SEARCH · Search bioRxiv

Results for “Developmental Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,477 records · Page 82Linked to original sources

bioRxiv2019

Mask family proteins ANKHD1 and ANKRD17 regulate YAP nuclear import, stability and phase separation.

The Mask family of multiple ankyrin repeat and KH domain proteins were discovered in Drosophila to promote the activity of the transcriptional coactivator Yorkie (Yki), the sole fly homolog of mammalian YAP (YAP1) and TAZ (WWTR1). The molecular function of Mask, or its mammalian homologs Mask1 (ANKHD1) and Mask2 (ANKRD17), remains unclear. Mask family proteins contain two Ankyrin repeat domains that bind Yki/YAP as well as a conserved nuclear localisation sequence (NLS) and nuclear export sequence (NES), suggesting a role in nucleo-cytoplasmic transport. Here we show that Mask acts to promote nuclear import of Yki, and that addition of an ectopic NLS to Yki is sufficient to bypass the requirement for Mask in Yki-driven tissue growth. Mammalian Mask1/2 proteins also promote nuclear import of YAP, as well as stabilising YAP and driving colloidal phase separation into large liquid droplets. Mask1/2 and YAP normally colocalise in a granular fashion in both nucleus and cytoplasm, and are co-regulated during mechanotransduction. Our results suggest that Mask family proteins promote YAP nuclear import and phase separation to regulate YAP stability and transcriptional activity.

developmental biology↗

bioRxiv2019

Complement in human pre-implantation embryos: attack and defense

It is essential for early human life that immunological responses to developing embryos are tightly regulated. An imbalance in the activation and regulation of the human complement system occurs in pregnancy complications, such as pre-eclampsia and recurrent miscarriage. We hereby present the first full analysis of the expression and deposition of complement molecules in human pre-implantation embryos. Thus far, immunological imbalance has been considered in stages of pregnancy following implantation. We here show that complement activation and deposition takes place on developing human embryos already at the pre-implantation stage. Using confocal microscopy, we observed deposition of activation products such as C1q, C3 and C5 on healthy developing embryos, which highlights the need for strict complement regulation. The early embryos express the complement membrane inhibitors CD46, CD55 and CD59 and bind the soluble regulators C4bp and factor H. These findings show that complement targets human embryos, and indicate potential adverse pregnancy outcomes, if regulation of activation fails. In addition, single-cell RNA sequencing of embryos at oocyte, zygote, 4-cell and 8-cell stages showed expression of complement genes, e.g. C1s, C2, C3, C5, factor B and factor D. This shows that the embryonic cells themselves have the capacity to express C3 and C5, which may become activated and function as mediators of cellular signaling. The specific local embryonic expression of complement components, regulators, and deposition of activation products on the surface of embryos suggests that complement has immunoregulatory functions and may impact cellular homeostasis and differentiation at the earliest stage of human life.\n\nStatement of significanceWhile canonical functions of the complement system relate to pathogen-defence, it is known to drive certain immune pathologies. The work here described shows, for the first time, expression and localization of a full range of complement molecules in human pre-implantation embryos. We demonstrate complement attack against early embryos, and show presence of embryonic defence mechanisms. Furthermore, we reveal early embryonic production of complement activators, suggesting non-canonical roles in cell signalling and development. Our findings thus reveal a fundamental role for complement at the earliest stages of human embryogenesis. Our data opens up for future studies into the role of complement, both in relation to infertility and pregnancy complications, as well as basic cellular processes during early human development.

developmental biology↗

bioRxiv2019

Tissue specific vulnerability to mitotic defects caused by mutations in the Drosophila ASPM homologue, Asp

Misregulation of candidate stem cell marker ASPM, and its Drosophila homologue Asp, leads to either tumour formation or microcephaly, but the functional roles contributing to each are not understood. We reverse-engineered flies to express a version of Asp (AspLIE), predicted to have lost its ability to bind the phosphatase PP2A-B. Although AspLIE flies were viable, they exhibited splayed neural stem cell spindle poles under stress, and development was substantially delayed. A tissue-level analysis of microcephaly and midgut abnormalities in Asp mutants with a compromised spindle assembly checkpoint (SAC) demonstrates tissue-specific vulnerability to mitotic defects.

developmental biology↗

bioRxiv2019

Suppression of transcytosis regulates zebrafish blood-brain barrier development

As an optically transparent model organism with an endothelial blood-brain barrier (BBB), zebrafish offer a powerful tool to study the vertebrate BBB. However, the precise developmental profile of functional zebrafish BBB acquisition and the subcellular and molecular mechanisms governing the zebrafish BBB remain poorly characterized. Here we find a spatiotemporal gradient of barrier acquisition. Moreover, we capture the dynamics of developmental BBB leakage using live imaging, revealing a combination of steady accumulation in the parenchyma and sporadic bursts of tracer leakage. Electron microscopy studies further reveal that this steady accumulation results from high levels of transcytosis that are eventually suppressed, sealing the BBB. Finally, we demonstrate a key mammalian BBB regulator Mfsd2a, which inhibits transcytosis, plays a conserved role in zebrafish. Mfsd2aa mutants display increased larval and adult BBB permeability due to increased transcytosis. Our findings indicate a conserved developmental program of barrier acquisition between zebrafish and mice.

developmental biology↗

bioRxiv2019

A Single-cell Transcriptomic Atlas of the Developing Chicken Limb

BackgroundThrough precise implementation of distinct cell type specification programs, differentially regulated in both space and time, complex patterns emerge during organogenesis. Thanks to its easy experimental accessibility, the developing chicken limb has long served as a paradigm to study vertebrate pattern formation. Through decades worth of research, we now have a firm grasp on the molecular mechanisms driving limb formation at the tissue-level. However, to elucidate the dynamic interplay between transcriptional cell type specification programs and pattern formation at its relevant cellular scale, we lack appropriately resolved molecular data at the genome-wide level. Here, making use of droplet-based single-cell RNA-sequencing, we catalogue the developmental emergence of distinct tissue types and their transcriptome dynamics in the distal chicken limb, the so-called autopod, at cellular resolution.\n\nResultsUsing single-cell RNA-sequencing technology, we sequenced a total of 17,628 cells coming from three key developmental stages of chicken autopod patterning. Overall, we identified 23 cell populations with distinct transcriptional profiles. Amongst them were small, albeit essential populations like the apical ectodermal ridge, demonstrating the ability to detect even rare cell types. Moreover, we uncovered the existence of molecularly distinct sub-populations within previously defined compartments of the developing limb, some of which have important signaling functions during autopod pattern formation. Finally, we inferred gene co-expression modules that coincide with distinct tissue types across developmental time, and used them to track patterning-relevant cell populations of the forming digits.\n\nConclusionsWe provide a comprehensive functional genomics resource to study the molecular effectors of chicken limb patterning at cellular resolution. Our single-cell transcriptomic atlas captures all major cell populations of the developing autopod, and highlights the transcriptional complexity in many of its components. Finally, integrating our data-set with other single-cell transcriptomics resources will enable researchers to assess molecular similarities in orthologous cell types across the major tetrapod clades, and provide an extensive candidate gene list to functionally test cell-type-specific drivers of limb morphological diversification.

developmental biology↗

bioRxiv2019

RUNX1 safeguards the identity of the fetal ovary through an interplay with FOXL2

Sex determination of the gonads begins with fate specification of gonadal supporting cells into either ovarian granulosa cells or testicular Sertoli cells. This process of fate specification hinges on a balance of transcriptional control. We discovered that expression of the transcription factor RUNX1 is enriched in the fetal ovary in rainbow trout, turtle, mouse, goat and human. In the mouse, RUNX1 marks the supporting cell lineage and becomes granulosa cell-specific as the gonads differentiate. RUNX1 plays complementary/redundant roles with FOXL2 to maintain fetal granulosa cell identity, and combined loss of RUNX1 and FOXL2 results in masculinization of the fetal ovaries. At the chromatin level, RUNX1 occupancy overlaps partially with FOXL2 occupancy in the fetal ovary, suggesting that RUNX1 and FOXL2 target a common set of genes. These findings identify RUNX1, with an ovary-biased pattern conserved across species, as a novel regulator in securing the identity of ovarian supporting cells and the ovary.

developmental biology↗

bioRxiv2019

Nanog regulates Pou3f1 expression and represses anterior fate at the exit from pluripotency.

Pluripotency is regulated by a network of transcription factors that maintains early embryonic cells in an undifferentiated state while allowing them to proliferate. NANOG is a critical factor for maintaining pluripotency and its role in primordial germ cell differentiation has been well described. However, Nanog is expressed during gastrulation across all the posterior epiblast, and only later in development its expression is restricted to primordial germ cells. In this work, we unveiled a previously unknown mechanism by which Nanog specifically represses the anterior epiblast lineage. Analysis of transcriptional data from both embryonic stem cells and gastrulating mouse embryos revealed Pou3f1 expression to be negatively correlated with that of Nanog during the early stages of differentiation. We have functionally demonstrated Pou3f1 to be a direct target of NANOG by using a dual transgene system for the controlled expression of Nanog. Use of Nanog null ES cells further demonstrated a role for Nanog in repressing anterior neural genes. Deletion of a NANOG binding site (BS) located nine kilobases downstream of the transcription start site of Pou3f1 revealed this BS to have a specific role in the regionalization of the expression of this gene in the embryo. Our results indicate an active role of Nanog inhibiting the neural fate by repressing Pou3f1 at the onset of gastrulation.

developmental biology↗

bioRxiv2019

The RNA binding protein DAZL functions as repressor and activator of maternal mRNA translation during oocyte maturation

Deleted in azoospermia like (DAZL) is an RNA-binding protein playing critical function during gamete development. In fully-grown oocytes, DAZL protein is detected in prophase and levels increase four to five fold during reentry into the meiotic cell cycle. Here, we have investigated the functional significance of this DAZL accumulation in maturing oocytes. Oocyte depletion of DAZL prevents progression to MII. This maturation block is associated with widespread disruption in the pattern of maternal transcripts loading on ribosomes and their translation measured using a RiboTag IP/RNASeq or qPCR strategy. In addition to decreased ribosome loading of a subset of transcripts, we found that DAZL depletion causes also translational activation of distinct subset of mRNAs. DAZL binds to mRNAs whose translation is both repressed and activated during oocyte maturation. Unexpectedly, DAZL depletion also causes increased ribosome loading of a subset of mRNAs in quiescent GV-arrested oocytes. This dual role of repression and activation is recapitulated by using YFP reporters including the 3UTR of DAZL targets. Injection of recombinant DAZL protein in DAZL-depleted oocytes rescues the translation of these targets as well as maturation to MII. Mutagenesis of putative DAZL-binding sites in these candidate mRNAs mimics the effect of DAZL depletion. These findings demonstrate that DAZL regulates translation of maternal mRNAs in mature oocytes, functioning both as translational repressor and activator.

developmental biology↗

bioRxiv2019

Drosophila ribosomal protein S5b is essential for oogenesis and interacts with distinct RNAs

In Drosophila melanogaster there are two genes encoding ribosomal protein S5, RpS5a and RpS5b. Here, we demonstrate that RpS5b is required for oogenesis. Females lacking RpS5b produce ovaries with numerous developmental defects that undergo widespread apoptosis in mid-oogenesis. Females lacking germline RpS5a are fully fertile, but germline expression of interfering RNA targeting germline RpS5a in an RpS5b mutant background worsened the RpS5b phenotype and blocked oogenesis before egg chambers form. A broad spectrum of mRNAs co-purified in immunoprecipitations with RpS5a, while RpS5b-associated mRNAs were specifically enriched for GO terms related to mitochondrial electron transport and cellular metabolic processes. Consistent with this, RpS5b mitochondrial fractions are depleted for proteins linked to oxidative phosphorylation and mitochondrial respiration, and RpS5b mitochondria tended to form large clusters and had more heterogeneous morphology than those from controls. We conclude that RpS5b-containing ribosomes preferentially associate with particular mRNAs and serve an essential function in oogenesis.

developmental biology↗

bioRxiv2019

NOTCH1 signaling establishes the medullary thymic epithelial cell progenitor pool during mouse fetal development

The cortical and medullary thymic epithelial cell (cTEC and mTEC) lineages are essential for inducing T cell lineage commitment, T cell positive selection and the establishment of self-tolerance, but the mechanisms controlling their fetal specification and differentiation are poorly understood. Here, we show that Notch signaling is required to specify and expand the mTEC lineage. Notch1 is expressed by and active in TEC progenitors. Deletion of Notch1 in TECs resulted in depletion of mTEC progenitors and dramatic reductions in mTECs during fetal stages, consistent with defects in mTEC specification and progenitor expansion. Conversely, forced Notch signaling in all TEC resulted in widespread expression of mTEC progenitor markers and profound defects in TEC differentiation. In addition, lineage-tracing analysis indicated that all mTECs have a history of receiving a Notch signal, consistent with Notch signaling occurring in mTEC progenitors. Interestingly, this lineage analysis also showed that cTECs are divided between Notch lineage-positive and lineage-negative populations, identifying a previously unknown complexity in the cTEC lineage.

developmental biology↗

bioRxiv2019

Canonical NOTCH signaling controls the early progenitor state and emergence of the medullary epithelial lineage in fetal thymus development.

Thymus function depends on the epithelial compartment of the thymic stroma. Cortical thymic epithelial cells (cTECs) regulate T cell lineage commitment and positive selection, while medullary (m) TECs impose central tolerance on the T cell repertoire. During thymus organogenesis, these functionally distinct sub-lineages are thought to arise from a common thymic epithelial progenitor cell (TEPC). The mechanisms controlling cTEC and mTEC production from the common TEPC are not however understood. Here, we show that emergence of the earliest mTEC lineage-restricted progenitors requires active NOTCH signaling in progenitor TEC and that, once specified, further mTEC development is NOTCH-independent. In addition, we demonstrate that persistent NOTCH activity favors maintenance of undifferentiated TEPC at the expense of cTEC differentiation. Finally, we uncover a direct interaction between NOTCH and FOXN1, the master regulator of TEC differentiation. These data establish NOTCH as a potent regulator of TEPC and mTEC fate during fetal thymus development and are thus of high relevance to strategies aimed at generating/regenerating functional thymic tissue in vitro and in vivo.

developmental biology↗

bioRxiv2019

A distinct cardiopharyngeal mesoderm genetic hierarchy establishes antero-posterior patterning of esophagus striated muscle

In most vertebrates, the upper digestive tract is composed of muscularised jaws linked to the esophagus that permit food uptake and swallowing. Masticatory and esophagus striated muscles (ESM) share a common cardiopharyngeal mesoderm (CPM) origin, however ESM are unusual among striated muscles as they are established in the absence of a primary skeletal muscle scaffold. Using mouse chimeras, we show that the transcription factors Tbx1 and Isl1 are required cell-autonomously for myogenic specification of ESM progenitors. Further, genetic loss-of-function and pharmacological studies point to Met/HGF signalling for antero-posterior migration of esophagus muscle progenitors, where HGF ligand is expressed in adjacent smooth muscle cells. These observations highlight the functional relevance of a smooth and striated muscle progenitor dialogue for ESM patterning. Our findings establish a Tbx1-Isl1-Met genetic hierarchy that uniquely regulate esophagus myogenesis and identify distinct genetic signatures that can be used as a framework to interpret pathologies arising within CPM derivatives.

developmental biology↗

bioRxiv2019

Apoptosis in the fetal testis eliminates developmentally defective germ cell clones

Many germ cells (GCs) are eliminated during development, long before differentiating to egg or sperm, but it is not clear why. Here, we examined how GC composition in the mouse fetal testis is altered by scheduled apoptosis during sex differentiation. Multicolored-lineage tracing revealed that apoptosis affects clonally-related GCs, suggesting that this fate decision occurs autonomously based on shared intrinsic properties. We identified extensive transcriptional heterogeneity among fetal GCs including an apoptosis-susceptible subpopulation delineated by high Trp53 and deviant differentiation. Alternatively, the GC subpopulation most likely to survive was advanced in differentiation. These results indicate that GC developmental fate is based upon discrete and cell-heritable fitnesses and imply that a dichotomy between sex-differentiation and apoptosis coordinates the removal of developmentally incompetent cells to improve gamete quality. Evidence that GC subpopulations are in different epigenetic states suggests that errors in epigenetic reprogramming form the basis of aberrant differentiation and apoptotic selection.\n\nOne sentence summaryGerm cells undergo autonomous selection in the fetal testis to promote male differentiation

developmental biology↗

bioRxiv2019

Identification of a basal stem cell subpopulation in the prostate via functional, lineage tracing and single-cell RNA-seq analyses

The basal cell compartment in many epithelial tissues such as the prostate, bladder, and mammary gland are generally believed to serve as an important pool of stem cells. However, basal cells are heterogenous and the stem cell subpopulation within basal cells is not well elucidated. Here we uncover that the core epithelial-to-mesenchymal transition (EMT) inducer Zeb is exclusively expressed in a prostate basal cell subpopulation based on both immunocytochemical and cell lineage tracing analysis. The Zeb1+ prostate epithelial cells are multipotent prostate basal stem cells (PBSCs) that can self-renew and generate functional prostatic glandular structures with all three epithelial cell types at the single-cell level. Genetic ablation studies reveal an indispensable role for Zeb1 in prostate basal cell development. Utilizing unbiased single cell transcriptomic analysis of over 9000 mouse prostate basal cells, we find that Zeb1+ basal cell subset shares gene expression signatures with both epithelial and mesenchymal cells and stands out uniquely among all the basal cell clusters. Moreover, Zeb1+ epithelial cells can be detected in mouse and clinical samples of prostate tumors. Identification of the PBSC and its transcriptome profile is crucial to advance our understanding of prostate development and tumorigenesis.

developmental biology↗

bioRxiv2019

Ancestral role of Fat-like cadherins in planar cell polarity

Fat family cadherins are enormous proteins that regulate planar cell polarity (PCP) and cell adhesion in bilaterian animals. Their evolutionary origin can be traced back to prebilaterian species, but their ancestral function(s) are unknown. We identified Fat-like and Dachsous cadherins in Hydra, a member of the early-diverging metazoan phylum Cnidaria. Hydra has a simple body plan with only two epithelial layers and radial symmetry. We find that Hydra homologues of Fat-like (HyFat) and Dachsous (HyDs) co-localize at the apico-lateral membrane of ectodermal epithelial cells. Remarkably, HyFat is planar polarized perpendicular to the oral-aboral axis of the animal. Using knockdown approaches we found that HyFat is involved in the regulation of local cell alignment, but is dispensable for the global alignment of ectodermal myonemes along the oral-aboral axis. The intracellular domain (ICD) of HyFat is involved in the morphogenesis of ectodermal myonemes. Thus, Fat family cadherins have ancient, prebilaterian functions in cell adhesion, tissue organization and planar polarity.

developmental biology↗

bioRxiv2019

A Comprehensive Molecular Portrait of Human Urine-derived Renal Progenitor Cells

BackgroundHuman urine is now recognised as a non-invasive source of stem cells with regeneration potential. These cells are mesenchymal stem cells but their detailed molecular and cellular identities are poorly defined. Furthermore, unlike the mouse, the gene regulatory network driving self-renewal and differentiation into functional renal cells in vitro remain unresolved.\n\nMethodsWe isolated urine stem cells from 10 individuals from both genders and distinct ages, characterized them as renal progenitor cells and explored the gene regulatory network sustaining self-renewal.\n\nResultsThese cells express pluripotency-associated proteins-TRA-1-60, TRA-1-81, SSEA4, C-KIT and CD133. Expression of pluripotency-associated proteins enabled rapid reprogramming into iPSCs using episomal-based plasmids without pathway perturbations. Transcriptome analysis revealed expression of a plethora of nephrogenesis-related genes such as SIX2, OSR1, CITED1, NPHS2, NPHS1, PAX2, SALL1, AQP2, EYA1, SLC12A1 and UMOD. As expected, the cells transport Albumin by endocytosis. Based on this, we refer to these cells as urine derived renal progenitor cells-UdRPCs. Associated GO-term analysis of UdRPCs and UdRPC-iPSCs underlined their renal identity and functionality. Upon differentiation by WNT activation using the GSK3{beta}-inhibitor (CHIR99021), transcriptome and KEGG pathway analysis revealed upregulation of WNT-associated genes-AXIN2, JUN and NKD1. Protein interaction network identified JUN- a downstream target of the WNT pathway in association with STAT3, ATF2 and MAPK1 as a putative regulator of self-renewal and differentiation in UdRPCs. Furthermore, like pluripotent stem cells, self-renewal is maintained by FGF2-driven TGF{beta}-SMAD2/3 pathway.\n\nConclusionThis in vitro model and the data presented should lay the foundation for studying nephrogenesis in man.\n\nSignificance StatementHuman urine is a non-invasive source of stem cells with regeneration potential. Here, we investigated the cellular and molecular identities, and the gene regulation driving self-renewal and differentiation of these cells in vitro. These cells express pluripotency-associated markers enabling easy reprogramming. Based on the expression of renal associated genes, proteins and functionality, we refer to these cells as urine derived renal progenitor cells-UdRPCs. CHIR99021-induced differentiation of UdRPCs activated WNT-related genes-AXIN2, JUN and NKD1. Protein interaction network identified JUN as a putative regulator of differentiation whereas self-renewal is maintained by FGF2-driven TGF{beta}-SMAD2/3. Our data will enhance understanding of the molecular identities of UdRPCs, and enable the generation of renal disease models in vitro and eventually kidney-associated regenerative therapies.

developmental biology↗

bioRxiv2019

Single cell transcriptome analysis reveals markers of naive and lineage-primed hematopoietic progenitors derived from human pluripotent stem cells.

During embryogenesis the hematopoietic system develops through distinct waves that generate progenitors with increasing lineage potential, ultimately producing haematopoietic stem cells (HSCs). In vitro differentiation of human pluripotent stem cells (hPSCs) follows the early steps of haematopoietic development but the production of HSCs has proven more challenging. To study the dynamics and heterogeneity of hematopoietic progenitor cells generated in vitro from hPSCs, we performed RNA sequencing of over 10000 CD235a-CD43+single cells. We identified the transcriptome of naive progenitors and those primed toward erythroid, megakaryocyte and leukocyte lineages, and revealed their markers by clustering, trajectory analyses and functional assays. CD44 marks naive clonogenic progenitors that express the transcription factor, LMO4 and can be expanded upon BMP4 stimulation. Naive progenitors give rise to primed CD326+erythroid, ICAM2+CD9+megakaryocyte, and monocyte, neutrophil and eosinophil progenitors. We have generated an online dataset of human hematopoietic progenitors and their transcriptional remodelling upon lineage priming.

developmental biology↗

bioRxiv2019

N-Glycome regulation limits the transdifferentiation of endothelial cells into hematopoietic stem cells

Hematopoietic stem and progenitor cells (HSPCs) that establish and maintain the blood system in adult vertebrates arise from the transdifferentiation of hemogenic endothelial cells (hemECs) during embryogenesis. This endothelial-to-hematopoietic transition (EHT) is tightly regulated, but the mechanisms are poorly understood. Here, we show that microRNA (miR)-223-mediated regulation of N-glycan biosynthesis in endothelial cells (ECs) regulates EHT. Single cell RNA-sequencing revealed that miR-223 is enriched in hemECs and in oligopotent nascent HSPCs. miR-223 restricts the EHT of lymphoid/myeloid lineages by suppressing the expression of mannosyltransferase alg2 and sialyltransferase st3gal2, two enzymes involved in N-linked protein glycosylation. High-throughput glycomics of ECs lacking miR-223 showed a decrease of high mannose versus sialylated complex/hybrid sugars on N-glycoproteins involved in EHT such as the metalloprotease Adam10. Endothelial-specific expression of an N-glycan Adam10 mutant or of the N-glycoenzymes phenocopied the aberrant HSPC production of miR-223 mutants. Thus, the N-glycome plays a previously unappreciated role as an intrinsic regulator of EHT, with specific mannose and sialic acid modifications serving as key endothelial determinants of their hematopoietic fate. One Sentence SummaryThe N-glycan "sugar code" governs the hematopoietic fate of endothelial cells and regulates blood stem cell production in vivo.

developmental biology↗