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Results for “developmental biology”

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bioRxiv2021

Contrasting impacts of environmental variability on the breeding biology of two sympatric small procellariiform seabirds in south-eastern Australia

Seabirds play a vital role in marine ecosystems and are determinant sentinels of the productivity of their environments. The long-term study of their breeding biology and their responses to environmental variations can be used to monitor the effects of climate change on marine fauna. However, the ecological and physiological differences among seabirds induce a large range of responses complicating our understanding of the effects of environmental changes on marine ecosystems. The present study investigated the impact of environmental variability on breeding biology in two sympatric small Procellariiform species, the fairy prion (Pachyptila turtur) and the common diving petrel (Pelecanoides urinatrix), over four reproductive seasons (2017-2020) in Bass Strait, south-eastern Australia. Marine heatwaves had a negative effect on chick growth, breeding success, and induced a delay in laying dates in both species. While fairy prions maintained a relatively high breeding success and broadly constant breeding phenology, common diving petrels delayed the start of the breeding season by up to 50 days and experienced dramatic collapses in breeding success in years of high marine heat wave occurrence. The high wing loading and absence of stomach oils in the common diving petrel are likely to have limited the capacity of this species to increase foraging effort in years of low food availability.

developmental biology↗

bioRxiv2021

A powerful and versatile new fixation protocol for immunohistology and in situ hybridization that preserves delicate tissues in planaria

Whole-mount in situ hybridization (WISH) is a powerful and widely used technique to visualize the expression pattern of genes in different biological systems. Here we describe a new protocol for ISH and immunostaining in the planarian Schmidtea mediterranea. The new Nitric Acid/Formic Acid (NAFA) protocol is compatible with both assays and prevents degradation of the epidermis or blastema. Instead of proteinase K digestion, formic acid treatment is used to permeabilize tissues and preserve antigen epitopes. We show that the NAFA protocol successfully permits development of chromogenic and fluorescent signals in situ, while preserving the anatomy of the animal. Further, the immunostaining of different proteins was compatible with the NAFA protocol following fluorescent in situ hybridization. Finally, we demonstrate with high resolution confocal imaging that the regeneration blastema is preserved when using the new method. This new NAFA protocol will be a valuable technique to study the process of wounding response and regeneration.

developmental biology↗

bioRxiv2024

Application of Elevated Atmospheric Pressure and Hypoxia Enhance Pluripotency and Stem Cell Differentiation

Physical forces regulate stem cell differentiation in-vivo, however few simple and precise methods exist to better understand this biology in-vitro. Here we describe the use of a novel bioreactor that enables addition of physical force in the form of elevated atmospheric pressure during reprogramming of human fibroblasts and culture of human induced pluripotent stem cell (iPSC) and neural stem cell (NSC) lines. We demonstrate that elevated atmospheric pressure and hypoxia can positively regulate reprogramming of human fibroblasts to iPSCs across multiple donors. Prolonged culture of iPSCs in elevated atmospheric pressure (+ 2 PSI) and 15% oxygen exhibited progressive differentiation with concomitant metabolic and epigenetic gene expression changes. Furthermore, elevated atmospheric pressure positively regulates differentiation of iPSCs to neural-ectodermal and hematopoietic lineages when combined with appropriate soluble factors and oxygen concentration. In summary, these results demonstrate the significance of applied atmospheric pressure for stem cell applications and warrants further investigation.

developmental biology↗

bioRxiv2025

The Calcium Pump ATP2B1/PMCA1 Regulates CNS Vascular Development by Facilitating Norrin- and WNT7A/B-induced Frizzled4 signaling

Frizzled4 (FZD4) is a receptor for Norrin and WNT7A/B ligands, is expressed in endothelial cells (ECs), is required for endothelial blood-central nervous system (CNS) barrier function as well as CNS angiogenesis, and transduces {beta}-catenin-dependent signaling. Despite its fundamental importance in neurovascular biology, including as a drug target, the molecular mechanisms governing FZD4 regulation remain poorly understood. Here, we employed proximity biotinylation to identify proteins that regulate FZD4. We identified ATPase Plasma Membrane Ca{superscript 2} Transporting 1 (ATP2B1, also known as PMCA1) as a FZD4 proximity interactor. Functional analyses revealed that ATP2B1 depletion increased EC Ca2+, activated NFAT, and significantly attenuated Norrin/Frizzled4 signaling. Endothelial-specific Atp2b1 deletion caused CNS vascular phenotypes consistent with compromised Norrin/Frizzled4 signaling. This study identifies ATP2B1 as a novel regulator of Norrin- and WNT7A/B-induced FZD4 signaling and suggests that in pathological contexts with elevated EC Ca2+-levels, EC function may be modulated by suppression of {beta}-catenin-dependent signaling.

developmental biology↗

bioRxiv2018

Functional Role of Annexins in Zebrafish Caudal Fin Regeneration: A Gene Knockdown Approach in Regenerating Tissue.

Regeneration is an adaptive phenomenon with wide biological implications spread heterogeneously in almost all the organism including human beings. The ability of regeneration varies from species to species for its impediment. Epimorphic regeneration of zebrafish caudal fin tissue is most widely studied regeneration mechanism for its discrete and rapid regenerative capability. Several genes/proteins were found to be associated with zebrafish caudal fin tissue regeneration. Here, we have evaluated the functional role of Annexin 2a and 2b genes in adult zebrafish caudal fin tissue undergoing regeneration using a novel CRSISPR-Cas9 gene knock down approach. Knock down of both the genes individually elicited in decelerated regeneration and down regulation of target genes and its partner genes/proteins such as ANXA1a, ANXA5b and ANXA13. This study validates a novel gene targeting approach and the possible role of annexin in regeneration mechanism.

developmental biology↗

bioRxiv2019

A Single-Cell Atlas and Lineage Analysis of the Adult Drosophila Ovary

The Drosophila ovary is a widely used model for germ cell and somatic tissue biology. We have used single-cell RNA-sequencing to build a comprehensive cell atlas of the adult Drosophila ovary containing unique transcriptional profiles for every major cell type in the ovary, including the germline and follicle stem cells. Using this atlas we identify novel tools for identification and manipulation of known and novel cell types and perform lineage tracing to test cellular relationships of previously unknown cell types. By this we discovered a new form of cellular plasticity in which inner germarial sheath cells convert to follicle stem cells in response to starvation.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC=\"FIGDIR/small/798223v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (60K):\norg.highwire.dtl.DTLVardef@b4c486org.highwire.dtl.DTLVardef@1d24e4aorg.highwire.dtl.DTLVardef@1217adorg.highwire.dtl.DTLVardef@1d09a63_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2024

Neural tube organoid generation: a robust and reproducible protocol from single mouse embryonic stem cells

The development of mammals is a highly complex process, characterized by the necessity for precise concentration- and time-dependent signaling for correct pattern formation and morphogenesis. Despite considerable technological advancements and knowledge gathered, numerous aspects of mammalian development remain elusive. When examining the entire organism, it becomes challenging to disentangle the effects of individual pathways or the mechanism by which external stimuli guide the interference of surrounding tissues and factors. In addressing this complexity, three-dimensional (3D) in vitro models such as organoids have emerged as valuable tools. Organoids, derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), exhibit tissue-like features that closely resemble their in vivo counterparts in terms of expression patterns and functionality. Importantly, they offer accessibility for manipulation and extensive biological studies within a controlled experimental setting. Despite originating from pluripotent cultures, organoid systems often exhibit heterogeneity and substantial variability, limiting their utility when studying complex and intricate biological questions. Therefore, there is a pressing need for detailed protocols aimed at harmonizing procedures that result in high-quality reproducible data, reduction of materials used and which importantly permit the investigation of convoluted phenomena. In this context, we present an optimized protocol for the cultivation of neural tube organoids (NTOs) in vitro. By producing stable culture conditions and offering comprehensive troubleshooting strategies, this protocol enables the reliable and reproducible generation of NTOs which serve as an adequate model to study relevant scientific questions. SUMMARYThree-dimensional neural tube organoids (NTOs) derived from mouse embryonic stem cells are valuable tools to study the central nervous system during early development. Here, we present a step-by-step demonstration of an optimized protocol for cultivating NTOs in vitro, providing stable culture conditions and troubleshooting strategies for reliable and reproducible NTO generation.

developmental biology↗

bioRxiv2025

Obesogenic diet alters decidual differentiation and cell-cell communication in the mouse uterus

Maternal obesity is associated with increased risk of infertility, implantation failure, miscarriage, and other pregnancy complications. While prior studies have linked obesity to uterine dysfunction and impaired endometrial biology, how obesity alters the cellular and molecular landscape of the early pregnant endometrium remains poorly understood. Here, we perform single-cell RNA sequencing of embryonic day 5.5 uterus from control and obesogenic mice to generate a cellular atlas of the early decidualizing endometrium. We identify obesity-associated transcriptional changes across multiple Endometrial Stromal Cell (ESC) states and innate immune populations, including uterine natural killer cells and macrophages. Computational modeling reveals that maternal obesity disrupts distinct routes of ESC differentiation during decidualization and leads to shifts in ESC-derived cues known to impact innate immune responses. These findings provide a comprehensive single-cell resource of the post-implantation mouse endometrium while simultaneously generating critical insight into how maternal obesity reprograms the maternal-fetal interface in early pregnancy.

developmental biology↗

bioRxiv2019

Regulatory Feedbacks on Receptor and Non-receptor Synthesis for Robust Signaling

Elaborate feedback regulatory processes are thought to make biological developments robust, i.e., resistant to changes induced by genetic or environmental perturbations. How this might be done is still not completely understood. Previous numerical simulations on reaction-diffusion models of Dpp gradients in Drosophila wing imaginal disc showed that feedback (of the Hills function type) on (signaling) receptors and/or non-(signaling) receptors are of limited effectiveness in promoting robustness. Spatial nonuniformity of the feedback processes is thought to lead to serious shape distortion and a principal cause for ineffectiveness. Through mathematical modeling of a spatially uniform nonlocal feedback mechanism, the present paper provides a theoretical support of these observations. More significantly, the new approach also enables us to uncover in this paper a new, theory-based multi-feedback instrument for broadly effective promotion of robust signaling gradients.

developmental biology↗

bioRxiv2026

Segmented wavetrains and sites of reversal in the mouse seminiferous tubules

Mammalian spermatogenesis occurs in the seminiferous tubules, which exhibit unique spatiotemporal differentiation patterns known as cellular association patterns. In mice, these patterns can be regarded as one-dimensional wavetrains that consistently propagate inward from both ends, resulting in one or more "sites of reversal." Segmented wavetrain pattern, in which the wave propagation direction spatially switches, was observed in our previous three-species reaction-diffusion model for interspecific species difference in spermatogenic waves (Kawamura et al., 2021). However, the biological mechanisms of the formation of sites of reversal and of this directional bias, as well as the principle of pattern formation, remain unknown. Here, we refined our previous model to match the actual biological spatiotemporal scale and examined its dynamics through extensive numerical simulations. The modified model frequently generated segmented wavetrain patterns, corresponding to the sites of reversal, but without directional bias. We systematically examined possible biological mechanisms for the bias and found that tubule elongation, especially near the rete testis, most effectively accounts for the bias among the tested. Extensive simulations revealed that the segmented pattern is numerically stable, emerges more frequently in longer domains, and shows an exponential segment size distribution with a lower limit for the stably existing segment length. These explorations imply that locally emerged unidirectional wavetrains serve as building blocks to generate the stable segmented wavetrains through their interactions. HighlightsO_LISegmented wavetrains reflect sites of reversal in seminiferous tubules. C_LIO_LISegmented patterns frequently emerge but show no inherent directional bias. C_LIO_LITubule elongation may contribute to inward propagation near the rete testis. C_LIO_LISegmented wavetrains are numerically stable and more frequent in longer domains. C_LIO_LIInteractions of local unidirectional wavetrains generate stable segmented structures. C_LI

developmental biology↗

bioRxiv2023

Single-cell transcriptional landscapes of bovine peri-implantation development

Supporting healthy pregnancy outcomes requires a comprehensive understanding of the cellular hierarchy and underlying molecular mechanisms during peri-implantation development. Here, we present a single-cell transcriptome-wide view of the bovine peri-implantation embryo development at day 12, 14, 16 and 18, when most of the pregnancy failure occurs in cattle. We defined the development and dynamic progression of cellular composition and gene expression of embryonic disc, hypoblast, and trophoblast lineages during bovine peri-implantation development. Notably, the comprehensive transcriptomic mapping of trophoblast development revealed a previously unrecognized primitive trophoblast cell lineage that is responsible for pregnancy maintenance in bovine prior to the time when binucleate cells emerge. We analyzed novel markers for the cell lineage development during bovine early development. We also identified cell-cell communication signaling underling embryonic and extraembryonic cell interaction to ensure proper early development. Collectively, our work provides foundational information to discover essential biological pathways underpinning bovine peri-implantation development and the molecular causes of the early pregnancy failure during this critical period. Significance StatementPeri-implantation development is essential for successful reproduction in mammalian species, and cattle have a unique process of elongation that proceeds for two weeks prior to implantation and represents a period when many pregnancies fail. Although the bovine embryo elongation has been studied histologically, the essential cellular and molecular factors governing lineage differentiation remain unexplored. This study profiled the transcriptome of single cells in the bovine peri-implantation development throughout day 12, 14, 16, and 18, and identified peri-implantation stage-related features of cell lineages. The candidate regulatory genes, factors, pathways and embryonic and extraembryonic cell interactions were also prioritized to ensure proper embryo elongation in cattle.

developmental biology↗

bioRxiv2025

Chromatin remodeling integrates vertebrate body axis elongation and cell fate determination

The effective elongation of the vertebrate body axis depends on the precise coordination of cell movements and cell fate determination within the posterior tailbud region. However, the specific biological determinant that integrates these cellular processes during vertebrate tail bud elongation has remained unclear. Here, we demonstrate that the spatiotemporal heterogeneity of chromatin condensation states governs both cell fate and cell motility within the mesodermal progenitor zone (MPZ) and the presomitic mesoderm (PSM) during zebrafish tail bud elongation. By integrating biological experiments with computational modeling, we reveal that nuclear volume heterogeneity, mediated by chromatin remodeling, acts as an active driver of collective cell migration. Reducing chromatin condensation heterogeneity in the MPZ through modulation of chromatin modifications impairs collective cell movements and delays tailbud elongation. Single cell ATAC-seq and RNA-seq analyses reveal that global chromatin accessibility heterogeneity and region-specific chromatin remodeling differences in the PSM and MPZ are functionally associated with gene expression pathways involving in cell lineage specification. These findings establish a molecular connection between cell fate determination and tissue-scale cellular dynamics. Collectively, our findings unveil a unifying framework for understanding the coordination of cell fate determination and large-scale cellular movements during tissue morphogenesis.

developmental biology↗

bioRxiv2018

Ordered arrangement of dendrites within a C. elegans sensory nerve bundle

Biological systems are organized into well-ordered structures and can evolve new patterns when perturbed. To identify principles underlying biological order, we turned to C. elegans for its simple anatomy and powerful genetics. We developed a method to quantify the arrangement of three dendrites in the main sensory nerve bundle, and found that they exhibit a stereotyped arrangement throughout larval growth. Dendrite order does not require prominent features including sensory cilia and glial junctions. In contrast, loss of the cell adhesion molecule (CAM) CDH-4/Fat-like cadherin causes dendrites to be ordered randomly, despite remaining bundled. Loss of the CAMs PTP-3/LAR or SAX-7/L1CAM causes dendrites to adopt an altered order, which becomes increasingly random as animals grow. Misexpression of SAX-7 leads to subtle but reproducible changes in dendrite order. Our results suggest that differential expression of CAMs allows dendrites to self-organize into a stereotyped arrangement that readily gives rise to new patterns when perturbed.

developmental biology↗

bioRxiv2019

zGrad: A nanobody-based degron system to inactivate proteins in zebrafish

The analysis of protein function is essential to modern biology. While protein function has mostly been studied through gene or RNA interference, more recent approaches to degrade proteins directly have been developed. Here, we adapted the anti-GFP nanobody-based system deGradFP from flies to zebrafish. We named this system zGrad and show that zGrad efficiently degrades transmembrane, cytosolic and nuclear GFP-tagged proteins in zebrafish in an inducible and reversible manner. Using tissue-specific and inducible promoters in combination with functional GFP-fusion proteins, we demonstrate that zGrad can inactivate transmembrane, cytosolic and nuclear proteins globally, locally and temporally with different consequences. Global protein depletion results in phenotypes similar to loss of gene activity while local and temporal protein inactivation yields more restricted and novel phenotypes. Thus, zGrad is a versatile tool to study the spatial and temporal requirement of proteins in zebrafish.

developmental biology↗

bioRxiv2017

A toolkit for tissue-specific protein degradation in C. elegans

Proteins essential for embryo production, cell division, and early embryonic events are frequently re-utilized later in embryogenesis, during organismal development, or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that utilizes expression of a fusion between a GFP-targeting nanobody and SOCS-box containing ubiquitin ligase adaptor to target GFP tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissue-specific protein ablation. We show that this approach works in multiple tissues--the epidermis, intestine, body wall muscle, sensory neurons, and touch neurons--where it recapitulates expected loss-of-function mutant phenotypes. The transgene toolkit and the strain set described here will complement existing approaches to enable routine analysis of the tissue-specific roles of C. elegans proteins.

developmental biology↗

bioRxiv2021

DeepProjection: Rapid and structure-specific projections of tissue sheets embedded in 3D microscopy stacks using deep learning

The efficient extraction of local high-resolution content from massive amounts of imaging data remains a serious and unsolved problem in studies of complex biological tissues. Here we present DeepProjection, a trainable projection algorithm based on deep learning. This algorithm rapidly and robustly extracts image content contained in curved manifolds from time-lapse recorded 3D image stacks by binary masking of background content, stack by stack. The masks calculated for a given movie, when predicted, e.g., on fluorescent cell boundaries on one channel, can subsequently be applied to project other fluorescent channels from the same manifold. We apply DeepProjection to follow the dynamic movements of 2D-tissue sheets in embryonic development. We show that we can selectively project the amnioserosa cell sheet during dorsal closure in Drosophila melanogaster embryos and the periderm layer in the elongating zebrafish embryo while masking highly fluorescent out-of-plane artifacts.

developmental biology↗

bioRxiv2023

Spatio-temporal remodeling of extracellular matrix orients epithelial sheet folding

Biological systems are inherently noisy; however, they produce highly stereotyped tissue morphology. Drosophila pupal wings show a highly stereotypic folding through uniform expansion and subsequent buckling of wing epithelium within a surrounding cuticle sac. The folding pattern produced by buckling is generally stochastic; it is thus unclear how buckling leads to stereotypic tissue folding of the wings. We found that the extracellular matrix (ECM) protein, Dumpy, guides the position and direction of buckling-induced folds. Dumpy anchors the wing epithelium to the overlying cuticle at specific tissue positions. Tissue-wide alterations of Dumpy deposition and degradation yielded different buckling patterns. In summary, we propose that spatio-temporal ECM remodeling shapes stereotyped tissue folding through dynamic interactions between the epithelium and its external structures. One-Sentence SummaryRemodeling of extracellular matrix protein, Dumpy, guides epithelial tissue folding during Drosophila wing development.

developmental biology↗

bioRxiv2024

MRCK-1 activates non-muscle myosin for outgrowth of a unicellular tube in Caenorhabditis elegans

The formation and patterning of unicellular biological tubes is essential for metazoan development. It is well established that vascular tubes and neurons use similar guidance cues to direct their development, but the downstream mechanisms that promote the outgrowth of biological tubes are not well characterized. We show that the conserved kinase MRCK-1 and its substrate the regulatory light chain of non-muscle myosin, MLC-4, are required for outgrowth of the unicellular excretory canal in C. elegans. Structure-function analysis of MRCK-1 indicates that the kinase domain, but not the small GTPase-binding CRIB domain, is required for canal outgrowth. Expression of a phosphomimetic form of MLC-4 rescues canal truncations in mrck-1 mutants and shows enrichment at the growing canal tip. Specific depletion of MRCK-1 or MLC-4 in the canal causes severe truncations, with unlumenized projections of the basal membrane. Moreover, our work reveals a novel function for non-muscle myosin downstream of MRCK-1 in excretory canal outgrowth that may be conserved in the development of seamless tubes in other organisms.

developmental biology↗