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bioRxiv2019

STOCHASTIC SINGLE CELL BEHAVIOUR LEADS TO ROBUST HORIZONTAL CELL LAYER FORMATION IN THE VERTEBRATE RETINA

Developmental programs that arrange cells and tissues into patterned organs are remarkably robust. In the developing vertebrate retina for example, neurons reproducibly assemble into distinct layers giving the mature organ its overall structured appearance. This stereotypic neuronal arrangement, termed lamination, is important for efficient neuronal connectivity. While retinal lamination is conserved in many vertebrates including humans, how it emerges from single cell behaviour is not fully understood. To shed light on this question, we here investigated the formation of the retinal horizontal cell layer. Using in vivo light sheet imaging of the developing zebrafish retina, we generated a comprehensive quantitative analysis of horizontal single cell behaviour from birth to final positioning. Interestingly, we find that all parameters analyzed including cell cycle dynamics, migration paths and kinetics as well as sister cell dispersal are very heterogeneous. Thus, horizontal cells show individual non-stereotypic behaviour before final positioning. Yet, these initially stochastic cell dynamics always generate the correct laminar pattern. Consequently, our data shows that lamination of the vertebrate retina contains a yet underexplored extent of single cell stochasticity.

developmental biology↗

bioRxiv2019

Thyroid hormone coordinates developmental trajectories but does not underlie developmental truncation in Danionins

BackgroundChanges in post-embryonic developmental trajectories can profoundly alter adult phenotypes and life history transitions. In vertebrate clades with complex, biphasic life cycles, thyroid hormone (TH) regulates metamorphosis, and alterations to TH metabolism underlie famous cases of paedomorphosis. We tested the requirement for TH in multiple post-embryonic developmental processes in the zebrafish, and asked if TH loss was associated with paedomorphosis in miniaturized Danionellas. ResultsWe found that TH regulates the relative growth of different body parts in zebrafish, inhibiting head growth at juvenile stages. The lateral line also showed sensitivity to the hormone: head neuromasts were inhibited by TH, while trunk neuromasts required TH for their proliferation. While several aspects of Danionella morphology resembled that of larval zebrafish, these species did not resemble hypothyroid zebrafish in their shape or lateral line, and these fish showed functional thyroid follicles. ConclusionsAlthough zebrafish do not undergo a discrete ecological transformation, we found that multiple tissues go through a protracted metamorphosis, and that TH ushers in growth patterns and processes typical of juveniles. We found no evidence that compromised TH metabolism is responsible for paedomorphosis in Danionellas. Nonetheless, modulations to TH-sensitive pathways affect evolutionarily-relevant traits, and are likely important targets for adaptation. Bullet PointsO_LIThyroid hormone regulates shifts in relative growth trajectories in different zebrafish tissues C_LIO_LIThyroid hormone inhibits head growth in juvenile zebrafish, and regulates juvenile growth patterns C_LIO_LIThyroid hormone stimulates formation of neuromast canals in the head and stitches on the trunk of zebrafish C_LIO_LIDanionella, the miniaturized sister group to Danio morphologically resemble larval zebrafish and do not form neuromast canals C_LIO_LIHypothyroidism is not the cause of paedomorphosis in Danionella C_LI Grant SponsorsNIH R00GM105874 NIH R03HD091634 Burroughs Wellcome Collaborative Research Travel Grant 1017439

developmental biology↗

bioRxiv2019

A critical but divergent role of PRDM14 in human primordial germ cell fate revealed by inducible degrons

PRDM14 is a crucial regulator of mouse primordial germ cells (mPGC), epigenetic reprogramming and pluripotency, but its role in the evolutionarily divergent regulatory network of human PGCs (hPGCs) remains unclear. Besides, a previous knockdown study indicated that PRDM14 might be dispensable for human germ cell fate. Here, we decided to use inducible degrons for a more rapid and comprehensive PRDM14 depletion. We show that PRDM14 loss results in significantly reduced specification efficiency and an aberrant transcriptome of human PGC-like cells (hPGCLCs) obtained in vitro from human embryonic stem cells (hESCs). Chromatin immunoprecipitation and transcriptomic analyses suggest that PRDM14 cooperates with TFAP2C and BLIMP1 to upregulate germ cell and pluripotency genes, while repressing WNT signalling and somatic markers. Notably, PRDM14 targets are not conserved between mouse and human, emphasising the divergent molecular mechanisms of PGC specification. The effectiveness of degrons for acute protein depletion is widely applicable in various developmental contexts.

developmental biology↗

bioRxiv2019

A conserved requirement for Fbxo7 during male germ cell cytoplasmic remodelling

Summary statementFbxo7 is the substrate-recognition subunit of an SCF-type ubiquitin E3 ligase complex. It has physiologically important functions in regulating mitophagy, proteasome activity and the cell cycle in multiple cell types, like neurons, lymphocytes and erythrocytes. Here we show that in addition to the previously-known Parkinsonian and haematopoietic phenotypes, Fbxo7-deficient male mice are completely sterile. In these males, despite successful meiosis, nuclear elongation and eviction of histones from chromatin, the developing spermatids are phagocytosed by Sertoli cells during late spermiogenesis, as the cells undergo cytoplasmic remodelling. Surprisingly, despite the loss of all germ cells, there was no evidence of the symplast formation and cell sloughing that is typically associated with spermatid death in other mouse sterility models, suggesting that novel cell death and/or cell disposal mechanisms may be engaged in Fbxo7-deficient males. Mutation of the Drosophila Fbxo7 orthologue, nutcracker (ntc) was previously shown to cause sterility at a similar stage of germ cell development, indicating that the requirement for Fbxo7 is conserved. The ntc phenotype was attributed to proteasome mis-regulation via an interaction with the proteasome regulator, DmPI31. Our data suggest rather that in mice, the requirement for Fbxo7 is either independent of its interaction with PI31, or relates specifically to cytoplasmic proteasome activity during spermiogenesis.

developmental biology↗

bioRxiv2019

Sex differences in gene expression and proliferation are dependent on the epigenetic modifier HP1γ

Sex differences in growth rate in very early embryos have been recognized in a variety of mammals and attributed to sex-chromosome complement effects as they occur before overt sexual differentiation. We previously found that sex-chromosome complement, rather than sex hormones regulates heterochromatin-mediated silencing of a transgene and autosomal gene expression in mice. Here, sex dimorphism in proliferation was investigated. We confirm that male embryonic fibroblasts proliferate faster than female fibroblasts and show that this proliferation advantage is completely dependent upon heterochromatin protein 1 gamma (HP1{gamma}). To determine whether this sex-regulatory effect of HP1{gamma} was a more general phenomenon, we performed RNA sequencing on MEFs derived from males and females, with or without HP1{gamma}. Strikingly, HP1{gamma} was found to be crucial for regulating nearly all sexually dimorphic autosomal gene expression because deletion of the HP1{gamma} gene in males abolished sex differences in autosomal gene expression. The identification of a key epigenetic modifier as central in defining gene expression differences between males and females has important implications for understanding physiological sex differences and sex bias in disease.

developmental biology↗

bioRxiv2019

Pluripotency factors regulate the onset of Hox cluster activation in the early embryo

Pluripotent cells are a transient population present in the early mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while simultaneously suppressing lineage specification. Interestingly, these factors are not exclusive to uncommitted cells, but are also expressed during early phases of differentiation. However, their role in the transition from pluripotency to lineage specification is largely unknown. Using genetic models for controlled Oct4 or Nanog expression during postimplantation stages, we found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that the HoxB cluster is coordinately regulated in this way by OCT4 binding sites located at the 3 end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the pre-committed epiblast, but are also necessary for the proper deployment of subsequent developmental programs.

developmental biology↗

bioRxiv2019

In vivo study of gene expression with an enhanced dual-color fluorescent transcriptional timer

Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to significant reduction of signal intensities. To overcome this limitation, we enhanced translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila. In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns.

developmental biology↗

bioRxiv2019

Neph/Nephrin-like adhesion and tissue level pulling forces regulate cell intercalation during Drosophila retina development.

Intercalation between neighboring cells contributes to shaping epithelial tissues and is regulated by the contractile actomyosin cytoskeleton. While intercalation typically occurs over minutes, instances of much slower cell intercalation have been reported during organogenesis. This is observed, for example, for the four glial-like cone cells (CC) that intercalate during Drosophila retinal patterning. Here we show that Myosin-II activity in the CCs is largely dispensable for their intercalation. Instead, we find that differential activity of the Notch-signaling pathway within the CC quartet regulates intercalation, which also depends on the cell adhesion proteins Roughest and Hibris. In addition, mathematical modeling predicts that forces external to the intercalating CC quartet are necessary for intercalation. Consistent with this prediction we show that the surrounding primary pigment cells are under significant contractile tension. Altogether, our work elucidates a novel mode of cell intercalation that relies on Neph/Nephrin-like adhesion and forces external to the intercalating cells.

developmental biology↗

bioRxiv2019

A smooth muscle-like niche facilitates lung epithelial regeneration

The mammalian lung is a highly complex organ due to its branched, tree-like structure and diverse cellular composition. Recent efforts using state-of-the-art genetic lineage tracing and single-cell transcriptomics have helped reduce this complexity and delineate the ancestry and fate of various cell subpopulations during organogenesis, homeostasis and repair after injury. However, mesenchymal cell heterogeneity and function in development and disease remain a longstanding issue in the lung field. In this study, we break down smooth muscle heterogeneity into the constituent subpopulations by combining in vivo lineage tracing, single-cell RNA sequencing and in vitro organoid cultures. We identify a repair-supportive mesenchymal cell (RSMC) population that is distinct from pre-existing airway smooth muscle cells (ASMC) and is critical for regenerating the conducting airway epithelium. Progenitors of RSMCs are intertwined with airway smooth muscle, undergo active WNT signaling, transiently acquire the expression of the smooth muscle marker ACTA2 in response to epithelial injury and are marked by PDGFR expression. Our data simplify the cellular complexity of the peribronchiolar domain of the adult lung and represent a forward step towards unraveling the role of mesenchymal cell subpopulations in instructing epithelial behavior during repair processes.

developmental biology↗

bioRxiv2019

A pleiotropic role for FGF signaling in mammary gland stromal fibroblasts

Fibroblast growth factor (FGF) signaling is crucial for mammary gland development. While multiple roles for FGF signaling in the epithelium were described, the function of FGF signaling in mammary stroma has not been elucidated. In this study, we investigated FGF signaling in mammary fibroblasts. We found that mammary fibroblasts express FGF receptors 1 and 2 and respond to FGF ligands. In particular, FGF2 and FGF9 induce sustained ERK1/2 signaling and promote fibroblast proliferation and migration in 2D. Intriguingly, only FGF2 induces fibroblast migration in 3D extracellular matrix (ECM) through regulation of actomyosin cytoskeleton and promotes force-mediated collagen remodeling by mammary fibroblasts. Moreover, FGF2 regulates production of ECM proteins by mammary fibroblasts, including collagens, fibronectin, osteopontin, and matrix metalloproteinases. Finally, we show that FGF2 signaling in mammary fibroblasts enhances fibroblast-induced branching of mammary epithelium. Our results demonstrate a pleiotropic role for FGF signaling in mammary fibroblasts with implications for regulation of mammary stromal functions and epithelial branching morphogenesis. Summary statementFGF signaling in mammary fibroblasts regulates fibroblast proliferation, migration, extracellular matrix production and remodeling, and fibroblast-mediated mammary epithelial branching morphogenesis.

developmental biology↗

bioRxiv2019

The Marginal Cells of the Caenorhabditis elegans Pharynx Scavenge Cholesterol and Other Hydrophobic Small Molecules

The nematode worm Caenorhabditis elegans is a bacterivore filter feeder. Through the contraction of the worms pharynx, a bacterial suspension is sucked into the pharynxs lumen. Excess liquid is then shunted out of the buccal cavity through ancillary channels that are made from specialized pharyngeal cells called marginal cells. Through the characterization of our library of worm-bioactive small molecules (a.k.a. wactives), we found that more than one third of wactives visibly accumulate inside of the marginal cells as crystals or globular spheres. Wactives that visibly accumulate are typically more hydrophobic than those that do not. To understand why wactives accumulate specifically in marginal cells, we performed a forward genetic screen for mutants that resist the lethality associated with one crystallizing wactive. We identified a presumptive sphingomyelin-synthesis pathway that is necessary for crystal and sphere accumulation. Sphingomyelin is a phospholipid that is enriched in the outer leaflet of the plasma membranes of most metazoans. We find that the predicted terminal enzyme of this pathway, sphingomyelin synthase 5 (SMS-5), is expressed in the pharynx, contributes to sphingomyelin abundance, and that its expression in marginal cells is sufficient for wactive accumulation. We also find that the expression of SMS-5 in the marginal cells is necessary for the proper absorption of exogenous cholesterol, without which C. elegans cannot develop. We conclude that the sphingomyelin-rich plasma membrane of the marginal cells acts as a sink to scavenge important hydrophobic nutrients from the filtered liquid that might otherwise be shunted back into the environment. One sentence summaryThe anterior pharynx of C. elegans is a Sink for Hydrophobic Small Molecules

developmental biology↗

bioRxiv2019

Distinct RhoGEFs activate apical and junctional actomyosin contractility under control of G proteins during epithelial morphogenesis

Small RhoGTPases and Myosin-II direct cell shape changes and movements during tissue morphogenesis. Their activities are tightly regulated in space and time to specify the desired pattern of contractility that supports tissue morphogenesis. This is expected to stem from polarized surface stimuli and from polarized signaling processing inside cells. We examined this general problem in the context of cell intercalation that drives extension of the Drosophila ectoderm. In the ectoderm, G protein coupled receptors (GPCRs) and their downstream heterotrimeric G proteins (G and G{beta}{gamma}) activate Rho1 both medial-apically, where it exhibits pulsed dynamics, and at junctions, where its activity is planar polarized (Kerridge et al., 2016; Munjal et al., 2015). However, the mechanisms responsible for polarizing Rho1 activity are unclear. In particular, it is unknown how Rho1 activity is controlled at junctions. We report a division of labor in the mechanisms of Rho1 activation in that distinct guanine exchange factors (GEFs), that serve as activators of Rho1, operate in these distinct cellular compartments. RhoGEF2 acts uniquely to activate medial-apical Rho1. Although RhoGEF2 is recruited both medial-apically and at junctions by G12/13-GTP, also called Concertina (Cta) in Drosophila, its activity is restricted to the medial-apical compartment. Furthermore, we characterize a novel RhoGEF, p114RhoGEF/Wireless (Wrl), and report its requirement for cell intercalation in the extending ectoderm. p114RhoGEF/Wireless activates Rho1 specifically at junctions. Strikingly it is restricted to adherens junctions and is under G{beta}13F/G{gamma}1 control. G{beta}13F/G{gamma}1 activates junctional Rho1 and exerts quantitative control over planar polarization of Rho1. In particular, overexpression of G{beta}13F/G{gamma}1 leads to hyper planar polarization of Rho1 and MyoII. Finally, we found that p114RhoGEF/Wireless is absent in the mesoderm, arguing for a tissue-specific control over junctional Rho1 activity. These results shed light on the mechanisms of polarization of Rho1 activity in different cellular compartments and reveal that distinct GEFs are sensitive tuning parameters of cell contractility in remodeling epithelia.

developmental biology↗

bioRxiv2019

Analysis of single nucleotide variants in CRISPR-Cas9 edited zebrafish embryos shows no evidence of off-target inflation

Therapeutic applications of CRISPR-Cas9 gene editing have spurred innovation in Cas9 enzyme engineering and single guide RNA (sgRNA) design algorithms to minimize potential off-target events. While recent work in rodents outlines favorable conditions for specific editing and uses a trio design to control for the contribution of natural genome variation, the potential for CRISPR-Cas9 to induce de novo mutations in vivo remains a topic of interest. In zebrafish, we performed whole exome sequencing (WES) on two generations of offspring derived from the same founding pair: 54 exomes from control and CRISPR-Cas9 edited embryos in the first generation (F0), and 16 exomes from the progeny of inbred F0 pairs in the second generation (F1). We did not observe an increase in the number of transmissible variants in edited individuals in F1, nor in F0 edited mosaic individuals, arguing that in vivo editing does not precipitate an inflation of deleterious point mutations.

developmental biology↗

bioRxiv2019

In the chick embryo, estrogen can induce chromosomally male ZZ left gonad epithelial cells to form an ovarian cortex which supports oogenesis

In chickens, as in most amniotes, the first morphogenetic event in ovary differentiation is the formation of two distinct domains: a steroidogenic core, the medulla, overlain by the germ cell niche, the cortex. This process normally starts immediately after sex determination in the ZW embryos, substantially before the progression of germ cells into meiosis. In order to address the extent to which the cortical domain depends on intrinsic or extrinsic factors, we generated models of gonadal intersex by mixing ZW (female) and ZZ (male) cells in gonadal chimeras, or by altering estrogen levels of ZZ and ZW embryos in ovo. We found that both chomosomally female and male cells can be induced to form the cortical domain and that this can form relatively independently of the phenotypic sex of the medulla as long as estrogen is provided. We also show that the cortex promoting activity of estrogen signalling is mediated via Estrogen Receptor alpha within the left gonad epithelium. Therefore, either a ZW or ZZ cortical domain can provide an adequate niche to allow the germ cells to progress into meiosis. However, the presence of a medulla with an \"intersex\" or male phenotype may compromise this progression, causing cortical germ cells to remain in an immature state in the embryo.

developmental biology↗

bioRxiv2019

DUX is a non-essential synchronizer of zygotic genome activation

Some of the earliest transcripts produced in fertilized human and mouse oocytes code for DUX, a double homeodomain protein that promotes embryonic genome activation (EGA). Deleting Dux by genome editing at the 1- to 2-cell stage in the mouse impairs EGA and blastocyst maturation. Here, we demonstrate that mice carrying homozygous Dux deletions display markedly reduced expression of DUX target genes and defects in both pre- and post-implantation development, with notably a disruption of the pace of the first few cell divisions and significant rates of late embryonic mortality. However, some Dux-/- embryos give raise to viable pups, indicating that DUX is important but not strictly essential for embryogenesis.\n\nSummary statementMurine DUX regulates transcription in the first embryonic cell divisions but its not necessary for embryogenesis

developmental biology↗

bioRxiv2019

GCNA interacts with Spartan and Topoisomerase II to regulate genome stability.

GCNA proteins are expressed across eukarya in pluripotent cells and have conserved functions in fertility. GCNA homologs Spartan/DVC-1 and Wss1 resolve DNA-protein crosslinks (DPCs), including Topoisomerase-DNA adducts, during DNA replication. We show that GCNA and Topoisomerase 2 (Top2) physically interact and colocalize on condensed chromosomes during mitosis, when Spartan is not present. We show that C. elegans gcna-1 mutants are sensitive to Top2 poison and accumulate mutations consistent with low fidelity repair of DNA damage, leading to loss of fitness and fertility over generations. We also demonstrate that mouse GCNA interacts with TOP2, and Gcna-mutant mice exhibit abnormalities consistent with the inability to process DPCs, including chromatin condensation and crossover defects. Together, our findings provide evidence that GCNA maintains genomic integrity by processing Top2 DPCs in the germline and early embryo, where the genome is challenged with an increased DPC burden.

developmental biology↗

bioRxiv2019

Interkinetic nuclear migration in the zebrafish retina as a diffusive process

A major hallmark of neural development is the oscillatory movement of nuclei between the apical and basal surfaces of the neuroepithelium during the process of interkinetic nuclear migration (IKNM). Here, we employ long-term, rapid lightsheet and two-photon imaging of Zebrafish retinas in vivo during early development to uncover the physical processes that govern the behavior of nuclei during IKNM. These images allow the capture of reliable tracks of nuclear movements and division during early retinogenesis for many tightly packed nuclei. These tracks are then used to create and test a theory of retinal IKNM as a diffusive process across a nuclear concentration gradient generated by the addition of new nuclei at the apical surface. The analysis reveals the role of nuclear packing at the apical surface on the migration dynamics of nuclei, provides a robust quantitative explanation for the distribution of nuclei across the retina, and may have implications for stochastic fate choice in this system.

developmental biology↗

bioRxiv2019

GCNA preserves genome integrity and fertility across species

The propagation of species depends on the ability of germ cells to protect their genome in the face of numerous exogenous and endogenous threats. While these cells employ a number of known repair pathways, specialized mechanisms that ensure high-fidelity replication, chromosome segregation, and repair of germ cell genomes remain incompletely understood. Here, we identify Germ Cell Nuclear Acidic Peptidase (GCNA) as a highly conserved regulator of genome stability in flies, worms, zebrafish, and humans. GCNA contains a long acidic intrinsically disordered region (IDR) and a protease-like SprT domain. In addition to chromosomal instability and replication stress, GCNA mutants accumulate DNA-protein crosslinks (DPCs). GCNA acts in parallel with a second SprT domain protein Spartan. Structural analysis reveals that while the SprT domain is needed to limit meiotic and replicative damage, most of GCNAs function maps to its IDR. This work shows GCNA protects germ cells from various sources of damage, providing novel insights into conserved mechanisms that promote genome integrity across generations.\n\nHighlightsGCNA ensures genomic stability in germ cells and early embryos across species\n\nGCNA limits replication stress and DNA double stranded breaks\n\nGCNA restricts DNA-Protein Crosslinks within germ cells and early embryos\n\nThe IDR and SprT domains of GCNA govern distinct aspects of genome integrity\n\nGraphic Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC=\"FIGDIR/small/570804_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (54K):\norg.highwire.dtl.DTLVardef@1928d82org.highwire.dtl.DTLVardef@885ff2org.highwire.dtl.DTLVardef@15322c9org.highwire.dtl.DTLVardef@110c615_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗