Biology subjects

Blanchard, G. B.

Publications and source records attributed to Blanchard, G. B..

7 recordsLinked to original sources

bioRxiv2023

Drosophila axis extension is robust to an orthogonal pull by invaginating mesoderm

As tissues grow and change shape during animal development, they physically pull and push on each other and these mechanical interactions can be important for morphogenesis. During Drosophila gastrulation, mesoderm invagination temporally overlaps with the extension of the ectodermal germband; the latter is caused primarily by Myosin II-driven polarised cell intercalation. Here we investigate the impact of mesoderm invagination on ectoderm extension, examining possible mechanical and mechanotransductive effects on Myosin II recruitment and polarised cell intercalation. We find that the germband ectoderm is deformed by the mesoderm pulling in the orthogonal direction, showing mechanical coupling between these tissues. However, we do not find a significant change in Myosin II planar polarisation in response to mesoderm invagination, nor an effect on the rate of junction shrinkage leading to cell intercalation events. We find some impact on the orientation of neighbour exchange events, and an increased rate of growth of new cell junctions, but this makes little difference to the rate of cell intercalation. We conclude that the cellular mechanisms of axis extension are robust to the mechanical pull of mesoderm invagination.

developmental biology↗

bioRxiv2021

Different temporal requirements for the LRR transmembrane receptors Tartan and Toll-2 in the formation of contractile interfaces at compartmental boundaries

Compartmental boundaries physically separate groups of epithelial cells, a property fundamental for the organization of the body plan in both insects and vertebrates. In many examples, this physical separation has been shown to be the consequence of a regulated increase in contractility of the actomyosin cortex at boundary cell-cell interfaces, a property important in developmental morphogenesis beyond compartmental boundary formation. In this study, we took an unbiased screening approach to identify cell surface receptors required for actomyosin enrichment and polarisation at parasegmental boundaries (PSBs) in early Drosophila embryos, leading us to uncover different temporal requirements for two LRR receptors, Tartan and Toll-2. First, we find that Tartan is required during germband extension for actomyosin enrichment at PSBs, confirming an earlier report. Next, by following in real time the dynamics of loss of boundary straightness in tartan mutant embryos compared to wildtype and ftz mutant embryos, we show that Tartan is not required beyond germband extension. At this stage, actomyosin enrichment at PSBs becomes regulated by Wingless signalling. We find that Wingless signalling regulates Toll-2 expression and we show that Toll-2 is required for planar polarization of actomyosin after the completion of germ-band extension. Thus the formation of contractile interfaces at PSBs depends on a dynamic set of LRR receptors cues. Our study also suggests that the number of receptor cues is small and that the receptors are interchangeable.

developmental biology↗

bioRxiv2021

Embryo-scale epithelial buckling forms a propagating furrow that initiates gastrulation

Cell apical constriction driven by actomyosin contraction forces is a conserved mechanism during tissue folding in embryo development. While much effort has been made to better understand the molecular mechanisms responsible for apical constriction, it is still not clear if apical actomyosin contraction forces are necessary or sufficient per se to drive tissue folding. To tackle this question, we use the Drosophila embryo model system that forms a furrow on the ventral side, initiating mesoderm internalization. Past computational models support the idea that cell apical contraction forces may not be sufficient and that active or passive cell apico-basal forces may be necessary to drive cell wedging and tissue furrowing. By using 3D computational modelling and in toto embryo image analysis and manipulation, we now challenge this idea and show that embryo-scale force balance of the tissue surface, rather than cell-autonomous shape changes, is necessary and sufficient to drive a buckling of the epithelial surface forming a furrow which propagates and initiates embryo gastrulation.

developmental biology↗

bioRxiv2020

Correct regionalisation of a tissue primordium is essential for coordinated morphogenesis

During organ development, tubular organs often form from flat epithelial primordia. In the placodes of the forming tubes of the salivary glands in the Drosophila embryo, we previously identified spatially defined cell behaviours of cell wedging, tilting and cell intercalation that are key to the initial stages of tube formation. Here we address what the requirements are that ensure the continuous formation of a narrow symmetrical tube from an initially asymmetrical primordium whilst overall tissue geometry is constantly changing. We are using live-imaging and quantitative methods to compare wild-type and mutants that either show disrupted cell behaviours or an initial symmetrical placode organisation, with both resulting in severe impairment of the invagination. We find that early transcriptional patterning of key morphogenetic transcription factors drives the selective activation of downstream morphogenetic modules, such as GPCR signalling that activates apical-medial actomyosin activity to drive cell wedging at the future asymmetrically-placed invagination point. Over time, transcription of key factors expands across the rest of the placode and cells switch their behaviour from predominantly intercalating to predominantly apically constricting as their position approaches the invagination pit. Misplacement or enlargement of the initial invagination pit leads to early problems in cell behaviours that eventually result in a defective organ shape. Our work illustrates that the dynamic patterning of the expression of transcription factors and downstream morphogenetic effectors ensures positionally fixed areas of cell behaviour with regards to the invagination point. This patterning in combination with the asymmetric geometrical set-up ensures functional organ formation.

developmental biology↗

bioRxiv2020

Embryonic stem cells commit to differentiation by symmetric divisions following a variable lag period

Mouse embryonic stem (ES) cells are derived from the epiblast of the preimplantation embryo and retain the capacity to give rise to all embryo lineages. ES cells can be released into differentiation from a near-homogeneous maintenance condition. Exit from the ES cell state can be accurately monitored using the Rex1-GFPd2 transgenic reporter, providing a powerful system for examining a mammalian cell fate transition. Here, we performed live-cell imaging and tracking of ES cells during entry into differentiation for 48 hours in defined conditions. We observed a greater cell surface area and a modest shortening of the cell cycle prior to exit and subsequently a reduction in cell size and increase in motility. We did not see any instance of cells regaining ES cell identity, consistent with unidirectional developmental progression. Transition occurred asynchronously across the population but genealogical tracking revealed a high correlation in cell-cycle length and Rex1-GFPd2 expression between daughter cells. A population dynamics model was consistent with symmetric divisions during exit from naive pluripotency. Collapse of ES cell identity occurred acutely in individual cells but after a variable delay. The variation in lag period can extend up to three generations, creating marked population asynchrony.

developmental biology↗

bioRxiv2019

The tricellular vertex-specific adhesion molecule Sidekick facilitates polarised cell intercalation during Drosophila axis extension

In epithelia, tricellular vertices are emerging as important sites for the regulation of epithelial integrity and function. Compared to bicellular contacts, however, much less knowledge is available. In particular, resident proteins at tricellular vertices were identified only at occluding junctions, with none known at adherens junctions. In a previous study, we discovered that in Drosophila embryos, the adhesion molecule Sidekick (Sdk), well known in invertebrates and vertebrates for its role in the visual system, localises at tricellular vertices at the level of adherens junctions. Here, we survey a wide range of Drosophila epithelia and establish that Sdk is a resident protein at tricellular adherens junctions, the first of its kind. Clonal analysis suggests that pair-wise homophilic adhesion is necessary and sufficient for Sdk tricellular vertex localisation. Super-resolution imaging using structured illumination reveals that Sdk proteins form string-like structures at vertices. Postulating that Sdk may have a role in epithelia where adherens junctions are actively remodelled, we analysed the phenotype of sdk null mutant embryos during Drosophila axis extension, using quantitative methods. We find that apical cell shapes are strikingly abnormal in sdk mutants. Moreover, adhesion at apical vertices is compromised in rearranging cells, with holes forming and persisting throughout axis extension. Finally, we show that polarized cell intercalation is decreased and abnormal in sdk mutants. Mathematical modeling of the cell behaviours supports the conclusion that the T1 transitions of polarized cell intercalation are delayed in sdk mutants. We propose that this delay, in combination with a change in the mechanical properties of the converging and extending tissue, causes the striking cell shape phenotype of sdk mutant embryos.

developmental biology↗

bioRxiv2018

Radially-patterned cell behaviours during tube budding from an epithelium

The budding of tubular organs from flat epithelial sheets is a vital morphogenetic process. Cell behaviours that drive such processes are only starting to be unraveled. Using live imaging and novel morphometric methods we show that in addition to apical constriction, radially oriented directional intercalation of placodal cells plays a major contribution to the early stages of invagination of the salivary gland tube in the Drosophila embryo. Extending analyses in 3D, we find that near the pit of invagination, isotropic apical constriction leads to strong cell wedging, and further from the pit cells interleave circumferentially, suggesting apically driven behaviours. Supporting this, junctional myosin is enriched in, and neighbour exchanges biased towards the circumferential orientation. In a mutant failing pit specification, neither are biased due to an inactive pit. Thus, tube budding depends on a radially polarised pattern of apical myosin leading to radially oriented 3D cell behaviours, with a close mechanical interplay between invagination and intercalation.

developmental biology↗