different from the previous report on the promoter of HSC70 gene in Penaeus monodon (pmhsc70), where only positive transcription elements were indicated. The sequences that are not conserved between the promoters of lvhsc70 and pmhsc70 might contribute to the differences. Finally, we tested the effect of a putative "NF-κb binding site" in the promoter and, surprisingly, found that deletion of this site would result in a significantly enhancement of the expression of reporter genes, while the underlying mechanisms remain unrevealed. Our results would provide supports for future studies to identify the functional transcription elements in the lvhsc70 promoter and to expand our knowledge on regulation of innate immune genes in penaeid shrimp.

Early mesodermal development in the patellogastropod Lottia goshimai.

Mesodermal development is essential to explore the interlineage variations in the development of spiralians. Compared with model mollusks such as Tritia and Crepidula, knowledge about the mesodermal development of other molluscan lineages is limited. Here, we investigated early mesodermal development in the patellogastropod Lottia goshimai, which shows equal cleavage and has a trochophore larva. The endomesoderm derived from the 4d blastomere, that is, the mesodermal bandlets, was situated dorsally and showed a characteristic morphology. Investigations of the potential mesodermal patterning genes revealed that twist1 and snail1 were expressed in a proportion of these endomesodermal tissues, while all of the five genes we investigated (twist1, twist2, snail1, snail2, and mox) were expressed in ventrally located ectomesodermal tissues. Relatively dynamic snail2 expression suggests additional roles in various internalization processes. By tracing snail2 expression in early gastrulae, the 3a211 and 3b211 blastomeres were suggested to be the precursors of the ectomesoderm, which elongated to become internalized before division. These results help to understand the variations in the mesodermal development of different spiralians and explore the different mechanisms by which ectomesodermal cells are internalized, which has important evolutionary implications.

Shell field morphogenesis in the polyplacophoran mollusk Acanthochitona rubrolineata.

BACKGROUND: The polyplacophoran mollusks (chitons) possess serially arranged shell plates. This feature is unique among mollusks and believed to be essential to explore the evolution of mollusks as well as their shells. Previous studies revealed several cell populations in the dorsal epithelium (shell field) of polyplacophoran larvae and their roles in the formation of shell plates. Nevertheless, they provide limited molecular information, and shell field morphogenesis remains largely uninvestigated. RESULTS: In the present study, we investigated shell field development in the chiton Acanthochitona rubrolineata based on morphological characteristics and molecular patterns. A total of four types of tissue could be recognized from the shell field of A. rubrolineata. The shell field comprised not only the centrally located, alternatively arranged plate fields and ridges, but also the tissues surrounding them, which were the precursors of the girdle and we termed as the girdle field. The girdle field exhibited a concentric organization composed of two circularly arranged tissues, and spicules were only developed in the outer circle. Dynamic engrailed expression and F-actin (filamentous actin) distributions revealed relatively complicated morphogenesis of the shell field. The repeated units (plate fields and ridges) were gradually established in the shell field, seemingly different from the manners used in the segmentation of Drosophila or vertebrates. The seven repeated ridges also experienced different modes of ontogenesis from each other. In the girdle field, the presumptive spicule-formation cells exhibited different patterns of F-actin aggregations as they differentiate. CONCLUSIONS: These results reveal the details concerning the structure of polyplacophoran shell field as well as its morphogenesis. They would contribute to exploring the mechanisms of polyplacophoran shell development and molluscan shell evolution.

Functional evidence that FGFR regulates MAPK signaling in organizer specification in the gastropod mollusk Lottia peitaihoensis.

The D-quadrant organizer sets up the dorsal-ventral (DV) axis and regulates mesodermal development of spiralians. Studies have revealed an important role of mitogen-activated protein kinase (MAPK) signaling in organizer function, but the related molecules have not been fully revealed. The association between fibroblast growth factor receptor (FGFR) and MAPK signaling in regulating organizer specification has been established in the annelid Owenia fusiformis. Now, comparable studies in other spiralian phyla are required to decipher whether this organizer-inducing function of FGFR is prevalent in Spiralia. Here, we indicate that treatment with the FGFR inhibitor SU5402 resulted in deficiency of organizer specification in the mollusk Lottia peitaihoensis. Subsequently, the bone morphogenetic protein (BMP) signaling gradient and DV patterning were disrupted, suggesting the roles of FGFR in regulating organizer function. Changes in multiple aspects of organizer function (the morphology of vegetal blastomeres, BMP signaling gradient, expression of DV patterning markers, etc.) indicate that these developmental functions have different sensitivities to FGFR/MAPK signaling. Our results reveal a functional role of FGFR in organizer specification as well as DV patterning of Lottia embryos, which expands our knowledge of spiralian organizers. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00194-x.