Saied-Santiago K, Baxter M, Mathiaparanam J
… +1 more, Granato M
Development
· 2025 Jul · PMID 40521655
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Optic nerve (ON) regeneration in mammalian systems is limited by an overshadowing dominance of inhibitory factors. This has severely hampered the identification of pro-regenerative pathways. Here, we take advantage of th...Optic nerve (ON) regeneration in mammalian systems is limited by an overshadowing dominance of inhibitory factors. This has severely hampered the identification of pro-regenerative pathways. Here, we take advantage of the regenerative capacity of larval zebrafish to identify pathways that promote ON regeneration. From a small molecule screen, we identified modulators of serotonin (5-HT) signaling that inhibit ON regeneration. We find that several serotonin type-1 (5-HT1) receptor genes are expressed in retinal ganglion cells during regeneration and that inhibiting 5-HT1 receptors or components of the 5-HT pathway selectively impedes ON regeneration. We show that 5-HT1 receptor signaling is dispensable during ON development yet is required for regenerating axons to emerge from the injury site. Blocking 5-HT receptors once ON axons have crossed the chiasm does not inhibit regeneration, suggesting a selective role for 5-HT receptor signaling early during ON regeneration. Finally, we show that agonist-mediated activation of 5-HT1 receptors leads to enhanced and ectopic axonal regrowth. Combined, our results provide evidence for mechanisms through which serotonin-dependent neuromodulation directs ON regeneration in vivo.
Development
· 2025 Jul · PMID 40512118
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Gene regulation by transcription factors (TFs) binding cognate sequences is of paramount importance. For example, the TFs Zelda (Zld) and GAGA factor (GAF) are widely acknowledged for pioneering gene activation during zy...Gene regulation by transcription factors (TFs) binding cognate sequences is of paramount importance. For example, the TFs Zelda (Zld) and GAGA factor (GAF) are widely acknowledged for pioneering gene activation during zygotic genome activation (ZGA) in Drosophila. However, quantitative dose/response relationships between bulk TF concentration and DNA binding, an event tied to transcriptional activity, remain elusive. Here, we map these relationships during ZGA: a crucial step in metazoan development. To map the dose/response relationship between nuclear concentration and DNA binding, we performed raster image correlation spectroscopy, a method that can measure biophysical parameters of fluorescent molecules. We found that, although Zld concentration increases during nuclear cycles 10 to 14, its binding in the transcriptionally active regions decreases, consistent with its function as an activator for early genes. In contrast, GAF-DNA binding is nearly linear with its concentration, which sharply increases during the major wave, implicating its involvement in the major wave. This study provides key insights into the properties of the two factors and puts forward a quantitative approach that can be used for other TFs to study transcriptional regulation.
Sakai S, Maeda Y, Saeki M
… +6 more, Konno D, Kawaji K, Matsuzaki F, Suzuki Y, Gotoh Y, Kishi Y
Development
· 2025 Jul · PMID 40501413
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During neuronal differentiation, gene transcription patterns change in response to both intrinsic and extrinsic cues. Chromatin regulation at regulatory elements plays a key role in this process. However, how chromatin a...During neuronal differentiation, gene transcription patterns change in response to both intrinsic and extrinsic cues. Chromatin regulation at regulatory elements plays a key role in this process. However, how chromatin accessibility evolves in vivo in cortical neurons remains unclear. Here, we established a method for labeling differentiating neurons with specific birthdates. Using this method, we traced the 4-day differentiation process of in vivo deep-layer excitatory neurons in the mouse embryonic cortex and examined changes in the genome-wide transcription pattern and chromatin accessibility using RNA sequencing and DNase sequencing, respectively. We found that genomic regions of genes linked to mature neuronal functions, including deep layer-specific and stimulus-responsive genes, became accessible even at the embryonic stage. Additionally, our results indicated the involvement of bivalent marks in neural precursor/stem cells and Dmrt3 and Dmrta2 in the regulation of chromatin accessibility during neuronal differentiation. These findings highlight the importance of chromatin regulation in embryonic neurons, enabling the timely activation of neuronal genes during maturation.
Development
· 2025 Jun · PMID 40495676
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A phenotypic screen of fungal filtrates on developing zebrafish embryos identified metabolites from the fungus Ceratocystis populicola to induce ectopic tail formation, including a split notochord and a duplicated caudal...A phenotypic screen of fungal filtrates on developing zebrafish embryos identified metabolites from the fungus Ceratocystis populicola to induce ectopic tail formation, including a split notochord and a duplicated caudal fin. Chemical analyses led to the identification of monoterpene alcohols, in particular geraniol, as the active compounds. Tüpfel long fin zebrafish embryos were more susceptible to geraniol-induced ectopic tail formation than Wild Indian Karyotpe zebrafish embryos. RNA-sequencing on tail buds of 15-somite-stage embryos revealed downregulation of essential genes of the retinoic acid signaling pathway. Differential expression of cyp26a1, fgf8a and downstream hox-genes was validated. Time-lapse imaging revealed that Kupffer's vesicle-derived cells failed to migrate after Kupffer's vesicle collapse upon geraniol treatment. These cells failed to merge with progenitors from the tail bud and contributed to an ectopic tail, expressing markers for presomitic mesoderm, somite and notochord tissue. Strikingly, ablation of Kupffer's vesicle by tbxta-morpholino injection rescued ectopic tail formation. Taken together, our data suggest that Kupffer's vesicle cells harbor tail progenitor capacity, and proper migration of these cells is essential for normal tail morphogenesis.
Beckman WF, Parkinson LM, Chaytor L
… +1 more, Philpott A
Development
· 2025 Jun · PMID 40452575
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ASCL1 is a potent proneural factor with paradoxical functions during development, promoting both progenitor pool expansion and neuronal differentiation. How a single factor executes and switches between these potentially...ASCL1 is a potent proneural factor with paradoxical functions during development, promoting both progenitor pool expansion and neuronal differentiation. How a single factor executes and switches between these potentially opposing functions remains to be understood. Using human neuroblastoma cells as a model system, we show that ASCL1 exhibits cell cycle phase-dependent chromatin binding patterns. In cycling cells, S/G2/M phase-enriched binding occurs at promoters of transcribed pro-mitotic genes, while G1 phase-enriched binding of ASCL1 is associated with the priming of pro-neuronal enhancer loci. Prolonged G1 arrest is further required to activate these ASCL1-bound and primed neuronal enhancers to drive neuronal differentiation. Thus, we reveal that the same transcription factor can control distinct transcriptional programmes at different cell cycle stages, and demonstrate how lengthening of G1 allows engagement of a differentiation programme by turning unproductive factor binding into productive interactions.
Tang Q, Li L, Li Y
… +8 more, Wang A, Li H, Wang L, Gu C, Lee JM, Wu Z, Kwon HE, Chen Y
Development
· 2025 Jun · PMID 40446216
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The canonical bone morphogenetic protein (BMP) signaling pathway plays a crucial regulatory role in tooth development by activating Smad proteins to regulate gene expression. We previously identified an atypical canonica...The canonical bone morphogenetic protein (BMP) signaling pathway plays a crucial regulatory role in tooth development by activating Smad proteins to regulate gene expression. We previously identified an atypical canonical BMP signaling in dental mesenchyme that is Smad4-independent but Smad1/5-dependent. This study demonstrates that phosphorylated Smad1/5 (pSmad1/5) and Smad4 regulate distinct gene sets in murine dental mesenchyme. Real-time monitoring of BMP-Smad transcriptional activity revealed that Smad4-dependent canonical BMP signaling is restricted to neurovascular cells surrounding the condensed dental mesenchymal cells where pSmad1/5 is present. Notably, pSmad1/5 in dental mesenchymal cells form complexes with pSmad3 to prevent canonical BMP signaling. CUT&RUN assays revealed genome-wide co-occupancy of pSmad1/5 and pSmad3, indicating their function as transcriptional regulation units. Integrative analyses demonstrated that this atypical canonical BMP signaling regulates tooth sensory innervation and maintains odontogenic inductive potential in dental mesenchyme, enabling the identification of crucial genes for maintaining tooth inductive capability. Our findings elucidate the operating mechanism of atypical canonical BMP signaling in dental mesenchymal cells and clarify how BMP-Smad signaling exerts diverse functions across different cell types, informing future tooth bioengineering strategies.
Tsukamoto T, Kwah JK, Zweifel ME
… +5 more, Courtemanche N, Gearhart MD, Walstrom KM, Jaramillo-Lambert A, Greenstein D
Development
· 2025 Jun · PMID 40446215
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Fertilization triggers the completion of female meiosis and launches the oocyte-to-embryo transition. Caenorhabditis elegans spe-11 is one of the few known paternal-effect embryonic lethal genes. We report that the sperm...Fertilization triggers the completion of female meiosis and launches the oocyte-to-embryo transition. Caenorhabditis elegans spe-11 is one of the few known paternal-effect embryonic lethal genes. We report that the sperm protein SPE-11 forms a complex with an oocyte protein, OOPS-1 (Oocyte Partner of SPE-11) at fertilization, and that the protein complex is required for the completion of meiosis, the block to polyspermy, and eggshell formation. Consistent with the molecular interaction of their encoded proteins, oops-1 and spe-11 exhibit indistinguishable null phenotypes in which fertilized oocytes arrest in meiosis I or meiosis II or fail to complete the actin-based process of meiotic cytokinesis. Biochemical analysis shows that the complex binds F-actin in the absence of other proteins and inhibits the nucleation of actin filaments at substoichiometric concentrations. Both OOPS-1 and SPE-11 are intrinsically disordered proteins that are highly phosphorylated, and biochemical and genetic experiments define interactions with the sperm-specific protein phosphatase 1 homologs GSP-3/4. Genetic results suggest that the cortical EGG complex recruits the OOPS-1-SPE-11 complex at fertilization, which promotes meiotic cytokinesis and in turn activates synthesis of the eggshell.
Gómez-Picos P, Ovens K, Ashique AM
… +3 more, Hassanzadeh M, McQuillan I, Eames BF
Development
· 2025 Jun · PMID 40440101
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The mechanisms by which crucial transcription factors of a gene regulatory network (GRN) interact continue to be revealed. In the vertebrate skeleton, SOX9 and RUNX2 combine to specify three different cell types. Sox9 dr...The mechanisms by which crucial transcription factors of a gene regulatory network (GRN) interact continue to be revealed. In the vertebrate skeleton, SOX9 and RUNX2 combine to specify three different cell types. Sox9 drives immature chondrocyte differentiation, Runx2 regulates osteoblast differentiation, and both Sox9 and Runx2 are somehow required for mature chondrocyte formation. To elucidate mechanisms of GRN regulation in mature chondrocytes, transcriptomic data were examined from all three skeletal cell types isolated by laser capture microdissection of embryonic mouse. Multiple bioinformatic analyses supported the hypothesis that SOX9 and RUNX2 operate two cross-inhibitory subnetworks of the skeletal cell GRN during immature chondrocyte and osteoblast formation, but mature chondrocyte differentiation involves cooperation between these subnetworks. Several mature chondrocyte gene clusters had expression levels that represented an averaging of SOX9 and RUNX2 subnetworks, while one cluster, containing the hallmark mature chondrocyte genes collagen type 10a1 and Indian hedgehog, suggested a synergistic interaction between subnetworks. Generally, this in vivo LCM-RNA-seq approach enabled new understanding of interactions between distinct GRN subnetworks during cell differentiation and can similarly reveal regulatory control of any developmental process.
Development
· 2025 May · PMID 40421980
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The Notch signaling pathway plays a crucial role in neurogenesis by regulating cell fate specification. However, its complexity poses challenges in uncovering the mechanisms underlying these decisions. This Review explor...The Notch signaling pathway plays a crucial role in neurogenesis by regulating cell fate specification. However, its complexity poses challenges in uncovering the mechanisms underlying these decisions. This Review explores the intricacies of the Notch pathway, including its diverse activation mechanisms and the influence of post-translational modifications of Notch receptors and ligands on pathway outcomes. We discuss how Notch signaling regulates embryonic neurogenesis via interactions with proneural genes and with other signaling pathways. We also examine the role of Notch in adult neurogenesis, and the therapeutic potential of leveraging Notch signaling to reprogram glia in the adult brain. Lastly, we highlight emerging technologies that measure Notch dynamics and discuss remaining knowledge gaps. Together, these insights underscore the multifaceted role of Notch signaling and outline key directions for future research.
Development
· 2025 Oct · PMID 40406997
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Botrylloides diegensis is a colonial ascidian that has been the focus of developmental, evolutionary and regeneration research. In this study, we performed single-cell RNA sequencing (scRNA-seq) of an entire B. diegensis...Botrylloides diegensis is a colonial ascidian that has been the focus of developmental, evolutionary and regeneration research. In this study, we performed single-cell RNA sequencing (scRNA-seq) of an entire B. diegensis colony, including zooids, buds and vascular tunics, to resolve cellular heterogeneity and to identify cell and tissue markers. We identified 29 major cell clusters within the colony and used in situ hybridization to examine the spatial expression of cluster marker genes. Numerous tissue types were identified at the molecular level, including blood cells and zooid tissues, such as the branchial epithelium, stomach and endostyle. Distinct cluster markers were identified for specific regions of the stomach epithelium, highlighting the specialization of these regions and the strength of using scRNA-seq to explore their functionality. Cell trajectory projections highlighted the early appearance of progenitor clusters, whereas more differentiated zooid-related tissues appeared later in the developmental path. This study provides a valuable resource for understanding the development, tissue function and regeneration of B. diegensis. It demonstrates the power of scRNA-seq to define cell types and tissues in complex colonial organisms.
Traba SA, Bacigalupo L, Fradin D
… +12 more, Talon I, Heidenreich AC, Muraro D, Garcia-Bernardo J, Gribben C, Lugtu F, Burgos JI, Romero A, Chhatriwala M, Pecci A, Vallier L, Rodríguez-Seguí SA
Development
· 2025 Jun · PMID 40351285
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Understanding pancreatic development is instrumental to diabetes research and β-cell replacement therapies. Here, we investigate glucocorticoid receptor (GR) signaling during early pancreas development in mice and humans...Understanding pancreatic development is instrumental to diabetes research and β-cell replacement therapies. Here, we investigate glucocorticoid receptor (GR) signaling during early pancreas development in mice and humans. Previous reports suggest that glucocorticoids do not play a significant role in mouse pancreas development before the second transition. In this study, we demonstrate that, under physiological conditions, the GR is selectively active in mouse pro-acinar and early endocrine cells from embryonic day 11.5, silenced in bipotent progenitors, and reactivated during endocrine commitment. In mouse pancreatic explants, ectopic GR activation globally promotes acinar fate. Surprisingly, GR activation in human in vitro-derived multipotent pancreatic progenitors steers lineage commitment toward a bipotent/endocrine trajectory and upregulates genes for which expression profiles resemble those of SOX9 and HES1 during human embryonic pancreatic bipotential and endocrine progenitor fate choice. Our combined epigenomic and single-cell transcriptomic analyses suggest that these newly identified marker genes may play important roles in human pancreas development. Taken together, our findings position the GR pathway as an endogenous developmental modulator of early-stage pancreatic progenitor cell differentiation and provide insights into the underlying transcriptional mechanisms involved.
Ostalé CM, Azpiazu N, Peropadre A
… +7 more, Martín M, Ruiz-Losada M, López-Varea A, Viales RR, Girardot C, Furlong EEM, de Celis JF
Development
· 2025 May · PMID 40326666
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The conserved Spalt proteins regulate gene expression and cell fate choices during multicellular development, generally acting as transcriptional repressors in different gene regulatory networks. In addition to their rol...The conserved Spalt proteins regulate gene expression and cell fate choices during multicellular development, generally acting as transcriptional repressors in different gene regulatory networks. In addition to their roles as DNA sequence-specific transcription factors, Spalt proteins show a consistent localization to heterochromatic regions. Vertebrate Spalt-like proteins can act through the nucleosome remodeling and deacetylase complex to promote closing of open chromatin domains, but their activities also rely on interactions with DNA methyltransferases or with the lysine-specific histone demethylase LSD1, suggesting that they participate in multiple regulatory mechanisms. Here, we describe several consequences of loss of Spalt function in Drosophila cells, including changes in chromatin accessibility, generation of DNA damage, alterations in the localization of chromosomes within the nucleus in the salivary glands and misexpression of transposable elements. We suggest that these effects are related to roles of Spalt proteins in the regulation of heterochromatin formation and chromatin organization. We propose that Drosophila Spalt proteins have two complementary functions, acting as sequence-specific transcriptional repressors on specific target genes and regulating more global gene silencing through the generation or maintenance of heterochromatic domains.
Romera-Branchat M, Pocard C, Vincent C
… +7 more, Cerise M, da Silveira Falavigna V, Pajoro A, Ding N, Gao H, Franzen R, Coupland G
Development
· 2025 May · PMID 40326559
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Arabidopsis florigen activation complex (FAC), formed by the interaction of the transcription factor FD and the florigen protein FT, activates gene expression in the shoot apical meristem to induce flowering. We show tha...Arabidopsis florigen activation complex (FAC), formed by the interaction of the transcription factor FD and the florigen protein FT, activates gene expression in the shoot apical meristem to induce flowering. We show that FD and its paralog FDP are also expressed in partially overlapping patterns in the floral meristem and floral organs, and that FT is present in floral meristems. The flowers of mutants for FT and its paralog TSF (ft tsf), and of fd fdp mutants show variable numbers of sepals and petals, and larger floral meristems than wild type. In the floral meristem, fd fdp and ft tsf mutants show a significant reduction in the expression of SEP and AG genes, which encode MADS-domain transcription factors, as well as increased expression of the homeobox gene WUS. Binding of FD to SEP genes suggests that diminished SEP gene expression is a primary defect in the mutants. We conclude that, beyond their role in floral transition, FAC components regulate floral homeotic gene expression to control floral meristem size, and influence floral organ development and identity.
Abid D, Murphy K, Murphy Z
… +3 more, Rahman N, Getman M, Steiner L
Development
· 2025 May · PMID 40260585
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Erythropoiesis is characterized by dramatic changes in gene expression in the context of a cell that is rapidly proliferating while simultaneously condensing its nucleus in anticipation of enucleation. The mechanisms tha...Erythropoiesis is characterized by dramatic changes in gene expression in the context of a cell that is rapidly proliferating while simultaneously condensing its nucleus in anticipation of enucleation. The mechanisms that maintain high level expression of erythroid genes and promote nuclear condensation remain poorly understood. Condensin II is a ring-like complex that promotes mitotic chromatin condensation and has roles in regulating interphase chromatin architecture and gene expression. We interrogated the role of Condensin II in erythropoiesis using an erythroid-specific deletion of the Condensin II subunit, Ncaph2. Ncaph2 loss resulted in severe anemia by embryonic day 12.5 with embryonic lethality. Ncaph2 mutant erythroid cells had dysregulated maturation and disrupted cell cycle progression, but surprisingly NCAPH2 was dispensable for nuclear condensation. Genomic studies revealed that NCAPH2 occupied the promoter of key erythroid and cell cycle genes that were downregulated following Ncaph2 loss. Together, our results demonstrate an essential role for NCAPH2 in the gene expression programs that regulate cell cycle progression and erythroid differentiation, and identify a role for the Condensin II complex in the regulation of a lineage-specific differentiation program.
Ranganathan R, Sari F, Wang SX
… +5 more, Thiery A, Buzzi AL, Guerra R, Moody SA, Streit A
Development
· 2025 Apr · PMID 40213817
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Branchio-otic (BOS) and branchio-oto-renal (BOR) syndromes are autosomal dominant disorders featuring multiple birth defects including ear, renal and branchial malformations. Mutations in the homeodomain transcription fa...Branchio-otic (BOS) and branchio-oto-renal (BOR) syndromes are autosomal dominant disorders featuring multiple birth defects including ear, renal and branchial malformations. Mutations in the homeodomain transcription factor SIX1 and its co-factor EYA1 have been identified in about 50% of individuals with BOS or BOR, while causative mutations are unknown in the other half. We hypothesise that SIX1 target genes represent new BOS and BOR candidates. Using published transcriptomic and epigenomic data from chick ear progenitors, we first identify putative Six1 targets. Next, we provide evidence that Six1 directly regulates some of these candidates: Six1 binds to their enhancers, and functional experiments in Xenopus and chick confirm that Six1 controls their expression. Finally, we show that most putative chick Six1 targets are also expressed in the human developing ear and are associated with known deafness loci. Together, our results not only characterise the molecular mechanisms that mediate Six1 function in the developing ear, but also provide new candidates for human congenital deafness.
Kanai SM, Garcia CR, Augustus MR
… +6 more, Sharafeldeen SA, Brooks EP, Sucharov J, Lencer ES, Nichols JT, Clouthier DE
Development
· 2025 Apr · PMID 40171762
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Vertebrate jaw development is coordinated by highly conserved ligand-receptor systems such as the peptide ligand Endothelin 1 (Edn1) and Endothelin receptor type A (Ednra), which are required for patterning of lower jaw...Vertebrate jaw development is coordinated by highly conserved ligand-receptor systems such as the peptide ligand Endothelin 1 (Edn1) and Endothelin receptor type A (Ednra), which are required for patterning of lower jaw structures. The Edn1/Ednra signaling pathway establishes the identity of lower jaw progenitor cells by regulating expression of numerous patterning genes, but the intracellular signaling mechanisms linking receptor activation to gene regulation remain poorly understood. As a first step towards elucidating this mechanism, we examined the function of the Gq/11 family of Gα subunits in zebrafish using pharmacological inhibition and genetic ablation of Gq/11 activity, and transgenic induction of a constitutively active Gq protein in edn1-/- embryos. Genetic loss of Gq/11 activity fully recapitulated the edn1-/- phenotype, with genes encoding G11 being most essential. Furthermore, inducing Gq activity in edn1-/- embryos not only restored Edn1/Ednra-dependent jaw structures and gene expression signatures but also caused homeosis of the upper jaw structure into a lower jaw-like structure. These results indicate that Gq/11 is necessary and sufficient to mediate the lower jaw patterning mechanism for Ednra in zebrafish.
Development
· 2025 Apr · PMID 40167323
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The urothelium is a stratified epithelium with an important barrier function in the urinary drainage system. The differentiation and maintenance of the three major urothelial cell types (basal, intermediate and superfici...The urothelium is a stratified epithelium with an important barrier function in the urinary drainage system. The differentiation and maintenance of the three major urothelial cell types (basal, intermediate and superficial cells) is incompletely understood. Here, we show that mice with a conditional deletion of the transcription factor gene peroxisome proliferator activated receptor gamma (Pparg) in the ureteric epithelium have a dilated ureter at postnatal stages with a urothelium consisting of a layer of undifferentiated luminal cells and a layer of proliferating basal cells. Molecular analysis of fetal stages revealed that the expression of a large number of genes is not activated in superficial cells and that of a few genes, including Shh, is not activated in intermediate and basal cells. Pharmacological activation of SHH signaling in explant cultures of perinatal Pparg-deficient ureters reduced ureteral width and urothelial cell number to normal levels, increased the number of intermediate cells and slightly reduced basal cell proliferation. Our data suggest that PPARG independently activates the expression of structural genes in superficial cells and of Shh in basal and intermediate cells, and that both functions contribute to urothelial integrity.
Wilhelm D, Perea-Gomez A, Newton A
… +1 more, Chaboissier MC
Development
· 2025 Mar · PMID 40162719
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Sex determination and differentiation are fundamental processes that are not only essential for fertility but also influence the development of many other organs, and hence, are important for species diversity and surviv...Sex determination and differentiation are fundamental processes that are not only essential for fertility but also influence the development of many other organs, and hence, are important for species diversity and survival. In mammals, sex is determined by the inheritance of an X or a Y chromosome from the father. The Y chromosome harbours the testis-determining gene SRY, and it has long been thought that its absence is sufficient for ovarian development. Consequently, the ovarian pathway has been treated as a default pathway, in the sense that ovaries do not have or need a female-determining factor. Recently, a female-determining factor has been identified in mouse as the master regulator of ovarian development. Interestingly, this scenario was predicted as early as 1983. In this Review, we discuss the model predicted in 1983, how the mechanisms and genes currently known to be important for sex determination and differentiation in mammals have changed or supported this model, and finally, reflect on what these findings might mean for sex determination in other vertebrates.
Li Y, Li Y, Huang B
… +4 more, Zhang R, He J, Luo L, Yang Y
Development
· 2025 Mar · PMID 40116142
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Cell labelling and lineage tracing are indispensable tools in developmental biology, offering powerful means with which to visualise and understand the complex dynamics of cell populations during embryogenesis. Tradition...Cell labelling and lineage tracing are indispensable tools in developmental biology, offering powerful means with which to visualise and understand the complex dynamics of cell populations during embryogenesis. Traditional cell labelling relies heavily on signal stability, promoter strength and stage specificity, limiting its application in long-term tracing. In this report, we optimise and reconfigure a perpetual cycling Gal4-UAS system employing a previously unreported Gal4 fusion protein and the autoregulatory Gal4 expression loop. As validated through heat-shock induction, this configuration ensures sustained transcription of reporter genes in target cells and their descendant cells while minimising cytotoxicity, thereby achieving long-term labelling and tracing. Further exploiting this system, we generate zebrafish transgenic lines with continuous fluorescent labelling specific to the endoderm, and demonstrate its effectiveness in long-term tracing by showing the progression of endoderm development from embryo to adult, providing visualisation of endodermal cells and their derived tissues. This continuous labelling and tracing strategy can span the entire process of endodermal differentiation, from progenitor cells to mature functional cells, and is applicable to studying endoderm patterning and organogenesis.
Yang W, Peng M, Wang Y
… +6 more, Zhang X, Li W, Zhai X, Wu Z, Hu P, Chen L
Development
· 2025 Apr · PMID 40110772
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Iron is essential for cell growth and hematopoiesis, which is regulated by hepcidin (hamp). However, the role of hamp in zebrafish hematopoiesis remains unclear. Here, we have created a stable hamp knockout zebrafish mod...Iron is essential for cell growth and hematopoiesis, which is regulated by hepcidin (hamp). However, the role of hamp in zebrafish hematopoiesis remains unclear. Here, we have created a stable hamp knockout zebrafish model using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 system (CRISPR/Cas9 system). Our study revealed that hamp deletion led to maternal iron overload in embryos, significantly downregulating hemoglobin genes and reducing hemoglobin content. Single-cell RNA sequencing identified abnormal expression patterns in blood progenitor cells, with a specific progenitor subtype showing increased ferroptosis and delayed development. By crossing hamp knockout zebrafish with a gata1+ line (blood cells labeled fish line), we confirmed ferroptosis in blood progenitor cells. These findings underscore the crucial role of hamp in iron regulation and hematopoiesis, offering novel insights into developmental biology and potential therapeutic targets for blood disorders.