Development
· 2026 Jul · PMID 42396865
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The term 'housekeeping gene' evokes a sense of banality. These are the boring genes that hum in the background, impervious to cell state or stress. For a developmental biologist captivated by the impact of differentiatio...The term 'housekeeping gene' evokes a sense of banality. These are the boring genes that hum in the background, impervious to cell state or stress. For a developmental biologist captivated by the impact of differentiation or environmental cues on gene expression, housekeeping genes find their use as loading controls. It is easy to lose sight of the fact that housekeeping genes are perhaps the most important genes - they code for the essential cellular factors: cytoskeleton components, basal metabolic enzymes, ribosomal proteins, etc. Therefore, understanding the basis of their extremely robust transcriptional regulation has critical implications for development and life in general.
Prahlad M, Feng W, Weigle IQ
… +4 more, Bat-Erdene O, Chen Y, Marques F, Kratsios P
Development
· 2026 Jul · PMID 42383485
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Motor neuron (MN) diversity is essential for generating animal movement, yet the molecular mechanisms specifying MN subtypes remain poorly understood. We investigate how Hox genes and their PBX co-factors establish choli...Motor neuron (MN) diversity is essential for generating animal movement, yet the molecular mechanisms specifying MN subtypes remain poorly understood. We investigate how Hox genes and their PBX co-factors establish cholinergic MN identity along the anterior-posterior axis of the Caenorhabditis elegans ventral nerve cord. In anterior MNs, the Hox genes ceh-13 (Lab/Hox1) and lin-39 (Scr/Dfd/Hox4-5) collaborate with the co-factor ceh-20 (Exd/Pbx1-4) and terminal selector unc-3 (Collier/Ebf1-4) to activate terminal identity genes. In posterior MNs, the Hox gene mab-5 (Antp/Hox6-8) represses terminal identity genes by antagonizing unc-3 in a ceh-20-dependent manner. Both mab-5 and ceh-20 are required not only during early development but also in later life stages to maintain posterior MN identity. In lumbar MNs, egl-5 (Abd-A/Abd-B/Hox9-13) collaborates with unc-3 to activate lumbar-specific identity genes independently of ceh-20. We also find that ceh-20 is required for Hox gene expression in ventral nerve cord MNs, supporting a model in which Hox-positive autoregulation depends on PBX activity. Together, these findings provide a conceptual framework for understanding how spatial patterning information is integrated with terminal selectors to generate and maintain neuronal subtype diversity.
Baldwin HS, Tompkins K, Gong Y
… +3 more, Helm B, Liu Q, Qu X
Development
· 2026 Jun · PMID 42367008
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Abnormal cardiac trabeculation often leads to congenital heart disease (CHD). TIE2-ANG1 signaling is a key regulator of endocardial growth during trabeculation. However, little is known about the underlying mechanisms. H...Abnormal cardiac trabeculation often leads to congenital heart disease (CHD). TIE2-ANG1 signaling is a key regulator of endocardial growth during trabeculation. However, little is known about the underlying mechanisms. Here we demonstrate that loss of endocardial Tie2 via Nfatc1Cre resulted in increased nuclear localization of Forkhead box O1 (FOXO1), reduced phosphorylation of AKT (the direct regulator of FOXO1) and reduced expression of several proliferation promoting genes in endocardial cells (EdCs). The cardiac defects seen in endocardial Tie2-deficient hearts were phenocopied by forced activation of FOXO1 within the endocardium and partially rescued by loss of FoxO1. Conversely, excessive PI3K signaling (the direct regulator of phosphorylation of AKT) in EdCs caused cardiac hypotrabeculation. Further, we identify that endocardial loss of Tie2 resulted in impaired cleavage of versican, which was associated with enhanced expression of ADAMTS endogenous inhibitor TIMP3 in Tie2-cko EdCs. Thus, our results suggest that TIE2 signaling in the early embryonic endocardium promotes endocardial growth via sequestering FOXO1 within the cytoplasm and modulates cardiomyocyte proliferation via stimulating versican cleavage to orchestrate myocardial trabeculation, providing insights into the pathogenesis of CHD.
Israeli A, Nir D, Shwartz I
… +5 more, Levy M, Schmid MW, Burko Y, Efroni I, Ori N
Development
· 2026 Jun · PMID 42343828
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Plant organ formation involves an initial initiation stage followed by growth, together determining organ shape. The plant hormone auxin regulates both initiation and growth. However, it is still not clear how auxin faci...Plant organ formation involves an initial initiation stage followed by growth, together determining organ shape. The plant hormone auxin regulates both initiation and growth. However, it is still not clear how auxin facilitates these two separate successive stages. Here, we show that in tomato compound-leaf development, auxin promotes leaflet initiation and growth via opposite effects on gibberellin (GA) metabolism. Short auxin treatment induced the expression of GA catabolism genes, while a longer increased auxin response induced the expression of GA biosynthesis genes. GA biosynthesis mutants suppressed auxin-induced blade growth. Leaflet number was reduced by increased GA activity, and local reduction of GA activity led to the formation of ectopic leaflets, indicating that leaflet initiation requires reduced GA. Expression of a GA catabolism gene in initiating leaflets was sufficient to induce ectopic leaflets in the adjacent intercalary region, suggesting a non-autonomous function of GA. Collectively, these results point to a context-dependent dual interaction between auxin and GA in organ formation: auxin reduces GA to enable initiation, and induces GA to promote growth.
Development
· 2026 Jun · PMID 42319758
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C.H. Waddington (1905-1975) was a British biologist and perhaps the major figure in developmental biology between the 1940s and 1960s. He is one of the few pre-molecular-age developmental biologists still widely remember...C.H. Waddington (1905-1975) was a British biologist and perhaps the major figure in developmental biology between the 1940s and 1960s. He is one of the few pre-molecular-age developmental biologists still widely remembered. This is for his pioneering work on using mutations to probe embryogenesis, for his deep analysis of how gene activity drives developmental change (summarised in his illustration of the epigenetic landscape), for his exploration of the mechanics of evolutionary change in the developing phenotype and for his terminology, much of which remains in common usage. Waddington died some 50 years ago, and this short article examines some of his key research papers that underpin these achievements and still remain significant. The supplementary material, available online, includes songs composed for Waddington's 50th birthday.
Mathias NA, Dolphin NM, LeFever NM
… +4 more, Amato E, Ybanez CS, Ruquet A, Forni PE
Development
· 2026 Jun · PMID 42299035
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The vomeronasal organ (VNO) is a specialized chemosensory structure that detects chemical cues involved in predator avoidance, social interactions and reproductive behaviors. In mice, the VNO contains distinct vomeronasa...The vomeronasal organ (VNO) is a specialized chemosensory structure that detects chemical cues involved in predator avoidance, social interactions and reproductive behaviors. In mice, the VNO contains distinct vomeronasal sensory neurons (VSNs) that express vomeronasal receptors (VRs) and formyl peptide receptors. Apical VSNs express Meis2, V1Rs and Gαi2, whereas basal VSNs express Tfap2e (also known as AP-2ε), V2Rs and Gαo. Type 2 VRs (V2Rs) are classified into families A-E, and basal neurons co-express a family C receptor with a VR from another family. Single-cell RNA-sequencing identified ∼980 genes differentially expressed between V1R- and V2R-expressing neurons, many of which are linked to endoplasmic reticulum (ER) functions. Notably, canopy 1 (Cnpy1) is highly enriched in V2R-expressing neurons. The VNO of Cnpy1 knockout mice develops normally but undergoes progressive loss of V2R-expressing neurons, which is associated with increased ER stress gene expression, upregulation of family C V2R mRNAs and reduced V2R protein levels. V2R-expressing VSNs in Cnpy1 knockouts fail to respond to pheromones, show altered guidance and adhesion gene expression, and display disrupted connectivity with the accessory olfactory bulb. These findings emphasize the role of cell-specific ER protein repertoires in maintaining neuronal function.
Development
· 2026 Aug · PMID 42290220
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The freshwater cnidarian Hydra vulgaris is a classic model for studying whole-body regeneration, but the underlying cell biology remains comparatively underexplored. Hydra has a simple body plan consisting of two epithel...The freshwater cnidarian Hydra vulgaris is a classic model for studying whole-body regeneration, but the underlying cell biology remains comparatively underexplored. Hydra has a simple body plan consisting of two epithelial monolayers separated by an extracellular matrix (ECM) containing conserved components such as collagen and laminin, making it well suited for investigating ECM function during regeneration. Following head amputation, collagen and laminin retract from the wound site, generating a region of low ECM accumulation that persists for several days. Multiple matrix metalloproteinase (MMP) genes are expressed in the endoderm during this process, and MMP inhibition reduces the size of the collagen-depleted region and disrupts head morphogenesis. In contrast, inhibition of collagen cross-linking also blocks regeneration, indicating that successful regeneration requires a balance between ECM degradation and deposition. Finally, invasion of interstitial stem cell progenitors from the ectoderm into the endoderm during regeneration is facilitated by MMP-driven reduction of Collagen I. Together, these findings identify ECM remodeling as a crucial and conserved feature of regeneration, and establish Hydra as a valuable model for studying its underlying mechanisms.
Ghatpande V, Lewis AJ, Windsor KE
… +6 more, Lee H, Perez KY, Alcala AJ, Menke DB, Cenik C, Vokes SA
Development
· 2026 Jun · PMID 42281473
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The vertebrate forelimb initiates as a localized swelling in the somatic lateral plate mesoderm (somatic LPM) in response to TBX5-dependent transcription. The molecular pathways driving limb morphogenesis have been exten...The vertebrate forelimb initiates as a localized swelling in the somatic lateral plate mesoderm (somatic LPM) in response to TBX5-dependent transcription. The molecular pathways driving limb morphogenesis have been extensively studied but the steps directly preceding limb bud formation remain poorly characterized. To address this, we defined the temporal onset of forelimb initiation in mouse embryos using sequencing based high-throughput approaches (RNA-seq, scRNA-seq, ChIP-seq, and Ribo-ITP) benchmarked to known features in forelimb development, identifying four distinct stages. Using scRNA-seq at the onset of forelimb-specific transcription, we determined the transcriptional profile of the somatic LPM and identified signature genes that distinguish the nascent forelimb from other cell types. This group includes multiple genes involved in neural projection as well as cell adhesion. Interestingly, these genes are highly enriched for TBX5 binding sites, suggesting they are candidate early transcriptional targets of TBX5. As TBX5 is essential for forelimb outgrowth, the identification of these genes suggests new mechanistic models for TBX5-driven limb initiation.
Development
· 2026 Jun · PMID 42267616
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Notch target genes HES1 and HES5 mutually repress one another, while also exhibiting oscillatory expression across tissues, to regulate cell fate specification, during mammalian neural tube development. In their work, Ve...Notch target genes HES1 and HES5 mutually repress one another, while also exhibiting oscillatory expression across tissues, to regulate cell fate specification, during mammalian neural tube development. In their work, Veronica Biga, Cerys Manning and colleagues show that there is a mechanistic link between oscillatory expression of the cross-repressive factors HES1 and HES5, and fate resolution in the developing spinal cord. To learn more about their work, we talked to the first author Veronica Biga, as well as the corresponding author, Cerys Manning, Presidential Fellow in the School of Medicine and Lecturer in Developmental Biology, Division of Developmental Biology and Medicine, University of Manchester, UK.
Spike CA, Parker DM, Tsukamoto T
… +6 more, Torres Mangual N, Tsukamoto EC, Coleman K, Gearhart MD, Greenstein D, Osborne Nishimura E
Development
· 2026 Jun · PMID 42226709
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The elimination of maternal mRNAs is an essential feature of the maternal-to-zygotic transition. We report an essential pathway that clears many maternal transcripts from early C. elegans embryos using the Rbfox-related...The elimination of maternal mRNAs is an essential feature of the maternal-to-zygotic transition. We report an essential pathway that clears many maternal transcripts from early C. elegans embryos using the Rbfox-related SPN-4 RNA-binding protein as a specificity factor and the CCR4-NOT deadenylase complex as an effector. We biochemically identified SPN-4-associated mRNAs from late-stage oocytes and found that the set of SPN-4-associated transcripts is enriched for maternal mRNAs that undergo early decay. Single-molecule fluorescence in situ hybridization experiments established that many SPN-4-associated mRNAs fail to be eliminated in the absence of SPN-4. In the 3'UTRs of two target mRNAs, we identified Rbfox motifs that are required for SPN-4-dependent clearance in vivo and bind SPN-4 in vitro. In a genetic screen to identify factors that work with SPN-4, we isolated mutant alleles of CCR4-NOT components. Auxin-induced degradation of the LET-711/NOT1 scaffold and the CCF-1 deadenylase disrupted clearance of two SPN-4-associated transcripts. Our results support a model in which SPN-4 initiates expression in late-stage oocytes, associates with maternal mRNA targets through RNA sequences in their 3'UTRs and promotes CCR4-NOT-mediated decay during early embryogenesis.
Development
· 2026 May · PMID 42224069
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In both mouse and humans, proper development of both the embryo and its supporting placenta is coordinated through gene expression programmes. However, the individual functions of many of these genes and consequences of...In both mouse and humans, proper development of both the embryo and its supporting placenta is coordinated through gene expression programmes. However, the individual functions of many of these genes and consequences of their loss remains unknown. In a new study, Taylor Guertin, Kimberley D. Tremblay and colleagues use mouse models to systematically analyse the effects of 22 individual gene knockouts during mid-gestation, when the placenta becomes mature enough to mediate maternal/foetal exchange. The results of this effort have illuminated different general principles of placental and embryo development, and have also been released as a browsable resource online: https://websites.umass.edu/kdtkomp/. To learn more about this work and the people behind it, we talked to first author Taylor Guertin and corresponding author Kimberley (Kim) Tremblay, Professor of Veterinary and Animal Sciences at the University of Massachusetts, Amherst, USA.
Development
· 2026 May · PMID 42189745
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Autosomal recessive primary microcephaly is a neurodevelopmental disorder characterised by a smaller brain size, resulting from a reduced number of neurons. In their work, Laura Ciapponi, Patrizia Somma and colleagues un...Autosomal recessive primary microcephaly is a neurodevelopmental disorder characterised by a smaller brain size, resulting from a reduced number of neurons. In their work, Laura Ciapponi, Patrizia Somma and colleagues uncover the function of two microcephaly-associated genes, asp and sas4, in chromatin epigenetic control and nuclear lamina structure. To learn more about their work, we talked to the first author, Degisew Yinur Mengistu, as well as the corresponding author, Laura Ciapponi, Associate Professor, Sapienza University of Rome, Italy.
Development
· 2026 Jun · PMID 42159204
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The presomitic mesoderm is a transient embryonic tissue patterned along the anterior-posterior axis of vertebrates that periodically generates somites at its anterior end. Somitogenesis is driven by waves of gene express...The presomitic mesoderm is a transient embryonic tissue patterned along the anterior-posterior axis of vertebrates that periodically generates somites at its anterior end. Somitogenesis is driven by waves of gene expression that propagate from the posterior to the anterior end of the presomitic mesoderm under the control of the segmentation clock, a molecular oscillator. Using a mouse primary culture system, we performed a small-scale chemical screen targeting pathways implicated in cellular mechanics and we identified Rac1-dependent signaling as a modulator of segmentation clock dynamics. Pharmacological perturbation of Rac1 signaling reproducibly slowed oscillatory dynamics without disrupting intercellular synchrony. These changes were reversible and associated with increased somite length and reduced somite number. In addition, inhibition of Rac1 signaling was associated with changes in the expression of selected Wnt and Notch target genes in tail explants. These findings identify Rac1-dependent signaling as a candidate modulator of segmentation clock dynamics and somite morphogenesis, linking mechanical and molecular aspects of segmentation dynamics.
Development
· 2026 May · PMID 42132012
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The Y chromosome harbours few protein-coding genes and their roles in early human development remain unclear. In their work, Tomohiko Akiyama and colleagues investigate the function of UTY in human pluripotent stem cells...The Y chromosome harbours few protein-coding genes and their roles in early human development remain unclear. In their work, Tomohiko Akiyama and colleagues investigate the function of UTY in human pluripotent stem cells and revisit the assumption that Y-linked genes play only limited roles in core developmental processes. To learn more about their work, we spoke with the first and corresponding author, Tomohiko Akiyama, Assistant Professor from Yokohama City University, Japan, in the Department of Molecular Biology, Graduate School of Medicine and the Department of Medicine.
Kawaguchi A, Igari M, Murayama Y
… +6 more, Iikawa H, Sakamoto M, Nakamura Y, Kuraku S, Ishihara K, Saito D
Development
· 2026 May · PMID 42113595
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Dynamic changes in chromatin accessibility at cis-regulatory elements underlie cell fate transitions during development. Primordial germ cells (PGCs) represent a rare population whose chromatin dynamics remain poorly und...Dynamic changes in chromatin accessibility at cis-regulatory elements underlie cell fate transitions during development. Primordial germ cells (PGCs) represent a rare population whose chromatin dynamics remain poorly understood compared to known epigenetic landscapes. Here, we utilized the chicken PGC model to bridge this gap, leveraging its capacity for in vitro expansion and in vivo colonization. We adapted the ATAC-seq workflow to obtain reproducible accessible chromatin region (ACR) profiles from as few as 200 cells, even after cryopreservation. Integrative analysis identified over 10,000 PGC-specific ACRs, many absent from somatic tissues, and revealed inherent Tn5 transposase sequence biases in the chicken genome. To validate these ACRs, we established an in vitro PGC differentiation system and utilized in vivo embryonic transplantation. Reporter assays confirmed enhancer activities in cultured PGCs, while transcriptome integration associated these ACRs with genes expressed at embryonic day 2.5. In vivo transplantation demonstrated that these enhancers exhibited early stage-specific activity, becoming silenced upon gonadal settlement. Our results provide a practical strategy for identifying functional regulatory elements from minimal starting material, facilitating the study of chromatin dynamics in rare cell populations.
Guertin TM, Sisson C, Gargiulo RM
… +7 more, Macrorie O, Pease EL, Garth HE, Vriens J, De Clercq K, Mager J, Tremblay KD
Development
· 2026 May · PMID 42093611
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The focus on assessing embryo-specific defects in lethal knockout (KO) mouse lines has resulted in an underrepresentation of documented placental abnormalities. Presented herein is a uniform analysis of 22 distinct KO mo...The focus on assessing embryo-specific defects in lethal knockout (KO) mouse lines has resulted in an underrepresentation of documented placental abnormalities. Presented herein is a uniform analysis of 22 distinct KO mouse lines that exhibit homozygous lethality in a narrow mid-gestation window. All genes altered by each KO have human orthologs, most are implicated in human disease, yet almost all are understudied. To unravel the role each plays in mammalian development, null embryonic and placental phenotype analysis was performed, and wild type (WT) expression of each KO gene was assessed. While the null phenotype of each KO line falls into two broad embryonic categories, the placental phenotypes are diverse and coincide with the onset of gross embryonic defects. The co-occurrence of null embryonic and placental defect onset coupled with WT gene expression highlights that many could be essential in either the embryo or placenta. This analysis serves to guide mid-gestation placental analysis, underscores the importance of routine analysis of the entire conceptus during mid-gestation lethality, and provides functional annotation for each understudied human ortholog.
Fan N, Gong H, Wang P
… +14 more, Bai X, He X, Chen N, Gao J, Li H, Wang L, Naren G, Gao F, Guo J, Liu T, Jiang N, Lin X, Li X, Nashun B
Development
· 2026 May · PMID 42083516
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Mammalian oogenesis is a precisely orchestrated developmental process, which depends on accurate chromatin remodeling and transcriptional regulation in the absence of DNA replication. The histone variant H2A.Z is require...Mammalian oogenesis is a precisely orchestrated developmental process, which depends on accurate chromatin remodeling and transcriptional regulation in the absence of DNA replication. The histone variant H2A.Z is required for oogenesis and embryogenesis, yet the chaperone directing its deposition has not been well characterized in mammalian oocytes. Here, we identify Znhit1, a core subunit of the SRCAP chromatin remodeling complex, as the essential factor mediating H2A.Z deposition in oocytes. Oocyte-specific depletion of Znhit1 impairs H2A.Z incorporation and leads to severe ovarian phenotype, characterized by follicle loss, homologous chromosome segregation defects and meiotic arrest, which ultimately leads to female infertility. On a molecular level, integrated Smart-seq2 and H2A.Z CUT&Tag analyses demonstrate that Znhit1 depletion severely reduces genome-wide H2A.Z deposition, particularly at promoter regions of key meiotic genes such as Aurkb, Tpm3 and Zar1, resulting in transcriptional dysregulation and aberrant meiotic gene expression. Our findings pinpoint Znhit1 as the histone chaperone essential for accurate deposition of histone variant H2A.Z, ensuring meiotic progression and oocyte development in mice.
Mengistu DY, Marzullo M, Pellacani C
… +5 more, Marchetti M, Terribili M, Montivero Morales E, Somma MP, Ciapponi L
Development
· 2026 May · PMID 42063344
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Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by reduced brain size and intellectual disability. Mutations in over 30 genes, nearly half linked to centrosome biogenesis or...Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by reduced brain size and intellectual disability. Mutations in over 30 genes, nearly half linked to centrosome biogenesis or microtubule (MT) dynamics, highlight spindle defects in disease aetiology, yet these alone do not fully explain MCPH. Here, we show that the Drosophila orthologs of ASPM/MCPH5 (asp) and CENPJ/MCPH6 (Sas4) contribute to safeguard nuclear architecture and chromatin organization during brain development. Loss of either gene perturbs MT organization and centromere clustering, leading to reduced Lamin and HP1α levels, and to deformed nuclear lamina. Mutants also display a global reduction in heterochromatin-associated histone marks, H3K9me2/3 and H3K27me3, along with an increase in the euchromatin-associated mark H3K4me3 and elevated DNA damage with delayed repair. Notably, inhibiting demethylases with methylstat restores H3K9me3 and nuclear morphology. These findings suggest a previously unreported role for centrosome proteins in regulating chromatin organization, providing new insights into the mechanisms underlying MCPH pathogenesis.
Development
· 2026 May · PMID 42063341
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LIN-28 is an evolutionarily conserved RNA-binding protein that is crucial for regulating pluripotency and cell fate determination during animal development. In Caenorhabditis elegans, lin-28 is an integral component of t...LIN-28 is an evolutionarily conserved RNA-binding protein that is crucial for regulating pluripotency and cell fate determination during animal development. In Caenorhabditis elegans, lin-28 is an integral component of the heterochronic (developmental timing) gene regulatory cascade. Loss-of-function mutations in lin-28 cause precocious cell fate determination during larval development. Previous studies indicate that proper progression of larval stage-specific cell fates relies on the downregulation of LIN-28, which is negatively regulated by the lin-4 microRNA through complementary sequences in the lin-28 3' untranslated region (UTR). This study employs CRISPR/Cas9 editing of the endogenous lin-28 locus to demonstrate that developmental downregulation of LIN-28 involves multiple inputs, including the action of the let-7 family and lin-4 microRNAs via adjacent complementary sites in the lin-28 3' UTR, along with post-translational inhibition of LIN-28 by the lep-5 long non-coding RNA, collectively accounting for nearly all LIN-28 repression. Additionally, systematic testing of truncations of the lin-28 3' UTR identifies three positive regulatory regions that enhance LIN-28 expression, counteracting the negative effects of the let-7 and lin-4 microRNAs and the lep-5 long non-coding RNA.
Yambe S, Sumiyama K, Watanabe H
… +5 more, Takimoto A, Sasaki T, Kondoh G, Docheva D, Shukunami C
Development
· 2026 May · PMID 42027071
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Scleraxis (Scx) is a basic helix-loop-helix transcription factor predominantly expressed in tendons, ligaments, and their attachment sites, where it regulates maturation. Here, we report a novel Scx enhancer that drives...Scleraxis (Scx) is a basic helix-loop-helix transcription factor predominantly expressed in tendons, ligaments, and their attachment sites, where it regulates maturation. Here, we report a novel Scx enhancer that drives tissue-specific expression. In transgenic reporter mice, a 5.3 kb downstream Scx enhancer (dSE) conferred robust, stable, and faithful reporter activity reproducing the endogenous Scx expression pattern. Within the dSE, we identified a 343 bp Scx enhancer (CSE), conserved from lobe-finned fishes to tetrapods, that recapitulates Scx expression in limbs. In developing limbs, CSE-deficient mice exhibited a marked reduction of endogenous Scx expression and a complete loss of the deltoid tuberosity (DT) supporting forelimb leverage, accompanied by attenuated BMP and TGFβ signaling, as in Scx-deficient mice. Precise enhancer activation within a particular developmental window was indispensable for DT formation. In contrast, tendon/ligament maturation retained prolonged plasticity, with Scx expression partially recovering later, likely through additional elements within the dSE. These findings uncover an essential role of the CSE in DT morphogenesis and reveal distinct temporal enhancer requirements for DT versus tendon/ligament formation, with additional enhancer dependence confined to tendons/ligaments.