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bioRxiv2020

C-Eci: A Cubic-Eci Combined Clearing Method For 3D Follicular Content Analysis In The Fish Ovary

Deciphering mechanisms of oocyte development in female fishes still remains challenging and a comprehensive overview of this process at the level of the organ is still needed. The recent development optical tissue clearing methods have tremendously boosted the 3D imaging of large size biological samples that are naturally opaque. However, no attempt of clearing on fish ovary that accumulates extremely high concentration of lipids within oocytes has been reported to date. To face with this ovarian-specific challenge, we combined two existing clearing methods, the non-toxic solvent-based Eci method for efficient clearing and the CUBIC method to enhance lipid removal and reduce non-specific staining. The methyl green fluorescent dye was used to stain nuclei and delineate follicles. Using this procedure (named C-Eci), ovaries of both medaka and trout could be imaged in 3D and all follicles analyzed. To our knowledge this is the first procedure elaborated for clearing and imaging fish ovary in 3D. The C-Eci methods thus provides an interesting tool for getting precise quantitative data on follicular content in fish ovary and promises to be useful for further morphological studies. SummaryA modified ethyl-cinnamate-based clearing method allows solving fish ovary-specific challenges for 3D imaging, including high lipid-contents, and analyzing the ovarian follicular content in medaka and trout.

developmental biology↗

bioRxiv2024

HemK2 functions for sufficient protein synthesis and RNA stability through eRF1 methylation during Drosophila oogenesis

HemK2 is a highly conserved methyltransferase spanning from yeast to humans. Despite its conservation, the identification of its genuine substrates has been controversial, and its biological importance in higher organisms remains unclear. In this study, we elucidate the role of HemK2 in the methylation of eukaryotic Release Factor 1 (eRF1), a process essential for female germline development in Drosophila melanogaster. Knockdown of hemK2 in the germline cells (hemK2-GLKD) induces apoptosis in these cells, accompanied by a pronounced decrease in both eRF1 methylation and protein synthesis. The overexpression of a methylation-deficient eRF1 variant recapitulates the defects observed in hemK2-GLKD, suggesting that eRF1 is a primary methylation target of HemK2. Furthermore, hemK2-GLKD leads to significant reduction mRNA levels in germline cell. We demonstrate that these defects in oogenesis and protein synthesis can be partially restored by inhibiting the No-Go Decay pathway. In addition, hemK2 knockdown is associated with increased disome formation, suggesting that disruptions in eRF1 methylation may provoke ribosomal stalling, which subsequently activates translation-coupled mRNA surveillance mechanisms that degrade actively-translated mRNAs. We propose that HemK2-mediated methylation of eRF1 is critical for ensuring efficient protein production and mRNA stability, which are vital for the generation of high-quality eggs.

developmental biology↗

bioRxiv2024

Deep-Tissue Spatial Omics: Imaging Whole-Embryo Transcriptomics and Subcellular Structures at High Spatial Resolution

The inherent limitations of fluorescence microscopy, notably the restricted number of color channels, have long constrained comprehensive spatial analysis in biological specimens. Here, we introduce cycleHCR technology that leverages multicycle DNA barcoding and Hybridization Chain Reaction (HCR) to surpass the conventional color barrier. cycleHCR facilitates high-specificity, single-shot imaging per target for RNA and protein species within thick specimens, mitigating the molecular crowding issues encountered with other imaging-based spatial omics techniques. We demonstrate whole-mount transcriptomics imaging of 254 genes within an E6.5[~]7.0 mouse embryo, achieving precise three-dimensional gene expression and cell fate mapping across a specimen depth of [~] 310 {micro}m. Utilizing expansion microscopy alongside protein cycleHCR, we unveil the complex network of 10 subcellular structures in primary mouse embryonic fibroblasts. Furthermore, in mouse hippocampal slice, we image 8 protein targets and profile the transcriptome of 120 genes, uncovering complex gene expression gradients and cell-type specific nuclear structural variances. cycleHCR provides a unifying framework for multiplex RNA and protein imaging, offering a quantitative solution for elucidating spatial regulations in deep tissue contexts for research and potentially diagnostic applications.

developmental biology↗

bioRxiv2024

Dual roles of EGO-1 and RRF-1 in regulating germline exo-RNAi efficiency in Caenorhabditis elegans

RNA interference (RNAi) is widely used in life science research and is critical for diverse biological processes, such as germline development and antiviral defense. In Caenorhabditis elegans, RNA-dependent RNA polymerases, with redundant involvement of EGO-1 and RRF-1, facilitate small RNA amplification in germline exogenous RNAi (exo-RNAi). However, their coordination during the regulation of exo-RNAi processes in the germline remains unclear. Here, we examined non-null mutants of the ego-1 gene and found that ego-1(S1198L) animals exhibited germline exo-RNAi defects with normal fertility, abnormalities in germ granules, and synthetic temperature-dependent sterility with rrf-1. The exo-RNAi defects in ego-1(S1198L) were partially restored by inhibiting hrde-1, cde-1, and znfx-1. Similar defects were observed in wild-type and ego-1(S1198L) heterozygous descendants derived from ego-1(S1198L), but these were suppressed by ancestral inhibition of rrf-1. These data reveal a dual role for EGO-1 in the positive regulation of germline exo-RNAi: it not only mediates target silencing through its RNA-dependent RNA polymerase activity, but also fine-tunes germ granule function or downstream processes, which are antagonized by RRF-1.

developmental biology↗

bioRxiv2019

Constraints on somite formation in developing embryos

Segment formation in vertebrate embryos is a stunning example of biological self-organisation. Here, we present an idealized model of the presomitic mesoderm (PSM) as a one-dimensional line of oscillators. We use the model to derive constraints that connect the size of somites, and the timing of their formation, to the growth of the PSM and the gradient of the somitogenesis clock period across the PSM. Our analysis recapitulates the observations made recently in ex-vivo cultures of mouse PSM cells, and makes predictions for how perturbations, such as increased Wnt levels, would alter somite widths. Finally, our model makes testable predictions for the shape of the phase profile and somite widths at different stages of PSM growth. In particular, we show that the phase profile is robustly concave when the PSM length is steady and slightly convex in an important special case when it is decreasing exponentially. In both cases, the phase profile scales with the PSM length; in the latter case, it scales dynamically. This has important consequences for the velocity of the waves that traverse the PSM and trigger somite formation, as well as the effect of errors in phase measurement on somite widths.

developmental biology↗

bioRxiv2023

Feedback control of organ size precision is mediated by BMP2-regulated apoptosis in the Drosophila eye

Biological processes are intrinsically noisy and yet, the result of development -like the species-specific size and shape of organs- is usually remarkably precise. This precision suggests the existence of mechanisms of feedback control that ensure that deviations from a target size are minimized. Still, we have very limited understanding of how these mechanisms operate. Here, we investigate the problem of organ size precision using the Drosophila eye. The size of the adult eye depends on the rates at which eye progenitor cells grow and differentiate. We first find that the progenitor net growth rate results from the balance between their proliferation and apoptosis, with this latter contributing to determining both final eye size and its variability. In turn, apoptosis of progenitor cells is hampered by Dpp, a BMP2/4 signaling molecule transiently produced by early differentiating retinal cells. Our genetic and computational experiments show how the status of retinal differentiation is communicated to progenitors through the differentiation-dependent production of Dpp which, by adjusting the rate of apoptosis, exerts a feedback control over the net growth of progenitors to reduce final eye size variability.

developmental biology↗

bioRxiv2025

Cytoneme-mediated signalling coordinates the development of glial cells and neurons in the Drosophila eye

Effective cell communication is essential for the development and maintenance of the nervous system, where neurons and glial cells must interact closely. While cytoneme-mediated signalling is well-documented in various biological contexts, its role in coordinating neuron-glia development remains poorly understood. In this study, we investigated the function of cytonemes in neuron-glia coordination using the Drosophila eye imaginal disc as a model. This is a well-established system for examining the orchestrated development of glial and neuronal cells. Our results reveal that glial cells produce two distinct types of cytonemes based on their spatial orientation: one set extends toward nascent photoreceptors, while the other targets the morphogenetic furrow (MF). We have characterised the dynamics of glial cytonemes and demonstrated that disrupting these structures has a significant impact on glial cell migration and differentiation. This highlights the critical role of cytoneme-mediated signalling in regulating glial behaviour. Our findings also demonstrate that cytoneme function is essential for activating the Hedgehog (Hh) pathway in glial cells, with Hh ligand produced by photoreceptors. This pathway is necessary for glial differentiation, uncovering a previously unrecognised role for Hh signalling in this process. Overall, our results suggest that cytoneme-mediated Hh signalling is key to coordinating the development of both glial and neuronal populations.

developmental biology↗

bioRxiv2025

Gadd45 regulates fate decisions of myeloid-type blood progenitor cells in Drosophila

Gadd45 is a highly conserved stress response protein that interacts with multiple cellular proteins, playing a crucial role in regulating the cell cycle and responding to various forms of stress. Altered expression of Gadd45 is linked to several solid tumors and hematopoietic malignancies. It is also identified as a gene crucial for cytokine-induced myeloid differentiation, although the precise molecular mechanisms underlying this process remain unclear. We utilized the Drosophila hematopoietic system to investigate the role of Gadd45 in the development of myeloid-type blood cells, employing in vivo genetics and cell biological methods. We show that the absence of Gadd45 reduces the number of crystal cells but does not significantly impact macrophage-like cell plasmatocyte differentiation. However, Gadd45 overexpression caused intermediate progenitors to differentiate into lamellocytes and plasmatocytes to transdifferentiate into lamellocytes, a response typically observed during larval stages in response to immune challenges and injury. Interestingly, Gadd45 overexpression initiated the lamellocyte differentiation program by activating a combination of Toll, JNK, and JAK/STAT signaling pathways. This closely resembles the process by which specialized macrophages are derived in mammals. Moreover, Gadd45 overexpression does not induce cell death but alters the cell cycle status of hematopoietic progenitor cells. This study enhances our understanding of how Gadd45 functions during myeloid cell development. Significance StatementO_LILoss of Gadd45 causes a reduction in crystal cells but does not significantly impact macrophage-like plasmatocyte differentiation. C_LIO_LIGadd45 overexpression causes myeloid-type progenitor cells to differentiate into lamellocyte, as well as plasmatocyte transdifferentiate to lamellocyte. Gadd45 triggers differentiation by activating a combination of Toll, JNK, and JAK/STAT signaling pathways. C_LIO_LIOverexpressing Gadd45 does not induce apoptosis but alters the cell cycle behavior of hematopoietic progenitor cells. C_LI

developmental biology↗

bioRxiv2020

Transcriptome analysis reveals evolutionary co-option of neural development and signaling genes for the wing pigmentation pattern of the polka-dotted fruit fly.

How evolutionary novelties have arisen is one of the central questions in evolutionary biology. Pre-existing gene regulatory networks or signaling pathways have been shown to be co-opted for building novel traits in several organisms. However, the structure of entire gene regulatory networks and evolutionary events of gene co-option for emergence of a novel trait are poorly understood. In this study, we used a novel wing pigmentation pattern of the polka-dotted fruit fly, and identified the complete set of genes for pigmentation pattern formation by de novo genome sequencing and transcriptome analyses. In pigmentation areas of wings, 151 genes were positively or negatively regulated by wingless, a master regulator of wing pigmentation. Genes for neural development, Wnt signaling, Dpp signaling, Zinc finger transcription factors, and effectors (such as enzymes) for melanin pigmentation were included among these 151 genes. None of the known regulatory genes that regulate pigmentation pattern formation in other fruit fly species were included. Our results suggest that the novel pigmentation pattern of the polka-dotted fruit fly emerged through multi-step co-options of multiple gene regulatory networks, signaling pathways, and effector genes, rather than recruitment of one large gene circuit.

developmental biology↗

bioRxiv2024

Decorin promotes cardiac organoid maturation by activating AMPK-PGC1A pathway to enhance cardiac metabolism and mitophagy

RationaleCardiac organoids (COs) are advanced models for investigating heart development and disease, while require maturation to resemble the structural and functional characteristics of the human heart. ObjectiveThis study reveals the role of Decorin (DCN) contributes to the mature and vascularized COs and assesses the biological mechanism responsible for CO maturation. Methods and ResultsDCN-treated COs exhibit structural maturation involving aligned sarcomere, mitochondria, and t-tubule structures, and vessel formations, as well as functional maturation involving synchronized contraction-relaxation, Ca2+ transient, and increases ion channel expressions. DCN-treated COs also show metabolic maturation, including enhanced fatty acid oxidation and increased mitophagy. Transcriptional profiling results indicate that DCN-treated COs have increased levels of AMPK signaling and mitophagy. In DCN-treated COs, AMPK knockdown affects mitochondrial biogenesis, cardiac metabolism, ion channels, and mitophagy. ConclusionsThese findings indicate that DCN is crucial for development of mature, vascularized COs and that CO maturation is primarily regulated through AMPK signaling, which is triggered by DCN. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/599970v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1333cfaorg.highwire.dtl.DTLVardef@e842baorg.highwire.dtl.DTLVardef@74ce1corg.highwire.dtl.DTLVardef@87ad38_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHIC ABSTRACTC_FLOATNO A graphic abstract is available for this article. DCN enhances metabolic maturation in COs by AMPK-triggered regulation of the glycolysis, fatty acid oxidation, and mitophagy. C_FIG

developmental biology↗

bioRxiv2025

Integrin promotes basement membrane crossing and branching of an invading intracellular tube

The narrowest biological tubes are comprised of cells that hollow to form an intracellular lumen. Here, we examine early lumenogenesis of the C. elegans excretory cell, which branches to form an H-shaped intracellular tube spanning the length of the worm. Using genetically paralyzed embryos to freeze movement, we describe lumen initiation and branching for the first time using time-lapse fluorescence microscopy. We show that the excretory cell lumen forms through a plasma membrane invasion mechanism when a nascent lumen grows from the plasma membrane into the cytoplasm. The lumen subsequently extends along the left-right axis before branching to form anterior-posterior projections. Through a genetic screen, we identify mutations in ina-1/-integrin and pat-3/{beta}-integrin that block lumenogenesis at the anterior-posterior branching step, and we show that integrin function is required within the excretory cell. Finally, we find that the excretory cell crosses the epidermal basement membrane where anterior-posterior branches form and demonstrate that basement membrane crossing fails in integrin mutant embryos. Our findings reveal how an intracellular lumen initiates and branches and identify integrins and basement membrane as key branching regulators.

developmental biology↗

bioRxiv2023

Clock driven waves of Tbx6 expression prefigure somite boundaries

The segmented body plan of vertebrates is established during embryogenesis by periodic and sequential formation of multi-cellular structures called somites. Somitogenesis is an example of patterning by a biological oscillator, the segmentation clock, which manifests as traveling waves of oscillating Hes/Her gene expression, reiterating during the formation of each1-3. How these waves are converted into the striped Mesp gene expression pattern that prefigures morphological somite boundaries4-8 remains unclear. Here, we image this conversion in real-time at single-cell resolution in zebrafish, using light-sheet microscopy of a novel reporter of Tbx6, a key activator of Mesp expression. We observe cellular oscillations and kinematic waves of Tbx6 expression that are driven by Hes/Her genes. Tbx6 waves arrest precisely in boundary cells that eventually express Mesp, thereby prefiguring the Mesp pattern, whereas Hes/Her waves do not. Although Hes/Her oscillations began before somitogenesis9-11, the first Tbx6 wave defines the boundary cells of the anterior-most somite, forming the head-trunk interface. Our findings imply that Tbx6 acts as a genetic clutch, converting Her/Hes pacemaker waves into Mesp stripes. We propose that this clock design shields the pacemaker from external perturbations, allowing flexible and robust patterning, making it of interest for organoids and tissue-engineering.

developmental biology↗

bioRxiv2024

Wnt/β-catenin signalling underpins juvenile Fasciola hepatica growth and development

Infection by the liver fluke, Fasciola hepatica, places a substantial burden on the global agri-food industry and poses a significant threat to human health in endemic regions. Widespread resistance to a limited arsenal of chemotherapeutics, including the frontline flukicide triclabendazole (TCBZ), renders F. hepatica control unsustainable and accentuates the need for novel therapeutic target discovery. A key facet of F. hepatica biology is a population of specialised stem cells which drive growth and development - their dysregulation is hypothesised to represent an appealing avenue for control. The exploitation of this system as a therapeutic target is impeded by a lack of understanding of the molecular mechanisms underpinning F. hepatica growth and development. Wnt signalling pathways govern a myriad of stem cell processes during embryogenesis and drive tumorigenesis in adult tissues. Here, we identify five putative Wnt ligands and five Frizzled receptors in liver fluke transcriptomic datasets and find that Wnt/{beta}-catenin signalling is most active in juveniles, the most pathogenic life stage. FISH-mediated transcript localisation revealed partitioning of the five Wnt ligands, with each displaying a distinct expression pattern, consistent with each Wnt regulating the development of different cell/tissue types. The silencing of each individual Wnt or Frizzled gene yielded significant reductions in juvenile worm growth and, in select cases, blunted the proliferation of neoblast-like cells. Notably, silencing FhCTNNB1, the key effector of the Wnt/{beta}-catenin signal cascade led to aberrant development of the neuromuscular system which ultimately proved fatal - the first report of a lethal RNAi-induced phenotype in F. hepatica. The absence of any discernible phenotypes following the silencing of the inhibitory Wnt/{beta}-catenin destruction complex components is consistent with low destruction complex activity in rapidly developing juvenile worms, corroborates transcriptomic expression profiles and underscores the importance of Wnt signalling as a key molecular driver of growth and development in early stage juvenile fluke. The pharmacological inhibition of Wnt/{beta}-catenin signalling using commercially available inhibitors phenocopied RNAi results and provides impetus for drug repurposing. Taken together, these data functionally and chemically validate the targeting of Wnt signalling as a novel strategy to undermine the pathogenicity of juvenile F. hepatica. AUTHOR SUMMARYThe liver fluke, Fasciola hepatica significantly undermines the health and welfare of livestock worldwide and causes fascioliasis, a neglected tropical disease of humans. The most damaging stage of liver fluke infection is caused by the migration of juvenile worms within the liver tissue. Of all drugs approved for liver fluke treatment, just one, triclabendazole (TCBZ), is active on this pathogenic juvenile stage. TCBZ resistance is now widespread rendering liver fluke control unsustainable. This highlights the need for novel drug target identification and validation. A key aspect of juvenile worm biology is their ability to rapidly grow and develop, processes driven by a population of specialised stem cells. As such, the dysregulation of stem cells represents an attractive avenue for liver fluke control. One molecular pathway known to regulate stem cell dynamics in higher organisms is the Wnt signalling pathway. Bioinformatic searches of gene sequence datasets identified all major signalling components of both canonical and non-canonical Wnt pathways in F. hepatica. The localisation of FhWnt pathway components revealed remarkably distinct and widespread expression patterns throughout the F. hepatica body. Gene silencing of putative FhWnt pathway components revealed that those involved in the Wnt/{beta}-catenin signal cascade are fundamental to juvenile growth and, in some cases, stem-like cell proliferation. The silencing of liver fluke {beta}-catenin led to aberrant neuromuscular development and proved lethal to juvenile fluke. Biweekly exposures to commercially available Wnt pathway inhibitory compounds phenocopied the delayed development observed in the gene silencing experiments. These data suggest that FhWnt pathway components represent attractive targets for the development of novel flukicides or indeed, the repurposing of existing Wnt antagonists for parasite control.

developmental biology↗

bioRxiv2020

Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus

Performing gene function analyses in a broad range of research organisms is crucial for understanding the biological functions of genes and their evolution. Recent studies have shown that short hairpin RNAs (shRNAs) can induce gene-specific knockdowns in two cnidarian species. We have developed a detailed, straightforward, and scalable method to deliver shRNAs into fertilized eggs of the hydrozoan cnidarian Hydractinia symbiolongicarpus via electroporation, yielding gene-targeted knockdowns that can be assessed throughout embryogenesis, larval settlement, and metamorphosis. Our electroporation protocol allows for the transfection of shRNAs into hundreds of fertilized H.symbiolongicarpus eggs simultaneously with minimal embryo death and no long-term harmful consequences on the developing animals. We show RT-qPCR and detailed phenotypic evidence of our method successfully inducing significant knockdowns of an exogenous gene (eGFP) and an endogenous gene (Nanos2). We also provide visual confirmation of successful shRNA transfection inside embryos through electroporation. This is the first time that electroporation as a delivery system has been developed for Hydractinia. Our detailed protocol for electroporation of shRNAs in H. symbiolongicarpus embryos constitutes an important experimental resource for the hydrozoan community while also serving as a successful model for the development of similar methods for interrogating gene function in other marine invertebrates.

developmental biology↗

bioRxiv2022

Core PCP mutations affect short time mechanical properties but not tissue morphogenesis in the Drosophila pupal wing

How morphogenetic movements are robustly coordinated in space and time is a fundamental open question in biology. We study this question using the wing of Drosophila melanogaster, an epithelial tissue that undergoes large-scale tissue flows during pupal stages. We showed previously (Etournay et al., 2015) that pupal wing morphogenesis involves both cellular behaviors that allow relaxation of mechanical tissue stress, as well as cellular behaviors that appear to be actively patterned. The core planar cell polarity (PCP) pathway influences morphogenetic cell movements in many other contexts, which suggests that it could globally pattern active cellular behaviors during pupal wing morphogenesis. We show here, however, that this is not the case: there is no significant phenotype on the cellular dynamics underlying pupal morphogenesis in mutants of core PCP. Furthermore, using laser ablation experiments, coupled with a rheological model to describe the dynamics of the response to laser ablation, we conclude that while core PCP mutations affect the fast timescale response to laser ablation, they do not affect overall tissue mechanics. In conclusion, our work shows that cellular dynamics and tissue shape changes during Drosophila pupal wing morphogenesis are independent of one potential chemical guiding cue, core PCP.

developmental biology↗

bioRxiv2024

Endothelial Rho kinase controls blood vessel integrity and angiogenesis

BackgroundThe Rho kinases 1 and 2 (ROCK1/2) are serine-threonine specific protein kinases that control actin cytoskeleton dynamics. They are expressed in all cells throughout the body, including cardiomyocytes, smooth muscle cells and endothelial cells, and intimately involved in cardiovascular health and disease. Pharmacological ROCK inhibition is beneficial in mouse models of hypertension, atherosclerosis, and neointimal thickening that display overactivated ROCK. However, the consequences of endothelial ROCK signaling deficiency in vivo remain unknown. To address this issue, we analyzed endothelial cell (EC) specific ROCK1 and 2 deletions. MethodsWe generated Cdh5-CreERT2 driven, tamoxifen inducible loss of function alleles of ROCK1 and ROCK2 and analyzed mouse survival and vascular defects through cellular, biochemical, and molecular biology approaches. ResultsWe observed that postnatal or adult loss of endothelial ROCK1 and 2 was lethal within a week. Mice succumbed to multi-organ hemorrhage that occurred because of loss of vascular integrity. ECs displayed deficient cytoskeletal actin polymerization that prevented focal adhesion formation and disrupted junctional integrity. Retinal sprouting angiogenesis was also perturbed, as sprouting vessels exhibited lack of polymerized actin and defective lumen formation. In a three-dimensional endothelial sprouting assay, combined knockdown of ROCK1/2 or knockdown or ROCK2 but not ROCK1 led to reduced sprouting, lumenization and cell polarization defects caused by defective actin and altered VE-cadherin dynamics. The isoform specific role of endothelial ROCK2 correlated with ROCK2 substrate specificity for FAK and LIMK. By analyzing single and three allele mutants we show that one intact allele of ROCK2 is sufficient to maintain vascular integrity in vivo. ConclusionEndothelial ROCK1 and 2 maintain junctional integrity and ensure proper angiogenesis and lumen formation. The presence of one allele of ROCK2 is sufficient to maintain vascular growth and integrity. These data indicate the need of careful consideration for the use of ROCK inhibitors in disease settings.

developmental biology↗

bioRxiv2022

Heritable changes in chromatin contacts linked to transgenerational obesity

Burgeoning evidence demonstrates that responses to environmental exposures can be transmitted to subsequent generations through the germline without DNA mutations1,2. This is controversial because underlying mechanisms remain to be identified. Therefore, understanding how effects of environmental exposures are transmitted to unexposed generations without DNA mutations is a fundamental unanswered question in biology. Here, we used an established murine model of transgenerational obesity to show that direct or ancestral exposure to the obesogen tributyltin (TBT) elicited persistent changes in topologically associating domains (TADs) in primordial germ cells (PGCs) isolated from embryos of exposed and subsequent unexposed generations. New TAD boundaries were formed within the Ide gene encoding insulin degrading enzyme in the exposed PGCs, then stably maintained in PGCs of the subsequent (unexposed) two generations. Concomitantly, Ide mRNA expression was decreased in livers of male descendants from the exposed dams. These animals were hyperinsulinemic and hyperglycemic, phenocopying Ide-deficient mice that are predisposed to adult-onset obesity. Creation of new TAD boundaries in PGCs, suppression of hepatic Ide mRNA, increased fat mass, hyperinsulinemia and hyperglycemia were male-specific. Our results provide a plausible molecular mechanism underlying transmission of the transgenerational predisposition to obesity caused by gestational exposure to an environmental obesogen. They also provide an entry point for future studies aimed at understanding how environmental exposures alter chromatin structure to influence physiology across multiple generations in mammals.

developmental biology↗

bioRxiv2024

Multiple Notch ligands in the synchronization of the segmentation clock

Notch signaling is a ubiquitous and versatile intercellular signaling system that drives collective behaviors and pattern formation in biological tissues. During embryonic development, Notch is involved in generation of collective biochemical oscillations that form the vertebrate body segments, and its failure results in embryonic defects. Notch ligands of the Delta family are key components of this collective rhythm, but it is unclear how different Delta ligands with distinct properties contribute to relaying information among cells. Motivated by the zebrafish segmentation clock, in this work we propose a theory describing interactions between biochemical oscillators, where Notch receptor is bound by both oscillatory and nonoscillatory Delta ligands. Based on previous in vitro binding studies, we first consider Notch activation by Delta dimers. This hypothesis is consistent with experimental observations in conditions of perturbed Notch signaling. Then we test an alternative hypothesis where Delta monomers directly bind and activate Notch, and show that this second model can also describe the experimental observations. We show that these two hypotheses assign different roles for a non-oscillatory ligand, as a binding partner or as a baseline signal. Finally, we discuss experiments to distinguish between the two scenarios. Broadly, this work highlights how a multiplicity of ligands may be harnessed by a signaling system to generate versatile responses.

developmental biology↗