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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

Regulation of Cell-Type-Specific Transcriptomes by miRNA Networks During Human Brain Development

MicroRNAs (miRNAs) regulate many cellular events by regulating hundreds of mRNA transcripts. However, it is unclear how miRNA-mRNA interactions are contextualized into the framework of transcriptional heterogeneity among closely related cells of the developing human brain. By combining the multiple complementary approaches, AGO2-HITS-CLIP, single-cell profiling and bipartite network analysis, we show that the miRNA-mRNA network operates as functional modules related to cell-type identities and undergo dynamic transitions during brain development.

developmental biology↗

bioRxiv2018

Evidence of functional long-range Wnt/Wg in the developing Drosophila wing epithelium

Wnts are secreted proteins that regulate cell fate specification during development of all metazoans. Wnt proteins were proposed to spread over several cell diameters to activate signalling directly at a distance. In the Drosophila wing epithelium, an extracellular gradient of Wingless (Wg, the homolog of Wnt1) was observed extending over several cells away from producing cells. However, it was also recently shown that a membrane-tethered Neurotactin-Wg fusion protein (NRT-Wg) can rescue the loss-of endogenous Wg, leading to proper patterning of the wing. Therefore, whether Wg spreading is required for correct tissue patterning during development remains controversial and the functional range of wild-type Wg is unclear. Here, by capturing secreted Wg on distally located cells we show that the Wg gradient acts directly up to eleven cell distances. Cells located outside the reach of extracellular Wg depend on the Frizzled2 receptor to maintain target gene expression. We find that NRT-Wg is not restricted to the producing cells and propose that it can rescue signalling defects by perdurance in the receiving cells. These results provide insight into the mechanisms by which Wnt proteins mediate patterning of a rapidly growing tissue.

developmental biology↗

bioRxiv2018

A tensile ring drives tissue flows to shape the gastrulating amniote embryo

Tissue morphogenesis is driven by local cellular deformations, themselves powered by contractile actomyosin networks. While it is well demonstrated that cell-generated forces at the microscopic scale underlie a variety of local morphogenetic processes (e.g. lengthening/ narrowing1-4, bending5-8, or folding9,10), how such local forces are transmitted across tissues to shape them at a mesoscopic scale remains largely unknown. Here, by performing a quantitative analysis of gastrulation in entire avian embryos, we show that the formation of the primitive streak and the associated large-scale rotational tissue flows (i.e. polonaise movements11,12) are integral parts of a global process that is captured by the laws of fluid mechanics. We identify a large-scale supracellular actomyosin ring (2 mm in diameter and 250 m thick) that shapes the embryo by exerting a graded tension along the margin between the embryonic and extra-embryonic territories. Tissue-wide flows arise from the transmission of these localized forces across the embryonic disk and are quantitatively recapitulated by a fluid-mechanical model based on the Stokes equations for viscous flow. We further show that cell division, the main driver of cell rearrangements at this stage13, is required for fluid-like behavior and for the progress of gastrulation movements. Our results demonstrate the power of a hydrodynamic approach to tissue-wide morphogenetic processes14-16 and provide a simple, unified mechanical picture of amniote gastrulation. A tensile embryo margin, in addition to directing tissue motion, could act as an interface between mechanical and molecular cues, and play a central role in embryonic self-organization.

developmental biology↗

bioRxiv2018

Heterogeneity of Sonic Hedgehog Response Dynamics and Fate Specification in Single Neural Progenitors

During neural tube patterning, a gradient of Sonic hedgehog (Shh) signaling specifies ventral progenitor fates. The cellular response to Shh is processed through a genetic regulatory network (GRN) to specify distinct fate decisions. This process integrates Shh response level, duration and other inputs and is affected by noise in signaling and cell position. How reliably Shh response profile predicts the fate choice of a single cell is unclear. Here we use live imaging to track neural progenitors in developing zebrafish and quantify both Shh and fate reporters in single cells over time. We found that there is significant heterogeneity between Shh response and fate choice in single cells. We quantitatively modeled reporter intensities to obtain single cell response levels over time and systematically evaluated their correlation with cell fate specification. Motor neuron progenitors (pMN) exhibit a high degree of variability in their Shh responses, which is particularly prominent in the posterior neural tube where the Shh response dynamics are similar to those of the more ventrally fated lateral floor plate cells (LFPs). Our results highlight the precision limit of morphogen-interpretation GRNs in small and dynamic target cell fields.

developmental biology↗

bioRxiv2018

Mechanical Coupling Coordinates the Co-elongation of Axial and Paraxial Tissues in Avian Embryos

Tissues undergoing morphogenesis impose mechanical effects on one another. How developmental programs adapt to or take advantage of these effects remains poorly explored. Here, using a combination of live imaging, modeling, and microsurgical perturbations, we show that the axial and paraxial tissues in the forming avian embryonic body coordinate their rates of elongation through mechanical interactions. First, a cell motility gradient drives paraxial presomitic mesoderm (PSM) expansion, resulting in compression of the axial neural tube and notochord; second, elongation of axial tissues driven by PSM compression and polarized cell intercalation pushes the caudal progenitor domain posteriorly; finally, the axial push drives progenitors to emigrate into the PSM to maintain tissue growth and cell motility. These interactions form an engine-like positive feedback loop, which ensures the tissue-coupling and self-sustaining characteristics of body elongation. Our results suggest a general role of inter-tissue forces in the coordination of complex morphogenesis involving distinct tissues.

developmental biology↗

bioRxiv2018

Onecut factors and Pou2f2 regulate diversification and migration of V2 interneurons in the mouse developing spinal cord

Acquisition of proper neuronal identity and position is critical for the formation of neural circuits. In the embryonic spinal cord, cardinal populations of interneurons diversify into specialized subsets and migrate to defined locations within the spinal parenchyma. However, the factors that control interneuron diversification and migration remain poorly characterized. Here, we show that the Onecut transcription factors are necessary for proper diversification and distribution of the V2 interneurons in the developing spinal cord. Furthermore, we uncover that these proteins restrict and moderate the expression of spinal isoforms of Pou2f2, a transcription factor known to regulate B-cell differentiation. By gain- or loss-of-function experiments, we show that Pou2f2 contribute to regulate the position of V2 populations in the developing spinal cord. Thus, we uncovered a genetic pathway that regulates the diversification and the distribution of V2 interneurons during embryonic development.\n\nSignificance statementIn this study, we identify the Onecut and Pou2f2 transcription factors as regulators of spinal interneuron diversification and migration, two events that are critical for proper CNS development.

developmental biology↗

bioRxiv2018

Modularity, criticality and evolvability of a developmental gene regulatory network

The existence of discrete phenotypic traits suggests that the complex regulatory processes which produce them are functionally modular. These processes are usually represented by networks. Only modular networks can be partitioned into intelligible subcircuits able to evolve relatively independently. Traditionally, functional modularity is approximated by detection of modularity in network structure. However, the correlation between structure and function is loose. Many regulatory networks exhibit modular behaviour without structural modularity. Here we partition an experimentally tractable regulatory network--the gap gene system of dipteran insects--using an alternative approach. We show that this system, although not structurally modular, is composed of dynamical modules driving different aspects of whole-network behaviour. All these subcircuits share the same regulatory structure, but differ in components and sensitivity to regulatory interactions. Some subcircuits are in a state of criticality while others are not, which explains the observed differential evolvability of the various expression features in the system.

developmental biology↗

bioRxiv2018

Checkpoint kinase 1 is essential for fetal haematopoiesis and hematopoietic stem cell survival

Checkpoint kinase 1 is critical for S-phase fidelity and preventing premature mitotic entry in the presence of DNA damage. Tumour cells have developed a strong dependence on CHK1 for survival and hence this kinase has developed into a popular drug-target. Chk1-deficiency in mice results in blastocyst death due to G2/M checkpoint-failure showing that it is an essential gene and may be difficult to target therapeutically without side-effects. Here, we show that chemical inhibition of CHK1 kills murine hematopoietic stem and progenitor cells (HSPCs) as well as human CD34+ HSPCs by the induction of BCL2-regulated but p53-independent apoptosis. Moreover, Chk1 is essential for stem cell survival and definite hematopoiesis in the mouse embryo. Remarkably though, cell death inhibition in hematopoietic stem cells (HSC) cannot restore blood cell formation in utero as Chk1 loss causes severe DNA damage that ultimately prevents HSC expansion. Our findings establish a previously unrecognized role for CHK1 in establishing hematopoiesis; they also suggest adverse effects of therapeutic CHK1-inhibtion, particularly under conditions forcing stem cells out of dormancy, such as chemotherapy-induced myelosuppression.

developmental biology↗

bioRxiv2018

Formation of the substantia nigra requires Reelin-mediated fast, laterally-directed migration of dopaminergic neurons

Midbrain dopaminergic (mDA) neurons migrate to form the laterally-located substantia nigra pars compacta (SN) and medially-located ventral tegmental area (VTA), but little is known about the underlying cellular and molecular processes. Reelin signaling regulates tangential migration of SN-mDA neurons, but whether Reelin acts directly on SN-mDA neurons and how it affects their cellular morphology and migratory behavior has not been explored. Here we visualize the dynamic cell morphologies of tangentially migrating SN-mDA neurons with 3D-time-lapse imaging and identify two distinct migration modes. Slow migration is the default mode in SN-mDA neurons, while fast, laterally-directed migration occurs infrequently and is strongly associated with bipolar cell morphology. By speci1cally inactivating Reelin signaling in mDA neurons we demonstrate its direct role in SN-mDA tangential migration. We show that Reelin signaling promotes laterally-biased movements in mDA neurons during their slow migration mode, stabilizes leading process morphology and increases the probability of fast, laterally-directed migration.

developmental biology↗

bioRxiv2018

Morphogenesis and differentiation of embryonic vascular smooth muscle cells in zebrafish

Despite the critical role of vascular mural cells (smooth muscle cells and pericytes) in supporting the endothelium of blood vessels, we know little of their early morphogenesis and differentiation. foxc1b:EGFP expressing cells in zebrafish associate with the vascular endothelium (kdrl) and co-express a smooth muscle marker (acta2), but not a pericyte marker (pdgfr{beta}). The expression of foxc1b in early peri-endothelial mesenchymal cells allows us to follow the morphogenesis of mesenchyme into acta2 expressing vascular smooth muscle cells. We show that mural cells expressing different markers associate with vessels of different diameters, depending on their embryonic location and developmental timing, suggesting marker expression is predictive of functional differences. We identify gene expression signatures for an enriched vascular smooth muscle cell population (foxc1b + acta2) and all smooth muscle (acta2) using fluorescence-activated cell sorting and RNA-Seq. Finally, we demonstrate that progressive loss of foxc1a/foxc1b results in decreased smooth muscle cell coverage. Together, our data highlight the early cellular dynamics and transcriptome profiles of smooth muscle cells in vivo, using foxc1b as a unique tool to probe vascular smooth muscle cell differentiation.\n\nSummary StatementTracing the morphogenesis and transcriptome of early vascular smooth muscle cells using foxc1b

developmental biology↗

bioRxiv2018

Myh10 deficiency leads to defective extracellular matrix remodeling and pulmonary disease

Impaired alveolar formation and maintenance are features of many pulmonary diseases that are associated with significant morbidity and mortality. In a forward genetic screen for modulators of mouse lung development, we identified the non-muscle myosin II heavy chain gene, Myh10. Myh10 mutant pups exhibit cyanosis and respiratory distress, and die shortly after birth from differentiation defects in alveolar epithelium and mesenchyme. From omics analyses and follow up studies, we find decreased Thrombospondin expression accompanied with increased matrix metalloproteinase activity in both mutant lungs and cultured mutant fibroblasts, as well as disrupted extracellular matrix (ECM) remodeling. Loss of Myh10 specifically in mesenchymal cells results in ECM deposition defects and alveolar simplification. Notably, MYH10 expression is down-regulated in the lung of emphysema patients. Altogether, our findings reveal critical roles for Myh10 in alveologenesis at least in part via the regulation of ECM remodeling, which may contribute to the pathogenesis of emphysema.

developmental biology↗

bioRxiv2018

Dynamin binding protein is required for Xenopus laevis kidney development

The adult human kidney contains over one million nephrons, with each nephron consisting of a tube containing segments that have specialized functions in nutrient and water absorption and waste excretion. The embryonic kidney of Xenopus laevis consists of a single functional nephron composed of regions that are analogous to those found in the human nephron, making it a simple model for the study of nephrogenesis. The exocyst complex, which traffics proteins to the cell membrane in vesicles via CDC42, is essential for normal kidney development. Here, we show that the CDC42-GEF, dynamin binding protein (Dnmbp/Tuba), is essential for nephrogenesis in Xenopus. dnmbp is expressed in Xenopus embryo kidneys during development, and knockdown of Dnmbp using two separate morpholino antisense oligonucleotides results in reduced expression of late pronephric markers, whereas the expression of early markers of nephrogenesis remains unchanged. A greater reduction in expression of markers of differentiated distal and connecting tubules was seen in comparison to proximal tubule markers, indicating that Dnmbp reduction may have a greater impact on distal and connecting tubule differentiation. dnmbp knockout using CRISPR results in a similar reduction of late markers of pronephric tubulogenesis. Overexpression of dnmbp in the kidney also resulted in disrupted pronephric tubules, suggesting that dnmbp levels in the developing kidney are tightly regulated, with either increased or decreased levels leading to developmental defects. Together, these data suggest that Dnmbp is required for nephrogenesis.

developmental biology↗

bioRxiv2018

Fate Before Function: Specification of the Hair Follicle Niche Occurs Prior to its Formation and Is Progenitor Dependent

Cell fate transitions are essential for specialization of stem cells and their niches, but the precise timing and sequence of molecular events during embryonic development are largely unknown. Here, we show that dermal condensates (DC), signaling niches for epithelial progenitors in hair placodes, are specified before niche formation and function. With 3D/4D microscopy we identify unclustered DC precursors. With population-based and single-cell transcriptomics we define a molecular time-lapse of dynamic niche signatures and the developmental trajectory as the DC lineage emerges from fibroblasts. Co-expression of downregulated fibroblast and upregulated DC genes in niche precursors reveals a transitory molecular state following a proliferation shutdown. Waves of transcription factor and signaling molecule expression then consolidate DC niche formation. Finally, ablation of epidermal Wnt signaling and placode-derived FGF20 demonstrates their requirement for DC-precursor specification. These findings uncover a progenitor-dependent niche precursor fate and the transitory molecular events controlling niche formation and function.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC=\"FIGDIR/small/414839_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (78K):\norg.highwire.dtl.DTLVardef@13fa73aorg.highwire.dtl.DTLVardef@1fbd280org.highwire.dtl.DTLVardef@1b8f66borg.highwire.dtl.DTLVardef@3a1ef9_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIPrecursors of the hair follicle niche are specified before niche cluster formation\nC_LIO_LIBulk/single cell RNA-seq defines early niche fate at molecular transitional state\nC_LIO_LISuccessive waves of transcription factor/signaling genes mark niche fate acquisition\nC_LIO_LINiche fate acquisition is not \"pre-programmed\" and requires FGF20 from progenitors\nC_LI

developmental biology↗

bioRxiv2018

HSCs contribute actively to native multilineage hematopoiesis but with reduced differentiation capacity upon aging

A hallmark of adult hematopoiesis is the continuous replacement of blood cells with limited lifespans. It is well established that adult hematopoietic stem cells (HSCs) are active contributors to these processes after transplantation, yet their role in native hematopoiesis has recently been called into question. Here, we use inducible lineage tracing from genetically marked adult HSCs to explore their roles in the steady state. We show that adult HSCs contribute robustly to all lineages via intermediate progenitor cells, but with neglible production of hematopoietic cells with a known fetal origin. We further reveal that the timing for regeneration of distinct blood lineages varies substantially. Finally, HSC contribution to multilineage hematopoiesis in aged animals declines with increasing age. Therefore, while HSCs are active contributors to native adult hematopoiesis, it appears that the numerical increase of HSCs is a physiologically relevant compensatory mechanism to account for a reduced differentiation capacity with age.

developmental biology↗

bioRxiv2018

The Central Nervous System Acts As A Transducer Of Stress-Induced Masculinization Through Corticotropin-Releasing Hormone B

Exposure to environmental stressors during early development has important implications for rescheduling many cellular and molecular mechanisms. In some fish species, environmental stressors, like high temperatures (HT), cause an increase in cortisol levels. In turn, this mechanism induces sex reversal of genotypic females, overriding genetic factors related to development of the gonad. However, the involvement of the brain in this process is not well clarified. In the present work, we investigated the mRNA levels of corticotropin-releasing hormone b (crhb) and its receptors (crhr1 and crhr2), and found out that they were up-regulated at HT during the critical period of gonadal sex determination in medaka (Oryzias latipes), i.e., when the gonadal primordium is sexually labile. In order to clarify their roles in sex reversal, biallelic mutants for crhr1 and crhr2 were produced by CRISPR/Cas9 technology. Remarkably, biallelic mutant of both loci (crhr1 and crhr2) did not undergo female-to-male sex reversal upon HT exposition, whereas mutants for either crhr1 or crhr2 showed partial, or intersex phenotypes, suggesting that both crh receptors are required for HT-induced masculinization. Inhibition of this process in double crhrs mutants could be successfully rescued through the administration of the downstream effector of the hypothalamic-pituitary interrenal axis, the cortisol. Taken together, these results revealed for the first time the participation of the central nervous system acting as a transducer of masculinization induced by thermal stress.

developmental biology↗

bioRxiv2018

Endothelial cells form transient Notch-dependent NO-containing cystic structures during zebrafish cerebrovascular development

Endothelial cell behaviour during blood vessel formation is highly complex and dynamic. Transgenic zebrafish have provided many new insights into these processes, due to their ability to provide detailed in vivo imaging.\n\nWe here report a previously undescribed endothelial cell behaviour during zebrafish embryonic development. Endothelial cells of the cerebral vessels of 3-5d post fertilisation embryos extruded large membranous spherical structures. These were only found on the cerebral vessels, and did not detach from the parent vessel, instead regressing back into the endothelial cell. These structures did not communicate with the vessel lumen, exhibited periodic oscillations in size and shape, and were enriched with filamentous actin at their neck. Due to their unknown nature and spherical appearance we termed these structures kugeln (German for sphere).\n\nPharmacological inhibition of vascular endothelial growth factor (VEGF) signalling significantly increased kugel number while Notch inhibition significantly reduced both kugel number and diameter. Kugeln contain little cytoplasm, but are highly positive for nitric oxide (NO) reactivity, suggesting they represent a novel NO containing organelle specific to the cerebral vessels.

developmental biology↗

bioRxiv2018

Stem cell mitotic drive ensures asymmetric epigenetic inheritance

Through the process of symmetric cell division, one mother cell gives rise to two identical daughter cells. Many stem cells utilize asymmetric cell division (ACD) to produce a self-renewed stem cell and a differentiating daughter cell. Since both daughter cells inherit the identical genetic information during ACD, a crucial question concerns how non-genic factors could be inherited differentially to establish distinct cell fates. It has been hypothesized that epigenetic differences at sister centromeres could contribute to biased sister chromatid attachment and segregation. However, direct in vivo evidence has never been shown. Here, we report that a stem cell-specific mitotic drive ensures biased sister chromatid attachment and segregation. We have found during stem cell ACD, sister centromeres become asymmetrically enriched with proteins involved in centromere specification and kinetochore function. Furthermore, we show that that temporally asymmetric microtubule activities direct polarized nuclear envelope breakdown, allowing for the preferential recognition and attachment of microtubules to asymmetric sister kinetochores and sister centromeres. This communication occurs in a spatiotemporally regulated manner. Abolishment of either the establishment of asymmetric sister centromeres or the asymmetric microtubule emanation results in randomized sister chromatid segregation, which leads to stem cell loss. Our results demonstrate that the cis-asymmetry at sister centromeres tightly coordinates with the trans-asymmetry from the mitotic machinery to allow for differential attachment and segregation of genetically identical yet epigenetically distinct sister chromatids. Together, these results provide the first direct in vivo mechanisms for partitioning epigenetically distinct sister chromatids in asymmetrically dividing stem cells, which opens a new direction to study how this mechanism could be used in other developmental contexts to achieve distinct cell fates through mitosis. One Sentence SummaryDuring Drosophila male germline stem cell asymmetric division, sister centromeres communicate with spindle microtubules for differential attachment and segregation of sister chromatids.

developmental biology↗

bioRxiv2018

foxc1a and foxc1b differentially regulate angiogenesis from arteries and veins by modulating Vascular Endothelial Growth Factor signalling

The forkhead transcription factors Foxc1 and Foxc2 are essential to establish intact vascular networks in mammals. How these genes interact with endothelial signalling pathways to exert their functions remains incompletely understood. We have generated novel zebrafish mutants in foxc1a and foxc1b, the zebrafish orthologues of mammalian Foxc1, to determine their function during angiogenesis. foxc1a mutants display abnormal formation of cranial veins including the primordial hindbrain channels (PHBC), reduced Vascular Endothelial Growth Factor (VEGF) receptor expression in these and loss of central arteries. foxc1b mutants are normal, whereas foxc1a; foxc1b double mutants exhibit ectopic angiogenesis from trunk segmental arteries. Dll4/Notch signalling is reduced in foxc1a; foxc1b double mutant arteries and ectopic angiogenesis can be suppressed by induction of Notch or inhibition of Vegfc signalling. We conclude that foxc1a and foxc1b play compensatory and context-dependent roles to co-ordinate angiogenesis by promoting venous sprouting via induction of VEGF receptor expression whilst antagonising arterial sprouting by inducing Dll4/Notch signalling. foxc1a/b mediated induction of both pro- and anti-angiogenic axes of VEGF-Dll4/Notch negative feedback imparts competition to balance arterial and venous angiogenesis within developing vascular beds.\n\nSummary Statementfoxc1a and foxc1b promote angiogenesis from veins and suppress angiogenesis from arteries by promoting competing pro-angiogenic Vascular Endothelial Growth Factor signalling, and anti-angiogenic Dll4/Notch signalling in zebrafish embryos.

developmental biology↗