Biology subjects

Wittbrodt, J.

Publications and source records attributed to Wittbrodt, J..

9 recordsLinked to original sources

bioRxiv2018

Retinal stem cells modulate proliferative parameters to coordinate post-embryonic morphogenesis in the eye of fish

A fundamental question in biology is how anatomically and functionally distinct tissues coordinate to direct growth and shape in complex organs. We address this question using as a model the eye of teleost fish, which grow while maintaining the precise shape needed for vision throughout the animals life.\n\nCombining clonal analysis in the eye of the teleost medaka (Oryzias latipes) with a computational agent based model, we find that the neural retina (NR) and retinal pigmented epithelium (RPE) differentially modulate cell divisions to coordinate their growth rates. Cell divisions in the NR are less stochastic, consistent with an upstream role as an inducer of growth in nearby tissues. Cells in the RPE display much higher stochasticity, consistent with a downstream role responding to inductive signals.\n\nOur simulation predicts that the segregation of stem- and progenitor cell domains in the retinal ciliary marginal zone niche is an emergent property, as the topology of the niche preconditions the system to undergo a spatially biased stochastic neutral drift. Clone properties in the NR support this prediction, and further suggest that NR cells control the direction of division axes to regulate organ shape and retinal cell topology.\n\nThis work highlights an as yet unappreciated mechanism for growth coordination in a complex organ, where one tissue integrates external and internal cues as a hub to synchronize growth rates in nearby tissues. In the eye of fish, proliferation parameters of neuroretinal stem cells are a minimal target node for evolution to exploit to adapt whole-organ morphogenesis in a complex vertebrate organ.

developmental biology↗

bioRxiv2018

Enhanced in vivo-imaging in fish by optimized anaesthesia, fluorescent protein selection and removal of pigmentation

Fish are ideally suited for in vivo-imaging due to their transparency at early stages combined with a large genetic toolbox. Key challenges to further advance imaging are fluorophore selection, immobilization of the specimen and approaches to eliminate pigmentation.\n\nWe addressed all three and identified the fluorophores and anaesthesia of choice by high throughput time-lapse imaging. Our results indicate that eGFP and mCherry are the best conservative choices for in vivo-fluorescence experiments, when availability of well-established antibodies and nanobodies matters. Still, mVenusNB and mGFPmut2 delivered highest absolute fluorescence intensities in vivo. Immobilization is of key importance during extended in vivo imaging. Here, traditional approaches are outperformed by mRNA injection of -Bungarotoxin which allows a complete and reversible, transient immobilization. In combination with fully transparent juvenile and adult fish established by the targeted inactivation of both, oca2 and pnp4a via CRISPR/Cas9-mediated gene editing in medaka we could dramatically improve the state-of-the art imaging conditions in post-embryonic fish, now enabling light-sheet microscopy of the growing retina, brain, gills and inner organs in the absence of side effects caused by anaesthetic drugs or pigmentation.

developmental biology↗

bioRxiv2018

Notch signalling patterns retinal composition by regulating atoh7 during post-embryonic growth

Patterning of a continuously growing naive field in the context of a life-long growing organ, the teleost eye is of highest functional relevance. Intrinsic and extrinsic signals were proposed to regulate lineage specification in progenitors that exit the stem cell niche in the ciliary marginal zone (CMZ). The proper cell type composition arising from those progenitors is prerequisite for retinal function. Our findings in the teleost medaka (Oryzias latipes) uncover that the Notch-Atoh7 axis continuously patterns the CMZ. The complement of cell-types originating from the two juxtaposed progenitors marked by Notch or Atoh7 activity contains all constituents of a retinal column. Modulation of Notch signalling specifically in Atoh7-expressing cells demonstrates the crucial role of this axis in generating the correct cell type proportions. After transiently blocking Notch signalling, retinal patterning and differentiation is reinitiated de novo. Taken together we show that Notch activity in the CMZ continuously structures the growing retina by juxtaposing Notch and Atoh7 progenitors giving rise to distinct, complementary lineages, revealing a coupling of de novo patterning and cell-type specification in the respective lineages.

developmental biology↗

bioRxiv2018

Efficient single-copy HDR by 5’ modified long dsDNA donors

CRISPR/Cas9 efficiently induces targeted mutations via non-homologous-end-joining but for genome editing, precise, homology-directed repair (HDR) of endogenous DNA stretches is a prerequisite. To favor HDR, many approaches interfere with the repair machinery or manipulate Cas9 itself. Using Medaka we show that the modification of 5 ends of long dsDNA donors strongly enhances HDR, favors efficient single-copy integration by retaining a monomeric donor conformation thus facilitating successful gene replacement or tagging.

genetics↗

bioRxiv2018

Pcdh18a-positive tip cells instruct notochord formation in zebrafish

The notochord defines the axial structure of all vertebrates during development. Notogenesis is a result of major cell reorganization in the mesoderm, the convergence and the extension of the axial cells. However, it is currently not known how these processes act together in a coordinated way during notochord formation. Analysing the tissue flow, we determined the displacement of the axial mesoderm and identified, relative to the ectoderm, an actively-migrating notochord tip cell population and a group of trailing notochordal plate cells. Molecularly, these tip cells express Protocadherin18a, a member of the cadherin superfamily. We show that Pcdh18a-mediated recycling of E-cadherin adhesion complexes transforms these tip cells into a cohesive and fast migrating cell group. In turn, these tip cells subsequently instruct the trailing mesoderm. We simulated cell migration during early mesoderm formation using a lattice-based mathematical framework, and predicted that the requirement for an anterior, local motile cell cluster could guide the intercalation of the posterior, axial cells. Indeed, grafting experiments validated the predictions and induced ectopic notochord-like rods. Our findings indicate that the tip cells influence the trailing mesodermal cell sheet by inducing the formation of the notochord.

developmental biology↗

bioRxiv2017

Activating the regenerative potential of Müller glia cells in a regeneration-deficient retina

Regeneration responses in animals are widespread across phyla. To identify molecular players that confer regenerative capacities to non-regenerative species is of key relevance for basic research and translational approaches. Here we report a differential response in retinal regeneration between medaka (Oryzias latipes) and zebrafish (Danio rerio). In contrast to zebrafish, medaka Muller glia (olMG) cells behave like progenitors and exhibit a restricted capacity to regenerate the retina. After injury, olMG cells proliferate but fail to self-renew and ultimately only restore photoreceptors.\n\nIn our injury paradigm, we observed that in contrast to zebrafish, proliferating olMG cells do not maintain sox2 expression. Sustained sox2 expression in olMG cells confers regenerative responses similar to those of zebrafish MG (drMG) cells. We show that a single, cell-autonomous factor reprograms olMG cells and establishes a regeneration-like mode. Our results position medaka as an attractive model to delineate key regeneration factors with translational potential.

developmental biology↗

bioRxiv2017

Optic Fissure Margin Morphogenesis Sets The Stage For Consecutive Optic Fissure Fusion, Pioneered By A Distinct Subset Of Margin Cells Using A Hyaloid Vessel As Scaffold

The optic fissure is a transient gap in the developing optic cup of vertebrates. Persisting optic fissures, coloboma, are a frequent reason for blindness in children. Although many genes have been linked to coloboma, it has remained unclear how the two bi-layered epithelia comprising the optic fissure margins are fusing to form a continuous neuroretina and retinal pigmented epithelium (RPE) respectively. Besides, highly variable morphologies of coloboma phenotypes strongly argue for a diverse set of underlying pathomechanisms.\n\nHere we investigated the contribution of the individual cell types with 4D in vivo time-lapse analyses using zebrafish (Danio rerio). This allowed defining the respective roles of the participating tissues and cell populations and their activities during fissure morphogenesis, contact formation between the margins as well as during fusion.\n\nWe show that optic fissure closure is initiated by a bilateral tissue flow partially in continuation of the dynamic optic cup morphogenesis but additionally including a tissue flow from the optic stalk. This process is followed by the setup of specific fissure margins by a distinct cell population translocating from of the optic stalk. The morphological fusion is triggered by in an EMT-like disassembly of the fissure margin driven by bi-potential pioneer cells that ultimately take the fate of both, neuroretina and RPE respectively. The consecutive fusion and re-epithelialization transforms the two initially separated epithelial bilayers into the two continuous layers of neuroretina and RPE. The processes described here in detail represents a fundamental mechanism of the seamless connection of adjacent multilayered epithelia and is highly reminiscent of other fusion processes, like palatal shelf fusion with key relevance for development and growth.

developmental biology↗

bioRxiv2017

TGF(beta) Mediated Structural Remodeling Facilitates Optic Fissure Fusion And The Necessity Of BMP Antagonism In This Process

The optic fissure is a transient gap in the developing vertebrate eye, which must be closed as development proceeds. A persisting optic fissure, coloboma, is a major cause for blindness in children. Multiple factors are genetically linked to coloboma formation. However, especially the process of optic fissure fusion is still largely elusive on a cellular and molecular basis.\n\nWe found a coloboma phenotype in mice with a targeted inactivation of the transforming growth factor 2 (TGF{beta}2). Here the optic fissure margins got in touch, however, failed to fuse. Transcriptomic analyses indicated TGF{beta} mediated ECM remodeling during optic fissure fusion. For functional analyses, we switched model systems and made use of zebrafish. We found TGF{beta} ligands expressed in the developing zebrafish eye, and the ligand binding receptor in the optic fissure. Using a new in vivo TGF{beta} signaling reporter, we also found active TGF{beta} signaling in the margins of the optic fissure. We addressed the function of Cadherin 6 (cdh6), one of the TGF{beta} regulated genes, by knock down experiments in zebrafish and found a prominent coloboma phenotype. Cdh6 was often found involved in processes of epithelial to mesenchymal transition (EMT), strengthening our hypothesis that an EMT-like process is also necessary for optic fissure fusion. Furthermore, we found Gremlin 2b (grem2b) and Follistatin a (fsta), homologs of TGF{beta} regulated bone morphogenetic protein (BMP) antagonists, expressed in the optic fissure margins, indicating the necessity of a localized inhibition of BMP signaling. Finally, we show that induced BMP expression is sufficient to inhibit optic fissure fusion. Together with our previous findings this indicates a dual role of BMP signaling during optic fissure closure.\n\nSummary statementTGF{beta} is crucial for optic fissure fusion, involving cdh6. TGF{beta} mediated optic fissure fusion is potentially hampered by BMP signaling, which is blocked by TGF{beta} induced BMP antagonists within the optic fissure margins.

developmental biology↗

bioRxiv2017

Dynamics of ASC speck formation during skin inflammatory responses in vivo

Activated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signalling platforms considered hallmarks of inflammasome activation. Because a lack of in vivo tools has prevented the study of endogenous ASC dynamics, we generated a live ASC reporter through CRISPR/Cas9 tagging of the endogenous gene in zebrafish. We see strong ASC expression in the skin and other epithelia that act as barriers to insult. A toxic stimulus triggered speck formation and rapid pyroptosis in keratinocytes in vivo. Macrophages engulfed and digested this speck-containing pyroptotic debris. A 3D ultrastructural reconstruction based on CLEM of in vivo assembled specks revealed a compact network of highly intercrossed filaments, whereas PYD or CARD alone formed filamentous aggregates. The effector caspase is recruited through PYD, whose overexpression induced pyroptosis, but after substantial delay. Therefore, formation of a single compact speck and rapid cell death induction in vivo requires full-length ASC.\n\nOne Sentence SummaryWith a new endogenous ASC real-time reporter we characterize speck dynamics in vivo as well as the concomitant pyroptosis speck formation causes in keratinocytes.

immunology↗