Phased ERK-responsiveness and developmental robustness regulate teleost skin morphogenesis.

Elongation of the vertebrate embryonic axis necessitates rapid expansion of the epidermis to accommodate the growth of underlying tissues. Here, we generated a toolkit to visualize and quantify signaling in entire cell populations of periderm, the outermost layer of the epidermis, in live developing zebrafish. We find that oriented cell divisions facilitate growth of the early periderm during axial elongation rather than cell addition from the basal layer. Activity levels of ERK, a downstream effector of MAPK pathway, gauged by a live biosensor, predicts cell cycle entry, and optogenetic ERK activation controls proliferation dynamics. As development proceeds, rates of peridermal cell proliferation decrease, ERK activity becomes more pulsatile and functionally transitions to promote hypertrophic cell growth. Targeted genetic blockade of cell division generates animals with oversized periderm cells, yet, unexpectedly, development to adulthood is not impaired. Our findings reveal stage-dependent differential responsiveness to ERK signaling and marked developmental robustness in growing teleost skin.

DeepProjection: specific and robust projection of curved 2D tissue sheets from 3D microscopy using deep learning.

The efficient extraction of image data from curved tissue sheets embedded in volumetric imaging data remains a serious and unsolved problem in quantitative studies of embryogenesis. Here, we present DeepProjection (DP), a trainable projection algorithm based on deep learning. This algorithm is trained on user-generated training data to locally classify 3D stack content, and to rapidly and robustly predict binary masks containing the target content, e.g. tissue boundaries, while masking highly fluorescent out-of-plane artifacts. A projection of the masked 3D stack then yields background-free 2D images with undistorted fluorescence intensity values. The binary masks can further be applied to other fluorescent channels or to extract local tissue curvature. DP is designed as a first processing step than can be followed, for example, by segmentation to track cell fate. We apply DP to follow the dynamic movements of 2D-tissue sheets during dorsal closure in Drosophila embryos and of the periderm layer in the elongating Danio embryo. DeepProjection is available as a fully documented Python package.

Aurora B-dependent polarization of the cortical actomyosin network during mitotic exit.

The isotropic metaphase actin cortex progressively polarizes as the anaphase spindle elongates during mitotic exit. This involves the loss of actomyosin cortex from opposing cell poles and the accumulation of an actomyosin belt at the cell centre. Although these spatially distinct cortical remodelling events are coordinated in time, here we show that they are independent of each other. Thus, actomyosin is lost from opposing poles in anaphase cells that lack an actomyosin ring owing to centralspindlin depletion. In examining potential regulators of this process, we identify a role for Aurora B kinase in actin clearance at cell poles. Upon combining Aurora B inhibition with centralspindlin depletion, cells exiting mitosis fail to change shape and remain completely spherical. Additionally, we demonstrate a requirement for Aurora B in the clearance of cortical actin close to anaphase chromatin in cells exiting mitosis with a bipolar spindle and in monopolar cells forced to divide while flat. Altogether, these data suggest a novel role for Aurora B activity in facilitating DNA-mediated polar relaxation at anaphase, polarization of the actomyosin cortex, and cell division.

Tissue Repair: A Tendon-see to Regenerate.

A new study establishes genetic tools to ablate tendon progenitor cells in zebrafish larvae, finding that larval tendons display high regenerative capacity. The authors employ this musculoskeletal repair model to explore the source of tendon progenitors by fate mapping and live imaging, as well as underlying molecular stimuli like BMP signaling.

Isotropic myosin-generated tissue tension is required for the dynamic orientation of the mitotic spindle.

The ability of cells to divide along their longest axis has been proposed to play an important role in maintaining epithelial tissue homeostasis in many systems. Because the division plane is largely set by the position of the anaphase spindle, it is important to understand how spindles become oriented. While several molecules have been identified that play key roles in spindle orientation across systems, most notably Mud/NuMA and cortical dynein, the precise mechanism by which spindles detect and align with the long cell axis remain poorly understood. Here, in exploring the dynamics of spindle orientation in mechanically distinct regions of the fly notum, we find that the ability of cells to properly reorient their divisions depends on local tissue tension. Thus, spindles reorient to align with the long cell axis in regions where isotropic tension is elevated, but fail to do so in elongated cells within the crowded midline, where tension is low, or in regions that have been mechanically isolated from the rest of the tissue via laser ablation. Importantly, these differences in spindle behavior outside and inside the midline can be recapitulated by corresponding changes in tension induced by perturbations that alter nonmuscle myosin II activity. These data lead us to propose that isotropic tension within an epithelium provides cells with a mechanically stable substrate upon which localized cortical motor complexes can act on astral microtubules to orient the spindle.

p120-catenin regulates WNT signaling and EMT in the mouse embryo.

Epithelial-to-mesenchymal transitions (EMTs) require a complete reorganization of cadherin-based cell-cell junctions. p120-catenin binds to the cytoplasmic juxtamembrane domain of classical cadherins and regulates their stability, suggesting that p120-catenin may play an important role in EMTs. Here, we describe the role of p120-catenin in mouse gastrulation, an EMT that can be imaged at cellular resolution and is accessible to genetic manipulation. Mouse embryos that lack all p120-catenin, or that lack p120-catenin in the embryo proper, survive to midgestation. However, mutants have specific defects in gastrulation, including a high rate of p53-dependent cell death, a bifurcation of the posterior axis, and defects in the migration of mesoderm; all are associated with abnormalities in the primitive streak, the site of the EMT. In embryonic day 7.5 (E7.5) mutants, the domain of expression of the streak marker Brachyury (T) expands more than 3-fold, from a narrow strip of posterior cells to encompass more than one-quarter of the embryo. After E7.5, the enlarged T+ domain splits in 2, separated by a mass of mesoderm cells. Brachyury is a direct target of canonical WNT signaling, and the domain of WNT response in p120-catenin mutant embryos, like the T domain, is first expanded, and then split, and high levels of nuclear ß-catenin levels are present in the cells of the posterior embryo that are exposed to high levels of WNT ligand. The data suggest that p120-catenin stabilizes the membrane association of ß-catenin, thereby preventing accumulation of nuclear ß-catenin and excessive activation of the WNT pathway during EMT.

Local actin nucleation tunes centrosomal microtubule nucleation during passage through mitosis.

Cells going through mitosis undergo precisely timed changes in cell shape and organisation, which serve to ensure the fair partitioning of cellular components into the two daughter cells. These structural changes are driven by changes in actin filament and microtubule dynamics and organisation. While most evidence suggests that the two cytoskeletal systems are remodelled in parallel during mitosis, recent work in interphase cells has implicated the centrosome in both microtubule and actin nucleation, suggesting the potential for regulatory crosstalk between the two systems. Here, by using both in vitro and in vivo assays to study centrosomal actin nucleation as cells pass through mitosis, we show that mitotic exit is accompanied by a burst in cytoplasmic actin filament formation that depends on WASH and the Arp2/3 complex. This leads to the accumulation of actin around centrosomes as cells enter anaphase and to a corresponding reduction in the density of centrosomal microtubules. Taken together, these data suggest that the mitotic regulation of centrosomal WASH and the Arp2/3 complex controls local actin nucleation, which may function to tune the levels of centrosomal microtubules during passage through mitosis.

Crumbs2 promotes cell ingression during the epithelial-to-mesenchymal transition at gastrulation.

During gastrulation of the mouse embryo, individual cells ingress in an apparently stochastic pattern during the epithelial-to-mesenchymal transition (EMT). Here we define a critical role of the apical protein Crumbs2 (CRB2) in the gastrulation EMT. Static and live imaging show that ingressing cells in Crumbs2 mutant embryos become trapped at the primitive streak, where they continue to express the epiblast transcription factor SOX2 and retain thin E-cadherin-containing connections to the epiblast surface that trap them at the streak. CRB2 is distributed in a complex anisotropic pattern on apical cell edges, and the level of CRB2 on a cell edge is inversely correlated with the level of myosin IIB. The data suggest that the distributions of CRB2 and myosin IIB define which cells will ingress, and we propose that cells with high apical CRB2 are basally extruded from the epiblast by neighbouring cells with high levels of apical myosin.

Coupling changes in cell shape to chromosome segregation.

Animal cells undergo dramatic changes in shape, mechanics and polarity as they progress through the different stages of cell division. These changes begin at mitotic entry, with cell-substrate adhesion remodelling, assembly of a cortical actomyosin network and osmotic swelling, which together enable cells to adopt a near spherical form even when growing in a crowded tissue environment. These shape changes, which probably aid spindle assembly and positioning, are then reversed at mitotic exit to restore the interphase cell morphology. Here, we discuss the dynamics, regulation and function of these processes, and how cell shape changes and sister chromatid segregation are coupled to ensure that the daughter cells generated through division receive their fair inheritance.

Protein O-Glucosyltransferase 1 (POGLUT1) Promotes Mouse Gastrulation through Modification of the Apical Polarity Protein CRUMBS2.

Crumbs family proteins are apical transmembrane proteins with ancient roles in cell polarity. Mouse Crumbs2 mutants arrest at midgestation with abnormal neural plate morphology and a deficit of mesoderm caused by defects in gastrulation. We identified an ENU-induced mutation, wsnp, that phenocopies the Crumbs2 null phenotype. We show that wsnp is a null allele of Protein O-glucosyltransferase 1 (Poglut1), which encodes an enzyme previously shown to add O-glucose to EGF repeats in the extracellular domain of Drosophila and mammalian Notch, but the role of POGLUT1 in mammalian gastrulation has not been investigated. As predicted, we find that POGLUT1 is essential for Notch signaling in the early mouse embryo. However, the loss of mouse POGLUT1 causes an earlier and more dramatic phenotype than does the loss of activity of the Notch pathway, indicating that POGLUT1 has additional biologically relevant substrates. Using mass spectrometry, we show that POGLUT1 modifies EGF repeats in the extracellular domain of full-length mouse CRUMBS2. CRUMBS2 that lacks the O-glucose modification fails to be enriched on the apical plasma membrane and instead accumulates in the endoplasmic reticulum. The data demonstrate that CRUMBS2 is the target of POGLUT1 for the gastrulation epithelial-to-mesenchymal transitions (EMT) and that all activity of CRUMBS2 depends on modification by POGLUT1. Mutations in human POGLUT1 cause Dowling-Degos Disease, POGLUT1 is overexpressed in a variety of tumor cells, and mutations in the EGF repeats of human CRUMBS proteins are associated with human congenital nephrosis, retinitis pigmentosa and retinal degeneration, suggesting that O-glucosylation of CRUMBS proteins has broad roles in human health.

Chip is required for posteclosion behavior in Drosophila.

Neurons acquire their molecular, neurochemical, and connectional features during development as a result of complex regulatory mechanisms. Here, we show that a ubiquitous, multifunctional protein cofactor, Chip, plays a critical role in a set of neurons in Drosophila that control the well described posteclosion behavior. Newly eclosed flies normally expand their wings and display tanning and hardening of their cuticle. Using multiple approaches to interfere with Chip function, we find that these processes do not occur without normal activity of this protein. Furthermore, we identified the nature of the deficit to be an absence of Bursicon in the hemolymph of newly eclosed flies, whereas the responsivity to Bursicon in these flies remains normal. Chip interacts with transcription factors of the LIM-HD (LIM-homeodomain) family, and we identified one member, dIslet, as a potential partner of Chip in this process. Our findings provide the first evidence of transcriptional mechanisms involved in the development of the neuronal circuit that regulates posteclosion behavior in Drosophila.