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bioRxiv2022

The Prmt5-Vasa module is essential for dichotomous spermatogenesis in Bombyx mori

Spermatogenesis is a key process for the sexual reproduction species. In lepidopteran insects, dichotomous spermatogenesis is a notable feature, which produces eupyrene (nucleate) and apyrene (anucleate) spermatozoa. Both sperm morphs are essential for fertilization, as eupyrene sperm fertilizes the egg, while apyrene sperm is necessary for eupyrene sperm migration. In Drosophila, Prmt5 acts as a type II arginine methyltransferase to catalyze the symmetrical dimethylation of arginine residues (sDMA) in Vasa. However, Prmt5 is involved in regulating spermatogenesis, but Vasa is not. To date, the functional genetic study on dichotomous spermatogenesis in the lepidopteran model Bombyx mori has been limited, thus the underlying mechanism remains largely unknown. In this study, we report that both BmPrmt5 and BmVasa act as essential components in the regulation of dichotomous spermatogenesis in Bombyx mori. The loss-of-function mutants of BmPrmt5 ({Delta}BmPrmt5) and BmVasa ({Delta}BmVasa) derived from CRISPR/Cas9-based gene editing show similar male and female-sterile phenotypes. Through immunofluorescence staining analysis, we found that the morphs of both{Delta} BmPrmt5 and{Delta} BmVasa sperm show severe defects, indicating an essential role for both BmPrmt5 and BmVasa in the regulation of dichotomous spermatogenesis. RNA-seq analyses indicate that the defects in dichotomous spermatogenesis observed for{Delta} BmPrmt5 and{Delta} BmVasa mutants could be attributed to the reduced expression of the spermatogenesis-related genes including Sex-lethal (BmSxl), implying that BmSxl may act downstream of Prmt5 and Vasa in regulating apyrene sperm development. These findings suggest that BmPrmt5 and BmVasa constitute an integral regulatory module essential for dichotomous spermatogenesis in Bombyx mori, in which BmPrmt5 may promote BmVasa activity through sDMA modification. Author SummaryDichotomous spermatogenesis is a notable feature in Lepidoptera insects, in which a single male produces nucleated and anucleated dimorphic sperm. Both sperm morphs are essential for fertilization, as the eupyrene sperm fertilizes the egg, while the apyrene sperm assists in transporting the eupyrene sperm to the female sperm storage organs. To date, the mechanism of dichotomous spermatogenesis is still unclear, as very limited components participating in this process have been reported. Here we defined the function of BmPrmt5 (Protein arginine methyltransferase 5) and its substrate BmVasa in dichotomous spermatogenesis in the lepidopteran insect Bombyx mori. Our genetic analyses revealed that both BmPrmt5 and BmVasa are essential for dichotomous spermatogenesis. Moreover, RNA-seq analyses, in conjunction with the genetic and immunofluorescence staining analyses, suggest a potential regulatory pathway consisting of the BmPrmt5-BmVasa module in dichotomous spermatogenesis in Bombyx mori. This study expands the knowledge of reproduction biology in insects, and may provide potential gene targets for pest control.

developmental biology↗

bioRxiv2023

The conserved wobble uridine tRNA thiolase Ctu1 is required to sustain development and differentiation

Recent studies have revealed that tRNA modification is an important epigenetic mechanism involved in gene expression. Cytosolic thiouridylase (consisting of Ctu1 and Ctu2 subunit) are the enzyme complex which catalyze the thio-modification at the 34th wobble uridine of the anticodon of tRNAGlnUUG, tRNAGluUUC, and tRNALysUUU. Besides introducing a thiol group at the C2 positions, those tRNAs were commonly modified with a methoxycarbonylmethyl at the C5 positions by Elongator and ALKBH8. tRNA-U34 modification, particularly the Elongator and ALKBH8, has been demonstrated to be involved in disease and development, however, the biological functional level of CTU in vertebrates remains elusive. Here, we found that in zebrafish, CTU may be an important regulatory factor in development and erythroid differentiation. By using morpholino targeting and knocking down CTU1, we observed that the loss of CTU1 led to impaired zebrafish larval development and blood vessel formation. Single-cell sequencing analysis showed that erythroid cell differentiation in the CTU1 knockdown group was blocked at an early stage, while the wild-type group exhibited mature erythroid cells. These findings suggest that CTU1 is involved in regulating erythrocyte development. These findings provide new insights into the biological function of CTU1.

developmental biology↗

bioRxiv2024

Derivation of human trophoblast stem cells from placentas at birth

Human trophoblast stem cells (hTSCs) have emerged as a powerful tool for modeling the placental cytotrophoblast (CTB) in vitro. hTSCs were originally derived from CTBs of the first trimester placenta or blastocyst-stage embryos in trophoblast stem cell medium (TSCM) that contains epidermal growth factor (EGF), the glycogen synthase kinase-beta (GSK3{beta}) inhibitor CHIR99021, the transforming growth factor-beta (TGF{beta}) inhibitors A83-01 and SB431542, valproic acid (VPA), and the Rho-associated protein kinase (ROCK) inhibitor Y-27632. Here we show that hTSCs can be derived from CTBs isolated from the term placenta, using TSCM supplemented with a low concentration of mitochondrial pyruvate uptake inhibitor UK5099 and lipid-rich albumin (TUA medium). Notably, hTSCs could not be derived from term CTBs using TSCM alone, or in the absence of either UK5099 or lipid-rich albumin. Strikingly, hTSCs cultured in TUA medium for a few passages could be transitioned into TSCM and cultured thereafter in TSCM. hTSCs from term CTBs cultured in TUA medium as well as those transitioned into and cultured in TSCM thereafter could be differentiated to the extravillous trophoblast and syncytiotrophoblast lineages and exhibited high transcriptome similarity with hTSCs derived from first trimester CTBs. We anticipate that these results will enable facile derivation of hTSCs from normal and pathological placentas at birth with diverse genetic backgrounds and facilitate in vitro mechanistic studies in trophoblast biology. Significance statementHuman trophoblast stem cells (hTSCs) derived from early pregnancy have emerged as a powerful tool for modeling early human placental development in vitro. However, restrictions on derivation of cell lines from fetal tissues limit the availability and genetic diversity of hTSC lines. Also, the associated pregnancy outcome of hTSCs derived from early gestation is unknown. Here we describe the derivation and characterization of hTSCs from placentas obtained at birth. Our results will enable derivation of genetically diverse hTSCs associated with normal and pathological pregnancies, enabling research in placental biology.

developmental biology↗

bioRxiv2024

Temporal dynamics of gene expression during metamorphosis in two distant Drosophila species

Complete metamorphosis of holometabolous insects is a complex biological process characterized by profound morphological, physiological, and transcriptional changes. To reveal the temporal dynamics of gene expression during this critical developmental transition, we conducted a detailed analysis of the developmental transcriptomes of two Drosophila species, Drosophila melanogaster and Drosophila virilis. We confirmed partial recapitulation of the embryonic transcriptional program in pupae, but unlike the traditional hourglass model, suggesting maximal conservation at mid-embryonic stages, at different stages of pupae we observed a more complicated pattern of alternating low and high diversity, resembling an inverted hourglass, or "spindle". This underscores the complexity of developmental processes during complete metamorphosis. Notably, recently formed genes (specific to insects) exhibit pronounced expression peaks during mid-pupal development, indicating their potential role in developmental transitions. Significance StatementThis study describes the transcriptomic dynamics of complete metamorphosis in holometabolous insects. By analyzing developmental transcriptomes of Drosophila melanogaster and Drosophila virilis, we characterize the spindle pattern of gene expression during pupal stages, contrasting with the classical hourglass model of embryogenesis. This alternating pattern of low and high transcriptional diversity highlights the unique complexity of the metamorphose processes.

developmental biology↗

bioRxiv2021

Manipulating the murine Lgr5 locus using a rapid, efficient and flexible CRISPR/Cas9 pipeline

The maintenance of adult epithelial tissues, such as the lining of the intestine, depends on their periodical renewal. This is achieved through small populations of adult stem cells. Lgr5 serves as a marker for these cells. Here we report a novel non-variegated Lgr5 mouse model, which was generated via the CRISPR/Cas9 system. We show that this Lgr5-2A-CreERT2-2A-mOrange2 mouse line can be used for lineage tracing, as well as for directing gene expression to Lgr5+ cells. The introduction of the transgene affects neither the expression, nor the function of endogenous Lgr5. Therefore, this new tool will serve to mark and manipulate intestinal stem cells to gain new biological insights.

developmental biology↗

bioRxiv2023

Global decoupling of cell differentiation from cell division in early embryo development

As tissues develop, cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation, we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single-cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. In the absence of cell division, differentiation slows down in some cell types, and cells exhibit global stress responses. While differentiation is robust to blocking cell division, the proportions of cells across cell states are not. This work simplifies our understanding of the role of cell division in development and showcases the utility of combining embryo-wide perturbations with single-cell RNA sequencing to uncover the role of common biological processes across multiple tissues.

developmental biology↗

bioRxiv2024

Mechanical control of the insect extracellular matrix nanostructure

Nanoscale modifications of apical extracellular matrix (ECM) have created a variety of functional surfaces with distinct physical properties, as exemplified by structural coloration and superhydrophobicity in animals and plants1. While the surface nanostructures of various organisms have inspired numerous biomimetic applications, the biological mechanisms underlying ECM patterning at the nanoscale remain largely unknown. Here, we investigated the morphogenesis of cuticular pores in Drosophila olfactory organs2,3. Hundreds of uniform-sized nanopores of [~]50 nm permit selective access of odorant molecules to olfactory neurons. We showed that matrices composed of the zona pellucida domain (ZPD) protein4-6 cover sensory organs in cell type-specific patterns and combinations. The ZPD proteins Dyl, Tyn, Mey, and Nyo form matrices with specific mixing and sorting properties, restricting cell growth and movement. The disruption of ZPD matrices leads to detachment of the envelope layer of the cuticle from the plasma membrane, and reduced numbers and irregularly sized nanopores. Our results suggest that compressive force from the ECM is essential for robust nanopore morphogenesis. This work reveals a previously unrecognized role for ZPD proteins as modular units that establish the mechanical environment required to modulate the nanoscale assembly of cuticle materials, opening a new biological context to these biomimetically important structures.

developmental biology↗

bioRxiv2025

Derivation of primed sheep embryonic stem cells and conversion to an intermediate naive-like state

Embryonic stem cells (ESCs) derived from the inner cell mass of embryos possess unlimited self-renewal and pluripotency, offering a powerful system to study early development and enable genetic and biotechnological innovation. Although several livestock ESC lines have been reported in recent years, defining culture conditions that support stable long-term self-renewal and controlled transitions across pluripotent states remains challenging. Here, we report the de novo derivation of sheep embryonic stem cells (sESCs) from in vivo blastocysts using a chemically defined culture system. The derived cells exhibit morphological and molecular features of primed pluripotency and can be propagated under both feeder-dependent and feeder-free conditions without loss of identity or karyotypic stability. Building on this foundation, we developed enhancer-driven reporter lines that faithfully reflect OCT4 and SOX2 transcriptional activity, enabling dynamic visualization of pluripotency and differentiation in live cultures. These reporter systems revealed the responsiveness of sESCs to signaling modulation and provided a functional readout of pluripotency state transitions. When cultured in defined media previously shown to stabilize naive pluripotency in human ESCs, sESCs adopted dome-shaped colony morphology, maintained OCT4, SOX2, and NANOG expression, retained differentiation potential, and exhibited a transcriptomic profile consistent with resetting to an intermediate pluripotent state with naive-like morphological features. These findings establish stable sheep ESC lines and demonstrate their plasticity across the pluripotency spectrum, providing a valuable platform for investigating ruminant stem cell biology and advancing livestock biotechnology.

developmental biology↗

bioRxiv2018

Xenopus hybrids provide insight into cell and organism size control

Determining how size is controlled is a fundamental question in biology that is poorly understood at the organismal, cellular and subcellular levels. The Xenopus species, X. laevis and X. tropicalis differ in size at all three of these levels. Despite these differences, fertilization of X. laevis eggs with X. tropicalis sperm gives rise to viable hybrid animals that are intermediate in size. We observed that although hybrid and X. laevis embryogenesis initiates from the same sized zygote and proceeds synchronously through development, hybrid animals were smaller by the tailbud stage, and a change in the ratio of nuclear size to cell size was observed shortly after zygotic genome activation (ZGA), suggesting that differential gene expression contributes to size differences. Transcriptome analysis at the onset of ZGA identified twelve transcription factors paternally expressed in hybrids. A screen of these X. tropicalis factors by expression in X. laevis embryos revealed that Hes7 and Ventx2 significantly reduced X. laevis body length size by the tailbud stage, although nuclear to cell size scaling relationships were not affected as in the hybrid. Together, these results suggest that transcriptional regulation contributes to biological size control in Xenopus.

developmental biology↗

bioRxiv2026

Genetic control of mitotic-to-meiotic transition regulates germline cell survival

AO_SCPLOWBSTRACTC_SCPLOWThe coordination between DNA damage repair and cell cycle progression is essential to ensure cell survival and organ homeostasis. This is particularly critical during gametogenesis, where germline cells first proliferate and then transition from mitosis to meiosis. Meiotic cells frequently undergo recombination, which itself implies the generation of severe DNA damage in the form of double-strand DNA breaks (DSBs) that ought to be repaired to preserve genome integrity. Here, we identify Drosophila bru1 as an essential factor in the mitotic-to-meiotic transition in the female germline. The RNA-binding protein Bru1 is a translational repressor of multiple targets, including cyclins, and is sharply upregulated at the mitotic-to-meiotic boundary. We show that loss of bru1 disrupts germline development by altering cell cycle regulation, increasing DNA damage and cellular stress, and triggering apoptosis. bru1 mutants display clear signs of accelerated mitotic activity leading to extra divisions in the germline, in agreement with their elevated Cyclin A and B levels. Importantly, slowing cell cycle progression in bru1 mutants via string/cdc25 knockdown decreases DNA damage and cell death. Mechanistically, bru1 regulates mei-W68 transcription, the topoisomerase responsible for DSB production in the germline. Higher Mei-W68 levels induce premature and ectopic DSBs, which persist longer in the mutant germline, indicating defective repair and potentially resulting in p53-mediated apoptosis. Our work classifies bru1 as a safeguard of genome integrity and germline survival during the early stages of Drosophila female gametogenesis. bru1 regulates Mei-W68 levels and DSB formation, and controls the mitotic-to-meiotic transition by influencing cell cycle progression. The existence of bru1 homologues in mammals with established roles in gametogenesis suggests a broader biological relevance of our discoveries.

developmental biology↗

bioRxiv2026

An alternative patterning mechanism for vertebrate tooth complexity

How development reliably generates complex patterns remains a central question in biology. Teeth provide an ideal system for addressing this problem, linking cusp-patterning mechanisms to diversification and the fossil record, yet the developmental basis of multicuspid crown patterning beyond mammals remains unresolved. In mammals, cusp initiation and spacing are controlled by transient epithelial signaling centers called enamel knots (EKs). Here, we combine comparative developmental analyses, 3D morphometrics, mutant perturbation, and Bayesian inverse modeling (BITES; Bayesian Inference for Tooth Emergence Simulation) to examine tooth morphogenesis across six squamate species spanning independent origins of multicuspid dentitions. We find that cusp patterning in these taxa does not rely on discrete EK organizers. Instead, cusps arise from a broad, persistent epithelial signaling field that deploys canonical tooth-patterning pathways but lacks the spatial restriction, apoptosis, and secondary organizers characteristic of the mammalian system. Elevated epithelial proliferation drives disproportionate expansion of this field during bud growth, enabling cusp addition. Quantitative variation in field extent and dynamics is sufficient to generate crown morphologies within and across species, providing a developmental basis for positional heterodonty and repeated gains and losses of multicuspidity in squamates. Our results identify an alternative epithelial patterning architecture for vertebrate cusp formation and suggest that the mammalian EK represents a derived, constrained implementation. Together, these findings revise how developmental complexity is encoded and show how shifts in patterning architecture can relax constraints and enable evolutionary convergence.

developmental biology↗

bioRxiv2016

An RNA binding polymer specifies nematode sperm fate

Metazoan germ cells develop as sperm or oocytes, depending on chromosomal sex, extrinsic signaling from somatic tissue and intrinsic factors within the germ cells. Gamete fate regulatory networks have been analyzed in nematodes, flies and mammals, but only in C. elegans have terminal intrinsic regulators been identified, which include a Tob/BTG protein family member, FOG-3. Canonical Tob/BTG proteins function as monomeric adaptor proteins that link RNA binding proteins to deadenylases. To ask if FOG-3 functions similarly, we first determined its crystal structure. FOG-3 harbors a classical Tob/BTG fold, but unlike other Tob/BTG proteins, FOG-3 dimerizes and these FOG-3 dimers assemble into polymers. The importance of FOG-3 polymers to sperm fate specification was confirmed in vivo using CRISPR/Cas9 gene editing to create mutations designed to disrupt the polymer interface. The FOG-3 surface potential is highly basic, suggesting binding to nucleic acid. We find that FOG-3 binds RNA directly with a strong preference for 3UTRs of oogenic mRNAs. Our results reveal a divergent but striking molecular assembly for proteins with a Tob/BTG fold, make key advances in understanding the mechanism of sperm fate specification and highlight the potential for undiscovered protein polymers in biology.

developmental biology↗

bioRxiv2023

Comparative Analysis of Ovarian Transcriptome Changes Across Gestational Stages in Kari Sheep

This study aimed to investigate the genetic determinants of gestation length in Kari sheep, employing RNA-Seq technology. Employing a comprehensive whole transcriptome analysis, we sought to pinpoint differentially expressed genes (DEGs) while also delving into gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway assessments. The analysis revealed the identification of a total of 19,546 genes expressed in ovary. While comparing the transcriptomes of Kari sheep with Balkhi, yielding 976 DEGs (p < 0.05, Log2fc>1, <-1). Notably, among these DEGs, an upregulation of genes was observed associated with Ubiquitin-protein transferase activity, such as CNOT4, RC3H1, and XIAP. Concurrently, DEGs like NFAT5, EPAS1, ZNF644, RBPJ, and FOXP2 exhibited associations with RNA polymerase II core promoter proximal region sequence-specific DNA binding. Conversely, downregulated genes, including EEA1, CNOT4, FGD4, MBNL1, ZRANB2, REV3L, XIAP, ATP13A3, RPAP2, FOXP2, and ADAMTS6, were implicated in the mRNA surveillance pathway. In addition, several Gene Ontology terms, such as GO:0001228 (transcriptional activator activity) and GO:0004842, along with GO:0000978 (transcriptional activator activity), were linked to the DEGs. KEGG pathways, including "Glycosaminoglycan biosynthesis - chondroitin sulfate/dermatan sulfate" (KEGG:532) and "basal cell carcinoma" (KEGG:5217), were associated with our findings. Our principal component analysis (PCA) demonstrated a cohesive clustering of gene expression profiles among the four samples, with subtle distinctions. Protein-protein interaction (PPI) analysis indicated the functional relationships among the DEGs. Notably, genes such as ABHD16B and NPBWR2 exhibited strong co-expression among the down-regulated DEGs, while DNAH7/TBC1D31 and MBNL1/NOVA1 displayed prominent co-expression among the up-regulated DEGs. Consequently, our study offers a comprehensive understanding of Kari sheep genetics and the pivotal genes involved in gestation length determinants. These findings carry significant genetic implications, enhancing genetic resources, furthering reproductive biology comprehension, and contributing to the advancement of sustainable sheep farming practices.

developmental biology↗

bioRxiv2024

Morphologically defined substages of tail tip morphogenesis in C. elegans males

BackgroundSex-specific morphogenesis occurs in C. elegans in the vulva of the hermaphrodite and in the male tail during the last larval stage. Temporal progression of vulva morphogenesis has been described in fine detail. However, a similar precise description of male tail morphogenesis was lacking. ResultsWe here describe morphogenesis of the male tail at time points matching vulva development with special focus on morphogenesis of the tail tip. Using fluorescent reporters, we follow changes in cell shapes, cell fusions, nuclear migration, modifications in the basement membrane and formation of a new apical extracellular matrix at the end of the tail. ConclusionOur analysis answers two open questions about tail tip morphogenesis (TTM) by showing that one of the four tail tip cells, hyp11, remains separate while the other cells fuse with each other and with two additional tail cells to form a ventral tail syncytium. This fusion begins early during TTM but is only completed towards the end of the process. This work provides a framework for future investigations of cell-biological factors that drive male tail morphogenesis.

developmental biology↗

bioRxiv2024

Spatiotemporal dynamics of NF-κB/Dorsal inhibitor IκBα/Cactus in Drosophila blastoderm embryos

The NF-{kappa}B signaling pathway is a key regulatory network in mammals that controls many cellular processes, including immunity and inflammation. Of particular note is the relationship between NF-{kappa}B and its inhibitor I{kappa}B, which sequesters NF-{kappa}B to the cytoplasm of cells until needed. It is also known that I{kappa}B can enter nuclei, disrupt NF-{kappa}B binding to DNA, and shuttle it out to again sequester NF-{kappa}B in the cytoplasm. In Drosophila melanogaster, a homologous system between the proteins Dorsal (homologous to NF-{kappa}B) and Cactus (homologous to I{kappa}B) is important in embryo development, specifically in establishment of the Dorsal nuclear concentration gradient. Previous work suggests Cactus also enters the nucleus; mathematical models of the Dorsal gradient fail to accurately predict the normal range of the gradient without nuclear Cactus. However, direct, in vivo visualization of Cactus spatiotemporal dynamics, including its localization to the nuclei, has been difficult to gather. Previously, imaging Cactus in live embryos was complicated by rapid protein turnover, preventing fluorescent protein fusions from fully maturing. To address this, we used the CRISPR/Cas9 system to tag Cactus with the recently developed "LlamaTag" (LT), a genetically encodable nanobody from llamas that dynamically binds to GFP in vivo. We then employed standard confocal imaging, as well as advanced optical techniques such as raster image correlation spectroscopy (RICS) and fluorescent recovery after photobleaching (FRAP) to investigate the spatiotemporal distribution of Cactus-LlamaTag in Drosophila embryos at the blastoderm stage. Our results demonstrate that Cactus can be found in the nuclei of early embryos, consistent with its role as a transcription factor regulator. Moreover, by using the data from FRAP and RICS, we were able to estimate biophysical parameters of Cactus dynamics in vivo, including its nuclear transport rate constants and fraction bound to GFP. These data were further used to constrain a mathematical model that allowed us to infer experimentally inaccessible biophysical parameters, such as the concentration of Cact protein and the dissociation constant of LT and GFP. Our study provides new insights into the regulation of the NF-{kappa}B pathway in early Drosophila embryos and highlights the power of advanced optical techniques for investigating complex biological dynamics.

developmental biology↗

bioRxiv2024

Dual nuclease single-cell lineage tracing by Cas9 and Cas12a

Single-cell lineage tracing based on CRISPR/Cas9 gene editing enables the simultaneous linkage of cell states and lineage history at a high resolution. Despite its immense potential in resolving the cell fate determination and genealogy within an organism, existing implementations of this technology suffer from limitations in recording capabilities and considerable barcode dropout. Here, we introduce DuTracer, a versatile tool, which utilizes two orthogonal gene editing systems to record deep cell lineage history at single-cell resolution in an inducible manner. DuTracer shows the ability of enhanced lineage recording with minimized target dropouts and deepened tree depth. Application of DuTracer in mouse embryoid bodies and neuromesodermal organoids illustrates the lineage relationship of different cell types and proposes potential lineage-biased molecular drivers. Moreover, we have developed an entropy-based approach to quantify the lineage recording ability of DuTracer in cell differentiation models. Together, DuTracer facilitates the precise and regulatory interrogation of cellular lineages of complex biological processes.

developmental biology↗

bioRxiv2025

NEDD4 promotes Sertoli cell proliferation and adult Leydig cell differentiation in the murine testis

Successful testis development relies on the coordinated differentiation and assembly of various cell types to establish both endocrine and reproductive functions. The ubiquitin ligase NEDD4 has emerged as a key player in murine testis development, with this enzyme being implicated in gonadal sex determination and spermatogonial stem cell differentiation. Here, we report hitherto uncharacterized roles of NEDD4 in postnatal testis development. Utilizing Nr5a1- and Amh-Cre drivers to conditionally ablate Nedd4 in testicular somatic cells, we show that NEDD4 promotes Sertoli cell proliferation through the modulation of the PI3K-AKT signaling pathway. This ubiquitin ligase also ensures proper differentiation of adult Leydig cells and may contribute to murine steroidogenesis. Furthermore, NEDD4 is essential for adrenal gland differentiation, as its loss results in adrenal dysgenesis. These findings highlight NEDD4 as a crucial factor in testis development, emphasizing the importance of ubiquitination and post-translational modifications in reproductive biology.

developmental biology↗

bioRxiv2019

NLRP7 Plays A Functional Role in Regulating BMP4 Signaling During Differentiation of Patient-Derived Trophoblasts

Complete Hydatidiform Mole (HM) is a gestational trophoblastic disease resulting in hyper proliferation of trophoblast cells and absence of embryo development. Mutations in the primate specific-maternal effect gene NLRP7 are the major cause of familial recurrent complete HM. Here, we established an in vitro model of HM using NLRP7 deficient patient-specific induced pluripotent stem cells (iPSCs) derived trophoblasts. Using whole transcriptome profiling during trophoblast differentiation, we showed that NLRP7 deficiency results in precocious downregulation of pluripotency factors, activation of trophoblast lineage markers and promotes maturation of differentiated extraembryonic cell types such as syncytiotrophoblasts. Interestingly, we found that these phenotypes are dependent on BMP4 signaling and BMP pathway inhibition corrected the excessive trophoblast differentiation of patient derived iPSCs. Our human iPSC model of a genetic placental disease recapitulates aspects of trophoblast biology, highlights the broad utility of iPSC-derived trophoblasts for modeling human placental diseases and identifies NLRP7 as an essential modulator of key developmental cell fate regulators.

developmental biology↗