Development
· 2026 Aug · PMID 42024002
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The development of the elaborate, calcified endoskeleton of sea urchin embryos is a model for understanding the dynamic nature of developmental gene regulatory networks and the control of biomineralization. While several...The development of the elaborate, calcified endoskeleton of sea urchin embryos is a model for understanding the dynamic nature of developmental gene regulatory networks and the control of biomineralization. While several signaling pathways have been shown to regulate gene expression and biomineral formation by sea urchin skeletogenic cells, important gaps in our understanding remain. Here, we focused on signals that regulate skeletogenesis along the dorsal-ventral axis of the late-stage embryo. We used a specific inhibitor of Type I BMP receptors, K02288, to show that BMP signaling regulates skeletal growth selectively in the dorsal region. K02288 treatment led to dorsal skeletal defects and inhibited the expression of genes typically expressed specifically in the dorsal skeletogenic cells, including biomineralization genes. Using RNA sequencing, we identified genes that were uniquely downstream of either the BMP or a ventral signaling pathway (the VEGF pathway) at late developmental stages and genes downstream of both pathways. Our findings establish BMP signaling as a key pathway regulating dorsal skeleton formation and show that BMP signaling functions in concert with VEGF signaling to define the dorsal-ventral axis of the skeleton.
Li W, Nakayama K, Sato R
… +3 more, Shimada R, Kubota Y, Mukouyama YS
Development
· 2026 May · PMID 42003386
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The von Hippel-Lindau (VHL) protein regulates cellular oxygen sensing by degrading hypoxia-inducible factors (HIFs) under normoxic conditions. VHL mutations show highly vascularized tumor across various organs due to HIF...The von Hippel-Lindau (VHL) protein regulates cellular oxygen sensing by degrading hypoxia-inducible factors (HIFs) under normoxic conditions. VHL mutations show highly vascularized tumor across various organs due to HIF activation and upregulation of HIF-target genes such as VEGF in non-endothelial cells (ECs), influencing neighboring ECs and triggering abnormal angiogenesis. Whether VHL mutations in ECs also contribute to abnormal angiogenesis remains unclear. To address this question, we utilized a well-characterized skin vasculature model that encompasses the processes of vascular patterning and arterial/venous development, to investigate vascular development in mice with an EC-specific Vhl deletion. The mutants exhibited abnormal vascular network formation and embryonic lethal. Mechanistically, the Vhl deletion led to ectopic expression of the chemokine receptor CXCR4 through HIF stabilization in ECs. Treatment with AMD3100, a CXCR4 antagonist, partially restored vascular abnormalities caused by Vhl deletion in ECs. Additionally, publicly available single-cell RNA-sequencing data from ECs of individuals with VHL syndrome supports our findings, indicating that VHL mutations in ECs contribute to abnormal angiogenesis through ectopic activation of the HIF-CXCR4 signaling axis.
Maillard L, Pimmett VL, Douaihy M
… +6 more, Garcia-Idieder P, Topno R, Brun A, Trullo A, Radulescu O, Lagha M
Development
· 2026 Apr · PMID 41978991
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Isogenic cells often exhibit variability in gene expression. Because of its stochastic nature, transcription is thought to lie at the heart of gene expression variability. While beneficial in some contexts, this transcri...Isogenic cells often exhibit variability in gene expression. Because of its stochastic nature, transcription is thought to lie at the heart of gene expression variability. While beneficial in some contexts, this transcriptional noise must be dampened to ensure the deployment of accurate gene expression programs. Here, we have investigated how the cis-regulatory code affects inter-nuclear variability in transcription, quantified at the level of individual RNA Polymerase II initiation events. Combining quantitative imaging in Drosophila embryos with mathematical modeling, we discovered that transcriptional noise is time dependent with two major components: mitotic exit and transcriptional bursting. Transcriptional noise peaks after mitosis due to the stochastic timing of postmitotic reactivation and the temporal heterogeneity of transcriptional bursting. When a steady-state regime is reached, transcriptional noise is then primarily driven by the kinetics of bursting. We demonstrate that the TATA box is necessary and sufficient to generate transcriptional noise, and assess the contribution of shadow enhancers to noise. Our work reveals the major contribution of the first polymerase passage after mitotic exit to transcriptional noise.
Miranda E, Slota-Burtt L, Berrio A
… +5 more, Bardhan A, Deiters A, Wray GA, Croce JC, McClay DR
Development
· 2026 Apr · PMID 41972364
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In the sea urchin embryo, as many as ten Wnts are expressed by cells that undergo gastrulation movements. Here, Wnt1, 5, 6, 7, 8 and 16 are examined to learn details of expression and function over time using results fro...In the sea urchin embryo, as many as ten Wnts are expressed by cells that undergo gastrulation movements. Here, Wnt1, 5, 6, 7, 8 and 16 are examined to learn details of expression and function over time using results from a temporal scRNA-seq analysis coupled with hybridization chain reaction in situ analysis in which multiple wnt genes are localized simultaneously in the same embryo. The data reveal that many cells express two or three wnt genes at the same time. A single sharp boundary of expression is seen just lateral to the blastopore. Other boundaries of expression are graded and change over developmental time. Functionally, experiments show that Wnts control expression and/or patterning of other wnt genes, and they control expression of marker genes during and following invagination of the archenteron. Stomodeum formation also requires Wnt signaling, indicating that the entire gut uses Wnt signaling for diversification of endodermal fates.
Zhang L, Zhang W, Lin D
… +3 more, Lai W, Zhang Z, Zhang Z
Development
· 2026 Apr · PMID 41958431
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Larval settlement is a crucial stage in the development of most marine benthic invertebrates, but the molecular mechanisms remain unclear. Here, we have functionally characterised FILa1, a mature peptide derived from the...Larval settlement is a crucial stage in the development of most marine benthic invertebrates, but the molecular mechanisms remain unclear. Here, we have functionally characterised FILa1, a mature peptide derived from the previously reported FILa precursor, and show that it promotes larval settlement. Sequence clustering and alignment analyses revealed that the FILa precursor shows similarity to annelid FLGa/FVLa and crustacean AST-A. Among its mature forms, FILa1 was the most effective inducer of settlement. Transcriptomic and qRT-PCR analyses showed that FILa1 rapidly activates cAMP-PKA signalling and is associated with altered expression of ciliary genes, including DYT2B, localised in the circumoral cilia. FILa1 inhibited DYT2B mRNA expression through the cAMP-PKA-CREB and Ca²+ pathways at early and later time points. Knockdown of DYT2B disrupted ciliary structure, reduced cilia number and length, and decreased beating frequency, demonstrating its essential role in ciliary function. Notably, larvae with reduced DYT2B expression exhibited significantly increased settlement rates at later stages. Our results demonstrate that FILa1 rapidly triggers larval settlement through cAMP-PKA signalling, while longer term regulation of ciliary genes contributes to proper ciliary function associated with successful settlement.
Development
· 2026 Aug · PMID 41924858
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Integrins mediate adhesion of basal keratinocytes to the underlying basement membrane. While high expression of integrins has been correlated with stemness, there is limited direct evidence that integrins mediate keratin...Integrins mediate adhesion of basal keratinocytes to the underlying basement membrane. While high expression of integrins has been correlated with stemness, there is limited direct evidence that integrins mediate keratinocyte retention within the basal layer. Here, we generate mosaic, epidermal-specific loss of integrin-β4 (encoded by Itgb4) or its ligand, laminin-α3β3γ2 (Lama3), in mouse using an in utero lentiviral-mediated approach. Although mutations in these genes cause postnatal skin blistering in mice and humans, we observe no evidence of dermal-epidermal separation embryonically. Despite no obvious alterations to apicobasal polarity, Itgb4-deficient basal cells show mild defects in oriented cell divisions, with increased oblique divisions and altered telophase correction. However, differentiation via delamination, whereby basal keratinocytes lose adhesion to the underlying basement membrane and transit into the suprabasal layer, is elevated upon Itgb4 or Lama3 loss. Notably, hyperactive Notch signaling both decreases integrin-β4 expression and increases delamination, while deletion of the Notch effector Rbpj has the opposite effect. These findings conclusively demonstrate a causal role for hemidesmosomes in regulating epidermal differentiation through both mitotic and non-mitotic mechanisms, and shed additional light on the programs regulating delamination.
Akiyama T, Nakahara T, Sato S
… +5 more, Ishiguro KI, Yukawa M, Yamamoto M, Takahashi H, Ko MSH
Development
· 2026 May · PMID 41906541
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The Y chromosome harbors few protein-coding genes, and their roles in early human development remain largely unclear. Here, we demonstrate that UTY, a Y-linked homolog of the histone demethylase UTX, plays a crucial deme...The Y chromosome harbors few protein-coding genes, and their roles in early human development remain largely unclear. Here, we demonstrate that UTY, a Y-linked homolog of the histone demethylase UTX, plays a crucial demethylase-independent role in maintaining pluripotency in human embryonic stem cells (hESCs). Despite its low expression and weak enzymatic activity, UTY co-occupies cis-regulatory elements with UTX and promotes transcription of key pluripotency genes, including NODAL, LEFTY1 and LEFTY2. Integrated analyses of single- and double-knockout hESCs reveal that UTY and UTX function redundantly to maintain chromatin accessibility and ensure proper recruitment of core transcription factors such as OCT4 and SOX2. The combined loss of UTY and UTX disrupts transcription factor localization, induces widespread gene expression changes and leads to a collapse of the pluripotent state, without detectable changes in H3K27 methylation. Instead, these defects are associated with impaired recruitment of ATP-dependent chromatin remodelers and reduced histone acetylation, suggesting a demethylase-independent mechanism. Our findings uncover an essential role for the evolutionarily conserved UTY in safeguarding enhancer function and transcription factor occupancy, highlighting Y-linked regulatory mechanisms crucial for human pluripotency.
Development
· 2026 Mar · PMID 41891748
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Transgenic reporter lines are essential experimental tools for studying molecular processes. Planarian flatworms have emerged as a model of remarkable regenerative abilities, but their use as models of regeneration has b...Transgenic reporter lines are essential experimental tools for studying molecular processes. Planarian flatworms have emerged as a model of remarkable regenerative abilities, but their use as models of regeneration has been limited by the difficulties in inducing transgenic reporter expression. In a recent study, Leonard Drees, Jochen Rink and colleagues show that the innate immune system of the planarian Schmidtea mediterranea is a key suppressor of transgene expression, and that repression of the conserved innate immunity kinase TBK1 can enhance reporter expression. To learn more about this work and the people behind it, we talked to corresponding author Jochen Rink and both first and corresponding author Leonard Drees, postdoc in the Rink Group at the Max Planck Institute for Multidisciplinary Sciences in Göttingen, Germany.
Hanzelova P, Baird C, Keshinro B
… +3 more, Kadhom R, Lalonde RL, Akimenko MA
Development
· 2026 Aug · PMID 41885168
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A key difference between tetrapod limb buds and teleost fin buds is the presence of rigid actinotrichia fibers that guide the migrating cells contributing to ray formation. Major structural components of actinotrichia ar...A key difference between tetrapod limb buds and teleost fin buds is the presence of rigid actinotrichia fibers that guide the migrating cells contributing to ray formation. Major structural components of actinotrichia are encoded by fish-specific actinodin (And) genes, which were lost in tetrapods. To investigate the consequences of this loss during the fin-to-limb transition, we generated deletions in zebrafish and1 and and2 using CRISPR/Cas9 mutagenesis. Double mutants (and1-/-and2-/-) lack actinotrichia. Embryos and larvae have reduced fin fold size, with disorganized cell migration. In adults, all fin fold-derived skeletal structures are disrupted, including the rays of all fins, as well as the caudal fin endoskeleton. Surprisingly, double mutant males fail to breed, despite being fertile. Video analysis revealed that defects in the fins of males impair their ability to stimulate egg release. Our findings highlight the role of actinotrichia in both fin patterning and zebrafish courtship. We propose that actinodin gene maintenance is under strong selection in fish with similar courtship. We speculate that the loss of actinodin genes and a shift in courtship strategy may have coincided during tetrapod evolution.
Li C, Zhao Y, Wang L
… +12 more, Li S, Li J, Gu Y, He S, Wang G, Lei F, Lu Y, Gu L, Xu S, Xiong W, Li B, Liu Z
Development
· 2026 Apr · PMID 41884985
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The inactivation of Rbm24, an RNA-binding protein, leads to cell death and hair bundle defects in cochlear outer hair cells (OHCs). However, the underlying molecular mechanisms remain unclear. To address this, we have pe...The inactivation of Rbm24, an RNA-binding protein, leads to cell death and hair bundle defects in cochlear outer hair cells (OHCs). However, the underlying molecular mechanisms remain unclear. To address this, we have performed comprehensive transcriptomic profiling of purified wild-type and Rbm24-/- mouse OHCs at postnatal day 7 (P7). Loss of Rbm24 perturbs numerous genes associated with hair bundle morphogenesis and delays the overall OHC differentiation program. Insm1, a key transcription factor normally downregulated by P2, remains aberrantly and persistently expressed in Rbm24-/- OHCs. Overexpression of Insm1 alone induces OHC death, whereas simultaneous inactivation of Rbm24 and Insm1 largely rescues OHC survival but only partially restores hair bundle morphology. It demonstrates that Rbm24 promotes OHC survival independently of its role in regulating hair bundle morphogenesis. Collectively, our findings establish Rbm24 as a dual-function regulator that ensures OHC survival by acting as a critical repressor of Insm1 expression, while independently orchestrating hair bundle assembly. These results highlight the central role of Rbm24 in coordinating OHC differentiation and structural maturation, and provide insights into potential molecular targets for hair cell regeneration.
Meyers J, Tower Z, Leighton O
… +2 more, Drolet B, Chang H
Development
· 2026 Apr · PMID 41866987
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Vascular anomalies are a group of congenital disorders characterized by structural abnormalities in the blood vessels. Over the past 20 years, genetic investigations have identified mosaic mutations in multiple genes in...Vascular anomalies are a group of congenital disorders characterized by structural abnormalities in the blood vessels. Over the past 20 years, genetic investigations have identified mosaic mutations in multiple genes in individuals with vascular anomalies, including G protein alpha subunit q/11-encoding genes GNAQ/GNA11. Here, we report a tunable and reversible mouse tool for studying the frequent GNAQ mutant, GNAQ R183Q. We have generated a transgenic mouse line carrying GNAQ R183Q under the control of the tet-responsive elements. The transgene expression level is tunable and can be induced at physiologically relevant ranges. We have shown that endothelial expression of GNAQ R183Q is sufficient to drive blood vessel malformation in developing mouse embryos. We have further identified multiple transcriptional changes caused by GNAQ R183Q in the skin using RNA-seq. This work strengthens the causal link between GNA mutations and vascular anomalies, demonstrates that endothelial cell-specific expression of GNAQ R183Q is sufficient to cause vascular defects during development, and offers valuable insights into the mechanisms underlying GNA mutation-associated diseases.
Petzold T, Lutes LK, Navarro i Batista K
… +7 more, Konle A, Baechler B, Jemelin S, Gerhardt H, Golub R, Scheiermann C, Bertrand JY
Development
· 2026 Apr · PMID 41853934
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Following specification in the dorsal aorta, haematopoietic stem and progenitor cells (HSPCs) proliferate in the HSPC niche, known as the caudal haematopoietic tissue (CHT) in zebrafish. Here, we demonstrate that bmal1a,...Following specification in the dorsal aorta, haematopoietic stem and progenitor cells (HSPCs) proliferate in the HSPC niche, known as the caudal haematopoietic tissue (CHT) in zebrafish. Here, we demonstrate that bmal1a, a core component of the circadian clock machinery, is expressed in CHT endothelial cells (ECs) and affects HSPCs in a non-cell autonomous manner. Using endothelial cell-specific dominant-negative Bmal1a zebrafish lines, we demonstrate a striking increase in HSPC numbers in the CHT, resulting from enhanced HSPC proliferation. RNA-sequencing of dominant-negative bmal1a ECs sorted from the CHT shows a downregulation of glud1a, resulting in increased glutamine levels in the CHT. This newly discovered bmal1a-glud1a-glutamine pathway fuels HSPC expansion. We demonstrate that this glutamine synthesis pathway controlling HSPC expansion is likely conserved in the mouse fetal liver (FL) niche, in which hepatocytes are the likely source of glutamine. Together, our data uncover a previously unreported mechanism of HSPC homeostasis, in which EC BMAL1, expressed by the niche, controls the amount of bioavailable glutamine for HSPCs by regulating the expression of genes involved in glutamine synthesis.
Development
· 2026 Apr · PMID 41822989
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In vertebrates, neural induction requires the inhibition of BMP signals by dorsal organizer-derived antagonists. However, the underlying mechanism is not completely understood. Here, using zebrafish as a model, we show t...In vertebrates, neural induction requires the inhibition of BMP signals by dorsal organizer-derived antagonists. However, the underlying mechanism is not completely understood. Here, using zebrafish as a model, we show that ADNP2 orthologs adnp2a and adnp2b are required for proper neural induction in gastrulating embryos. Deficiency of adnp2 causes deficits in neuroectoderm specification, showing reduced expression of both anterior and posterior neural markers. Adnp2 functions as a transcriptional repressor, and directly occupies and suppresses the BMP-related ved/vent/vox homeobox genes, thereby excluding their expression from dorsal territories, including the dorsal mesoderm and presumptive neural plate. adnp2-deficient larvae display aberrant behavioral alterations, which can be partially rescued by the administration of BMP antagonist Dorsomorphin or dnBmpr1 mRNAs. Our findings in the zebrafish model reveal an indirect role for Adnp2 in neuroectoderm formation by antagonizing the activities of BMP-related ventralizing genes, which will be useful for understanding neural disorders caused by ADNP2 dysfunction in human.
Abt KM, Bartholomew MA, Nixon AEK
… +4 more, Richman HE, Gura MA, Seymour KA, Freiman RN
Development
· 2026 Apr · PMID 41814948
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Female reproductive senescence results from the regulated depletion of a finite pool of oocytes called the ovarian reserve. This pool of oocytes is initially established during fetal development, but the oocytes that it...Female reproductive senescence results from the regulated depletion of a finite pool of oocytes called the ovarian reserve. This pool of oocytes is initially established during fetal development, but the oocytes that it consists of must remain quiescent for decades until they are activated during maturation in adulthood. In order for developmentally competent oocytes to populate the ovarian reserve, they must successfully initiate both meiosis and oogenesis. As the factors that regulate the timing and fidelity of these early events remain elusive, we assessed the precise function and timing of the transcriptional regulator TAF4b during meiotic prophase I progression in mouse fetal oocytes. Compared to matched controls, E14.5 Taf4b-deficient oocytes enter meiosis I in a timely manner; however, their subsequent progression through the pachytene-to-diplotene transition of meiotic prophase I is compromised. Moreover, this disruption of meiotic progression is associated with the reduced ability of Taf4b-deficient oocytes to repair double-strand DNA breaks. Transcriptional profiling of Taf4b-deficient oocytes reveals that between E16.5 and E18.5 these oocytes fail to properly coordinate the reduction of meiotic gene expression and the activation of oocyte differentiation genes.
Development
· 2026 Mar · PMID 41766387
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Retinas from mice with a targeted disruption of the gene encoding forkhead transcription factor Foxn3 contained additional displaced amacrine interneurons and retinal astrocytes in the inner plexiform and ganglion cell l...Retinas from mice with a targeted disruption of the gene encoding forkhead transcription factor Foxn3 contained additional displaced amacrine interneurons and retinal astrocytes in the inner plexiform and ganglion cell layers, as well as ectopic primary cilia on bipolar and amacrine interneurons. Foxn3 is a transcriptional repressor and numerous genes linked to cilia structure or assembly were upregulated in embryonic retinas with disrupted Foxn3. CUT&RUN analysis revealed that many upregulated retinal genes were bound by the Foxn3 and Rfx3 proteins. A short hydrophobic motif (LXXLXWL) shared by Foxn3, Foxn4 and Foxj1 was required for association with Rfx3 and for full transcriptional repression by Foxn3, as well as for full transcriptional activation by Foxj1 or Foxn4. AlphaFold 3 predicted interaction between the hydrophobic motif and the Rfx3 dimerization domain. Mutations in Rfx3 at the predicted interaction site disrupted association of Rfx3 with Foxn3, Foxn4 or Foxj1. These results reveal a new layer of transcriptional regulation of genes required for cilia, with Foxn3 functioning as a repressor of cilia genes and limiting primary cilia formation in the developing retina.
Tsuyuzaki K, Yaguchi J, Yamamoto T
… +2 more, Ikeo K, Yaguchi S
Development
· 2026 Feb · PMID 41744098
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We generated a developmental stage-specific single-cell RNA-sequencing atlas of the Western Pacific sea urchin Hemicentrotus pulcherrimus, uncovering new gene modules associated with neurogenesis and identifying Delta/No...We generated a developmental stage-specific single-cell RNA-sequencing atlas of the Western Pacific sea urchin Hemicentrotus pulcherrimus, uncovering new gene modules associated with neurogenesis and identifying Delta/Notch-sensitive regulators of neuronal differentiation. Pharmacological perturbation of this pathway revealed neurogenic genes that are inconspicuous under normal conditions and clarified the roles of regional specifiers in maintaining progenitor states. To promote broader accessibility, we developed an interactive web platform within HpBase with Kana, enabling gene expression exploration even for researchers without computational expertise. Furthermore, we performed a cell-type deconvolution method that links bulk RNA sequencing to the single-cell reference, allowing rapid visualization of cell-type composition changes from bulk data alone. These integrated resources and analytical tools not only provide mechanistic insights into echinoderm neurodevelopment but also establish a generalizable workflow for combining bulk and single-cell transcriptomics in non-model organisms, empowering developmental and evolutionary biologists with practical strategies for cell-type-level resolution in complex systems.
Development
· 2026 Mar · PMID 41742783
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The planarian flatworm Schmidtea mediterranea has become a powerful model for studying whole-body regeneration, tissue patterning, and stem cell regulation. Yet the absence of reliable tools for transgene expression stil...The planarian flatworm Schmidtea mediterranea has become a powerful model for studying whole-body regeneration, tissue patterning, and stem cell regulation. Yet the absence of reliable tools for transgene expression still limits the elucidation of molecular mechanisms in in this system. Here, we establish a proof-of-principle system for plasmid-based expression of NanoLuciferase (NanoLuc) in S. mediterranea, employing commercially available transfection reagents and a panel of endogenous promoter sequences. Despite successful delivery, reporter expression remained low and transient. To identify biological barriers to robust transgene expression, we investigated the role of innate immune pathways. Candidate gene searches and biochemical pull-down of cytoplasmic DNA coupled to mass spectrometry identified several planarian homologs of conserved immune regulators and putative DNA sensors. Through RNA interference screening of conserved innate immune components, we uncover roles for S. mediterranea homologs of Tank-binding kinase 1 (TBK1) and macrophage mannose receptor 1 (MRC1) as potent repressors of transgene expression. Transcriptomic and functional analyses further implicate TBK1 in regulating broad innate immune and stress-response programs, akin to its vertebrate function. Together, our findings demonstrate that innate immune signaling limits transgene expression in S. mediterranea and suggest that modulating these pathways may be key to enabling stable and efficient genetic manipulation in planarians.
Development
· 2026 Feb · PMID 41738557
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An optogenetic split-GAL4 system, ShineGAL4, allows genes to be manipulated with unprecedented spatiotemporal precision. Here, we convert a panel of 14 GAL4 drivers widely used in Drosophila research into their ShineGAL4...An optogenetic split-GAL4 system, ShineGAL4, allows genes to be manipulated with unprecedented spatiotemporal precision. Here, we convert a panel of 14 GAL4 drivers widely used in Drosophila research into their ShineGAL4 counterparts. Homology assisted CRISPR knock-in (HACK) is used to replace GAL4 with the GAL4 DNA binding domain fused to a Magnet photoswitch. We show that the resulting ShineGAL4 drivers enable gene expression to be rapidly induced by light specifically in fat body, muscles, enterocytes, oenocytes, Malpighian tubules, neurons, neuroblast lineages, glial subtypes or in all glia. We also develop an optogenetic cassette for photoactivation of GAL4 in 'silent' FLP-out clones. This panel of optogenetic tools will enable precise spatiotemporal control of gene expression in a wide range of different Drosophila tissues and cell-types.
Purvis IJ, Ochoa Olmos OE, Park KU
… +7 more, Kaufman ML, Henry CM, Schaaf C, Clise OJ, Tesdahl CD, Haas A, Brzezinski JA
Development
· 2026 Mar · PMID 41736562
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The transcription factor Otx2 is essential for photoreceptor and bipolar cell formation during retinal development. Otx2 expression is complex and underlies multiple cell fate decisions during development. To understand...The transcription factor Otx2 is essential for photoreceptor and bipolar cell formation during retinal development. Otx2 expression is complex and underlies multiple cell fate decisions during development. To understand how Otx2 expression is regulated, we explored the activity and function of three of its enhancers (DHS2, DHS4 and DHS15). Enhancer reporter assays and lineage tracing show that DHS4 initiates Otx2 expression while DHS2 and DHS15 maintain expression in photoreceptors. Matched CRISPR/Cas9 and CRISPR interference systems were used to mutate or epigenetically silence enhancers, respectively. CRISPR reduced OTX2 expression acutely, but failed to significantly alter cell fate choice over the long term. In contrast, CRISPR interference of these enhancers caused permanent OTX2 loss and corresponding cell fate changes. While these data suggest that each enhancer is needed for normal Otx2 expression, it also highlights that the enhancers can interact and substitute for each other during development. This cis-regulatory element flexibility likely promotes Otx2 expression robustness. Such robustness may enable complex genes, like Otx2, to resist environmental stressors and regulatory disruptions to promote reproducible developmental outcomes.
Bell CC, de Boer CG, Camellato BR
… +3 more, Delás MJ, Ibrahim DM, Montgomery MT
Development
· 2026 Feb · PMID 41733210
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The cis-regulatory code is exceedingly complex, involving many proteins acting in concert to interpret individual cis-regulatory DNA sequences, which themselves work together to regulate the expression of genes. The Comp...The cis-regulatory code is exceedingly complex, involving many proteins acting in concert to interpret individual cis-regulatory DNA sequences, which themselves work together to regulate the expression of genes. The Company of Biologists' 2024 Workshop 'Building to Understand: The Constructionist Approach to Studying Gene Regulation' brought together an international, interdisciplinary team of investigators studying how cis-regulatory elements control gene expression. Here, we summarise some of the Workshop's discussions and review how our understanding of genome regulation has been shaped by constructionist approaches, as well as where these approaches may lead in the future. An eventual goal is to be able to design cis-regulatory DNA to have desired functions; therefore, we provide guidance on their design based on current knowledge, while simultaneously highlighting what remains to be discovered.