Development

In ascidian embryos, gene expression from the zygotic genome begins between the 8- and 16-cell stages. While most zygotic genes are expressed in specific cell lineages at these stages, transcription factors that provide spatial cues for establishing specific expression patterns are insufficient to activate target genes at normal physiological levels. Gata.a is a transcription factor that provides spatial cues for targets expressed specifically in the animal hemisphere. Intriguingly, it is also required for physiological-level expression of many zygotic genes expressed in the vegetal hemisphere. In the present study, we found that Tbx21 and Klf6/7 augment the latter function of Gata.a. To determine the global extent of genes under control of these factors, we identified genes zygotically activated in early embryos using RNA-sequencing of BrU-labelled zygotic mRNAs. Our results revealed that approximately 80% of all zygotically activated genes were under control of these three factors. That is, together, Gata.a, Tbx21, and Klf6/7 are necessary to regulate target gene expression at physiological levels. This requirement for a specific set of broadly distributed factors resembles those of pioneer transcription factors that trigger zygotic genome activation (ZGA) in other animals, including flies and vertebrates. Regulatory factors involved in ZGA vary among animals, and our results indicate that ascidians use a distinct set of transcription factors for ZGA.

Germ granules are condensates in germ plasm, a specialized cytoplasm that segregates to the embryonic germline. In Drosophila, translation of nanos mRNA occurs at the surface of germ granules, suggesting that the granules promote translation. In C. elegans, however, germ (P) granules are not essential for Nanos expression. Using single-molecule imaging in C. elegans embryos, we map the distribution of translating and non-translating molecules of the Nanos homolog nos-2 and two other maternal mRNAs enriched in P granules. In early germline blastomeres, these mRNAs are not translated and distribute between the cytoplasm and P granules. At translation onset, mRNA molecules in the cytoplasm are translated, while most mRNA molecules in the P granules remain non-translating. nos-2 translation requires a rise in the concentration of the RNA-binding protein POS-1, which occurs independently of P granules. Consistent with low translation inside the granules, P granules are depleted of ribosomes and 43S pre-initiation complexes. Our observations suggest that germ granules promote Nanos protein expression by concentrating Nanos mRNA in germline precursors, but do not directly promote translation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/735846v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1875b06org.highwire.dtl.DTLVardef@16919f6org.highwire.dtl.DTLVardef@1278c27org.highwire.dtl.DTLVardef@1628645_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisGerm granules are condensates proposed to regulate the translation of mRNAs like Nanos that code for germ cell fate determinants. Using single-molecule imaging in C. elegans embryos, this study shows that P granule scaffolds concentrate mRNAs in germline precursors, but do not control the activity of translational regulators. - P granules concentrate mRNAs but are depleted of ribosomes and 43S pre-initiation complexes - Translation occurs mainly in the cytoplasm where ribosomes are most abundant - nanos translation onset is timed by a rise in POS-1, which counteracts the repressor SPN-4; both enrich in P granules but act independently.

A central unresolved question in development biology is how systems of overwhelming complexity arise from relatively few families of growth factors. Compounding this issue, signaling pathways often show signal convergence, where many ligands interact with fewer receptors, which then signal through a single second messenger complex. Here we investigate this question in the context of bone morphogenetic protein (BMP) signaling and its role directing dorsal spinal cord development, focusing on two receptor-regulated (R) Smads, Smad1 and Smad5. Multiple models have been proposed for their mode of action from acting redundantly through combined signal strength, to having distinct activities that drive different fate outcomes. We sought to distinguish between these models by generating CRISPR-edited Smad1 and Smad5 null mouse embryonic stem cell (ESC) lines to dissect the cell fate of activities of individual R-Smads, with a resolution not possible in vivo. Using a directed differentiation protocol for dorsal interneurons (dI), together with bioinformatic analyses, we have defined the roles of the R-Smads at key decision points along the dI specification timeline. Together, these findings support a model in which Smad1 and Smad5 play largely distinct roles in dorsal spinal cord development. While both R-Smads can activate canonical BMP-responsive transcriptional targets, they asymmetrically contribute to cell fate specification. Smad1 plays a restricted role, while Smad5 has a dominant role, regulating dorsal progenitor transcriptional dynamics and reiteratively directing the dorsal-most dI fates.

Vascular Endothelial Zinc Finger 1 (VEZF1) is essential for embryonic development, but its role in pluripotency exit remains unclear. Previous work showed that Vezf1-deficient ESCs exhibit impaired differentiation, reduced Dnmt3b expression, and genome-wide hypomethylation. Here, we show that Vezf1-/- ESCs fail to efficiently repress the pluripotency transcriptional program during differentiation, a defect that persists after ectopic Dnmt3b expression. Genome-wide analysis revealed VEZF1 occupancy at regulatory regions of genes involved in several developmental signaling pathways, including MAPK, WNT, and Hippo, as well as at some pluripotency-associated genes. Many VEZF1-bound MAPK genes showed reduced expression in undifferentiated Vezf1-/-ESCs, suggesting that VEZF1 activity contributes to transcriptional competence required for efficient pluripotency exit. VEZF1 loss also led to widespread acquisition of new CTCF sites associated with developmental signaling, a subset of which overlapped VEZF1-bound regulatory regions. CTCF depletion had only limited effects on the expression of the VEZF1-bound MAPK genes examined, indicating that increased CTCF occupancy alone is insufficient to explain their reduced expression in Vezf1-/- ESCs. Together, our findings identify a DNMT3B-independent function of VEZF1 in facilitating the exit from pluripotency and establishing transcriptional competence for differentiation, while revealing a potential role for VEZF1 in regulating CTCF occupancy during developmental state transitions.

Reelin is a secreted extracellular matrix protein that regulates neuronal migration and layer formation in the developing brain, yet its role in retinal development remains incompletely defined. Here, we investigated Reelin function in retinal lamination using wild-type and Reeler (Reln-/-) mice, combining stage-resolved RNA in situ hybridization, immunohistochemistry, and single-nucleus RNA sequencing. We show that Reln is dynamically expressed in ganglion cell layer and inner nuclear layer neurons during retinal development and persists in discrete adult neuronal populations. Loss of Reelin leads to widespread defects in retinal organization affecting both neurons and Muller glia. In Reln-/- retinas, Muller glia exhibit reduced Glul positive extensions, indicating impaired glial scaffold maturation. Early-born neuronal populations are also disrupted, with altered spatial organization markers associated with retinal ganglion cell differentiation within the ganglion cell layer at postnatal day 9. Horizontal cells are significantly reduced with dorsal-predominant vulnerability, while cone photoreceptors are generated in normal numbers but show incomplete positioning within the outer nuclear layer. Together, these findings identify Reelin as a key regulator of retinal lamination that coordinates neuronal positioning with Muller glia morphogenesis, extending its canonical role in brain development to the vertebrate retina.

MyoD plays a central role in determining the skeletal muscle lineage in vertebrate embryos. The core enhancer (CE) and distal regulatory region (DRR) are the only known MyoD enhancers, and together, they recapitulate all major aspects of MyoD expression in the embryo. However, knocking out each enhancer individually has only modest effects on MyoD expression. Here, we show that embryos lacking both enhancers maintain muscle-specific MyoD expression, indicating the existence of unknown MyoD regulatory elements. Precision run-on sequencing together with available ChIP-seq and DNase I hypersensitivity datasets identified three new candidate enhancer regions within 96 kb of MyoD 5 flanking sequences. Transgenic analysis revealed that DNA elements at -36 and -60 kb are active in all muscle-forming regions, each recapitulating aspects of endogenous MyoD expression. Enhancer activities in muscle regulatory factor-deficient mice suggest that they are components of the auto- and cross-regulatory circuitry that maintains MyoD expression. Analysis of Hi-C data showed that the entire -96 kb region constitutes a loop domain, within which multiple interactions between elements and with the MyoD gene were detected. A larger loop domain delimited by CTCF sites was also identified from -96 kb to +220 kb relative to the MyoD transcriptional start site. These data indicate that the newly identified enhancers are key components of a cis regulatory network that controls the activation and maintenance of MyoD expression in the embryo. One sentence summaryCis regulation of MyoD transcription during development

During development axial patterning of the otic vesicle sets up the blueprint for the adult inner ear. While human inner ear organoids recapitulate the initial steps of ear formation, whether and how vesicles are properly regionalised remains unknown. Here, we combine re-analysis of published single-cell transcriptomic datasets with 2D and 3D imaging to assess axial patterning in human stem-cell-derived otic vesicles using a new PAX2-reporter line. We find that the transcriptional signatures of individual cells do not conform to clearly defined regional characteristics, with some cells co-expressing conflicting regional markers, while markers that should be co-expressed are not. Some vesicles establish spatially segregated molecular domains. However, patterning is incomplete, stochastic, and restricted to a subset of vesicles. Within the same aggregates, patterned axes lack consistent orientation. Together, our findings point to incomplete segregation of transcriptional programs that are mutually exclusive in vivo and to the absence of morphogen gradients that are crucial for otic vesicle patterning. >One sentence summaryIn human inner ear organoids, otic vesicles show incomplete and disorganized regionalisation pointing to fundamental limits of self-organization, while suggesting new routes to increase cell complexity in vitro.

Fat and Dachsous cadherins are large transmembrane proteins that regulate tissue growth and planar cell polarity across species. In mammals, Fat4 is known to bind Dchs1, but whether it also binds Dchs2 is unclear. Here we show that Dchs2 binds Fat4 in a trans-heterophilic manner at cell-cell contacts, mirroring the Fat4-Dchs1 interaction. In the developing mouse retina, Fat4 and Dchs1 expression peaks embryonically and declines after birth, whereas Dchs2 expression begins postnatally and persists into adulthood, with all three co-expressed from birth through the second postnatal week. While Fat4 and Dchs1 mutants show no defects in retinal lamination or cell-type composition, loss of Dchs2 increases number of cones, reduces Muller glia number, and results in disorganization of photoreceptor outer segments. Electron microscopy shows that Dchs2 mutant photoreceptors fail to form the regular nuclear columns seen in controls, with disrupted stacking and breaks in the outer limiting membrane. These findings identify Dchs2 as a novel Fat4 ligand and reveal a distinct, non-redundant requirement for Dchs2 in photoreceptor organization and outer limiting membrane integrity in the mature retina.

Neural crest (NC) multipotent stem cells give rise to many tissues including most of the peripheral nervous system, pigment cells and the craniofacial mesenchyme and skeleton. During gastrulation and early neurulation, cranial NC cells are specified in the ectoderm territory located between the anterior neural plate ectoderm and the future pre-placodal and lateral non-neural ectoderm. At the end of neurulation, NC cells undergo an epithelial-to-mesenchymal transition and migrate to various locations in the developing embryo where they differentiate. While the fine-tuning of NC specification is increasingly being elucidated, many questions remain, including how microRNAs may govern expression of gene programs during these processes. MicroRNAs are short non-coding 20-22 nucleotides-long RNAs which regulate gene expression through post-transcriptional repression. We have identified miR-219 as a candidate regulator of Xenopus NC development. Here, miR-219-dependent molecular pathways were investigated by morpholino knock-down and reveal NC phenotypes. The development of the NC and adjacent ectoderm was evaluated using whole mount in situ hybridization of key markers (pax3, zic1, xhe2, sox10, snai2, sox2), alcian blue cartilage staining, phenotype analysis, RNA sequencing of microdissected dorsal ectoderm and microRNA rescue experiments. While neural induction is mainly unaffected, miR-219 depletion alters gene expression programs associated with neural border development, resulting in loss of NC specification. HighlightsO_LImiR-219 depletion expands the neural border territory and disrupts neural crest specification. C_LIO_LImiR-219 depletion phenotypes are rescued with miRNA mimics. C_LIO_LImiR-219 morphant neural border expansion is rescued by pax3 depletion. C_LIO_LIRNA-seq reveals specific gene program modulation in miR-219 morphant neural crest. C_LIO_LImiR-219 is predicted to directly downregulate the neural gene Hes5.3. C_LI

Background: During vertebrate embryogenesis, the isthmic region spans the midbrain hindbrain-boundary of the neural tube and includes an organizer (IsO) that is essential for proper formation of adjacent brain regions, yet the molecular and cellular composition of the isthmic region remains unresolved. Results: We employed combined single-nucleus ATAC-seq and RNA-seq (scMultiome) in 13 and 16 hours-post-fertilization zebrafish embryos to molecularly resolve cell populations in the isthmic region and validated our findings in vivo by RNA fluorescence in situ hybridization. We identified two distinct isthmic cell populations (isthmic midbrain [IsMB] and isthmic hindbrain [IsHB]) that share expression of canonical isthmic genes, but that differ in their expression of midbrain vs hindbrain genes. We also uncovered a previously unrecognized heterogeneity within the IsHB, reflecting a canonical fgf8-expressing population anteriorly (IsO/r0a), and a novel fgf8-negative population posteriorly (r0p). We find that inhibition of Fgf signaling disrupts formation of the isthmic region, leading to loss of isthmic cell populations except a residual population characterized by a mixed neural identity. Conclusions: Using transcriptional and epigenetic characterization, we expand on prior anatomical and genetic analyses of the isthmic region to refine our understanding of its cellular organization and demonstrate that it consists of several subdomains.

The morphogenesis of the optic cup provides a robust system for studying how two apposed epithelial monolayers with distinct properties fold and stretch in a coordinated manner to form the primordial eye. While much research has been conducted on the temporal dynamics of retinal neuroepithelium invagination, the spatial organization and stretching of the retinal pigment epithelium has received less attention. The fish species Astyanax mexicanus offers a unique model to examine the mechanisms of optic tissue morphogenesis through a comparative lens, as it exhibits natural variation in eye development between its river-dwelling and cave-adapted morphs. Using quantitative 3D imaging of optic cups from both morphs, we found that RPE morphogenesis involves transient, graded, and anisotropic cell stretching that patterns the epithelium during optic cup shaping. Analyses of RPE nuclear spacing and cell morphology showed that tissue stretching gradually increases along the proximo-distal axis, suggesting maximal tension in the elongated distal RPE cells aligned along the optic cup meridians. Furthermore, nuclear volumes and apical surface areas of RPE cells scaled spatially along the same axis, independently of endoreplication. In the cavefish natural mutant, RPE expansion was delayed by over six hours and proximal stretching exhibited altered isotropy, indicative of disrupted temporal coordination and suggesting modified mechanical constraints. These results demonstrate that RPE morphogenesis is a highly heterogeneous process from a spatiotemporal perspective, offering new insights into the study of the biomechanical principles of eye development in vertebrates. Summary statementThis study reveals the emergence of cell morphology gradients within the retinal pigment epithelium during morphogenesis of the eye in two distinct populations of the same species of fish.

Gastrulation is thought to be driven primarily by forces generated within individual cells. These cell-intrinsic forces collectively induce tissue-scale flows and transform the monolayered embryo into a multilayered structure. However, as the embryo constitutes a mechanically closed system, these flows must be balanced by regions of resistance or anchoring to enable asymmetric morphogenesis. In the Drosophila embryo, integrin-mediated attachment of the blastoderm to the vitelline envelope has been shown to stabilize germ band extension at the organismal scale. Disrupting such an attachment leads to a characteristic twisting phenotype. Yet, how this attachment shapes concurrent global morphogenetic events remains unclear. We discovered that the integrin -subunit scab, which mediates the attachment, is expressed in three different regions of the cellular blastoderm near prominent invagination events. Through a combination of light-sheet imaging, genetic and mechanical perturbations, we demonstrate that integrin-enhanced friction is essential for unidirectional tissue flows in those regions, with effects including cephalic furrow positioning and epithelial stability. Guided by a minimal physical model, we further show that multiple attachment sites enhance the robustness and reproducibility of global tissue movements. Together, our results indicate that Drosophila gastrulation emerges from a balance between cell-intrinsic force generation and spatially distributed adhesion to the surrounding envelope, which together shape tissue flows at the embryo scale.

Apical extracellular matrices (aECMs) line exposed body surfaces to shape tissues and protect them from the environment. These aECMs often organize into complex patterns and structures, but how such matrices assemble remains poorly understood. Caenorhabditis elegans cuticle patterns initiate within the transient pre-cuticle, which then helps direct the placement of cuticle collagens. Pre-cuticle patterns arise through post-secretory sorting, which must involve specific molecular interactions among them. Consistent with such a model, Alphafold3 predicts a high confidence physical interaction between two pre-cuticle proteins, the lipocalin LPR-3 and the Hedgehog-related protein WRT-10, with a conserved N-terminal region of LPR-3 forming a {beta}-strand that incorporates into the {beta}-barrel-like structure of the WRT-10 WRT domain. Genetic studies showed that WRT-10 requires this LPR-3 region in order to become properly patterned in the pre-cuticle matrix. Furthermore, WRT-10 and the LPR-3 {beta}-strand region are required to pattern a specific cuticle substructure, the lateral alae ridges, but not for other LPR-3-dependent matrix roles. These data indicate that LPR-3 and WRT-10 are functional partners and support a "landing pad" model whereby physical interactions between them allow LPR-3 to recruit WRT-10 to specific aECM regions. Similar mechanisms may explain how other members of the C. elegans Hh-r family associate with the aECM. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/739337v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@18c3601org.highwire.dtl.DTLVardef@2da36dorg.highwire.dtl.DTLVardef@4461d1org.highwire.dtl.DTLVardef@171e287_HPS_FORMAT_FIGEXP M_FIG C_FIG Article SummaryAll animal skin is covered by a set of proteins, sugars and lipids that comprise the apical extracellular matrix (aECM). These matrix components can be organized into patterned ridges and other distinctive structures. This study addresses how such patterns form in the developing cuticle of the nematode C. elegans. The study provides evidence for a regulatory mechanism that enables one matrix protein to establish a pattern and then recruit a second protein into the same pattern.

Adult schistosomes can survive for decades in the hostile niche of the host vasculature, yet the mechanisms that support tissue repair and resilience remain poorly understood. Here, we show that both mechanical and chemical injury trigger robust proliferative responses in adult parasites and display distinct spatial patterns. We identify a bHLH transcription factor that responds specifically to mechanical injury and is required for injury-induced stem cell proliferation and parenchymal cell renewal. Downstream, we find a WD40 repeat-containing protein that acts within a subset of stem cells to mediate this regenerative program. Together these findings detail an injury-responsive regenerative program in schistosomes that supports both their longevity and resilience in vivo.

Neural progenitor cell differentiation is a complex process requiring the proper integration of instructive and permissive factors. Instructive cues including signaling molecules and transcription factor networks have been well studied in this context, but permissive factors such as cell homeostasis have not. Cell homeostasis is critical to support the health and stability of a cell and enable the cell to act on instructive differentiation cues. Our study investigates a homeostasis protein, FAF2, and its function in neural progenitor cells. FAF2 is an adaptor protein involved in endoplasmic reticulum (ER) associated degradation to remove misfolded proteins and restore ER homeostasis. Here we show that knocking out Faf2 in neural progenitor cells results in increased ER stress signature at the protein and transcription level, indicating a conserved functional role in neural progenitor cells. Induced neural differentiation of FAF2 deletion cells shows a failure of neurite development but RNA-seq indicates genes that support neural differentiation are induced. Reducing ER stress in FAF2 knockout cells with a small molecule inhibitor can rescue neural differentiation, providing evidence that excess ER stress contributes to the inhibited differentiation. Taken together, these results reveal that FAF2 is a critical protein in neural progenitor cells for the maintenance of ER homeostasis and execution of neural differentiation. Highlights- FAF2 is required to regulate ER homeostasis in neural progenitor cells - FAF2 knockout blocks differentiation of neural progenitor cells to neurons at the cell morphological level, but does not inhibit the mounting of transcriptional programs associated with neural differentiation. - Excess ER stress due to FAF2 knockout contributes to blocked neural differentiation.

The olfactory epithelium of adult mammals contains two populations of stem cells that support its remarkable ability to regenerate neuronal and non-neuronal cell types throughout life. How olfactory epithelial cell types are established during development, however, is not well understood. Here, we use genetic lineage tracing and single-cell RNA sequencing of the perinatal mouse olfactory epithelium to construct a developmental trajectory consisting of multiple lineages. We identify transitional states and lineage relationships between individual cells and establish Ascl1+ cells as the primary multipotent progenitors in the perinatal olfactory epithelium. Further, Ascl1+ cells become progressively restricted in their cell fate capacity over developmental time and appear to be transcriptionally primed toward specific lineages. We also predict signaling pathways that may contribute to lineage plasticity and niche permissiveness. Together, these results contribute to our understanding of how cell-intrinsic and-extrinsic signals contribute to the establishment of a stem cell niche.

Epithelial tubes are an essential component of many organ systems. The formation of their lumens depends on the close coordination of epithelial polarity, the apical actin cytoskeleton, and apical junctions, yet the mechanisms that organize the apical cytoskeleton and junctions downstream of polarity remain poorly understood. Here, we identify the Ste20 family kinase GCK-4, the single C. elegans ortholog of the mammalian kinases LOK and SLK, as a critical regulator of intestinal lumen formation. GCK-4 localizes to the apical membrane during early lumen formation and to microvillar tips as these structures develop. Its loss results in a lethal cystic lumen phenotype, without disrupting the establishment of epithelial polarity, and in occasional failure to maintain attachment between the pharynx and intestine. Lumens in gck-4 mutant animals show irregular junction patterning, severely impaired apical accumulation of both actin and the membrane-actin linker Ezrin/Radixin/Moesin protein ERM-1, and microvilli atrophy. In Drosophila and mammalian cells, the GCK-4 orthologs are thought to organize the apical actin network largely through phosphorylation of ERM proteins. In contrast, ERM-1 phosphorylation is only partially reduced in gck-4 mutants and is not abolished, indicating that GCK-4 acts through additional targets and is not the principal ERM-1 kinase in the intestine. Together, these findings identify GCK-4 as a key regulator of apical actin and junction organization during lumen formation, and demonstrate that Ste20 kinases can control apical cytoskeletal architecture at least partially independently of ERM phosphorylation.

A critical event in the development of the highly structured cerebral cortex is the appropriate generation of differentiated daughter cell populations from asymmetric precursor cell divisions. Internalization of extracellular receptors and ligands in the process of endocytosis is key to the establishment of asymmetry. However, the detailed mechanisms mediating this exchange are incompletely understood. The dynamin family of membrane remodeling GTPases is considered critical for many forms of clathrin-mediated endocytosis (CME) but its role in brain development is unexplored. Here we found that dynamin 2 (Dnm2), a protein involved in CME through vesicle release from the plasma membrane, is essential for the maintenance of neural stem cells. Conditional deletion of Dnm2 leads to early exhaustion of neural precursor cells and premature neurogenesis, resulting in gross structural abnormalities, periventricular hemorrhaging and increased perinatal mortality. Notably, Notch ligands accumulate at the cell surface and cleaved NICD is reduced in neural stem cells, consistent with impaired ligand-mediated activation. In contrast, inhibition of CME in receptor-expressing cells increases cell autonomous Notch signaling, likely by promoting receptor accumulation at the plasma membrane. Further, dynamin 1, 2, and 3 can rescue loss of Dnm2 to different extents. Taken together, these findings reveal that Dnm2 is the essential isoform for physiological non-cell autonomous Notch-mediated neural stem cell maintenance, and that CME exerts fundamentally distinct roles in Notch signaling, promoting ligand activity while restricting receptor signaling through control of surface receptor abundance.

Lamins are the major structural components of the nuclear lamina with a variety of roles in development and organogenesis. However, the function of lamins during trophoblast development, the first lineage to differentiate during mouse embryogenesis, remains unexplored. By utilizing an in vitro trophoblast stem cell differentiation model in a lamin null setting, we uncover that lamins maintain expression of genes related to trophoblast differentiation, while repressing genes involved in maintaining trophoblast stem cell stemness and off-lineage development. By deleting different combinations of lamins in mice, we show that both lamin triple-knockout and lamin-A and -B1 (lamin-A/B1) double-knockout result in placental defects, including reduced placenta size and disrupted placental organogenesis at embryonic day (E)9.5. At this stage, lamin-A/B1 are expressed in trophoblast giant cells of the placenta, and lamin-A/B1 loss leads to their impaired maturation in vivo. Lamin-A/B1 double knockout trophoblast giant cells exhibit reduced nuclear size along with a reduction of DNA damage signaling foci, suggesting a role for lamins in supporting trophoblast giant cell polyploidization. Similar to the transcriptional dysregulation observed during differentiation of lamin triple knockout trophoblast stem cells in vitro, lamin-A/B1 knockout in vivo results in downregulation of genes related to trophoblast giant cell function and upregulation of off-lineage genes. Our results suggest lamins are required for placental organogenesis by maintaining polyploidization and lineage-associated transcriptional programs in trophoblast giant cells.

The mature kidney contains approximately one million nephrons, and defects arising during nephron development can result in lifelong renal impairment, often culminating in kidney failure and transplantation. Nephric tubule formation requires coordinated epithelial processes, including polarity, adhesion, signaling, and vesicle transport; however, how these processes are integrated during kidney morphogenesis remains unclear. Dynamin binding protein (Dnmbp) is a multi-domain scaffolding protein expressed in human kidneys that is involved in several cellular processes. Using the Xenopus embryonic kidney, we previously demonstrated that Dnmbp is essential for nephrogenesis, yet the mechanisms by which it influences nephron development remain undefined. Here, we identify Dnmbp as a novel interacting partner of the Wnt/planar cell polarity effector Daam1. The interaction between Daam1 and Dnmbp was independently identified in two yeast two-hybrid screens, biochemically verified, and supported by structural modeling predictions of a Daam1-Dnmbp complex. In developing Xenopus laevis kidneys, Dnmbp localized to punctate structures associated with E-cadherin-rich cell-cell contacts. Dnmbp depletion significantly reduced junctional E-cadherin localization in both epithelializing and mature nephric tubules without affecting total E-cadherin levels, indicating a role in E-cadherin recruitment or stabilization at adherens junctions. Furthermore, expression of human DNMBP rescued the junctional defects, confirming the specificity of the loss-of-function phenotype. Together, these findings identify Dnmbp as an essential regulator of kidney development and support a model in which Dnmbp provides a mechanistic link between Wnt/PCP signaling, Cdc42 activation, and adherens junction formation during nephrogenesis.