Developmental Biology

Eph and ephrin genes encode receptor-ligand pairs that mediate contact-dependent cell signaling and are essential for nervous system development. However, less is known about the role of Ephrin signaling during adult tissue homeostasis and regeneration. Here, we investigated the role of Ephrin signaling in neural patterning in the planarian Schmidtea mediterranea. We discovered that RNAi against the Eph receptor EphR1 led to striking ectopic expression of the mechanosensory neuron markers pkd1L-2 and hmcn-1-L, without obvious disruption of the overall architecture of the central nervous system. To investigate the basis of this phenotype, we identified additional Eph receptor homologs and four putative ephrin ligands and assessed their function. An RNAi screen revealed that ephrin-1 phenocopies the defects of EphR1 RNAi. Temporal analyses of EphR1 and ephrin-1 inhibition revealed a progressive increase in pkd1L-2+ and hmcn-1-L+ cells, indicating an unappreciated role for Ephrin signaling in regulating neural patterning and cell number during adult tissue homeostasis. Together, these findings provide a framework for dissecting Ephrin-dependent mechanisms in adult tissue maintenance and regeneration.

Amphibians develop both external and internal gills during ontogeny, offering an opportunity to investigate the developmental relationship between these positionally distinct respiratory organs. Although internal gills of vertebrates are widely accepted to arise from pharyngeal endoderm, external gills have long been regarded as purely ectodermal outgrowths, obscuring their relationship to other vertebrate gills. Here, we combine histological analysis with direct lineage tracing in the Mexican axolotl (Ambystoma mexicanum) and the African clawed frog (Xenopus laevis) to resolve the embryonic origin of amphibian gills. We show that the external gill develops as a continuous epithelial extension of the pharyngeal endoderm, which forms its basal epithelium and reaches the distal gill tip. In the frog, this extension remains continuous with the epithelium giving rise to the internal gills. Rather than representing separate epithelial structures, external and internal gills therefore arise from a shared epithelial domain of the pharyngeal endoderm. These findings resolve a longstanding question concerning the embryonic origin of amphibian gills and provide a developmental viewpoint for understanding how spatially diverse vertebrate gills can evolve through repeated modification of a conserved endodermal tissue.

Epithelial tube morphogenesis is critical for the function of many organs. Basement membranes underlie epithelia and their remodeling is a key step to reach the correct size and shape. Key regulators that mediate basement membrane remodeling for tube elongation and branching remain largely unknown. We analyze the expression and function of AdamTS-B, a matrix metalloprotease, in the respiratory system of Drosophila. Here we show, that AdamTS-B is expressed early in tracheal development during placode formation. We generated a mutant line of AdamTS-B, which is lethal. Analysis of trachea morphogenesis in this AdamTS-B mutant reveal a function in tube elongation and cell migration. Our results suggest that AdamTS-B control BM remodeling required for organ shape.

The laminar architecture of the mammalian neocortex depends on precise radial migration of newborn neurons to the appropriate cortical layers. This process is governed by the integration of extracellular signals with cell-intrinsic transcriptional programs. BMP signaling has been previously demonstrated to be essential for radial migration of late-born (E15.5) upper-layer cortical neurons. However, the gene expression programs downstream of BMP signaling that regulate this process remained unknown. To address this, we combined temporally targeted in utero electroporation with GeoMx Digital Spatial Profiling (DSP) to map BMP-responsive transcriptional programs in E15.5-born layer II/III neurons at two defined developmental timepoints: E17.5, when neurons actively migrate through the intermediate zone, and postnatal day 0 (P0), when they have completed migration and have attained their laminar position. BMP inhibition produced largely non-overlapping transcriptional changes at these two stages. At E17.5, chromatin-regulatory programs and ribosomal protein gene expression were collectively upregulated upon BMP inhibition. However, by P0, the same cohort of ribosomal genes exhibited downregulation while membrane lipid biosynthesis and synaptic specialization pathways became dominant, revealing a stage-dependent transcriptional switch. A subset of shared BMP-responsive genes was regulated in opposite directions at these two stages, which lent further support to the hypothesis that there is a temporal reorganization of BMP-dependent transcriptional outputs. We selected four candidates from among the BMP-responsive genes for functional studies, namely Mfap4, Olfm2, Adora1, and Arpp21, which belong to diverse functional categories, including extracellular matrix proteins, G protein-coupled receptors, secreted glycoproteins, and RNA-binding proteins. RNAi-mediated knockdown of all four candidates resulted in radial migration defects that closely phenocopied inhibition of BMP signaling, establishing these genes as functional effectors of the BMP signaling pathway regulating neuronal migration.

Renal cysts are categorized as non-pathogenic simple cysts and pathogenic malignant cysts based on their pathophysiological status. Cyst formation is divided by cyst initiation and cyst progression/promotion. Pathogenic cysts are thought to be developed through continuous initiation followed by progression until pathogenic status is achieved. Although many genetic and environmental factors are identified to cause pathogenic cyst formation, the mechanisms that discriminate cyst initiation and progression are poorly understood. Using genetic mutation models of ETV transcription factors, ETV1, ETV4, and ETV5, and a pharmacological inhibitor of hedgehog signaling, cyclopamine, we identified one of the mechanisms regulating cyst initiation and progression. Nephron specific deletion of ETV4 and ETV5 initiated cyst formation. However, cyst initiation did not continue as animals grow, and a limited number of the initial cysts underwent further growth. Additional deletion of ETV1 was required for continuous initiation in addition to promotion of cyst growth. Furthermore, administration of cyclopamine attenuated promotion of cyst progression but had little effect on cyst initiation. Therefore, we provide evidence that cyst initiation and progression is genetically and molecularly distinct and can be modulated. This information provides new insight into how to control renal cyst initiation and progression and can be used to suppress pathogenic cyst growth.

Zebrafish bone regeneration is a highly efficient process, enabling the complete restoration of an amputated fin within few weeks. The hallmark of this epimorphic regeneration is the formation of a blastema atop of a bony fin ray. Osteoblasts near the injury site dedifferentiate and migrate off the bone to contribute to the developing blastema. We show that an injury or a blastema alone is not sufficient to trigger off-bone migration of osteoblasts. Surprisingly, we found that blastema cells themselves possess intrinsic migratory properties. Moreover, when multiple injury sites are present, a preferential distal migration could be observed. We conclude that multiple injuries are hierarchical organized, and that injuries with the highest regenerative potential take priority.

HNF1B is a transcription factor required for proximal tubule (PT) specification during kidney development, but whether it is also required to maintain PT identity after differentiation remains unknown. Using PT-specific genetic deletion in mice, we found that loss of Hnf1b in differentiated PT cells causes cyst formation and early postnatal lethality. PT-specific transcriptomic analysis revealed downregulation of PT-specific gene programs, including Hnf4a and PT-enriched transport and metabolic genes. Strikingly, Hnf1b-deficient PT cells ectopically activated podocyte-specific genes, including Wt1 and Nphs1, demonstrating that PT cells retain the capacity to engage alternative nephron segment programs when identity-stabilizing mechanisms are disrupted. In addition, loss of Hnf1b disrupted epithelial integrity, as evidenced by reduced epithelial adhesion gene expression and induction of mesenchymal markers. Wnt/{beta}-catenin signaling was also aberrantly activated, suggesting broader dysregulation of epithelial homeostasis. These findings establish HNF1B as a critical post-specification regulator of PT identity that sustains PT-specific transcriptional programs and actively suppresses alternative segmental identity programs.

The Notch signaling pathway plays a central role in development and cell fate determination. Its function depends on tightly regulated intracellular trafficking of the Notch receptor and the Notch intracellular domain (NICD) after cleavage by {gamma}-secretase. Notch signaling is essential for principal cell differentiation within the renal collecting duct and for proximal-distal patterning during kidney development. Notch activity has also been shown to influence the trafficking of several membrane proteins, including nephrin in kidney cells and monocarboxylate transporter 1 in brain endothelial cells. Aquaporin-2 (AQP2) is the key vasopressin-regulated water channel in the collecting duct, and proper AQP2 trafficking and recycling are required for physiologically appropriate urine concentration. To determine whether and, if so, how Notch signaling modulates AQP2 trafficking, we performed studies using LLCPK1 renal epithelial cells stably expressing AQP2 (LLCPK1-AQP2). Exposing cells to 35 M DAPT (which inhibits y-secretase, preventing cleavage and activation of Notch receptor signaling) for 30 min significantly increased AQP2 membrane accumulation in LLCPK1-AQP2 cells as revealed by immunofluorescence staining. Using a rhodamine-transferrin internalization assay, we found that DAPT reduced clathrin-mediated endocytosis by 60%. This blockade increases AQP2 membrane accumulation by preventing the reinternalization of AQP2 that is delivered to the plasma membrane by exocytosis during its constitutive recycling pathway. Using an F-actin polymerization assay, we then found that Notch inhibition decreases F-actin polymerization by de-activating the small GTPase RhoA, using GSTRBD, a substrate that binds to active RhoA, as seen by western blotting using phospho-specific antibodies. Because actin polymerization is required for AQP2 endocytosis, RhoA inhibition by DAPT would result in the decreased internalization of AQP2 that we observed by immunofluorescence. While the mechanism by which DAPT inhibits RhoA activity remains to be determined, our study shows that AQP2 trafficking is regulated by the Notch signaling pathway in vitro and suggests that modulation of Notch signaling may represent a novel strategy to address water balance disorders that involve defects in the AQP2 trafficking process.

The Tbx1 gene is haploinsufficient in mice and in humans, where it causes a DiGeorge syndrome phenotype characterized by developmental deficits of the pharyngeal apparatus. TBX1 plays a critical role in the differentiation and regionalization of the cardiopharyngeal mesoderm lineage and its derivatives. Nevertheless, its regulation is incompletely understood. Here we used a combination of computational and wet-lab approaches to identify regulatory sequences of the Tbx1 gene, and we use single-cell molecular analysis as a read-out and to establish the consequences of their deletion. Results revealed a cluster of regulatory sequences with at least three distinct elements. Elimination of the entire cluster caused a near shut down of the gene, while individual deletions had milder, quantitative effects. Transcriptomic analyses of the deletion mutants revealed the down regulation of genes related to cardiopharyngeal lineage specification and, more surprisingly, up regulation and anteriorization of genes related to embryonic patterning, thereby providing a rationale for the severe dysmorphogenesis of the posterior pharyngeal apparatus observed in Tbx1 mutant mice.

Butterfly wings present a tremendous gallery of colorful patterns, offering a unique opportunity to study how developmental pattern formation processes evolve. We still do not understand the genetic basis of several key aspects of wing pattern development, however. Three paralogous POU domain transcription factors nubbin, ventral veinless (vvl), and pdm3 are all known wing development genes in Drosophila melanogaster. Here we combine gene expression and knockout approaches to show that each of these genes plays multiple novel wing patterning roles in the common buckeye butterfly, Junonia coenia. We found that nubbin controls eyespot pattern determination via a non-cell autonomous repressor-like effect originating at the wing veins, such that nubbin knockouts have larger eyespots. nubbin also regulates pigment identity and scale morphology across the wings. We also found that vvl regulates pigment identity of the discal bands and ventral hindwing. Last, we found that pdm3 is required for determining the outer rings of eyespot patterns, where it is co-expressed with spalt and the lncRNA ivory. pdm3 is also necessary for determining wing margin stripes, where it is again co-expressed with spalt, leading us to propose that the eyespot and wing margin gene regulatory networks could be homologous. Finally, pdm3 affects pigmentation of the ventral hindwing, phenocopying the seasonally-plastic color switch in J. coenia. Together, our work shows that POU domain transcription factors play diverse roles in butterfly wing pattern development and highlights nubbin as one of the first genes implicated in the repressive function of wing veins in color pattern determination. Highlights- Gene expression and knockouts reveal three POU factors regulate butterfly wing color pattern - nubbin regulates eyespot development, likely via a repressor from the wing veins - nubbin controls scale color and morphology across the wing - pdm3 coordinates eyespot development and is co-expressed with spalt and ivory - Expression of genes in the eyespot and wing margin suggests network homology

Neuromesodermal progenitors (NMPs) sustain vertebrate body-axis elongation by generating both neural and paraxial mesodermal tissues. Although signaling and metabolic pathways regulate NMP states, whether intercellular communication contributes to the coordination of progenitor behaviour and developmental timing remains unclear. Here, we identify GJA1, encoding connexin-43 (Cx43), as a gene dynamically enriched within the neuromesodermal competent domain of the chick embryo. Cx43-associated channels and hemichannels accumulate preferentially within the NMP population, and a photoactivatable tracer assay demonstrates enhanced connexin-mediated exchange within the posterior growth zone. Pharmacological inhibition of hemichannels or gap junctions revealed distinct contributions of these communication modes to transcriptional regulation across the NMP continuum. Gap-junction inhibition primarily altered SOX2 expression within progenitor populations, whereas hemichannel inhibition selectively affected TBXT expression in mesodermal cells. Connexin inhibition also reduced the relative size of the progenitor compartment and altered the spatial organization of newly formed somites. Strikingly, disruption of connexin-mediated communication impaired segmentation dynamics, leading to increased frequencies of off-pace segmentation events, accelerated segmentation timing and progressive deviation from the expected segmentation program. These defects emerged rapidly and accumulated over successive segmentation cycles, indicating a requirement for connexin activity in maintaining developmental robustness. Together, our findings identify connexin-43 as a regulator of neuromesodermal progenitor states and reveal a previously unrecognized link between intercellular communication and segmentation clock robustness during vertebrate axis elongation.

The conserved core planar cell polarity (PCP) pathway orients cells and subcellular structures within an epithelium through asymmetric protein localization and intercellular communication. In vestibular organs and lateral-line neuromasts, mechanosensory hair cells are interspersed among support cells and form opposing hair-bundle orientations along a shared axis, enabling bidirectional sensitivity to head motion and water flow, respectively. In zebrafish neuromasts, Notch-mediated lateral inhibition gives rise to two hair-cell populations, distinguished by differential Emx2 expression, that orient their cell-intrinsic polarity machinery differently relative to a PCP-dependent tissue-wide axis. However, it remains unclear how PCP proteins are organized across hair cells and support cells to achieve both opposing hair-bundle orientations and tissue-wide alignment, and whether PCP signaling remains required after hair-bundle polarity is established. Combining quantitative spatial mapping of the core PCP protein Vangl2 with cell-type-specific and temporally controlled protein degradation, we show that hair cells and support cells make distinct yet coordinated contributions to the polarized Vangl2 organization within neuromasts and to bidirectional hair-bundle polarity. Support-cell Vangl2 facilitates tissue-wide alignment of hair bundles along the anteroposterior axis, whereas hair-cell Vangl2 is required to generate opposing hair-bundle orientations along this axis. Vangl2 degradation after hair bundles have formed disrupts their tissue-wide alignment, showing that planar polarity is actively maintained rather than fixed after establishment. Together, these findings reveal how Vangl2-dependent PCP signaling is distributed across distinct cell types within a heterogeneous epithelium to generate opposing polarity outcomes and remains necessary to preserve tissue-level planar organization.

Mammalian preimplantation development requires precisely coordinated lineage decisions to establish the trophectoderm (TE), inner cell mass (ICM), epiblast (EPI), and primitive endoderm (PrE). Glucose metabolism and epigenetic regulation are increasingly recognised as key determinants of lineage specification during preimplantation development. However, how glucose-dependent metabolic cues interface with epigenetic mechanisms to regulate embryonic cell fate remains poorly understood. Here, we investigated the role of glucose in regulating protein methylation by protein arginine methyltransferases (PRMT) in bovine preimplantation development. PRMT1 and its associated histone mark H4R3me2a were detected throughout bovine oocyte maturation and embryo development. Pharmacological inhibition of Type I PRMTs using two structurally distinct inhibitors, GSK3368715 and MS023, markedly reduced global protein asymmetric dimethylarginine (ADMA) and H4R3me2a levels. PRMT inhibition impaired blastocyst cell proliferation, reduced total cell number, and disrupted both first and second lineage decisions, as demonstrated by decreased CDX2- and SOX2-positive TE and ICM cells and reduced NANOG- and GATA6-positive EPI and PrE cell allocation. Mechanistically, Type I PRMT inhibition downregulated key components of the Hippo-associated TE programme, including YAP, TEAD4, and TFAP2C. Consistent effects were observed in mouse embryos, where MS023 treatment reduced ADMA, CDX2, YAP, and TFAP2C expression and impaired TE and ICM allocation. Collectively, our findings identify Type I PRMT-mediated ADMA as an essential epigenetic regulator of early mammalian lineage specification and support a conserved ADMA-Hippo regulatory axis linking arginine methylation to embryonic cell fate decisions. In briefType I protein arginine methyltransferase (PRMT)-mediated asymmetric dimethylarginine (ADMA) is required for proper lineage specification during mammalian preimplantation development. ADMA depletion disrupts Hippo signalling, cell proliferation, and trophectoderm and inner cell mass allocation in bovine and mouse embryos.

Nitrogenous waste excretion is essential for all developmental stages of fish. Embryonic fish excrete urea, transitioning to cutaneous and later branchial ammonia excretion. In zebrafish, ammonia excretion involves rhesus glycoproteins Rhbg and Rhcgb in keratinocytes and ionocytes, but the developmental moment they appear in the gill remains unclear. Potential redundancy between Rhbg and Rhcgb in ammonia excretion is also not fully investigated, nor is the difference in response to low pH. We hypothesized that rhesus glycoproteins are partially redundant, and that they differ in their response to low pH as ammonia excretion enables ionocytes to exchange Na+ and H+ (Rh-NHE-metabolon). We predicted that a loss of rhbg or rhcgb induces compensatory responses. We characterized the transition from urea to branchial ammonia excretion from 0 to 8 days-post fertilization (dpf) and the response to pH 5.0 on the expression and localization of rhesus glycoproteins in control zebrafish and rhbg or rhcgb-crispants. Effects of high external ammonia (HEA, 500 M NH4Cl) and 10 mM HEPES-buffering were further characterized in rhcgb-crispants. Rhag and Rhbg appeared in the gill at 5 dpf, while Rhcgb appeared at 6 dpf. A loss of rhbg or rhcgb did not impact baseline N-excretion, illustrating that zebrafish can maintain ammonia excretion without the full complement of rhesus glycoproteins. We observed no compensatory increase in rhesus glycoproteins, but expression of the transporter hippocampus-abundant transcript 1b increased. HEA-exposed rhcgb-crispants switched to urea as primary nitrogen waste. Together, these findings underline the plasticity of the larval in dealing with nitrogenous waste.

Nuclear pore complexes (NPCs) are nuclear envelope (NE)-embedded protein assemblies that mediate nucleocytoplasmic exchange and interact with the genome, including binding of an NPC component Nup93 to Polycomb chromatin domains. Here, we investigated the in vivo relevance of this relationship in Drosophila, which unusually contains two distinct paralogs of Nup93. Interestingly, we identified a Nup93-2-specific tumorigenic phenotype in larval wings, where depletion of Nup93-2, but not Nup93-1, led to tumor-like overgrowth, reminiscent of Polycomb mutations. Consistently, our transcriptomic analysis revealed a wide-spread loss of gene silencing in Nup93-2-depleted wings, particularly in a Nup93-bound Polycomb domain spanning genes for activators of JAK/STAT signaling. Nup93 paralogs were not found to differ in their effect on NPC biogenesis but strikingly, showed differences in subnuclear localization patterns. While Nup93-1 co-localized exclusively with fully assembled NPCs, Nup93-2 exhibited only partial co-localization and was found at additional NE locations in a tissue-specific manner. Together, our results identify an in vivo silencing role of a Nup93 paralog and suggest that Nup93-2 may form a unique NE-associated complex that targets a subset of Polycomb domains containing growth-promoting genes.

Primary cilia coordinate signaling pathways that regulate tissue homeostasis and development, and defects in cilia contribute to numerous ciliopathies. However, the transcriptional consequences of disrupting ciliary protein localization remain poorly defined. ARL13B is a cilia-enriched regulatory GTPase required for ciliary trafficking and signaling. The ARL13BV358A variant is undetectable in cilia yet retains known biochemical functions, providing a unique model to investigate the functions of ciliary ARL13B independently of ciliogenesis. To define transcriptional programs associated with loss of ciliary ARL13B, we generated two independent Arl13bV358A/V358A kidney epithelial cell lines and matched rescue lines. The ARL13BV358A mutation did not affect ciliation frequency or cilia length but altered ciliary protein composition, including loss of ARL3 and INPP5E localization and increased accumulation of GPR161 and TULP3. RNA sequencing revealed expression changes in genes associated with ciliary biology, mechanotransduction, epithelial organization, and kidney-related phenotypes. Despite similar ciliary phenotypes, the independently-derived, mutant clones displayed substantial transcriptomic heterogeneity, highlighting a potential source of variation in CRISPR-based transcriptional studies. By integrating data from the independent mutant and rescue clones, we identified a high-confidence set of 131 genes whose expression reproducibly tracked with loss and restoration of ciliary ARL13B. Together, these findings demonstrate that ciliary ARL13B is required to maintain normal ciliary composition and gene expression programs and underscores the value of multi-clone, rescue-based experimental designs for robust transcriptomic analyses. Summary for ReviewersThis study examined how excluding the protein ARL13B from primary cilia affects kidney epithelial cells. The researchers created two independent cell lines carrying a modified form of ARL13B,along with matched rescue cell lines. The findings show that ciliary ARL13B helps maintain normal ciliary composition. By comparing the cell lines, the researchers identified a high-confidence set of genes associated with loss of ciliary ARL13B. By highlighting the importance of using independent gene-edited clones and rescue-based controls, these results advance understanding of how cilia regulate kidney cell function and provide guidance for designing robust transcriptomic analyses.

Satellite DNA is long arrays of tandem repetitive DNA located often near the centromeres of chromosomes, whose function, or lack of, has been debated since its discovery. Although situated in heterochromatin, satellite DNA may be expressed as long noncoding RNAs (lncRNAs). Although there are a few examples of satellite lncRNAs being characterized, and functions suggested, how widespread and functionally important they may be for developmental processes is not understood. Here, we take an evolutionary approach to investigate satellite lncRNA expression in Drosophila spp. ovaries, a tissue whose development is well-characterized but where satellite expression has only been minimally explored. Using a publicly-available total RNAseq dataset, we find that 118/156 surveyed satellite DNAs were expressed across 10 species, with 33 satellites having high expression over 20 RPM. However, all but two of these expressed satellites (AAACTAC in D. virilis and ACAGACAGACAGG in D. ananassae) had higher read counts in a sister smallRNA dataset, suggesting that most satellite transcripts primarily serve as precursors for piRNA biogenesis. The two "stand-alone" lncRNAs were highly strand-biased, with 96-97% of the total reads coming from one strand. We further investigated AAACTAC expression with RNA FISH and found the transcript is specifically present in the oocyte nucleus following a dynamic spatiotemporal pattern, with the highest expression in stage 3-5 oocytes. The transcription pattern of AAACTAC is conserved in the three other virilis clade species that contain this satellite DNA. Further, we found expression of unrelated satellites in more distantly related D. borealis and littoralis both in the oocyte and the nurse cells. Overall, our work identifies a novel lncRNA AAACUAC found in the early oocyte nucleus, which is conserved across ~5 MY of evolution, and is therefore a strong candidate for the discovery of novel functions of satellite lncRNAs in development.

Injuries to individual cells happen frequently as a result of physiological and environmental stresses during their normal daily functions that can lead to a ruptured cell cortex (plasma membrane and underlying cortical cytoskeleton). The capacity of cells to rapidly repair general daily injuries, as well as ones resulting from trauma, infection, or diseases/cancer, is essential for their survival. While we know the general cell biological outline of the highly-conserved physiological events taking place during cell wound repair, our knowledge of the molecular mechanisms governing the repair process is still fairly limited, due in large part to the lack of information regarding the molecules, machineries, and pathways involved. Here, we performed a genetic screen of 1322 fluorescent-tagged proteins to identify cell wound repair components that are recruited upon laser wounding or whose expression is lost and/or altered upon laser wounding. We identified 129 proteins that are recruited to wounds during the cell repair process through high resolution spatio-temporal expression analyses of these gene fusions in conjunction with a fluorescent actin reporter. Strikingly, we find that many members of the Rab family GTPases are recruited to wounds where, in addition to their well-known roles in intracellular membrane trafficking, they are affecting actin cytoskeletal organization and dynamics during the repair process. These studies are allowing us to define the earliest acting proteins, as well as those required at specific steps in the repair process based on their recruitment patterns and the precise timing of their recruitment to wounds. Thus, our imaging-based screen is providing us with a global view of the repair processes, as well as a large number of genes/gene families that provide new entry points for examining specific steps in the cell wound repair process. Author SummaryCells in our bodies get injured every day from normal activity, environmental stress, infection, or disease. To survive, they must quickly repair these injuries and restore normal function. While some molecules have been identified as key players of cell wound repair, many of the molecules involved and their roles remain unknown. In this study, we identified new molecules that are involved in different steps of cell wound repair. Using laser-induced injury in the Drosophila model, we examined 1322 proteins and observed their spatial and temporal dynamics in a cell after injury. From the 1322 proteins examined, we identified 129 proteins recruited to distinct regions around the damage site during cell wound repair, suggesting roles in specific steps of the repair process. Interestingly, a subset of these proteins are Rab family GTPase members, highlighting new roles for these proteins in regulating actin dynamics. By identifying new candidate repair molecules, we provide a foundation for understanding how cells maintain their integrity and how repair processes may be influenced by factors such as wound size, infection, aging, and disease.

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.

Controversy surrounds the lineage potential of cardiac cells, particularly epicardial cells, during heart development, largely due to the non-specific expression of epicardial marker genes and the resulting non-specific labeling in Cre-loxP mouse models. Using DARLIN mice, a CRISPR/Cas9-based lineage-tracing system independent of the Cre-loxP system, we analyzed the lineage development of embryonic cardiac cells in an unbiased manner and identified lineages shared among different cell types, such as epicardial cells and vascular endothelial cells (Vas_ECs). To further confirm the lineage potential of epicardial cells, we identified an epicardial cell-specific marker gene, Lrrn4, through analysis of a multi-staged single-cell mRNA-sequencing (scRNA-seq) dataset, and generated a corresponding Lrrn4-CreER mouse line. We then bred this line with a reporter mouse to confirm its specificity for labeling epicardial cells, and subsequently performed prolonged lineage tracing, which revealed specification of the labeled epicardial cells into Vas_ECs. Finally, Using this mouse line, we investigated epicardial cell function by selectively ablating these cells and by expressing TGF{beta} in epicardial cells to convert their lineage from Vas_ECs to fibroblasts. Both approaches resulted in significant developmental defects in embryonic hearts. Together, these results indicate that epicardial cells can give rise to Vas_ECs, and that the Lrrn4-CreER mouse model is a valuable tool for elucidating the role of the epicardium in heart development.