Vascular cells hold great potential not only for treating vascular disease but also for engineering vascularized organoids and tissues for transplantation. Both endothelial cells (ECs) and smooth muscle cells (SMCs) can be derived from human induced pluripotent stem cells (hiPSCs) via intermediate vascular progenitors (VPs). Our previous study showed that the VPs generated from hiPSCs using VEGF, FGF2, and BMP4 can efficiently differentiate into ECs. In this study, we found that BMP4 favored EC differentiation at the cost of SMC differentiation. Interestingly, inhibition of the TGF-β signaling by small molecule SB431542 or SB525334, could also induce EC differentiation while suppress SMC differentiation. In addition, both BMP4 and the TGF-β inhibitors activated SMAD1, suggesting that the TGF-β signaling counteracts with the BMP4 signaling. Furthermore, blocking the ERK signaling pathway with an inhibitor U0126 promoted SMC differentiation and inhibited EC differentiation likely through inhibiting the activation of SMAD1 and stimulating SMAD2. Indeed, the treatment of U0126 upregulated the expression of TGF-β2. Importantly, TGF-β inhibitor SB431542 effectively rescued EC differentiation impaired by ERK inhibition, likely through maintaining KDR-mediated VEGF signaling and restricting TGF-β2 signaling. Collectively, our findings highlight the competitive interplay among BMP4, TGF-β and ERK signaling pathways in determining EC versus SMC differentiation.

Megakaryocytes and platelets are essential for hemostasis, immune regulation and hematopoietic niche maintenance, while also promoting thrombosis. Comprehensive characterization of late-stage megakaryocyte maturation in mammals using single-cell RNA-sequencing (scRNA-Seq) remains challenging due to the large size of megakaryocytes and the low RNA content of platelets. Although zebrafish thrombocytes are functionally analogous and retain high transcriptional activity at maturity, their transcriptional similarities to mammals have not been systematically defined. To address this, we generated a single-cell transcriptomic atlas spanning the full developmental trajectory of zebrafish thrombocytes, demonstrating that lineage progression is continuous and functionally coordinated. Cross-species comparison with human and mouse datasets defined conserved differentiation stages and gene modules of the megakaryocyte-platelet lineage, indicating evolutionary conservation of lineage development at single-cell resolution. Moreover, we identified a gene set (M3pan-PLT) that marks a platelet-like transcriptional state in zebrafish thrombocytes, supporting their transcriptomic analogy to mammalian platelets. Finally, bulk transcriptomic analyses using these conserved gene signatures demonstrated their ability to capture physiological megakaryocyte states, as well as disease-associated transcriptional alterations in acute megakaryoblastic leukemia. Together, our study defines a conserved and continuous developmental framework of megakaryocytes at single-cell resolution and establishes zebrafish thrombocytes as a transcriptionally conserved model for dissecting late-stage megakaryocyte-platelet biology.

Signal transducer and transcription 1 (STAT1) exerts inhibitory effects on osteoclastogenesis and thereby alleviates bone loss. As a key modulator of apoptosis, STAT1 mediates apoptotic signaling in osteoclast precursors (OCPs). Our phosphoproteomic profiling revealed that RANKL induces STAT1 phosphorylation at the Ser727 residue in OCPs. The present study was designed to elucidate the regulatory interplay between RANKL-triggered STAT1-Ser727 phosphorylation, osteoclastogenesis, and OCP apoptosis. The results of this study demonstrated that RANKL upregulates STAT1 phosphorylation at Ser727 in OCPs. Knockdown of STAT1 significantly attenuates OCP apoptosis, while increasing both the number and size of mature osteoclasts. Notably, a Ser727-site mutation to Cys that lead to STAT1 dephosphorylation promotes osteoclastogenesis and suppresses OCP apoptosis. Mechanistic investigation revealed that STAT1 silencing eliminates RANKL-induced upregulation of Bax expression and the interaction between STAT1 and p53, and mutation for STAT1 dephosphorylation blocks both STAT1 overexpression-promoted Bax expression and the formation of the STAT1-p53 complex. Furthermore, the enhancement of Bax luciferase reporter activity induced by STAT1 overexpression was abrogated by introduction of the STAT1 dephosphorylation mutant, and no significant intergroup difference was observed after p53 silencing. Consistently, p53 silencing abrogated the regulatory effects of STAT1 overexpression on the apoptotic level of OCPs and osteoclast differentiation, while STAT1 dephosphorylation mutation exerted no significant effects on the aforementioned osteoclast-related parameters in the context of p53 silencing. Taken together, RANKL-induced STAT1 phosphorylation at Ser727 promotes OCP apoptosis by augmenting p53-mediated transcriptional activation of Bax, which acts as a negative regulator of RANKL-driven osteoclastogenesis. Based on these observations, we propose that targeted interventions aimed at enhancing STAT1 phosphorylation may serve as a promising therapeutic strategy for osteoclastogenic bone loss.

Transient plasma membrane domains called lipid rafts have emerged as important regulators of signal transduction. These territories are formed by lipid-lipid and lipid-protein interactions, and these local interactions can be scaffolded by resident lipid raft organizing protein family members. While roles for lipid rafts have been described for multiple signaling pathways in many contexts, their in vivo prevalence and roles during embryonic development remain incompletely understood. Here we examined gene expression for the Raftlin family of lipid raft organizing proteins, Raftlin-1 (RFTN1) and Raftlin-2 (RFTN2), over the course of early vertebrate development, with a focus on neural crest cell dynamics. By analyzing transcriptomic data across vertebrate species, we identified conserved patterns of RFTN1 and RFTN2 expression across species, where RFTN1 is broadly expressed at low levels, while RFTN2 is distinctly enriched in neural crest cells. We used fluorescent in situ hybridization to spatially define Raftlin gene family expression patterns in the early avian embryo. Our results show that RFTN1 is broadly expressed with periods of enrichment in the developing paraxial mesoderm. In contrast, RFTN2 expression is strongly enriched in neural crest cells, beginning during specification and persisting through migration, with additional expression in both the cranial and intermediate mesoderm. Together, these patterns suggest that Raftlin proteins may play important roles in regulating signaling during development with potential roles in somitogenesis and in neural crest and mesodermal cell migrations.

Cartilage and bone that comprise craniofacial structures as well as neurons and glia of the peripheral nervous system are derived from a multipotent population of cranial neural crest cells, that respond to both cell intrinsic and extrinsic cues to differentiate into precise cell states. Both a genetic and epigenetic regulatory network are required for each step in the differentiation process, involving transcription factors, histone modifiers and chromatin remodelers. Here, we examined the direct transcriptional targets of two histone methyltransferases, Prdm3 and Prdm16 in zebrafish neural crest cells at 48 h post fertilization in zebrafish. Using CUT&RUN, we examined both direct DNA binding and nucleosome association. At this stage of development, CUT&RUN fragment size analysis indicated that Prdm3 and Prdm16 are largely associated with nucleosomes. We further analyzed these nucleosome peak sets to identify 6 clusters where differential binding of Prdm3 and Prdm16 and differential enrichment of gene ontology terms for target genes was observed. We validated gene expression in each cluster by in situ hybridization chain reaction (HCR) at 48 hpf demonstrating that prdm3 and prdm16 mutants exhibit corresponding changes in gene expression of the putative gene targets identified. Finally, we performed CUT&RUN-qPCR in prdm3 and prdm16 mutant zebrafish embryos and demonstrated reduced binding at putative target loci. Together these data suggest that Prdm3 and Prdm16 regulate their transcriptional targets primarily by binding nucleosomes around their putative target loci to control downstream gene expression.

This article reports the joint meeting of the Indian Society of Developmental Biology (InSDB) and the International Society of Differentiation (ISD), held at the Indian Institute of Science Education and Research (IISER) Berhampur, India, from 14th to 17th December, 2025. The meeting brought together scientists from across the world and showcased the latest research in development and differentiation.

Early vertebrate development involves several highly orchestrated morphogenic movements. Two key movements are gastrulation, which organizes germ layer spatial arrangement, and neurulation, which results in the establishment of the neural tube. In this study, we demonstrate that the POZ-ZF transcription factor xZnf131 is required for proper morphogenetic movements during gastrulation and neurulation in Xenopus laevis. xZnf131 is expressed throughout early Xenopus development, with enriched localization in dorsal and anterior embryonic structures, including the neural plate. Morpholino oligonucleotide-mediated depletion of xZnf131 results in defects in axial mesoderm extension as well as neural tube formation. Together, these findings establish xZnf131 as a critical regulator required for the proper morphogenetic movements during early Xenopus development.

Endocardial cushions, derived from the atrioventricular canal (AVC) and outflow tract (OFT), serve as the primordia for the formation of the septum and valves. Improper morphogenesis of the endocardial cushion leads to septal and valvular abnormalities, which contribute to the largest proportion of congenital heart diseases (CHDs). The development and maturation of the endocardial cushion rely on endothelial-to-mesenchymal transition (EndMT), a process by which endocardial cells undergo morphological remodeling and develop into the endocardial cushion mesenchyme. This biological event is strictly governed by regional signals originating from the myocardium, endocardium, and mesenchyme. Various signaling pathways, including TGFβ/BMP, Wnt, Notch, Hippo/YAP, PI3K/AKT, VEGF, and mechanical stress, are involved in the modulation of endocardial cell proliferation, migration, and mesenchymal transition. Transcription factors, such as SMAD, SNAIL, TWIST, TBX2, SOX9, KLF2/4, and NFATc1, exert distinct effects on endocardial EndMT. Maternal environmental exposures can disrupt signaling pathways and transcriptional network during endocardial cushion development, thereby enhancing susceptibility to CHDs. This review summarizes the mechanistic pathways and transcriptional regulation in AVC endocardial cushion development and EndMT, highlighting their association with CHDs.

Human THP-1 monocyte-like cells are a commonly used model for monocyte-derived macrophages (MoDM) in vitro, and significant literature has demonstrated how MoDM phenotype depends on the type, concentration, and duration of differentiation stimuli. However, far less attention has been given to how the choice of differentiation protocols actively reprograms transcriptional signaling networks during the differentiation process. Monocytes begin transcriptional and signaling rewiring immediately upon exposure to differentiation cues, suggesting that these early events condition how macrophages later respond to inflammatory and polarizing stimuli. As a result, differentiation protocols should be considered not as equally neutral preparatory steps, but as biologically active variables that modulate downstream signaling behavior. We compared three differentiation stimuli: PMA, GM-CSF, and M-CSF, to investigate how each agent influenced the temporal activity of key transcription factors. We generated THP-1 cells expressing bioluminescent reporters for CMV, NF-κB and STAT6, enabling longitudinal, quantitative assessment of transcription factor activity throughout differentiation. We found that PMA and GM-CSF enhanced NF-κB activation, whereas M-CSF led to higher STAT6 activity. We also observed that PMA induced early spikes in NF-κB activity and GM-CSF delayed NF-κB activation at later differentiation stages, revealing distinct temporal signaling programs. Differentiation stimuli also induced early-stage downregulation of CMV promoter activity, underscoring global transcriptional remodeling during macrophage maturation. Cytokine assays following secondary polarization demonstrated that CSF-differentiated macrophages exhibited greater responsiveness and dynamic range compared with PMA-differentiated cells. Together, these findings demonstrate that THP-1 differentiation protocols encode stimulus- and time-dependent transcriptional states that directly impact macrophage function. This study establishes temporal transcription factor profiling as a powerful framework for selecting differentiation strategies and experimental timepoints, improving biological relevance and reproducibility in in vitro macrophage models.

Precise regulation of pre-mRNA splicing is essential for normal development, and its disruption represents an important but frequently underrecognized mechanism of human disease. The C-type natriuretic peptide (CNP) receptor NPR2 is a critical regulator of growth plate chondrocyte proliferation and differentiation, and loss-of-function variants in NPR2 cause acromesomelic dysplasia, Maroteaux type (AMDM). Here, we identify a homozygous synonymous NPR2 variant (NM_003995.4:c.2484C > T) in an individual with AMDM and demonstrate its pathogenic mechanism at the RNA level. Although predicted to be silent at the protein level, in silico analysis suggested splice donor gain. Functional analysis using patient-derived leukocyte RNA revealed aberrant splicing leading to partial exon truncation, frameshift, and premature termination of NPR2 which is predicted to trigger nonsense-mediated mRNA decay given its position upstream of multiple downstream exon-exon junctions. Heterozygous family members expressed both normal and aberrant transcripts, whereas the affected individual showed exclusive expression of the aberrant isoform, consistent with a dosage-dependent loss-of-function mechanism. These findings establish aberrant splicing induced by a synonymous variant as a disease-causing mechanism affecting a core developmental signaling pathway. Our study highlights the importance of transcript-level functional analysis in the interpretation of rare variants and underscores the central role of precise RNA processing in skeletal development and human disease.

Polycystic ovary syndrome (PCOS) is a complex heterogeneous endocrine disorder characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology. Emerging studies suggest that ovarian fibrosis, as an underrecognized pathological feature, may contribute to ovarian dysfunction and infertility in PCOS phenotypes. Available experimental and limited human studies indicate that hyperandrogenism, insulin resistance, chronic low-grade inflammation, oxidative and endoplasmic reticulum stress collectively promote extracellular matrix accumulation and stromal remodeling predominantly through transforming growth factor-β (TGF-β)-mediated signaling pathways. Additionally, fibrotic remodeling has been linked to other pathways, including the NLRP3 inflammasome, PI3K/Akt, Hippo/YAP, and epithelial-mesenchymal transition. Although standardised diagnostic criteria are currently lacking, new imaging modalities, such as shear-wave elastography, and circulating fibrosis-related biomarkers may offer non-invasive approaches to evaluating ovarian fibrotic alterations. Furthermore, several anti-fibrotic agents, metabolic modulators, phytochemicals, and stem cell-based therapies have been suggested to have potential therapeutic effects in ovarian fibrosis models of PCOS. Overall, ovarian fibrosis represents a developing aspect of PCOS pathophysiology that requires further clinical and translational investigation.

Palmatine, a protoberberine alkaloid with recognized anti-inflammatory and anti-resorptive activities, has recently attracted interest as a potential modulator of bone homeostasis; however, its direct osteo-inductive effects on human adipose-derived mesenchymal stem cells (hADMSCs) remain undefined. Given the accessibility, proliferative efficiency, and clinical relevance of hADMSCs, identifying natural small molecules capable of enhancing their osteogenic differentiation is of considerable therapeutic significance. This study investigated the osteogenic potential of Palmatine using a multi-level in vitro approach. Cytotoxicity assays established a non-toxic working concentration. Osteogenic differentiation was assessed by quantifying alkaline phosphatase (ALP) activity, calcium deposition, and matrix mineralization using Alizarin Red and von Kossa staining. Mechanistic insight was gained by evaluating antioxidant enzyme activity (superoxide dismutase, catalase) and profiling key osteogenic genes via real-time RT-PCR. Low-dose Palmatine (1 μM) enhanced hADMSC viability, whereas higher concentrations displayed dose-dependent cytotoxicity. Functional assays demonstrated that Palmatine significantly increased ALP activity and calcium content at both early (Day 7) and late (Day 14) stages. Transcriptionally, Palmatine promoted early upregulation of Runt-related transcription factor 2, osteocalcin, and osteonectin, followed by robust induction of ALP and collagen type I at later stages, reflecting canonical osteogenic progression. Furthermore, Palmatine augmented antioxidant capacity through early elevation of superoxide dismutase and later activation of catalase, suggesting a redox-associated mechanism that stabilizes osteogenic signaling and supports matrix maturation. Overall, these findings provide the first comprehensive evidence that Palmatine functions as a potent, multi-level osteo-inductive molecule in hADMSCs, highlighting its promise as a natural candidate for redox-modulated bone regenerative strategies.

Precise dorsoventral patterning in Xenopus embryos depends on coordinated transcriptional regulation between organizer and ventral genes. Goosecoid (Gsc) promotes dorsal identity, whereas Ventx1.1, a downstream effector of BMP signaling, specifies ventral fate. Although their antagonistic relationship has been described, the cis-regulatory basis underlying their reciprocal repression has not been clearly defined. Here, we demonstrate that Gsc and Ventx1.1 reciprocally repress each other's transcription through specific cis-regulatory elements within their promoters. Serial promoter deletions and site-directed mutagenesis identified a functional Goosecoid response element (GRE) in the ventx1.1 promoter and Ventx1.1 response elements (VREs) in the gsc promoter. Mutation of these elements markedly reduced reciprocal repression in luciferase assays, and Chromatin immunoprecipitation revealed enrichment at the corresponding promoter regions during gastrulation. In addition, the neuroectoderm-specific transcription factor Foxd4l1.1 also repressed gsc, indicating an additional layer of transcriptional control that refines spatial gene expression. Together, these findings identify a promoter-level reciprocal repression circuit between Gsc and Ventx1.1 and suggest that this circuitry contributes to the refinement of dorsoventral patterning in Xenopus gastrulae.