ß-H-Spectrin is a key component of an apical-medial hub of proteins during cell wedging in tube morphogenesis.

Coordinated cell shape changes are a major driver of tissue morphogenesis, with apical constriction of epithelial cells leading to tissue bending. During the tube budding of the salivary glands in the Drosophila embryo we previously identified a key interplay between the apical-medial actomyosin, driving apical constriction, with the underlying longitudinal microtubule array. At this microtubule-actomyosin-interface a hub of proteins accumulates: as shown before, the microtubule-actin-crosslinker Shot and the minus-end-binder Patronin, and now identified two actin-crosslinkers, ß-H-Spectrin and Filamin, and the multi-PDZ-protein Big-bang. We show that tissue-specific-degradation of ß-H-Spectrin led to reduction of apical-medial F-actin, Shot, Patronin and Big-bang and concomitant defects in apical constriction, but residual Patronin was still sufficient to assist microtubule reorganisation. Contrary to Patronin and Shot, neither ß-H-Spectrin nor Big bang required microtubules for their localisation. ß-H-Spectrin was instead recruited via binding to apical-medial phosphoinositides. Overexpression of ß-H-33 containing the PH domain displaced endogenous ß-H-Spectrin and led to strong morphogenetic defects. This protein hub therefore required the synergy and coincidence of membrane- and microtubule-associated components for its assembly and function in sustaining the apical constriction during tubulogenesis.

Identification of a core transcriptional program driving the human renal mesenchymal-to-epithelial transition.

During kidney development, nephron epithelia arise de novo from fate-committed mesenchymal progenitors through a mesenchymal-to-epithelial transition (MET). Downstream of fate specification, transcriptional mechanisms that drive establishment of epithelial morphology are poorly understood. We used human iPSC-derived renal organoids, which recapitulate nephrogenesis, to investigate mechanisms controlling renal MET. Multi-ome profiling via snRNA-seq and ATAC-seq of organoids identified dynamic changes in gene expression and chromatin accessibility driven by activators and repressors throughout MET. CRISPR interference identified that paired box 8 (PAX8) is essential for initiation of MET in human renal organoids, contrary to in vivo mouse studies, likely by activating a cell-adhesion program. While Wnt/ß-catenin signaling specifies nephron fate, we find that it must be attenuated to allow hepatocyte nuclear factor 1-beta (HNF1B) and TEA-domain (TEAD) transcription factors to drive completion of MET. These results identify the interplay between fate commitment and morphogenesis in the developing human kidney, with implications for understanding both developmental kidney diseases and aberrant epithelial plasticity following adult renal tubular injury.

Toll-like receptor signalling via IRAK4 affects epithelial integrity and tightness through regulation of junctional tension.

Toll-like receptors (TLRs) in mammalian systems are well known for their role in innate immunity. In addition, TLRs also fulfil crucial functions outside immunity, including the dorsoventral patterning function of the original Toll receptor in Drosophila and neurogenesis in mice. Recent discoveries in flies suggested key roles for TLRs in epithelial cells in patterning of junctional cytoskeletal activity. Here, we address the function of TLRs and the downstream key signal transduction component IRAK4 in human epithelial cells. Using differentiated human Caco-2 cells as a model for the intestinal epithelium, we show that these cells exhibit baseline TLR signalling, as revealed by p-IRAK4, and that blocking IRAK4 function leads to a loss of epithelial tightness involving key changes at tight and adherens junctions, such as a loss of epithelial tension and changes in junctional actomyosin. Changes upon IRAK-4 inhibition are conserved in human bronchial epithelial cells. Knockdown of IRAK4 and certain TLRs phenocopies the inhibitor treatment. These data suggest a model whereby TLR receptors near epithelial junctions might be involved in a continuous sensing of the epithelial state to promote epithelial tightness and integrity.

Zasp52 strengthens whole embryo tissue integrity through supracellular actomyosin networks.

During morphogenesis, large-scale changes of tissue primordia are coordinated across an embryo. In Drosophila, several tissue primordia and embryonic regions are bordered or encircled by supracellular actomyosin cables, junctional actomyosin enrichments networked between many neighbouring cells. We show that the single Drosophila Alp/Enigma-family protein Zasp52, which is most prominently found in Z-discs of muscles, is a component of many supracellular actomyosin structures during embryogenesis, including the ventral midline and the boundary of the salivary gland placode. We reveal that Zasp52 contains within its central coiled-coil region a type of actin-binding motif usually found in CapZbeta proteins, and this domain displays actin-binding activity. Using endogenously-tagged lines, we identify that Zasp52 interacts with junctional components, including APC2, Polychaetoid and Sidekick, and actomyosin regulators. Analysis of zasp52 mutant embryos reveals that the severity of the embryonic defects observed scales inversely with the amount of functional protein left. Large tissue deformations occur where actomyosin cables are found during embryogenesis, and in vivo and in silico analyses suggest a model whereby supracellular Zasp52-containing cables aid to insulate morphogenetic changes from one another.

Correct regionalization 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 behaviors 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 placodes and mutants that either show disrupted cell behaviors or an initial symmetrical placode organization, 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 signaling 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 behavior 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 behaviors 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 behavior with regards to the invagination point. This patterning in combination with the asymmetric geometrical setup ensures functional organ formation.

A release-and-capture mechanism generates an essential non-centrosomal microtubule array during tube budding.

Non-centrosomal microtubule arrays serve crucial functions in cells, yet the mechanisms of their generation are poorly understood. During budding of the epithelial tubes of the salivary glands in the Drosophila embryo, we previously demonstrated that the activity of pulsatile apical-medial actomyosin depends on a longitudinal non-centrosomal microtubule array. Here we uncover that the exit from the last embryonic division cycle of the epidermal cells of the salivary gland placode leads to one centrosome in the cells losing all microtubule-nucleation capacity. This restriction of nucleation activity to the second, Centrobin-enriched, centrosome is key for proper morphogenesis. Furthermore, the microtubule-severing protein Katanin and the minus-end-binding protein Patronin accumulate in an apical-medial position only in placodal cells. Loss of either in the placode prevents formation of the longitudinal microtubule array and leads to loss of apical-medial actomyosin and impaired apical constriction. We thus propose a mechanism whereby Katanin-severing at the single active centrosome releases microtubule minus-ends that are then anchored by apical-medial Patronin to promote formation of the longitudinal microtubule array crucial for apical constriction and tube formation.

Mesenchymal-to-Epithelial Transitions in Development and Cancer.

The evolutionary emergence of the mesenchymal phenotype greatly increased the complexity of tissue architecture and composition in early Metazoan species. At the molecular level, an epithelial-to-mesenchymal transition (EMT) was permitted by the innovation of specific transcription factors whose expression is sufficient to repress the epithelial transcriptional program. The reverse process, mesenchymal-to-epithelial transition (MET), involves direct inhibition of EMT transcription factors by numerous mechanisms including tissue-specific MET-inducing transcription factors (MET-TFs), micro-RNAs, and changes to cell and tissue architecture, thus providing an elegant solution to the need for tight temporal and spatial control over EMT and MET events during development and adult tissue homeostasis.

"Neur"al brain wave: Coordinating epithelial-to-neural stem cell transition in the fly optic lobe.

In the Drosophila larval optic lobe, the generation of neural stem cells involves an epithelial-to-mesenchymal-like transition of a continuous stripe of cells that sweeps across the neuroepithelium, but the dynamics at cell and tissue level were unknown until now. In this issue, Shard et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.202005035) identify that Neuralized controls a partial epithelial-to-mesenchymal transition through regulation of the apical Crumbs complex and through the coordination of cell behaviors such as apical constriction and cell alignment.

Microtubules enter centre stage for morphogenesis.

Cell shape changes are key to observable changes at the tissue level during morphogenesis and organ formation. The major driver of cell shape changes in turn is the actin cytoskeleton, both in the form of protrusive linear or branched dynamic networks and in the form of contractile actomyosin. Over the last 20 years, actomyosin has emerged as the major cytoskeletal system that deforms cells in epithelial sheets during morphogenesis. By contrast, the second major cytoskeletal system, microtubules, have so far mostly been assumed to serve 'house-keeping' functions, such as directed transport or cell division, during morphogenetic events. Here, I will reflect on a subset of studies over the last 10 years that have clearly shown a major direct role for the microtubule cytoskeleton in epithelial morphogenesis, suggesting that our focus will need to be widened to give more attention and credit to this cytoskeletal system in playing an active morphogenetic role. This article is part of a discussion meeting issue 'Contemporary morphogenesis'.

Control of cell shape during epithelial morphogenesis: recent advances.

Morphogenesis is an essential process by which a given tissue, organ or organism acquires its final shape. A select number of mechanisms are used in order to drive epithelial morphogenesis, including cell shape changes as well as cell death or cell division. A cell's shape results from the combination of intrinsic properties of the actomyosin and microtubule (MTs) cytoskeletons, and extrinsic properties due to physical interactions with the neighbouring environment. While we now have a good understanding of the genetic pathways and some of the signalling pathways controlling cell shape changes, the mechanical properties of cells and their role in morphogenesis remain largely unexplored. Recent improvements in microscopy techniques and the development of modelling and quantitative methods have enabled a better understanding of the bio-mechanical events controlling cell shape during morphogenesis. This review aims to highlight recent findings elegantly unravelling and quantifying the contribution of mechanical forces during morphogenesis.

Rho-Kinase Planar Polarization at Tissue Boundaries Depends on Phospho-regulation of Membrane Residence Time.

The myosin II activator Rho-kinase (Rok) is planar polarized at the tissue boundary of the Drosophila embryonic salivary gland placode through a negative regulation by the apical polarity protein Crumbs that is anisotropically localized at the boundary. However, in inner cells of the placode, both Crumbs and Rok are isotropically enriched at junctions. We propose that modulation of Rok membrane residence time by Crumbs' downstream effectors can reconcile both behaviors. Using FRAP combined with in silico simulations, we find that the lower membrane dissociation rate (koff) of Rok at the tissue boundary with low Crumbs explains this boundary-specific effect. The S/T kinase Pak1, recruited by Crumbs and Cdc42, negatively affects Rok membrane association in vivo and in vitro can phosphorylate Rok near the pleckstrin homology (PH) domain that mediates membrane association. These data reveal an important mechanism of the modulation of Rok membrane residence time via affecting the koff that may be widely employed during tissue morphogenesis.

Quantitative Imaging and the Effect of Tissue Topology on Morphogenesis.

Recent improvements in live-imaging and quantitative morphometric approaches have illustrated how the topology of a tissue primordium plays a key role in the type and coordination of morphogenetic behaviors taking place.

Alignment of cytoskeletal structures across cell boundaries generates tissue cohesion during organ formation.

One of the most fascinating aspects of development is the complexity and diversity of tissues and organs that are formed from simple primordia, involving complex coordination between large groups of cells. Lack of coordination leads to developmental defects and failure in organ formation. The simple primordia are often polarised epithelial sheets, with cells connected to neighbours apically via Cadherin-based cell-cell junctions that intracellularly link to the cytoskeleton. Coordination of cells in epithelia during morphogenesis occurs in part at these junctions. Furthermore, in many tissues a striking supracellular order and alignment of cytoskeletal structures can be observed, likely playing an important part in the coordination of cells. Here, we will introduce examples of morphogenetic events where this supracellular order of the cytoskeleton is very apparent and will discuss recent advances in understanding the generation and function of this order.

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 cells plays a major contribution to 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. Further from the pit cells interleave circumferentially, suggesting apically driven behaviours. Supporting this, junctional myosin is enriched in, and neighbour exchanges are biased towards the circumferential orientation. In a mutant failing pit specification, neither are biased due to an inactive pit. Thus, tube budding involves radially patterned pools of apical myosin, medial as well as junctional, and radially patterned 3D-cell behaviours, with a close mechanical interplay between invagination and intercalation.

Squeezing out in a "tug of war": The role of myosin in neural stem cell delamination.

Neural stem cells or neuroblasts in the Drosophila melanogaster embryo delaminate as single cells from the embryonic epidermis to give rise to the nervous system. Using this accessible system to examine the molecular mechanisms of cell ingression at a high temporal and spatial resolution, in this issue, Simões et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201608038) reveal that myosin-driven anisotropic junction loss and apical constriction are the main drivers of this process.

The spectraplakin Short stop is an essential microtubule regulator involved in epithelial closure in Drosophila.

Dorsal closure of the Drosophila embryonic epithelium provides an excellent model system for the in vivo analysis of molecular mechanisms regulating cytoskeletal rearrangements. In this study, we investigated the function of the Drosophila spectraplakin Short stop (Shot), a conserved cytoskeletal structural protein, during closure of the dorsal embryonic epithelium. We show that Shot is essential for the efficient final zippering of the opposing epithelial margins. By using isoform-specific mutant alleles and genetic rescue experiments with truncated Shot variants, we demonstrate that Shot functions as an actin-microtubule cross-linker in mediating zippering. At the leading edge of epithelial cells, Shot regulates protrusion dynamics by promoting filopodia formation. Fluorescence recovery after photobleaching (FRAP) analysis and in vivo imaging of microtubule growth revealed that Shot stabilizes dynamic microtubules. The actin- and microtubule-binding activities of Shot are simultaneously required in the same molecule, indicating that Shot is engaged as a physical crosslinker in this process. We propose that Shot-mediated interactions between microtubules and actin filaments facilitate filopodia formation, which promotes zippering by initiating contact between opposing epithelial cells.

The Gas2 family protein Pigs is a microtubule +TIP that affects cytoskeleton organisation.

Coordination between different cytoskeletal systems is crucial for many cell biological functions, including cell migration and mitosis, and also plays an important role during tissue morphogenesis. Proteins of the class of cytoskeletal crosslinkers, or cytolinkers, have the ability to interact with more than one cytoskeletal system at a time and are prime candidates to mediate any coordination. One such class comprises the Gas2-like proteins, combining a conserved calponin-homology-type actin-binding domain and a Gas2 domain predicted to bind microtubules (MTs). This domain combination is also found in spectraplakins, huge cytolinkers that play important roles in many tissues in both invertebrates and vertebrates. Here, we dissect the ability of the single Drosophila Gas2-like protein Pigs to interact with both actin and MT cytoskeletons, both in vitro and in vivo, and illustrate complex regulatory interactions that determine the localisation of Pigs to and its effects on the cytoskeleton.

Integration of cell-cell adhesion and contractile actomyosin activity during morphogenesis.

During embryonic development, cells become organized into complex tissues. Cells need to adhere and communicate with their immediate and remote neighbors to allow morphogenesis to take place in a coordinated way. Cell-cell adhesion, mediated by transmembrane adhesion receptors such as Cadherins and their intracellular interaction partners, is intimately linked to cell contractility that drives cell shape changes. Research in recent years has revealed that the contractile machinery responsible for cell shape changes, actomyosin, can in fact be organized into a number of different functional assemblies such as cortical-junctional actomyosin, apical-medial actomyosin, supracellular actomyosin cables as well as basal actomyosin networks. During coordinated shape changes of a tissue, these assemblies have to be functionally and mechanically linked between cells through cell-cell junctions. Although many actin-binding proteins associated with adherens junctions have been identified, which specific factors are required for the linkage of particular actomyosin assemblies to junctions is not well understood. This review will summarize our current knowledge, based mainly on the in vivo study of morphogenesis in the fruit fly Drosophila melanogaster.

A dynamic microtubule cytoskeleton directs medial actomyosin function during tube formation.

The cytoskeleton is a major determinant of cell-shape changes that drive the formation of complex tissues during development. Important roles for actomyosin during tissue morphogenesis have been identified, but the role of the microtubule cytoskeleton is less clear. Here, we show that during tubulogenesis of the salivary glands in the fly embryo, the microtubule cytoskeleton undergoes major rearrangements, including a 90° change in alignment relative to the apicobasal axis, loss of centrosomal attachment, and apical stabilization. Disruption of the microtubule cytoskeleton leads to failure of apical constriction in placodal cells fated to invaginate. We show that this failure is due to loss of an apical medial actomyosin network whose pulsatile behavior in wild-type embryos drives the apical constriction of the cells. The medial actomyosin network interacts with the minus ends of acentrosomal microtubule bundles through the cytolinker protein Shot, and disruption of Shot also impairs apical constriction.