The C. elegans TspanC8 tetraspanin TSP-14 exhibits isoform-specific localization and function.

Tetraspanin proteins are a unique family of highly conserved four-pass transmembrane proteins in metazoans. While much is known about their biochemical properties, the in vivo functions and distribution patterns of different tetraspanin proteins are less understood. Previous studies have shown that two paralogous tetraspanins that belong to the TspanC8 subfamily, TSP-12 and TSP-14, function redundantly to promote both Notch signaling and bone morphogenetic protein (BMP) signaling in C. elegans. TSP-14 has two isoforms, TSP-14A and TSP-14B, where TSP-14B has an additional 24 amino acids at its N-terminus compared to TSP-14A. By generating isoform specific knock-ins and knock-outs using CRISPR, we found that TSP-14A and TSP-14B share distinct as well as overlapping expression patterns and functions. While TSP-14A functions redundantly with TSP-12 to regulate body size and embryonic and vulva development, TSP-14B primarily functions redundantly with TSP-12 to regulate postembryonic mesoderm development. Importantly, TSP-14A and TSP-14B exhibit distinct subcellular localization patterns. TSP-14A is localized apically and on early and late endosomes. TSP-14B is localized to the basolateral cell membrane. We further identified a di-leucine motif within the N-terminal 24 amino acids of TSP-14B that serves as a basolateral membrane targeting sequence, and showed that the basolateral membrane localization of TSP-14B is important for its function. Our work highlights the diverse and intricate functions of TspanC8 tetraspanins in C. elegans, and demonstrates the importance of dissecting the functions of these important proteins in an intact living organism.

Physical interactions facilitate sex change in the protogynous orange-spotted grouper, Epinephelus coioides.

Sex change in teleost fishes is commonly regulated by social factors. In species that exhibit protogynous sex change, such as the orange-spotted grouper Epinephelus coioides, when the dominant males are removed from the social group, the most dominant female initiates sex change. The aim of this study was to determine the regulatory mechanisms of socially controlled sex change in E. coioides. We investigated the seasonal variation in social behaviours and sex change throughout the reproductive cycle of E. coioides, and defined the behaviour pattern of this fish during the establishment of a dominance hierarchy. The social behaviours and sex change in this fish were affected by season, and only occurred during the prebreeding season and breeding season. Therefore, a series of sensory isolation experiments was conducted during the breeding season to determine the role of physical, visual and olfactory cues in mediating socially controlled sex change. The results demonstrated that physical interactions between individuals in the social groups were crucial for the initiation and completion of sex change, whereas visual and olfactory cues alone were insufficient in stimulating sex change in dominant females. In addition, we propose that the steroid hormones 11-ketotestosterone and cortisol are involved in regulating the initiation of socially controlled sex change.

Natural sex change in mature protogynous orange-spotted grouper (Epinephelus coioides): gonadal restructuring, sex hormone shifts and gene profiles.

Sexual patterns of teleosts are extremely diverse and include both gonochorism and hermaphroditism. As a protogynous hermaphroditic fish, all orange-spotted groupers (Epinephelus coioides) develop directly into females, and some individuals change sex to become functional males later in life. This study investigated gonadal restructuring, shifts in sex hormone levels and gene profiles of cultured mature female groupers during the first (main) breeding season of 2019 in Huizhou, China (22° 42' 02.6″ N, 114° 32' 10.1″ E). Analysis of gonadal restructuring revealed that females with pre-vitellogenic ovaries underwent vitellogenesis, spawning and regression and then returned to the pre-vitellogenic stage in the late breeding season, at which point some changed sex to become males via the intersex gonad stage. A significant decrease in the level of serum 17ß-estradiol (E2) was observed during ovary regression but not during sex change, whereas serum 11-ketotestosterone (11-KT) concentrations increased significantly during sex change with the highest concentration in newly developed males. Consistent with serum hormone changes, a significant decrease in cyp19a1a expression was observed during ovary regression but not during sex change, whereas the expression of cyp11c1 and hsd11b2 increased significantly during sex change. Interestingly, hsd11b2 but not cyp11c1 was significantly upregulated from the pre-vitellogenic ovary stage to the early intersex gonad stage. These results suggest that a decrease in serum E2 concentration and downregulation of cyp19a1a expression are not necessary to trigger the female-to-male transformation, whereas increased 11-KT concentration and upregulation of hsd11b2 expression may be key events for the initiation of sex change in the orange-spotted grouper.

MicroRNA-29b modulates the innate immune response by suppressing IFNγs production in orange-spotted grouper (Epinephelus coioides).

Interferon-γ (IFNγ), a type II interferon, is essential to host resistance against various infections. Unlike other vertebrates, fish have two types of IFNγs, IFNγ1 (also named IFNγ-rel) and IFNγ2. MicroRNAs (miRNAs) regulate multiple biological processes by suppressing mRNA translation or inducing mRNA degradation. Among them, miR-29 can directly target IFNγ and affact innate and adaptive immune responses in mice. There are five members of the miR-29 family in orange-spotted grouper (Epinephelus coioides), which share the same miRNA seed region. However, whether miR-29 directly targets E. coioides IFNγs and regulate IFNγ production is still unknown. In the present study, the negative correlation between miR-29b and both IFNγs in immune tissues of healthy E. coioides and grouper spleen cells (GS cells) stimulated with LPS or poly I:C was demonstrated. Moreover, dual-luciferase reporter assays and western blotting were performed to demonstrate that miR-29b suppressed E. coioides IFNγ production. Studies of NO production in GS cells after miR-29b transfection revealed that miR-29b overexpression affected NO production through the downregulation of IFNγ expression. Taken together, our results suggest that miR-29b may directly target E. coioides IFNγs and modulate IFNγ-mediated innate immune responses by suppressing E. coioides IFNγs production.

Tetraspanins TSP-12 and TSP-14 function redundantly to regulate the trafficking of the type II BMP receptor in Caenorhabditis elegans.

Tetraspanins are a unique family of 4-pass transmembrane proteins that play important roles in a variety of cell biological processes. We have previously shown that 2 paralogous tetraspanins in Caenorhabditis elegans, TSP-12 and TSP-14, function redundantly to promote bone morphogenetic protein (BMP) signaling. The underlying molecular mechanisms, however, are not fully understood. In this study, we examined the expression and subcellular localization patterns of endogenously tagged TSP-12 and TSP-14 proteins. We found that TSP-12 and TSP-14 share overlapping expression patterns in multiple cell types, and that both proteins are localized on the cell surface and in various types of endosomes, including early, late, and recycling endosomes. Animals lacking both TSP-12 and TSP-14 exhibit reduced cell-surface levels of the BMP type II receptor DAF-4/BMPRII, along with impaired endosome morphology and mislocalization of DAF-4/BMPRII to late endosomes and lysosomes. These findings indicate that TSP-12 and TSP-14 are required for the recycling of DAF-4/BMPRII. Together with previous findings that the type I receptor SMA-6 is recycled via the retromer complex, our work demonstrates the involvement of distinct recycling pathways for the type I and type II BMP receptors and highlights the importance of tetraspanin-mediated intracellular trafficking in the regulation of BMP signaling in vivo. As TSP-12 and TSP-14 are conserved in mammals, our findings suggest that the mammalian TSP-12 and TSP-14 homologs may also function in regulating transmembrane protein recycling and BMP signaling.

Retinoic acid and androgen influence germ cells development and meiotic initiation in juvenile orange-spotted grouper, Epinephelus coioides.

Meiosis is essential for germ cells development for all sexually reproducing species. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in mammals, birds and tetrapods. Here, we investigated the effects of RA on meiotic initiation and sex determination in protogynous hermaphrodite orange-spotted grouper (Epinephelus coioides). Expression profile investigations of meiotic marker genes during gonadal development indicated that germ cells undergone meiosis approximately at 180 days after hatching in the orange-spotted grouper. RA synthase inhibitor treatments on juvenile orange-spotted groupers resulted in impeded germ cells development and delayed meiotic initiation with simultaneous down-regulation of vasa, dazl, sycp3 and rec8, which was rescued by exogenous RA administration. Additionally, exogenous androgen treated fish showed a delayed meiotic initiation consistent with decreased sycp3 and rec8 expression and were directed to a spermiogenesis fate. Our results imply that meiotic initiation in the orange-spotted grouper is strongly influenced by RA and androgen, and the regulation of meiotic initiation may involve in the spermatogenesis induced by exogenous androgen.

Effect of starvation and refeeding on growth, gut microbiota and non-specific immunity in hybrid grouper (Epinephelus fuscoguttatus♀×E. lanceolatus♂).

Environmental changes can lead to food deprivation among aquatic animals. The main objective of this present research was to assess the effect of starvation and refeeding on growth, gut microbiota and non-specific immunity in a hybrid grouper (Epinephelus fuscoguttatus♀×E. lanceolatus♂). A total of 120 fish with an average weight of 74.16 ± 12.08 g were randomly divided into two groups (control group and fasted-refed group). The control group was fed until satiation for 60 days, while the fasted-refed group was fasted for 30 days and then fed to satiation for 30 days. The results showed that starvation led to a significantly decreased growth performance parameters [weight gain rate (WGR) and specific weight gain rate (SGR), while the feeding rate (FR) ] increased during the refeeding, non-specific immunity was significantly improved (p < 0.05) during the first 15 days of starvation, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), lysozyme (LYM) and catalase (CAT). However, non-specific immunity decreased at 30 days of starvation, the expression of genes related to immunity, such as TNF-α, was upregulated (p < 0.05) during starvation, while the expression levels of IL-17 and IFN-γ was reduced (p < 0.05). The expression of IFN-γ and IL-1ß peaked during refeeding. Starvation led to significantly decreased abundance and diversity of intestinal microflora, with a higher abundance of Vibrio and a lower abundance of Brevibacillus, Bifidobacterium, Alloprevotella in the fasted-refed group during refeeding than in the control group. The above results reveal that starvation stimulates changes in growth, non-specific immunity, and the gut microbiota, providing new insights for the study of fish habitat selection and adaptability to environmental changes.

The Caenorhabditis elegans SMOC-1 Protein Acts Cell Nonautonomously To Promote Bone Morphogenetic Protein Signaling.

Bone morphogenetic protein (BMP) signaling regulates many different developmental and homeostatic processes in metazoans. The BMP pathway is conserved in Caenorhabditis elegans, and is known to regulate body size and mesoderm development. We have identified the C. elegans smoc-1 (Secreted MOdular Calcium-binding protein-1) gene as a new player in the BMP pathway. smoc-1(0) mutants have a small body size, while overexpression of smoc-1 leads to a long body size and increased expression of the RAD-SMAD (reporter acting downstream of SMAD) BMP reporter, suggesting that SMOC-1 acts as a positive modulator of BMP signaling. Using double-mutant analysis, we showed that SMOC-1 antagonizes the function of the glypican LON-2 and acts through the BMP ligand DBL-1 to regulate BMP signaling. Moreover, SMOC-1 appears to specifically regulate BMP signaling without significant involvement in a TGFß-like pathway that regulates dauer development. We found that smoc-1 is expressed in multiple tissues, including cells of the pharynx, intestine, and posterior hypodermis, and that the expression of smoc-1 in the intestine is positively regulated by BMP signaling. We further established that SMOC-1 functions cell nonautonomously to regulate body size. Human SMOC1 and SMOC2 can each partially rescue the smoc-1(0) mutant phenotype, suggesting that SMOC-1's function in modulating BMP signaling is evolutionarily conserved. Together, our findings highlight a conserved role of SMOC proteins in modulating BMP signaling in metazoans.

Expression profiles of dmrts and foxls during gonadal development and sex reversal induced by 17α-methyltestosterone in the orange-spotted grouper.

The orange-spotted grouper, Epinephelus coioides, is a marine protogynous hermaphrodite fish of commercial importance. There are many examples of sex change species among marine fish, but the molecular basis for the sex change is still unknown. Gonadal expression patterns of the dmrts and foxls genes in E. coioides have pointed to sexual dimorphism in this species and it has been shown that mRNA levels of dmrts and foxls to vary significantly during reproduction cycles. The steroid 17α-methyltestosterone was used to induce sex reversal in these fish, during which dmrts and foxls levels changed significantly and subsequently reverted to normal when 17α-methyltestosterone was withdrawn. Interestingly, the expression of dmrt2b and dmrt3 was not affected by this steroid. We speculate that the role of foxl2 in reproduction may be conserved via regulation of early differentiation of the ovary by the hypothalamus-pituitary-gonad axis, and dmrt2 may have a significant role in premature ovarian differentiation and maintenance in E. coioides. dmrt1 and foxl3 played a role in the development of the testes and are believed to be potential male regulatory genes.

The forkhead transcription factor UNC-130/FOXD integrates both BMP and Notch signaling to regulate dorsoventral patterning of the C. elegans postembryonic mesoderm.

The proper development of a multicellular organism requires precise spatial and temporal coordination of cell intrinsic and cell extrinsic regulatory mechanisms. Both Notch signaling and bone morphogenetic protein (BMP) signaling function to regulate the proper development of the C. elegans postembryonic mesoderm. We have identified the C. elegans FOXD transcription factor UNC-130 as a major target functioning downstream of both BMP signaling and Notch signaling to regulate dorsoventral patterning of the postembryonic mesoderm. We showed that unc-130 expression in the postembryonic M lineage is asymmetric: its absence of expression in the dorsal side of the M lineage requires the antagonism of BMP signaling by the zinc finger transcription factor SMA-9, while its expression in the ventral side of the M lineage is activated by LIN-12/Notch signaling. We further showed that the regulation of UNC-130 expression by BMP signaling and Notch signaling is specific to the M lineage, as the ventral expression of UNC-130 in the embryonically-derived bodywall muscles was not affected in either BMP pathway or Notch pathway mutants. Finally, we showed that the function of UNC-130 in the M lineage is independent of UNC-129, a gene previously shown to function downstream of and be repressed by UNC-130 for axon guidance. Our studies uncovered a new function of UNC-130/FOXD in the C. elegans postembryonic mesoderm, and identify UNC-130 as a critical factor that integrates two independent spatial cues for the proper patterning and fate specification of the C. elegans postembryonic mesoderm.

Unc93b1 is essential for cytokine activation of five PAMPs in the orange-spotted grouper (Epinephelus coioides).

Toll-like receptors (TLRs) are important innate immune receptors that recognize multiple pathogen-associated molecular patterns (PAMPs) and activate the immune responses to resist the invasion of pathogens. Many TLRs need assistance from trafficking chaperones to transport to the specific cell compartments and then are processed before they are activated. In this study, we identified an important trafficking chaperone, Unc-93 homolog B1 (unc93b1), from the Epinephelus coioides (orange-spotted grouper). The deduced protein sequence of Eco.unc93b1 was 632 amino acids, containing 12 transmembrane domains, consistent with other UNC93B1 proteins from other species. Phylogenetic analysis showed that Eco.Unc93b1 was clustered with teleost Unc93b1 and had the closest relationship with Larimichthys crocea (large yellow croaker) Unc93b1. Eco.unc93b1 was expressed the highest in the spleen, and its protein was co-localized with the endoplasmic reticulum and early endosomes in both human embryonic kidney 293T cells and grouper spleen cells (GS cells). Moreover, the stimulation of lipopolysaccharide (LPS), high-molecular-weight poly (I:C) (HMW), imidazoquinoline (R848), polyadenylic-polyuridylic acid (poly AU), and 19-mer Staphylococcus aureus 23S rRNA-derived oligoribonucleotide (ORN Sa 19) promoted the mRNA expression of unc93b1 in GS cells with different patterns. Furthermore, the cytokine expression induced by these PAMPs was suppressed, while Eco.unc93b1 was knocked down, by small interfering RNA. In conclusion, these results suggest that Eco.unc93b1 plays an essential role in several PAMP-induced immune responses.

The C. elegans Spalt-like protein SEM-4 functions through the SoxC transcription factor SEM-2 to promote a proliferative blast cell fate in the postembryonic mesoderm.

Proper development of a multicellular organism relies on well-coordinated regulation of cell fate specification, cell proliferation and cell differentiation. The C. elegans postembryonic mesoderm provides a useful system for uncovering factors involved in these processes and for further dissecting their regulatory relationships. The single Spalt-like zinc finger containing protein SEM-4/SALL is known to be involved in specifying the proliferative sex myoblast (SM) fate. We have found that SEM-4/SALL is sufficient to promote the SM fate and that it does so in a cell autonomous manner. We further showed that SEM-4/SALL acts through the SoxC transcription factor SEM-2 to promote the SM fate. SEM-2 is known to promote the SM fate by inhibiting the expression of two BWM-specifying transcription factors. In light of recent findings in mammals showing that Sall4, one of the mammalian homologs of SEM-4, contributes to pluripotency regulation by inhibiting differentiation, our work suggests that the function of SEM-4/SALL proteins in regulating pluripotency versus differentiation appears to be evolutionarily conserved.

Two Paralogous Tetraspanins TSP-12 and TSP-14 Function with the ADAM10 Metalloprotease SUP-17 to Promote BMP Signaling in Caenorhabditis elegans.

The highly conserved bone morphogenetic protein (BMP) signaling pathway regulates many developmental and homeostatic processes. While the core components of the BMP pathway have been well studied, much research is needed for understanding the mechanisms involved in the precise spatiotemporal control of BMP signaling in vivo. Here, we provide evidence that two paralogous and evolutionarily conserved tetraspanins, TSP-12 and TSP-14, function redundantly to promote BMP signaling in C. elegans. We further show that the ADAM10 (a disintegrin and metalloprotease 10) ortholog SUP-17 also functions to promote BMP signaling, and that TSP-12 can bind to and promote the cell surface localization of SUP-17. SUP-17/ADAM10 is known to be involved in the ligand-induced proteolytic processing of the Notch receptor. We have evidence that the function of SUP-17, and of TSP-12/TSP-14 in BMP signaling is independent of their roles in Notch signaling. Furthermore, presenilins, core components of the γ-secretase complex involved in processing Notch, do not appear to play a role in BMP signaling. These studies established a new role of the TSP-12/TSP-14/SUP-17 axis in regulating BMP signaling, in addition to their known function in the Notch signaling pathway. We also provide genetic evidence showing that a known BMP signaling modulator, UNC-40/neogenin/DCC, is one of the substrates of SUP-17/ADAM10 in the BMP signaling pathway.

Promotion of bone morphogenetic protein signaling by tetraspanins and glycosphingolipids.

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor ß (TGFß) superfamily of secreted molecules. BMPs play essential roles in multiple developmental and homeostatic processes in metazoans. Malfunction of the BMP pathway can cause a variety of diseases in humans, including cancer, skeletal disorders and cardiovascular diseases. Identification of factors that ensure proper spatiotemporal control of BMP signaling is critical for understanding how this pathway is regulated. We have used a unique and sensitive genetic screen to identify the plasma membrane-localized tetraspanin TSP-21 as a key new factor in the C. elegans BMP-like "Sma/Mab" signaling pathway that controls body size and postembryonic M lineage development. We showed that TSP-21 acts in the signal-receiving cells and genetically functions at the ligand-receptor level. We further showed that TSP-21 can associate with itself and with two additional tetraspanins, TSP-12 and TSP-14, which also promote Sma/Mab signaling. TSP-12 and TSP-14 can also associate with SMA-6, the type I receptor of the Sma/Mab pathway. Finally, we found that glycosphingolipids, major components of the tetraspanin-enriched microdomains, are required for Sma/Mab signaling. Our findings suggest that the tetraspanin-enriched membrane microdomains are important for proper BMP signaling. As tetraspanins have emerged as diagnostic and prognostic markers for tumor progression, and TSP-21, TSP-12 and TSP-14 are all conserved in humans, we speculate that abnormal BMP signaling due to altered expression or function of certain tetraspanins may be a contributing factor to cancer development.

A role of the LIN-12/Notch signaling pathway in diversifying the non-striated egg-laying muscles in C. elegans.

The proper formation and function of an organ is dependent on the specification and integration of multiple cell types and tissues. An example of this is the Caenorhabditis elegans hermaphrodite egg-laying system, which requires coordination between the vulva, uterus, neurons, and musculature. While the genetic constituents of the first three components have been well studied, little is known about the molecular mechanisms underlying the specification of the egg-laying musculature. The egg-laying muscles are non-striated in nature and consist of sixteen cells, four each of type I and type II vulval muscles and uterine muscles. These 16 non-striated muscles exhibit distinct morphology, location, synaptic connectivity and function. Using an RNAi screen targeting the putative transcription factors in the C. elegans genome, we identified a number of novel factors important for the diversification of these different types of egg-laying muscles. In particular, we found that RNAi knockdown of lag-1, which encodes the sole C. elegans ortholog of the transcription factor CSL (CBF1, Suppressor of Hairless, LAG-1), an effector of the LIN-12/Notch pathway, led to the production of extra type I vulval muscles. Similar phenotypes were also observed in animals with down-regulation of the Notch receptor LIN-12 and its DSL (Delta, Serrate, LAG-2) ligand LAG-2. The extra type I vulval muscles in animals with reduced LIN-12/Notch signaling resulted from a cell fate transformation of type II vulval muscles to type I vulval muscles. We showed that LIN-12/Notch was activated in the undifferentiated type II vulval muscle cells by LAG-2/DSL that is likely produced by the anchor cell (AC). Our findings provide additional evidence highlighting the roles of LIN-12/Notch signaling in coordinating the formation of various components of the functional C. elegans egg-laying system. We also identify multiple new factors that play critical roles in the proper specification of the different types of egg-laying muscles.

The neogenin/DCC homolog UNC-40 promotes BMP signaling via the RGM protein DRAG-1 in C. elegans.

The deleted in colorectal cancer (DCC) homolog neogenin functions in both netrin- and repulsive guidance molecule (RGM)-mediated axon guidance and in bone morphogenetic protein (BMP) signaling. How neogenin functions in mediating BMP signaling is not well understood. We show that the sole C. elegans DCC/neogenin homolog UNC-40 positively modulates a BMP-like pathway by functioning in the signal-receiving cells at the ligand/receptor level. This function of UNC-40 is independent of its role in netrin-mediated axon guidance, but requires its association with the RGM protein DRAG-1. We have identified the key residues in the extracellular domain of UNC-40 that are crucial for UNC-40-DRAG-1 interaction and UNC-40 function. Surprisingly, the extracellular domain of UNC-40 is sufficient to promote BMP signaling, in clear contrast to the requirement of its intracellular domain in mediating axon guidance. Mouse neogenin lacking the intracellular domain is also capable of mediating BMP signaling. These findings reveal an unexpected mode of action for neogenin regulation of BMP signaling.

The C. elegans SoxC protein SEM-2 opposes differentiation factors to promote a proliferative blast cell fate in the postembryonic mesoderm.

The proper development of multicellular organisms requires precise regulation and coordination of cell fate specification, cell proliferation and differentiation. Abnormal regulation and coordination of these processes could lead to disease, including cancer. We have examined the function of the sole C. elegans SoxC protein, SEM-2, in the M lineage, which produces the postembryonic mesoderm. We found that SEM-2/SoxC is both necessary and sufficient to promote a proliferating blast cell fate, the sex myoblast fate, over a differentiated striated bodywall muscle fate. A number of factors control the specific expression of sem-2 in the sex myoblast precursors and their descendants. This includes direct control of sem-2 expression by a Hox-PBC complex. The crucial nature of the HOX/PBC factors in directly enhancing expression of this proliferative factor in the C. elegans M lineage suggests a possible more general link between Hox-PBC factors and SoxC proteins in regulating cell proliferation.

The RGM protein DRAG-1 positively regulates a BMP-like signaling pathway in Caenorhabditis elegans.

The bone morphogenetic protein (BMP) signaling pathway regulates multiple developmental and homeostatic processes. Mutations in the pathway can cause a variety of somatic and hereditary disorders in humans. Multiple levels of regulation, including extracellular regulation, ensure proper spatiotemporal control of BMP signaling in the right cellular context. We have identified a modulator of the BMP-like Sma/Mab pathway in C. elegans called DRAG-1. DRAG-1 is the sole member of the repulsive guidance molecule (RGM) family of proteins in C. elegans, and is crucial in regulating body size and mesoderm development. Using a combination of molecular genetic and biochemical analyses, we demonstrate that DRAG-1 is a membrane-associated protein that functions at the ligand-receptor level to modulate the Sma/Mab pathway in a cell-type-specific manner. We further show that DRAG-1 positively modulates this BMP-like pathway by using a novel Sma/Mab-responsive reporter. Our work provides a direct link between RGM proteins and BMP signaling in vivo and a simple and genetically tractable system for mechanistic studies of RGM protein regulation of BMP pathways.

The FoxF/FoxC factor LET-381 directly regulates both cell fate specification and cell differentiation in C. elegans mesoderm development.

Forkhead transcription factors play crucial and diverse roles in mesoderm development. In particular, FoxF and FoxC genes are, respectively, involved in the development of visceral/splanchnic mesoderm and non-visceral mesoderm in coelomate animals. Here, we show at single-cell resolution that, in the pseudocoelomate nematode C. elegans, the single FoxF/FoxC transcription factor LET-381 functions in a feed-forward mechanism in the specification and differentiation of the non-muscle mesodermal cells, the coelomocytes (CCs). LET-381/FoxF directly activates the CC specification factor, the Six2 homeodomain protein CEH-34, and functions cooperatively with CEH-34/Six2 to directly activate genes required for CC differentiation. Our results unify a diverse set of studies on the functions of FoxF/FoxC factors and provide a model for how FoxF/FoxC factors function during mesoderm development.