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Results for “developmental biology”

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bioRxiv2026

HoloBio: A Holographic Microscopy Tool for Quantitative Biological Analysis

Holographic imaging in microscopy enables label-free quantitative information of biological specimens and has found applications across a wide range of biomedical studies, from cell morphology to particle dynamics; yet its widespread adoption is often limited by the lack of accessible and standardized analysis software. We present HoloBio, an open-source, Python-based graphical user interface developed to address this issue. This software offers two primary operational modes: a Real-Time mode that enables live processing of holograms at video frame rates, and an Offline mode designed for post-processing previously recorded holograms. HoloBio is compatible with holograms recorded using both lens-based and lensless systems, supporting off-axis architectures in telecentric and non-telecentric configurations, as well as slightly off-axis and in-line optical setups. The software incorporates tools for cell tracking, phase profiling, thickness estimation, and morphological analysis, including cell counting and object area quantification. HoloBio is designed to be accessible for users without coding expertise, offering a reproducible, high-throughput environment tailored for researchers in biology, biophotonics, and biomedical imaging.

developmental biology↗

bioRxiv2026

Cancer cachexia overrides sex-specific traits in Drosophila

Cancer cachexia, a metabolic syndrome characterized by pathologic weight loss, dramatically limits therapeutic options and reduces survival. While the mechanisms by which tumors induce wasting of distant organs (particularly muscle and adipose tissues) remain incompletely understood, the influence of biological sex on cachexia progression is even less clear. Here we investigated phenotypic and molecular manifestations of cachexia in both sexes using a model of inducible intestinal tumors in Drosophila. We show that tumor progression leads to the development of severe cachexia both in males and in females manifested by organ atrophies and an inflammatory state. Nevertheless, both phenotypical and molecular cachexic alterations exhibit striking differences between sexes. Transcriptomic analysis show that many genes differentially expressed at high levels in males under normal conditions become downregulated during cachexia, while their expression is upregulated in cachexic females, and reciprocally. Together, sex-specific deregulation in gene expression and metabolic profiles ultimately converge towards a molecular cachexic state that erase or diminish many sex-specific differences otherwise characteristic of healthy males and females. These findings highlight the interplay between oncogenic stress and biological sex in shaping cachexia phenotypes, shedding light on how sex influences the development of cancer cachexia.

developmental biology↗

bioRxiv2023

The emergence of human gastrulation upon in vitro attachment

While studied extensively in model systems, human gastrulation remains obscure. This process starts upon blastocyst implantation into the uterine wall, which is assumed to occur after 14 days post-fertilization. The scarcity and limited access to fetal biological material as well as ethical considerations limit our understanding of the cellular and molecular portrait of human gastrulation. In vitro culture of natural human blastocysts shed light on the second week of human development, unveiling an unexpected level of self-organization embedded in the pre-gastrulating embryo, yet whether they can undergo gastrulation upon in vitro attachment remains elusive. Blastocyst models called blastoids, which are derived from human pluripotent stem cells, provide the opportunity to reconstitute post-implantation human development in vitro with unlimited biological material. Here we show that human blastoids break symmetry and undergo gastrulation upon in vitro attachment. scRNA-seq of these models replicate the transcriptomic signature of the natural human gastrula, recapitulating aspects of the second to the third week of human development. Surprisingly, analysis of developmental timing reveals that in both blastoid models and natural in vitro attached human embryos, the onset of gastrulation as defined by molecular makers, can be traced to time scales equivalent to 12 days post-fertilization, which appeals for a reconsideration of the onset of human gastrulation upon extended in vitro culture.

developmental biology↗

bioRxiv2024

Monocytes-derived cxcl12 guides a directional migration of blood vessels in zebrafish

BackgroundSprouting blood vessels, reaching the aimed location, and establishing the proper connections are vital for building vascular networks. Such biological processes are subject to precise molecular regulation. So far, the mechanistic insights into understanding how blood vessels grow to the correct position are limited. In particular, the guiding cues and the signaling-originating cells remain elusive. MethodsLive imaging analysis was used to observe the vascular developmental process of zebrafish. Whole-mount in situ hybridization and fluorescent in situ hybridization were used to detect the expression profiles of the genes. Single-cell sequencing analysis was conducted to identify the guiding protein and its originating cells. ResultsTaking advantage of live imaging analysis, we described a directional blood vessel migration in the vascularization process of zebrafish pectoral fins. We demonstrated that pectoral fin vessel c (PFVc) migrated over long distances and was anastomosed with the second pair of intersegmental vessels (ISVs). Furthermore, we found the cxcl12a-cxcr4a axis specifically guided this long-distance extension of PFVc-ISV, and either inhibition or over-expression of cxcl12a-cxcr4a signaling both mislead the growth of PFVc to ectopic areas. Finally, based on an analysis of single-cell sequencing data, we revealed that a population of monocytes expresses the Cxcl12a, which guides the migration of the vascular sprout. ConclusionsOur study identified Cxcl12a as the signaling molecule for orchestrating organotypic-specific long-distance migration and anastomosis of the pectoral fin vessel and ISVs in zebrafish. We discovered a specific cluster of gata1-positive monocytes that are responsible for expressing Cxcl12a. The findings offer novel insights into the mechanisms underlying organotypic vascularization in vertebrates.

developmental biology↗

bioRxiv2017

A novel flatworm-specific gene family implicated in reproduction in Macrostomum lignano

Free-living flatworms, such as the planarian Schmidtea mediterranea, are extensively used as model organisms to study stem cells and regeneration. The majority of studies in planarians so far focused on broadly conserved genes. However, investigating what makes these animals different might be equally informative for understanding its biology. Here, we present a re-analysis of neoblast and germline transcriptional signatures in the flatworm M. lignano and combine it with the whole-animal electron microscopy atlas (nanotomy) as a reference platform for ultrastructural studies in M. lignano. We show that germline-enriched genes have a high fraction of flatworm-specific genes and identify Mlig-sperm1 gene as a member of a novel gene family conserved only in free-living flatworms and essential for producing healthy spermatozoa. This work demonstrates that investigation of flatworm-specific genes is crucial for understanding flatworm biology and establishes a basis for future research in this direction in M. lignano.

developmental biology↗

bioRxiv2023

Parallel genome-scale CRISPR screens distinguish pluripotency and self-renewal

Pluripotent stem cells are defined by their self-renewal capacity, which is the ability of the stem cells to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into any somatic cell lineage. However, understanding the mechanisms that control stem cell fitness versus the pluripotent cell identity is challenging. To investigate the interplay between these two aspects of pluripotency, we performed four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSC self-renewal conditions, and the dissolution of the primed pluripotency identity during early differentiation. Comparative analyses led to the discovery of genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further discovered a core set of factors that control both stem cell fitness and pluripotent identity, including a network of chromatin factors that safeguard pluripotency. Our unbiased and systematic screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide rich datasets for exploring pluripotent cell identity versus cell fitness, and offer a valuable model for categorizing gene function in broad biological contexts.

developmental biology↗

bioRxiv2025

Identification and classification of abundant RNA-binding proteins in the mouse lens and interactions of Carhsp1, Igf2bp1/ZBP1, and Ybx1 with crystallin and β-actin mRNAs

RNA-binding proteins (RBPs) are critical regulators of mRNAs controlling all processes such as RNA transcription, transport, localization, translation, mRNA:ncRNA interactions, and decay. Cellular differentiation is driven by tissue-specific and/or tissue-preferred expression of proteins needed for the optimal function of mature cells, tissues and organs. Lens fiber cell differentiation is marked by high levels of expression of crystallin genes encoding critical proteins for lens transparency and light refraction. Herein we performed proteomic and transcriptomic analyses of RBPs in differentiating mouse lenses to identify the most abundant RBPs and establish dynamic changes of their expression in differentiating lens. Expression analyses include highly abundant RBPs, including Carhsp1, Igf2bp1/ZBP1, Ybx1, Pabpc1, Ddx39, and Rbm38. Binding sites of Carhsp1, Ybx1, and Igf2bp1/ZBP1 were predicted in various crystallin and {beta}-actin mRNAs. Immunoprecipitations using antibodies against Carhsp1, Igf2bp1/ZBP1, and Ybx1 confirmed their interactions with A-, B-, and {gamma}A-crystallin mRNAs. A combination of single molecule RNA FISH (smFISH) and immunofluorescence was used to probe in vivo interactions of these RBPs with A-, B-crystallin, and {beta}-actin mRNAs in cytoplasm and nucleoplasm of cultured mouse lens epithelial cells. Together, these results open new avenues to perform comprehensive genetic, cell, and molecular biology studies of individual RBPs in the lens.

developmental biology↗

bioRxiv2025

Asymmetric Histone Inheritance Regulates Differential Transcription Re-initiation and Cell Fate Decisions in Mouse Olfactory Horizontal Basal Cells

The olfactory epithelium (OE) possesses an adult stem cell population, the horizontal basal cells (HBCs), to permit lifelong tissue regeneration. Here we show that HBCs exhibit asymmetric inheritance of histone H4 but not H2A-H2B during OE regeneration in mice. Primary HBC cultures further revealed asymmetric histone inheritance for H3 and H3.3. Upon mitotic exit, asymmetric histone inheritance correlates with asynchronous transcription re-initiation and differential enrichment of p63, a key transcription factor for HBC cell fate. Disruption of asymmetric histone inheritance abolished these asymmetric cellular features and attenuated OE regeneration and smell behavior recovery. Single-cell RNA sequencing of paired HBC daughters in culture further supported asymmetric multilineage cell fate priming. Together, these findings reveal asymmetric histone inheritance in a mammalian adult stem cell lineage and highlight its biological significance in neural tissue regeneration and animal behavior.

developmental biology↗

bioRxiv2021

Genetic, Genomic and Biophysical Case Study: Familial 46, XY Sex Reversal due to a Novel Inherited Mutation in Human Testis-Determining Factor SRY

ObjectiveTo describe the clinical, histopathological and molecular features of a novel inherited SRY allele (p.Met64Val; consensus box position 9) observed within an extensive pedigree: two 46, XY sisters with primary amenorrhea (16 and 14 years of age; probands P1 and P2), their normal father and brother, and an affected paternal XY grandaunt. Design, Setting, Participants and Outcome MeasurementsFollowing DNA sequencing to identify the SRY mutation, hormonal studies of the probands and histopathological examination of their gonads were performed. Functional consequences of p.Met64Val (and other mutations at this site) were also investigated. ResultsBreast development in P1 and P2 was Tanner II and IV, respectively. Mullerian structures and gonads resembling ovaries were found in each sister. Histopathology revealed gonadal dysgenesis, gonadablastoma and dysgerminoma. AMH/MIS, P450 SCC, and P450 aromatase were expressed in gonadoblastoma tissues. Genomic sequencing revealed no candidate mutations in other genes related to sexual differentiation. Variant p.Met64Val impaired Sox9 transcriptional activation associated with attenuated occupancy of the testis-specific enhancers Enh13 and TESCO. Biophysical studies indicated that the mutant HMG box retains specific DNA binding and DNA bending but with accelerated rate of protein-DNA dissociation. ConclusionThe partial biological activity of p.Met64Val SRY, maintained at the threshold of SRY function, rationalizes opposing paternal and proband phenotypes (the "the father-daughter paradox"). Steroidal biosynthesis by gonadoblastoma may delay genetic diagnosis and recognition of gonadal tumors. Quantitative assessment of inherited SRY alleles highlights the tenuous transcriptional threshold of developmental decision-making in the bipotential gonadal ridge.

developmental biology↗

bioRxiv2020

In vivo characterization of chick embryo mesoderm by optical coherence tomography assisted microindentation

Embryos are growing organisms with highly heterogeneous properties in space and time. Understanding the mechanical properties is a crucial prerequisite for the investigation of morphogenesis. During the last ten years, new techniques have been developed to evaluate the mechanical properties of biological tissues in vivo. To address this need, we employed a new instrument that, via the combination of micro-indentation with Optical Coherence Tomography (OCT), allows us to determine both, the spatial distribution of mechanical properties of chick embryos and the structural changes in real-time provided by OCT. We report here the stiffness measurements on live chicken mesoderm during somite formation, from the mesenchymal tailbud to the epithelialized somites. The storage modulus of the mesoderm increases from (176{+/-}18) Pa in the tail up to (716{+/-}117) Pa in the somitic region. The midline has a storage modulus of (947{+/-}111) Pa in the caudal presomitic mesoderm, indicating a stiff rod along the body axis, which thereby mechanically supports the surrounding tissue. The difference in stiffness between midline and presomitic mesoderm decreases as the mesoderm forms somites. The viscoelastic response of the somites develops further until somite IV, which is commensurate with the slow process of epithelization of somites between S0 and SIV. Overall, this study provides an efficient method for the biomechanical characterization of soft biological tissues in vivo and shows that the mechanical properties strongly relate to different morphological features of the investigated regions.

developmental biology↗

bioRxiv2020

A metabolic coincidence mechanism controls winter photoperiodism in plants

Plants have served as a preeminent study system for photoperiodism because of their propensity to flower in concordance with the seasons. A nearly singular focus on understanding seasonal flowering has been to the detriment of discovering other photoperiod measuring mechanisms that may be necessary for vegetative health. Here we use bioinformatics to identify a group of winter photoperiod-induced genes in Arabidopsis and show that one, PP2-A13, is critical for fitness and survival, exclusively in winter-like photoperiods. We create a real-time photoperiod reporter, using the PP2-A13 promoter driving luciferase, and show that winter photoperiod genes are regulated independent of the canonical CO/FT mechanism for photoperiodic flowering. The reporter then allows us to identify the first genetic and cellular drivers of winter photoperiodism and reveal a mechanism that relies on coincidence between light capture through photosynthesis and rhythmic metabolism. This work demonstrates that plants have distinct photoperiod measuring mechanisms that enact critical biological and developmental processes in different seasons.

plant biology↗

bioRxiv2018

Embryo timelapses can be compiled and quantified to understand canonical histone dynamics across multiple cell cycles.

In the last decade, computational analysis of big datasets has facilitated the processing of unprecedented quantities of collected biological data. Thus, automations and big data analyses have been revolutionary in detecting and quantifying subtle phenotypes in cell biological contexts. Analyzing similar quantities of data in larger and more complicated biological systems such as live embryos has been more challenging due to experimental necessities impeding both compilations of data collection and informative analysis. Here we present a streamlined workflow that can quantify cell cycle dynamics in early developing embryos using fluorescently labeled proteins. We benchmark this pipeline using Caenorhabditis elegans (nematode) embryonic development and a fluorescently labeled histone. Using our pipeline, we find that histone proteins are broadly stable in early embryonic development. In sum, we have utilized the large biological and experimental variation associated with quantification of fluorescent proteins in embryonic systems, to quantify nuclear accumulation rate, chromatin incorporation, and turnover/stability of canonical histones during early development.

developmental biology↗

bioRxiv2026

Pre-cuticle DPY 6 acts as a blueprint for aECM periodic organization in C. elegans

Apical extracellular matrices (aECMs) are essential for tissue integrity and function in multicellular organisms, but there is limited understanding of how such matrices are assembled and organized in the extracellular environment. The Caenorhabditis elegans cuticle, a model aECM that undergoes morphogenesis during each of the worms four larval molts, requires periodic circumferential furrows for structural integrity and immune regulation. Here, we show that furrow collagens must be cleaved from their N-terminal transmembrane domain for secretion and depend on the mucin-like pre-cuticle protein DPY-6 for their periodic assembly. While DPY-6 is dispensable for initial embryonic furrow formation, it acts as a mold during subsequent molts, ensuring pattern replication via its C-terminal cysteine cradle domain. These results reveal a central role for a transient matrix factor in organizing a complex periodically structured aECM. Author SummaryIn multicellular organisms, the extracellular matrix (ECM) provides structural support and regulates tissue function. Using the free-living worm Caenorhabditis elegans, we investigated how its apical ECM, the cuticle, forms a precise, repeating pattern of ridges called furrows. The cuticle is rebuilt at each of the worms four larval stages, providing a unique opportunity to study matrix morphogenesis in real time. We discovered that a transient protein, DPY-6, acts as a molecular mold to guide the self-organization of the matrix outside the epidermal cells. DPY-6 ensures that newly secreted proteins assemble into the correct periodic pattern during each rebuilding phase. Without DPY-6, the furrows lose their organization, leading to structural defects and immune system activation. Our findings reveal how a temporary scaffold can template the assembly of a complex, self-organizing structure. This work provides new insights into how biological matrices are built and maintained, with broader implications for understanding ECM assembly in health and disease.

developmental biology↗

bioRxiv2020

Human brain organoids reveal accelerated development of cortical neuron classes as a shared feature of autism risk genes

Genetic risk for autism spectrum disorder (ASD) has been associated with hundreds of genes spanning a wide range of biological functions. The phenotypic alterations in the human brain resulting from mutations in ASD risk genes remain unclear, and the level at which these alterations converge on shared disease pathology is poorly understood. Here, we leveraged reproducible organoid models of the human cerebral cortex to identify cell type-specific developmental abnormalities associated with haploinsufficiency in three ASD risk genes, SUV420H1 (KMT5B), PTEN, and CHD8. We performed comprehensive single-cell RNA-sequencing (scRNA-seq) of over 400,000 cells, and proteomic analysis on individual organoids sampled at different developmental stages to investigate phenotypic convergence among these genes. We find that within a defined period of early cortical development, each of the three mutations demonstrates accelerated development of cortical neurons. Notably, they do so by affecting different neuronal populations: excitatory deep layer (SUV420H1) and callosal (PTEN) neurons, and inhibitory interneurons (CHD8). This work shows that haploinsufficiency in ASD risk genes converge on early developmental defects in the generation of neurons of the cortical microcircuit.

developmental biology↗

bioRxiv2018

Acidic microenvironment shaped by lactate accumulation promotes pluripotency through multiple mechanisms

Enhanced glycolysis is a distinct feature associated with numerous stem cells and cancer cells. However, little is known about its regulatory roles in gene expression and cell fate determination. Here we show that acidic environment shaped by lactate accumulation promotes the self-renewal and pluripotency of both mouse and human embryonic stem cells (ESCs). Mechanistically, acidic pH reduces the tri-methylation of H3K27 globally at transcriptional start sites to partially prevent ESC differentiation. In addition, acidic pH stabilizes a large number of mRNAs including pluripotency genes. Furthermore, we found that AGO1 protein is downregulated at acidic conditions, leading to the de-repression of a subset of microRNA targets in low-pH treated ESCs. Altogether, our study provides insights into mechanisms whereby acidic microenvironment produced by enhanced glycolysis regulates gene expression to determine cell fate and has broad implications in the fields of regenerative medicine and cancer biology.

developmental biology↗

bioRxiv2022

Relationship between thyroid hormones and associated metabolites and gene expression bioindicators in the serum of Rana catesbeiana tadpoles and frogs during metamorphosis

Anuran metamorphosis is characterized by profound morphological changes including remodeling of tissues and organs. This transition is initiated by thyroid hormones (THs). However, the current knowledge of changing levels of THs during metamorphosis relies on pooled samples using methods known for high variability with sparse reporting of measured variation. Moreover, establishing a clear linkage between key gene expression bioindicators and TH levels throughout the metamorphic process is needed. Using state-of-the-art ultra-high performance liquid chromatography isotope-dilution tandem mass spectrometry, we targeted 12 THs and metabolites in the serum of Rana [Lithobates] catesbeiana (n=5-10) across seven distinct postembryonic stages beginning with premetamorphic tadpoles (Gosner stage 31-33) and continuing through metamorphosis to a juvenile frog (Gosner stage 46). TH levels were related to TH-relevant gene transcripts (thra, thrb, and thibz) in back skin of the same individual animals. Significant increases from basal levels were observed for thyroxine (T4) and 3,3,5-triiodothyronine (T3) at Gosner stage 41, reaching maximal levels at Gosner stage 44 (28{+/-}10 and 2.3{+/-}0.5 ng/mL, respectively), and decreasing to basal levels in juvenile frogs. In contrast, 3,5-diiodothyronine (T2) increased significantly at Gosner stage 40 and was maintained elevated until stage 44. While thra transcript levels remained constant and then decreased at the end of metamorphic climax, thrb and thibz were induced to maximal levels at Gosner stage 41, followed by a decrease to basal levels in the froglet. This exemplifies the exquisite timing of events during metamorphosis as classic early response genes are transcribed in anticipation of peak TH concentrations. The distinct T2 concentration profile suggests a biological role of this biomolecule in anuran postembryonic development and an additional aspect that may be a target of anthropogenic chemicals that can disrupt anuran metamorphosis and TH signalling. Hence, as a second aim of the study, we set out to find additional bioindicators of metamorphosis, which can aid future investigations of developmental disruption. Using a sensitive nanoLC-Orbitrap system an untargeted analysis workflow was applied. Among 6,062 endogenous metabolites, 421 showed metamorphosis-dependent concentration dynamics. These potential bioindicators included several carnitines, prostaglandins and some steroid hormones.

developmental biology↗

bioRxiv2020

An atlas of cell types in the mammalian epididymis and vas deferens

Following spermatogenesis in the testis, mammalian sperm continue to mature over the course of approximately 10 days as they transit a long epithelial tube known as the epididymis. The epididymis is comprised of multiple segments/compartments that, in addition to concentrating sperm and preventing their premature activation, play key roles in remodeling the protein, lipid, and RNA composition of maturing sperm. In order to understand the complex roles for the epididymis in reproductive biology, we generated a single cell atlas of gene expression from the murine epididymis and vas deferens. We recovered all the key cell types of the epididymal epithelium, including principal cells, clear cells, and basal cells, along with associated support cells that include fibroblasts, smooth muscle, macrophages and other immune cells. Moreover, our data illuminate extensive regional specialization of principal cell populations across the length of the epididymis, with a substantial fraction of segment-specific genes localized in genomic clusters of functionally-related genes. In addition to the extensive region-specific specialization of principal cells, we find evidence for functionally-specialized subpopulations of stromal cells, and, most notably, two distinct populations of clear cells. Analysis of ligand/receptor expression reveals a network of potential cellular signaling connections, with several predicted interactions between cell types that may play roles in immune cell recruitment and other aspects of epididymal function. Our dataset extends on existing knowledge of epididymal biology, and provides a wealth of information on potential regulatory and signaling factors that bear future investigation.

developmental biology↗

bioRxiv2020

Functional specialization of human salivary glands and origins of proteins intrinsic to human saliva

Salivary proteins are essential for maintaining health in the oral cavity and proximal digestive tract and serve as a diagnostic window into human disease. However, their precise organ origins remain unclear. Through transcriptomic analysis of major adult and fetal salivary glands, and integration with the saliva proteome and transcriptomes of 28+ organs, we linked human saliva proteins to their source, identified salivary gland-specific genes, and uncovered fetal- and adult-specific gene repertoires. Our results also provide new insights into the degree of gene retention during maturation and suggest that functional diversity between adult gland-types is driven by specific dosage combinations of hundreds of transcriptional regulators rather than a few gland-specific factors. Finally, we demonstrate the hitherto unrecognized heterogeneity of the human acinar cell lineage. Our results pave the way for future investigations into glandular biology and pathology, as well as salivas use as a diagnostic fluid.

developmental biology↗