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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

Molecular determinants of WNT9b responsiveness in nephron progenitor cells

Primed nephron progenitor cells (NPCs) appear in metanephric mesenchyme by Ell.5 and differentiate in response to the inductive WNT9b signal from the ureteric bud. However, the NPC WNT-receptor complex is unknown. We obtained M15 cells from E10.5 mesonephric mesenchyme and systematically analyzed components required for canonical WNT9b-responsiveness. When M15 cells were transfected with a ({beta}-catenin luciferase reporter plasmid, exposure to recombinant WNT9b resulted in minimal luciferase activity. We then analyzed mRNA-expression of WNT-pathway components and identified Fzdl-6 and Lrp6 transcripts but not RSPO1. When M15 cells were treated with recombinant RSPO1 the response to transfected WNT9b was augmented 4.8-fold. Co-transfection of M15 cells with Fzd5 (but no other Fzd family member) further increased the WNT9b signal to 16.8-fold and siRNA knockdown of Fzd5 reduced the signal by 52%. Knockdown of Lrp6 resulted in 60% WNT9b signal reduction. We confirmed Fzd5, Lrp6 and RSPO1 rrtRNA expression in CITED1(+) NPCs from E15.5 embryonic mouse kidney. Thus, while many WNT signaling-pathway components are present by E10.5, optimum responsiveness of Ell.5 cap mesenchyme requires that NPCs acquire RSPO1, FZD5 and LRP6.\n\nSummary StatementResponsiveness to the inductive WMT9b signal from ureteric bud is crucial for nephrogenesis. Here we analyze the molecules needed to prime nephron progenitor cells in embryonic mouse kidney.

developmental biology↗

bioRxiv2018

Genomic Analysis of a Transcriptional Networks Directing Progression of Cell States During MGE development.

BackgroundHomeodomain (HD) transcription factor (TF) NKX2-1 critical for the regional specification of the medial ganglionic eminence (MGE) as well as promoting the GABAergic and cholinergic neuron fates via the induction of TFs such as LHX6 and LHX8. NKX2-1 defines MGE regional identity in large part through transcriptional repression, while specification and maturation of GABAergic and cholinergic fates is mediated in part by transcriptional activation via TFs such as LHX6 and LHX8. Here we analyze the signaling and TF pathways, downstream of NKX2-1, required for GABAergic and cholinergic neuron fate maturation.\n\nMethodsDifferential ChIP-seq analysis was used to identify regulatory elements (REs) where chromatin state was sensitive to change in the Nkx2-1cKO MGE at embryonic day (E) 13.5. TF motifs in the REs were identified using RSAT. CRISPR-mediated genome editing was used to generate enhancer knockouts. Differential gene expression in these knockouts was analyzed through RT-qPCR and in situ hybridization. Functional analysis of motifs within hs623 was analyzed via site directed mutagenesis and reporter assays in primary MGE cultures.\n\nResultsWe identified 4782 activating REs (aREs) and 6391 repressing REs (rREs) in the Nkx2-1 conditional knockout (Nkx2-1cKO) MGE. aREs are associated with basic-Helix-Loop-Helix (bHLH) TFs. Deletion of hs623, an intragenic Tcf12 aRE, caused a reduction of Tcf12 expression in the sub-ventricular zone (SVZ) and mantle zone (MZ) of the MGE. Mutation of LHX, SOX and octamers, within hs623, caused a reduction of hs623 activity in MGE primary cultures.\n\nConclusionsTcf12 expression in the sub-ventricular zone (SVZ) of the MGE is mediated through aRE hs623. The activity of hs623 is dependent on LHX6, SOX and octamers. Thus, maintaining the expression of Tcf12 in the SVZ involves on TF pathways parallel and genetically downstream of NKX2-1.

developmental biology↗

bioRxiv2018

Molecular mechanism of symmetry breaking in a 3D model of a human epiblast

Breaking the anterior-posterior (AP) symmetry in mammals takes place at gastrulation. Much of the signaling network underlying this process has been elucidated in the mouse, however there is no direct molecular evidence of events driving axis formation in humans. Here, we use human embryonic stem cells to generate an in vitro 3D model of a human epiblast whose size, cell polarity, and gene expression are similar to a 10-day human epiblast. A defined dose of bone mor-phogenetic protein 4 (BMP4) spontaneously breaks axial symmetry, and induces markers of the primitive streak and epithelial to mesenchymal transition. By gene knockouts and live-cell imaging we show that, downstream of BMP4, WNT3 and its inhibitor DKK1 play key roles in this process. Our work demonstrates that a model human epiblast can break axial symmetry despite no asymmetry in the initial signal and in the absence of extraembryonic tissues or maternal cues. Our 3D model opens routes to capturing molecular events underlying axial symmetry breaking phenomena, which have largely been unexplored in model human systems.

developmental biology↗

bioRxiv2018

Hhex regulates the specification and growth of the hepatopancreatic ductal system

Significant efforts have advanced our understanding of foregut-derived organ development; however, little is known about the molecular mechanisms that underlie the formation of the hepatopancreatic ductal (HPD) system. Here, we report a role for the homeodomain transcription factor Hhex in directing HPD progenitor specification in zebrafish. Loss of Hhex function results in impaired HPD system formation. We found that Hhex specifies a distinct population of HPD progenitors that gives rise to the cystic duct, common bile duct, and extra-pancreatic duct. Since hhex is not uniquely expressed in the HPD region but is also expressed in endothelial cells and the yolk syncytial layer (YSL), we tested the role of blood vessels as well as the YSL in HPD formation. We found that blood vessels are required for HPD patterning, but not for HPD progenitor specification. In addition, we found that Hhex is required in both the endoderm and the YSL for HPD development. Our results shed light on the mechanisms necessary to direct endodermal progenitors towards the HPD fate and also advance our understanding of HPD system formation.

developmental biology↗

bioRxiv2018

Nr5a1 suppression during the fetal period optimizes ovarian development by fine-tuning of Notch signaling

The nuclear receptor NR5A1 (also known as Ad4BP, or SF1) is essential for the initial steps of mammalian gonadal development. The Nr5a1 gene is equally expressed in XX and XY gonadal primordia, but after sex determination, is up-regulated in XY and down-regulated in XX gonads. We recently reported a case of 46, XX disorder of sex development (DSD) in which ectopically expressed NR5A1 in XX gonads led to an ovo-testicular phenotype, suggesting that excess NR5A1 can direct the development of immature XX gonads towards testicular formation. However, a direct causal relationship has not been demonstrated in an animal model. Here, using a Wt1-BAC (bacterial artificial chromosome) transgene system, we generated two lines of mice overexpressing Nr5a1 in the fetal gonads at different levels. One of these lines (Tg-S), highly expressing Nr5a1, revealed that enforced Nr5a1 expression alone is insufficient to switch the fate of the 46,XX gonads toward testicular formation in mice. In the other line (Tg-A) expressing Nr5a1 at lower level, ovarian development was compromised, with multi-oocyte follicles, reduced number of matured follicles, and impaired expression of Wnt4, resulting in late onset infertility at 20 weeks after birth. The phenotype was similar to that of genetically modified mice with impaired Notch signaling. Indeed, the expression level of Notch2 and 3 was significantly reduced in Tg-A mice, and the ovarian phenotype in Tg-A mice was almost completely rescued by in utero treatment with a Notch2 agonist HMN2-29. We conclude that suppression of Nr5a1 during the fetal period optimizes ovarian development by fine tuning of Notch signaling levels.\n\nAUTHOR SUMMARYSexual development is a process of differentiation from undifferentiated bipotential gonads, and insight into sexual differentiation will bring important new knowledge to our understanding of organogenesis. The nuclear receptor NR5A1 which is essential for mammalian gonadal development, is equally expressed in both gonadal primordia, but after sex determination, is up-regulated in XY and down-regulated in XX gonads. We have recently demonstrated that this down-regulation is mediated by ovarian transcription factor, Forkhead box L2 (FOXL2). This finding raised two key questions, whether Nr5a1 can function as a male sex-determining factor, and whether the repression is essential for appropriate ovarian development. By generating two lines Tg mice in XX gonads with different enforced expression levels of Nr5a1, our present study revealed that alterations in Nr5a1 dosage, either reduced or excessive, result in pathological effects in ovarian development and female fertility, indicating that the precise control of Nr5a1 at the transcriptional level is essential for optimal ovarian development. We envisage that the improved understanding of how this pathway regulates ovarian development and female fertility would aid the development of artificial somatic ovarian cells, which in turn may provide a valuable treatment option in reproductive medicine.\n\nABBREVIATIONS

developmental biology↗

bioRxiv2018

Dynamic control of proinflammatory cytokines Il-1β and Tnf-α by macrophages is necessary for functional spinal cord regeneration in zebrafish

Spinal cord injury leads to a massive response of innate immune cells (microglia, macrophages, neutrophils) both, in non-regenerating mammals and in successfully regenerating zebrafish, but the role of these immune cells in functional spinal cord regeneration in zebrafish has not been addressed. Here we show that inhibiting inflammation reduces and promoting it accelerates axonal regeneration in larval zebrafish. Mutant analyses show that peripheral macrophages, but not neutrophils or microglia, are necessary and sufficient for full regeneration. Macrophage-less irf8 mutants show prolonged inflammation with elevated levels of Il-1{beta} and Tnf-. Decreasing Il-1{beta} levels or number of Il-1{beta}+ neutrophils rescues functional regeneration in irf8 mutants. However, during early regeneration, interference with Il-1{beta} function impairs regeneration in irf8 and wildtype animals. Inhibiting Tnf- does not rescue axonal growth in irf8 mutants, but impairs it in wildtype animals, indicating a pro-regenerative role of Tnf-. Hence, inflammation is tightly and dynamically controlled by macrophages to promote functional spinal cord regeneration in zebrafish.

developmental biology↗

bioRxiv2018

Actomyosin dynamics, Bmp and Notch signaling pathways drive apical extrusion of proepicardial cells

The epicardium, the outer mesothelial layer enclosing the myocardium, plays key roles in heart development and regeneration. During embryogenesis it arises from the proepicardium (PE), a cell cluster that appears in the dorsal pericardium close to the venous pole of the heart. Little is known about how the PE emerges from the pericardial mesothelium. Using the zebrafish model and a combination of genetic tools, pharmacological agents and quantitative in vivo imaging we reveal that a coordinated collective movement of the dorsal pericardium drives PE formation. We found that PE cells are apically extruded in response to actomyosin activity. Our results reveal that the coordinated action of Notch/Bmp pathways is critically needed for apical extrusion of PE cells. More generally, by comparison to cell extrusion for the elimination of unfit cells from epithelia, our results describe a unique mechanism where extruded cell viability is maintained.

developmental biology↗

bioRxiv2018

Hmgcr promotes a long-range signal to attract germ cells which is aided by Wunens but independent of hh

Summary StatementMigrating Drosophila germ cells are attracted by a long range Hmgcr mediated signal which is aided and acts simultaneously with Wunens suggesting that these pathways converge on a single chemoattractant.\n\nAbstractIn a developing embryo, many cell types migrate from their point of specification to their final position. This usually involves highly stereotyped routes which are determined through deployment of cell surface or secreted guidance molecules. Whilst genetic techniques have been successful in identifying these molecules, the distances over which such signals operate in their native context can be difficult to determine. Here we have quantified the range of an attractive signal for the migration of Drosophila germ cells. Their migration is guided by an attractive signal generated by the expression of genes in the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (Hmgcr) pathway, and by a repulsive signal generated by the expression of Wunens. We demonstrate that the attractive signal downstream of Hmgcr operates over a long range and is sufficient to reach germ cells for the entirety of their migration. Furthermore, Hmgcr-mediated attraction and Wunen-mediated repulsion can operate simultaneously ruling out a model in which these pathways operate consecutively. Indeed, we show that Hmgcr-mediated attraction is boosted by Wunens suggesting the action of these two pathways is linked. Lastly, several papers have pointed to the secreted molecule Hedgehog (Hh) as being the germ cell attractant, whose secretion is increased by hmgcr. In this paper, we provide evidence that Hh is not downstream of hmgcr in germ cell migration.

developmental biology↗

bioRxiv2018

WNT signaling memory is required for ACTIVIN to function as a morphogen in human gastruloids

Self-organization of discrete fates in human gastruloids is mediated by a hierarchy of signaling pathways. How these pathways are integrated in time, and whether cells maintain a memory of their signaling history remains obscure. Here, we dissect the temporal integration of two key pathways, WNT and ACTIVIN, which along with BMP control gastrulation. CRISPR/Cas9 live reporters of SMAD1, 2 and 4 demonstrate that in contrast to the stable signaling by SMAD1, signaling and transcriptional response by SMAD2 is transient, and while necessary for pluripotency, it is insufficient for differentiation. Pre-exposure to WNT, however, endows cells with the competence to respond to graded levels of ACTIVIN, which induces differentiation without changing SMAD2 dynamics. This cellular memory of WNT signaling is necessary for ACTIVIN morphogen activity. A re-evaluation of the evidence gathered over decades in model systems, re-enforces our conclusions and points to an evolutionarily conserved mechanism.

developmental biology↗

bioRxiv2018

Commensal bacteria act as a broad genetic buffer in Drosophila during chronic under-nutrition

Eukaryotic genomes encode several well-studied buffering mechanisms that robustly maintain invariant phenotypic outcome despite fluctuating environmental conditions. Here we show that the gut microbiota, represented by a single Drosophila facultative symbiont, Lactobacillus plantarum (LpWJL), acts also as a broad genetic buffer that masks the contribution of the cryptic genetic variations in the host under nutritional stress. During chronic under-nutrition, LpWJL consistently reduces variation in different host phenotypic traits and ensures robust organ patterning; LpWJL also decreases genotype-dependent expression variation, particularly for development-associated genes. We further demonstrate that LpWJL buffers via reactive oxygen species (ROS) signaling whose inhibition severely impairs microbiota-mediated phenotypic robustness. We thus identified an unexpected contribution of facultative symbionts to Drosophila fitness by assuring developmental robustness and phenotypic homogeneity in times of nutritional stress.

developmental biology↗

bioRxiv2018

Chorioamnionitis Is a Risk Factor for Intraventricular Hemorrhage in Preterm Infants: A Systematic Review and Meta-Analysis

Although chorioamnionitis (CA) is a well-known risk factor for white matter disease of prematurity, the association with intraventricular hemorrhage (IVH) is controversial and has not been yet systematically reviewed. We performed a systematic review and meta-analysis of studies exploring the association between CA and IVH. A comprehensive literature search was conducted using PubMed/MEDLINE and EMBASE, from their inception to 1 July 2017. Studies were included if they examined preterm infants and reported primary data that could be used to measure the association between exposure to CA and the presence of IVH. A random-effects model was used to calculate odds ratios (OR) and 95% confidence intervals (CI). We found 1284 potentially relevant studies, of which 85 met the inclusion criteria (46,244 infants, 13,432 CA cases). Meta-analysis showed that CA exposure was significantly associated with all grades IVH (OR 1.88, 95% CI 1.61-2.19), with grades 1-2 IVH (OR 1.69, 95% CI 1.22-2.34), and with grades 3-4 IVH (OR 1.62, 95% CI 1.42-1.85). Both clinical and histological CA were associated with an increased risk for developing IVH in very preterm infants. In contrast, the presence of funisitis did not increase IVH risk when compared to CA in the absence of funisitis (OR 1.22, 95% CI 0.89-1.67). Further meta-analyses confirmed earlier findings that CA-exposed infants have significantly lower gestation age (GA; mean difference [MD] -1.20 weeks) and lower birth weight (BW; MD -55g) than the infants not exposed to CA. However, meta-regression and subgroup analysis could not demonstrate an association between the lower GA and BW and the risk of IVH in the CA-exposed infants. In conclusion, our data show that CA is a risk factor for IVH, but also a risk factor for greater prematurity and more clinical instability. In contrast to other complications of prematurity, such as patent ductus arteriosus, retinopathy of prematurity, or bronchopulmonary dysplasia, the effect of CA on IVH appears to be independent of CA as causative factor for very preterm birth.

developmental biology↗

bioRxiv2018

CRISPR/Cas9-mediated knock-out of dUTPase in mice leads to early embryonic lethality

Sanitization of nucleotide pools is essential for genome maintenance. Among the enzymes significant in this mechanism, deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) performs cleavage of dUTP into dUMP and inorganic pyrophosphate. By this reaction the enzyme efficiently prevents uracil incorporation into DNA and provides dUMP, the substrate for de novo thymidylate biosynthesis. Despite its physiological significance, knock-out models of dUTPase have not yet been investigated in mammals, only in unicellular organisms, such as bacteria and yeast. Here we generate CRISPR/Cas9-mediated dUTPase knock-out in mice. We find that heterozygous dut +/-animals are viable while the decreased dUTPase level is clearly observable. We also show that the enzyme is essential for embryonic development. Based on the present results, early dut -/-embryos can still reach the blastocyst stage, however, they die shortly after implantation. Analysis of preimplantion embryos indicate perturbed growth of both inner cell mass (ICM) and trophectoderm (TE). We conclude that dUTPase is indispensable for post-implantation development in mice. The gene targeting model generated in the present study will allow further detailed studies in combination with additional gene knock-outs.

developmental biology↗

bioRxiv2018

Genome-Wide Identification of HES1 Target Genes Uncover Novel Roles for HES1 in Pancreatic Development

Notch signalling and the downstream effector HES1 is required for multiple pancreatic cell fate choices during development, but the direct target genes remain poorly characterised. Here we identify direct HES1 target genes on a genome-wide scale using ChIP-seq and RNA-seq analyses combined with human embryonic stem cell (hESC) directed differentiation of CRISPR/Cas9-generated HES1-/- mutant hESC lines. We found that HES1 binds to a distinct set of endocrine-specific genes, a set of genes encoding basic Helix-Loop-Helix (bHLH) proteins not normally expressed in the pancreas, genes in the Notch pathway, and the known HES1 target NEUROG3. RNA-seq analysis of wild type, HES1-/-, NEUROG3-/-, and HES1-/-NEUROG3-/- mutant hESC lines allowed us to uncover NEUROG3-independent, direct HES1 target genes. Among the HES1 bound genes that were derepressed in HES1-/-NEUROG3-/- cells compared to NEUROG3-/- cells, we found members of the endocrine-specific gene set, the Notch pathway genes DLL1, DLL4, and HEY1, as well as the non-pancreatic bHLH genes ASCL1 and ATOH1. We also found a large number of transcripts specific to the intestinal secretory lineage to be increased in HES1-/-NEUROG3-/- cells. Together, our data reveal that HES1 employs a multi-layered control of endocrine differentiation, controls Notch ligand expression independent of NEUROG3, and prevents initiation of ectopic intestinal transcriptional programmes in pancreas progenitors.

developmental biology↗

bioRxiv2018

Kinetic sculpting of the seven stripes of the Drosophila even-skipped gene

We used live imaging to visualize the transcriptional dynamics of the Drosophila melanogaster even-skipped gene at single-cell and high temporal resolution as its seven stripe expression pattern forms, and developed tools to characterize and visualize how transcriptional bursting varies over time and space. We find that despite being created by the independent activity of five enhancers, even-skipped stripes are sculpted by the same kinetic phenomena: a coupled increase of burst frequency and amplitude. By tracking the position and activity of individual nuclei, we show that stripe movement is driven by the exchange of bursting nuclei from the posterior to anterior stripe flanks. Our work provides a conceptual, theoretical and computational framework for dissecting pattern formation in space and time, and reveals how the coordinated transcriptional activity of individual nuclei shape complex developmental patterns.

developmental biology↗

bioRxiv2018

Notch Controls Multiple Pancreatic Cell Fate Regulators Through Direct Hes1-mediated Repression

Notch signaling and its effector Hes1 regulate multiple cell fate choices in the developing pancreas, but few direct target genes are known. Here we use transcriptome analyses combined with chromatin immunoprecipitation with next-generation sequencing (ChIP-seq) to identify direct target genes of Hes1. ChIP-seq analysis of endogenous Hes1 in 266-6 cells, a model of multipotent pancreatic progenitor cells, revealed high-confidence peaks associated with 354 genes. Among these were genes important for tip/trunk segregation such as Ptf1a and Nkx6-1, genes involved in endocrine differentiation such as Insm1 and Dll4, and genes encoding non-pancreatic basic-Helic-Loop-Helix (bHLH) factors such as Neurog2 and Ascl1. Surprisingly, we find that Hes1 binds a large number of loci previously reported to bind Ptf1a, including a site downstream of the Nkx6-1 gene. Notably, we find a number of Hes1 bound genes that are upregulated by {gamma}-secretase inhibition in pancreas explants independently of Neurog3 function, including the tip progenitor/acinar genes; Ptf1a, Gata4, Bhlha15, and Gfi1. Together, our data suggest that Notch signaling suppress the tip cell fate by Hes1-mediated repression of the tip-specific gene regulatory network module that includes transcriptional regulators such as Ptf1a, Gata4, Mist1, and Gfi1. Our data also uncover new molecular targets of Notch signaling that may be important for controlling cell fate choices in pancreas development.

developmental biology↗

bioRxiv2018

Turing-like patterns can arise from purely bioelectric mechanisms

Turing-like patterns can potentially occur in non-neural (non-excitable) tissues through strictly bioelectric processes, without involving transcriptional (gene) regulation, cell migration, or traditional reaction-diffusion mechanisms. Small molecules that gate transmembrane ion channels are often charged and capable of passing through intercellular gap junctions, and their transport under the influence of trans-junction electric fields furnishes a bioelectric feedback loop. We develop an analytically tractable, circuit-based model of this phenomenon and show that it can lead to spontaneous formation of spatial patterns in ligand density and membrane voltage under physiologically plausible conditions. The process is distinct from Turings reaction-diffusion paradigm but closely analogous to the spontaneous formation of patterns in colonies of chemotactic bacteria.

developmental biology↗

bioRxiv2018

Dll1 and Jag1 are Differentially Required to Specify Proximal and Distal Pancreatic Duct Compartments

Notch signaling controls proliferation of multipotent pancreatic progenitor cells (MPCs) and their segregation into bipotent progenitors (BPs) and unipotent pro-acinar cells (PACs). Here we uncover fast ultradian oscillations in the ligand Dll1, and the transcriptional effector Hes1, which proved crucial for MPC expansion. Conversely Jag1, a uniformly expressed ligand, curbed MPC growth, but as expression later segregated to PACs it proved critical for specifying all but the most proximal 5% of BPs, while BPs were entirely lost in Jag1, Dll1 double mutants. Moreover, experimentally induced changes in Hes1 oscillation parameters was associated with selective adoption of BP or PAC fates. Anatomically, ductal morphogenesis and organ architecture is minimally perturbed in Jag1 mutants until later stages, when ductal remodeling fails and signs of acinar-to-ductal metaplasia appear. Our study uncovers oscillating Notch activity in the developing pancreas, which along with modulation by Jag1 is required to coordinate MPC growth and fate.

developmental biology↗

bioRxiv2018

β-cyclocitral is a natural root growth regulator

Natural compounds capable of increasing root depth and branching are desirable tools for enhancing stress tolerance in crops. We devised a sensitized screen to identify natural metabolites capable of regulating root traits in Arabidopsis. {beta}-cyclocitral, an endogenous root compound, was found to promote cell divisions in root meristems and stimulate lateral root branching. {beta}-cyclocitral rescued meristematic cell divisions in ccd1ccd4 biosynthesis mutants and {beta}-cyclocitral-driven root growth was found to be independent of auxin, brassinosteroid, and ROS signaling pathways. {beta}-cyclocitral had a conserved effect on root growth in tomato and rice and generated significantly more compact crown root systems in rice. Moreover, {beta}-cyclocitral treatment enhanced plant vigor in rice plants exposed to salt-contaminated soil. These results indicate that {beta}-cyclocitral is a broadly effective root growth promoter in both monocots and eudicots and could be a valuable tool to enhance crop vigor under environmental stress.\n\nOne Sentence Summary{beta}-cyclocitral is a metabolite of {beta}-carotene that was identified using a sensitized chemical screen and acts broadly across plants to enhance root growth and branching.

developmental biology↗