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The beating of motile cilia requires to coordinated action of diverse machineries that include not only the axonemal dynein arms, but also the central apparatus, the radial spokes, and the microtubule inner proteins. These machines exhibit complex radial and proximodistal patterns in mature axonemes, but little is known about the interplay between them during motile ciliogenesis. Here, we describe and quantify the relative rates of axonemal deployment for these diverse cilia beating machineries during the final stages of differentiation of Xenopus epidermal multiciliated cells.

This data includes XL/MS and structural models for the Tetrahymena motile cilia interactome.

DIFFRAC is a powerful method for systematically comparing proteome content and organization between samples in a high-throughput manner. By subjecting control and experimental protein extracts to native chromatography and quantifying the contents of each fraction using mass spectrometry, it enables the quantitative detection of alterations to protein complexes and abundances. Here, we applied DIFFRAC to investigate the consequences of genetic loss of Ift122, a subunit of the intraflagellar transport-A (IFT-A) protein complex that plays a vital role in the formation and function of cilia and flagella, on the proteome ofTetrahymena thermophila. A single DIFFRAC experiment was sufficient to detect changes in protein behavior that mirrored known effects of IFT-A loss and revealed new biology. We uncovered several novel IFT-A-regulated proteins, which we validated through live imaging inXenopusmulticiliated cells, shedding new light on both the ciliary and non-ciliary functions of IFT-A. Our findings underscore the robustness of DIFFRAC for revealing proteomic changes in response to genetic or biochemical perturbation.

Ependymal cells, lining brain ventricular walls, display tufts of cilia that beat in concert promoting laminar Cerebrospinal fluid (CSF) flow within brain ventricles. The ciliary axonemes of multiciliated ependymal cells display a 9+2 microtubule array common to motile cilia. Dyneins and kinesins are ATPase microtubule motor proteins that promote the rhythmic beating of cilia axonemes. Despite common consensus about the importance of axonemal dynein motor proteins, little is known about how Kinesin motors contribute to cilia motility. Here, we define the function of Kinesin family member 6 (Kif6) using a mutation that lacks a highly conserved C-terminal tail domain (Kif6p.G555fs) and which displays progressive hydrocephalus in mice. An analogous mutation was isolated in a proband displaying macrocephaly, hypotonia, and seizures implicating an evolutionarily conserved function for Kif6 in neurodevelopment. We find that loss of Kif6 function caused decreased ependymal cilia motility and subsequently decreased fluid flow on the surface of brain ventricular walls. Kif6 protein was localized at ependymal cilia and displayed processive motor movement (676 nm/s) on microtubulesin vitro. Loss of the Kif6 C-terminal tail domain did not affect the initial ciliogenesisin vivo, but did result in defects in cilia orientation, the formation of robust apical actin networks, and stabilization of basal bodies at the apical surface. This suggests a novel role for the Kif6 motor in maintenance of ciliary homeostasis of ependymal cells.Summary statementWe found that Kif6 is localized to the axonemes of ependymal cells. In vitro analysis shows that Kif6 moves on microtubules and that its loss mice decrease cilia motility and cilia-driven flow, resulting in hydrocephalus.

Animals are shaped through the movement of large cellular collectives. Such morphogenetic processes require cadherin-based cell adhesion to maintain tissue cohesion and planar cell polarity to coordinate movement. Despite a vast literature surrounding cadherin-based adhesion and planar cell polarity it is unclear how these molecular networks interface. Here we investigate the relationship between cadherins and planar cell polarity during gastrulation cell movements inXenopus laevis. We first assessed the bulk cadherin localization and found that cadherins were enriched at a specific subset of morphogenetically active cell-cell junctions. We then found that disruption of planar cell polarity desynchronized temporal cadherin and actin dynamics. Next, using super-resolution time-lapse miscopy and quantitative image analysis we were able to measure the lifespan and size of individual cadherin clusters. Finally, we show that planar cell polarity not only controls the size of cadherin clusters but, more interestingly, regulates cluster stability. These results reveal an intriguing link between two essential cellular properties, adhesion and planar polarity, and provide insight into the molecular control of morphogenetic cell movements.HighlightsPlanar cell polarity is required for cadherin spatiotemporal dynamics.Super-resolution imaging reveals the spatiotemporal patterns of cadherin clustering.Cadherin cluster lifespan and size are highly heterogenous.Planar cell polarity controls cadherin cluster stability and size.

Convergent extension (CE) is an evolutionarily conserved developmental process whereby polarized collective cell movements drive the elongation of tissues or organs, and defects in CE are associated with multiple human birth defects. Here, we combined embryology with cell biology and newly developed biomechanical tools to interrogate the link between cell behavior and tissue-scale CE. We found that unique patterns of polarized cortex tension are required to resolve individual cell intercalation events, and this in turn maintains normal cell packing configurations that favor the planar propagation of the cellular forces across the tissue, and thus normal CE. Our data suggest that planar polarized force propagation plays a critical role in the propagation of cell behaviors via cellular mechanoreciprocity, underpinning the propagation of tissue-scale CE.

Female fertility in mammals requires iterative remodeling of the entire adult female reproductive tract across the menstrual/estrous cycle. However, while transcriptome dynamics across the estrous cycle have been reported in human and bovine models, no global analysis of gene expression across the estrous cycle has yet been reported for the mouse. Here, we examined the cellular composition and global transcriptional dynamics of the mouse oviduct along the anteroposterior axis and across the estrous cycle. We observed robust patterns of differential gene expression along the anteroposterior axis, but we found surprisingly few changes in gene expression across the estrous cycle. Notable gene expression differences along the anteroposterior axis included a surprising enrichment for genes related to embryonic development, such as Hox and Wnt genes. The relatively stable transcriptional dynamics across the estrous cycle differ markedly from other mammals, leading us to speculate that this is an evolutionarily derived state that may reflect the extremely rapid five-day mouse estrous cycle. This dataset fills a critical gap by providing an important genomic resource for a highly tractable genetic model of mammalian female reproduction.

The adult uterus regenerates in the human during the menstrual cycle, and remodels in the mouse during the estrous cycle. Decades of work has demonstrated that this process is controlled by cycling steroid hormones, estrogen and progesterone. However, downstream signaling pathways that link hormonal action to this regeneration and remodeling are yet to be identified in the cycling uterus. We set out to identify these pathways, with the overarching hypothesis that developmental signaling pathways are redeployed in the adult uterus to control remodeling in the mouse. We were surprised to find that the majority of Hedgehog signaling components were transcriptionally co-regulated throughout the estrous cycle. To test the role of Hh signaling in cyclical uterine remodeling, we conditionally knocked out the major activator of the pathway,smoothened(Smo) using theprogesterone receptor cre(PR-Cre). In the absence of Hh signaling, the uterus no longer remodels throughout the estrous cycle. We also show that the smooth muscle fibers of the uterus are significantly larger in the conditional knockouts compared to the controls suggesting hypertrophy of the smooth muscle. Our findings support the possibility that this smooth muscle homeostasis may underlie important aspects of uterine function such as contractility during late-stage pregnancy or the development of uterine smooth muscle tumors.

During neural tube closure, neural ectoderm cells constrict their apical surfaces to bend and fold the tissue into a tube that will become the central nervous system. Recent data from mice and humans with neural tube defects suggest that key genes required for neural tube closure can exert non-cell autonomous effects on cell behavior, but the nature of these effects remains obscure. Here, we coupled tissue-scale, high-resolution time-lapse imaging of the closing neural tube of Xenopus to multivariate regression modeling, and we show that medial actin accumulation drives apical constriction non-autonomously in neighborhoods of cells, rather than solely in individual cells. To further explore this effect, we examined mosaic crispant embryos and identified both autonomous and non-autonomous effects of the apical constriction protein Shroom3.

Apical constriction results in apical surface reduction in epithelial cells and is a widely used mechanism for epithelial morphogenesis during embryo development. Both medioapical and junctional actomyosin remodeling are involved in apical constriction, but the deployment of medial versus junctional actomyosin in specific developmental processes has not been fully described. Additionally, genetic regulation of actomyosin dynamics during apical constriction is poorly understood in vertebrate systems. In this study, we investigate actomyosin dynamics and their regulation by the RhoGEF protein Plekhg5 in Xenopus bottle cells. Using live imaging and quantitative image analysis, we show that bottle cells assume different shapes, with rounding bottle cells constricting earlier in small clusters followed by fusiform bottle cells forming between the clusters. Though both medioapical and junctional actomyosin accumulate as surface area decreases, apical constriction is better correlated with medioapical actomyosin localization, which may promote formation of microvilli in the apical membrane. Knockdown of plekhg5 disrupts both medioapical actomyosin activity and apical constriction but does not affect initial F-actin dynamics. Taken together, our study reveals distinct cell morphologies, uncovers actomyosin behaviors, and demonstrates the crucial role of a RhoGEF protein in controlling actomyosin dynamics during apical constriction of bottle cells in Xenopus gastrulation.

Cilia are multifunctional organelles that originated with the last eukaryotic common ancestor and play central roles in the life cycles of diverse organisms. The motile flagella that move single cells like sperm or unicellular organisms, the motile cilia on animal multiciliated cells that generate fluid flow in organs, and the immotile primary cilia that decorate nearly all cells in animals share many protein components in common, yet each also requires specialized proteins to perform their specialized functions. Despite a now-advanced understanding of how such proteins are transported within cilia, we still know very little about how they are transported from their sites of synthesis through the cytoplasm to the ciliary base. Here, we review the literature concerning this underappreciated topic in ciliary cell biology. We discuss both general mechanisms, as well as specific examples of motor-driven active transport and passive transport via diffusion-and-capture. We then provide deeper discussion of specific, illustrative examples, such as the diverse array of protein subunits that together comprise the intraflagellar transport (IFT) system and the multi-protein axonemal dynein motors that drive beating of motile cilia. We hope this Review will spur further work, shedding light not only on ciliogenesis and ciliary signaling, but also on intracellular transport in general.

Intraflagellar transport (IFT) is a conserved process of cargo transport in cilia that is essential for development and homeostasis in organisms ranging from algae to vertebrates. In humans, variants in genes encoding subunits of the cargo-adapting IFT-A and IFT-B protein complexes are a common cause of genetic diseases known as ciliopathies. While recent progress has been made in determining the atomic structure of IFT-B, little is known of the structural biology of IFT-A. Here, we combined chemical cross-linking mass spectrometry and cryo-electron tomography with AlphaFold2-based prediction of both protein structures and interaction interfaces to model the overall architecture of the monomeric six-subunit IFT-A complex, as well as its polymeric assembly within cilia. We define monomer-monomer contacts and membrane-associated regions available for association with transported cargo, and we also use this model to provide insights into the pleiotropic nature of human ciliopathy-associated genetic variants in genes encoding IFT-A subunits. Our work demonstrates the power of integration of experimental and computational strategies both for multi-protein structure determination and for understanding the etiology of human genetic disease.SummaryThe 3D structure of the six-subunit complex and its polymeric assembly gives insights into cargo transport in cilia and how specific mutations in these genes lead to ciliopathy birth defects.

During neural tube closure, neural ectoderm cells constrict their apical surfaces to bend and fold the tissue into a tube that will become the central nervous system. These cells are physically interconnected via N-cadherin, and mutation of critical genes within relatively small numbers of cells can result in neural tube closure defects due to non-cell autonomous cell behavior defects. Despite this finding, we have a poor understanding of how neuroepithelial cells interact during apical constriction. In our previous paper, we introduced an imaging and analysis paradigm for tracking and quantifying apical constriction during neural tube closure, while also using CRISPR/Cas9 to generate mosaic loss of function of the apical constriction gene shroom3. Here we analyze the behaviors of cells along the mosaic interface of our shroom3 crispant clones, and find that Shroom3 non-cell autonomously regulates apical constriction and N-cadherin localization. Control cells along the interface constrict less, while shroom3 crispant cells along the interface constrict more. Finally, we construct a partial least squares regression (PLSR) model to estimate how both autonomous and non-cell autonomous dynamics of actin and N-cadherin affect apical surface area in both control and shroom3 crispant cells. Overall, our results demonstrate a previously unidentified non-cell autonomous role for Shroom3 in neural tube closure.

Cell-type specific RNA-associated proteins are essential for development and homeostasis in animals. Despite a massive recent effort to systematically identify RNA-associated proteins, we currently have few comprehensive rosters of cell-type specific RNA-associated proteins in vertebrate tissues. Here, we demonstrate the feasibility of determining the RNA-interacting proteome of a defined vertebrate embryonic tissue using DIF-FRAC, a systematic and universal (i.e., label-free) method. Application of DIF-FRAC to cultured tissue explants of Xenopus mucociliary epithelium identified dozens of known RNA-associated proteins as expected, but also several novel RNA-associated proteins, including proteins related to assembly of the mitotic spindle and regulation of ciliary beating. In particular, we show that the inner dynein arm tether Cfap44 is an RNA-associated protein that localizes not only to axonemes, but also to liquid-like organelles in the cytoplasm called DynAPs. This result led us to discover that DynAPs are generally enriched for RNA. Together, these data provide a useful resource for a deeper understanding of mucociliary epithelia and demonstrate that DIF-FRAC will be broadly applicable for systematic identification of RNA-associated proteins from embryonic tissues.

In mammals, multiciliated cells (MCCs) line the lumen of the trachea, oviduct, and brain ventricles, where they drive fluid flow across the epithelium. Each MCC population experiences vastly different local environments that may dictate differences in their lifetime and turnover rates. However, with the exception of MCCs in the trachea, the turnover rates of these multiciliated epithelial populations at extended time scales are not well described. Here, using genetic lineage-labeling techniques we provide a direct comparison of turnover rates of MCCs in these three different tissues. We find that oviduct turnover is similar to that in the airway (∼6 months), while multiciliated ependymal cells turnover more slowly.

Apical constriction refers to the active, actomyosin-driven process that reduces apical cell surface area in epithelial cells. Apical constriction is utilized in epithelial morphogenesis during embryonic development in multiple contexts, such as gastrulation, neural tube closure, and organogenesis. Defects in apical constriction can result in congenital birth defects, yet our understanding of the molecular control of apical constriction is relatively limited. To uncover new genetic regulators of apical constriction and gain mechanistic insight into the cell biology of this process, we need reliable assay systems that allow real-time observation and quantification of apical constriction as it occurs and permit gain- and loss-of-function analyses to explore gene function and interaction during apical constriction. In this chapter, we describe using the early Xenopus embryo as an assay system to investigate molecular mechanisms involved in apical constriction during both gastrulation and neurulation.

SummaryConvergent extension (CE) is a collective cell movement required for embryonic axis elongation, neural tube closure, and kidney tubule elongation. During CE, iterative cell intercalations drive the elongation of a tissue 1. In order to move, cells reorganize their actin cytoskeleton; the implication of actin regulators during CE, then, is expected. Interestingly, two other groups of proteins have been shown to be required for this cell behavior: Planar Cell Polarity proteins (PCP)2-4 and Septins5, both of which interact with the actin cytoskeleton 6-14. Disruption of any of these systems have substantial overlap in terms of embryonic phenotypes, but how they interact to govern CE is largely unknown15-17. Here, we find that PCP proteins and Septins sequester actomyosin activity, create flows of actin across the anterior side of cells, and bundle actin into a node and cable system that displays a planar polarized gradient of stiffness.Key findings-PCP- and Septin-dependent actin node and cable structures form and become anteriorized over time-Actin cytoskeletal reorganization correlates with PCP protein polarization.-Actomyosin flows are directed to anterior PCP puncta and PCP movement relies on actin turnover.-Septins localize to anterior nodes and bundle actin filaments into anterior actin cable.-PCP- and Septin-dependent anterior actin cables increase local cellular stiffness and create a planar polarized stiffness gradient across the cell.

Most motile cilia have a stereotyped structure of nine microtubule outer doublets and a single central pair of microtubules. The central pair microtubules are surrounded by a set of proteins, termed the central pair apparatus. A specific kinesin, Klp1 projects from the central pair and contributes to ciliary motility in Chlamydomonas. The vertebrate orthologue, Kif9 is required for beating in mouse sperm flagella, but the mechanism of Kif9/Klp1 function remains poorly defined. Here, using Xenopus epidermal multiciliated cells, we show that Kif9 is necessary for ciliary motility as well as leads to defects in the distal localization of not only central pair proteins, but also radial spokes and dynein arms. In addition, single-molecule assays in vitro revealed that Xenopus Kif9 is a processive motor, though like axonemal dyneins it displays no processivity in ciliary axonemes in vivo. Thus, our data suggest that Kif9 plays both indirect and direct role in ciliary motility.

Frogs are an ecologically diverse and phylogenetically ancient group of living amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., centric) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding, surrounded by pericentromeric LINE/L1 elements. We explored chromosome structure across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (HiC) data for all species. Abundant satellite repeats occupy the unusually long (∼20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible association of centromeric chromatin, and of telomeres, reflecting a Rabl configuration similar to the “bouquet” structure of meiotic cells. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.

Humans have sought to understand the embryo for millennia. Paradoxically, even as technical and intellectual innovations bring us ever closer to a transformative understanding of developmental biology, our discipline faces an "image problem." We should face this problem by acknowledging that developmental biology is fundamental to the human experience.

Oscillatory flows of actomyosin play a key role in the migration of single cells in culture and in collective cell movements in Drosophila embryos. In vertebrate embryos undergoing convergent extension (CE), the Planar Cell Polarity (PCP) pathway drives the elongation of the body axis and shapes the central nervous system, and mutations of the PCP genes predispose humans to various malformations including neural tube defects. However, the spatiotemporal patterns of oscillatory actomyosin contractions during vertebrate CE and how they are controlled by the PCP signaling remain unknown. Here, we address these outstanding issues using a combination of in vivo imaging and mathematical modeling. We find that effective execution of CE requires alternative oscillations of cortical actomyosin across cell membranes of neighboring cells within an optimal frequency range. Intriguingly, temporal and spatial clustering of the core PCP protein Prickle 2 (Pk2) is correlated to submembranous accumulations of F-actin, and depletion of Pk2 perturbs the oscillation of actomyosin contractions. These findings shed light on the significance of temporal regulation of actomyosin contraction by the PCP pathway during CE, in addition to its well-studied spatial aspects.

Thiabendazole (TBZ) is an FDA-approved benzimidazole widely used for its antifungal and antihelminthic properties. We showed previously that TBZ is also a potent vascular disrupting agent and inhibits angiogenesis at the tissue level by dissociating vascular endothelial cells in newly formed blood vessels. Here, we uncover TBZ’s molecular target and mechanism of action. Using human cell culture, molecular modeling, and humanized yeast, we find that TBZ selectively targets only 1 of 9 human β-tubulin isotypes (TUBB8) to specifically disrupt endothelial cell microtubules. By leveraging epidemiological pesticide resistance data and mining chemical features of commercially used benzimidazoles, we discover that a broader class of benzimidazole compounds, in extensive use for 50 years, also potently disrupt immature blood vessels and inhibit angiogenesis. Thus, besides identifying the molecular mechanism of benzimidazole-mediated vascular disruption, this study presents evidence relevant to the widespread use of these compounds while offering potential new clinical applications.

Morphogenesis is governed by the interplay of molecular signals and mechanical forces across multiple length scales. The last decade has seen tremendous advances in our understanding of the dynamics of protein localization and turnover at subcellular length scales, and at the other end of the spectrum, of mechanics at tissue-level length scales. Integrating the two remains a challenge, however, because we lack a detailed understanding of the subcellular patterns of mechanical properties of cells within tissues. Here, in the context of the elongating body axis of Xenopus embryos, we combine tools from cell biology and physics to demonstrate that individual cell-cell junctions display finely-patterned local mechanical heterogeneity along their length. We show that such local mechanical patterning is essential for the cell movements of convergent extension and is imparted by locally patterned clustering of a classical cadherin. Finally, the patterning of cadherins and thus local mechanics along cell-cell junctions are controlled by Planar Cell Polarity signaling, a key genetic module for CE that is mutated in diverse human birth defects.

Intraflagellar transport (IFT) is essential for construction and maintenance of cilia. IFT proteins concentrate at the basal body where they are thought to assemble into trains and bind cargoes for transport. To study the mechanisms of IFT recruitment to this peri-basal body pool, we quantified protein dynamics of eight IFT proteins, as well as five other basal body localizing proteins using fluorescence recovery after photobleaching in vertebrate multiciliated cells. We found that members of the IFT-A and IFT-B protein complexes show distinct turnover kinetics from other basal body components. Additionally, known IFT subcomplexes displayed shared dynamics, suggesting shared basal body recruitment and/or retention mechanisms. Finally, we evaluated the mechanisms of basal body recruitment by depolymerizing cytosolic MTs, which suggested that IFT proteins are recruited to basal bodies through a diffusion-to-capture mechanism. Our survey of IFT protein dynamics provides new insights into IFT recruitment to basal bodies, a crucial step in ciliogenesis and ciliary signaling.

It has not gone unnoticed in recent times that historical writing about science is heavily Eurocentric. A striking example can be found in the history of developmental biology: textbooks and popular science writing frequently trace an intellectual thread from the Greek philosopher Aristotle through 19th century embryology to 20th century genetics. Few in our field are aware of the depth and breadth of early embryological thinking outside of Europe. Here, I provide a series of vignettes highlighting the rich history of embryological thinking in Asia and Latin America. My goal is to provide an entertaining, even provocative, synopsis of this important but under-studied topic. It is my hope that this work will spur others to carry out more thorough investigations, with the ultimate goal of building a more inclusive discipline., Summary: This Review highlights the rich history of embryological thinking in Asia and Latin America, challenging the European-centric view of the history of scientific investigation and discovery in developmental biology.

The design of an animal's body plan is encoded in the genome, and the execution of this program is a mechanical progression involving coordinated movement of proteins, cells, and whole tissues. Thus, a challenge to understanding morphogenesis is connecting events that occur across various length scales. Here, we describe how a poorly characterized adhesion effector, Arvcf catenin, controls Xenopus head-to-tail axis extension. We find that Arvcf is required for axis extension within the intact organism but not within isolated tissues. We show that the organism-scale phenotype results from a defect in tissue-scale force production. Finally, we determine that the force defect results from the dampening of the pulsatile recruitment of cell adhesion and cytoskeletal proteins to membranes. These results provide a comprehensive understanding of Arvcf function during axis extension and produce an insight into how a cellular-scale defect in adhesion results in an organism-scale failure of development.

Background Explanted tissues from vertebrate embryos reliably develop in culture and have provided essential paradigms for understanding embryogenesis, from early embryological investigations of induction, to the extensive study of Xenopus animal caps, to the current studies of mammalian gastruloids. Cultured explants of the Xenopus dorsal marginal zone ("Keller" explants) serve as a central paradigm for studies of convergent extension cell movements, yet we know little about the global patterns of gene expression in these explants. Results In an effort to more thoroughly develop this important model system, we provide here a time-resolved bulk transcriptome for developing Keller explants. Conclusions The dataset reported here provides a useful resource for those using Keller explants for studies of morphogenesis and provide genome-scale insights into the temporal patterns of gene expression in an important tissue when explanted and grown in culture.

Recent studies have revealed that pathogenic mutations in the endocytic receptor LRP2 in humans are associated with severe neural tube closure defects (NTDs) such as anencephaly and spina bifida. Here, we combined analysis of neural tube closure in mouse and in the African Clawed FrogXenopus laevisto elucidate the etiology of Lrp2-related NTDs.Lrp2loss-of-function (LOF) impaired neuroepithelial morphogenesis, culminating in NTDs that impeded anterior neural plate folding and neural tube closure in both model organisms. Loss of Lrp2 severely affected apical constriction as well as proper localization of the core planar cell polarity (PCP) protein Vangl2, demonstrating a highly conserved role of the receptor in these processes essential for neural tube formation. In addition, we identified a novel functional interaction of Lrp2 with the intracellular adaptor proteins Shroom3 and Gipc1 in the developing forebrain. Our data suggest that during neurulation, motifs within the intracellular domain of Lrp2 function as a hub that orchestrates endocytic membrane removal for efficient apical constriction as well as PCP component trafficking in a temporospatial manner.Summary statementAnalysis of neurulation in mouse andXenopusreveals novel roles for Lrp2-mediated endocytosis in orchestrating apical constriction and planar cell polarity essential for neural tube closure.

Failures of neural tube closure are common and serious birth defects, yet we have a poor understanding of the interaction of genetics and cell biology during neural tube closure. Additionally, mutations that cause neural tube defects (NTDs) tend to affect anterior or posterior regions of the neural tube but rarely both, indicating a regional specificity to NTD genetics. To better understand the regional specificity of cell behaviors during neural tube closure, we analyzed the dynamic localization of actin and N-cadherin via high-resolution tissue-level time-lapse microscopy during Xenopus neural tube closure. To investigate the regionality of gene function, we generated mosaic mutations in shroom3, a key regulator or neural tube closure. This new analytical approach elucidates several differences between cell behaviors during cranial/anterior and spinal/posterior neural tube closure, provides mechanistic insight into the function of shroom3, and demonstrates the ability of tissue-level imaging and analysis to generate cell biological mechanistic insights into neural tube closure.

Failures of neural tube closure are common and serious birth defects, yet we have a poor understanding of the interaction of genetics and cell biology during neural tube closure. Additionally, mutations that cause neural tube defects (NTDs) tend to affect anterior or posterior regions of the neural tube but rarely both, indicating a regional specificity to NTD genetics. To better understand the regional specificity of cell behaviors during neural tube closure, we analyzed the dynamic localization of actin and N-cadherin via high-resolution tissue-level time-lapse microscopy duringXenopusneural tube closure. To investigate the regionality of gene function, we generated mosaic mutations inshroom3, a key regulator or neural tube closure This approach elucidates new differences between cell behaviors during cranial/anterior and spinal/posterior neural tube closure, provides mechanistic insight into the function ofshroom3and demonstrates the ability of tissue-level imaging and analysis to generate cell-biological mechanistic insights into neural tube closure.

SummaryAxis extension is a fundamental biological process that shapes multicellular organisms. The design of an animal’s body plan is encoded in the genome and execution of this program is a multiscale mechanical progression involving the coordinated movement of proteins, cells, and whole tissues. Thus, a key challenge to understanding axis extension is connecting events that occur across these various length scales. Here, we use approaches from proteomics, cell biology, and tissue biomechanics to describe how a poorly characterized cell adhesion effector, the Armadillo Repeat protein deleted in Velo-Cardio-Facial syndrome (Arvcf) catenin, controls vertebrate head-to-tail axis extension. We find that Arvcf catenin is required for axis extension within the intact organism but is not required for extension of isolated tissues. We then show that the organism scale phenotype is caused by a modest defect in force production at the tissue scale that becomes apparent when the tissue is challenged by external resistance. Finally, we show that the tissue scale force defect results from dampening of the pulsatile recruitment of cell adhesion and cytoskeletal proteins to cell membranes. These results not only provide a comprehensive understanding of Arvcf function during an essential biological process, but also provide insight into how a modest cellular scale defect in cell adhesion results in an organism scale failure of development.

Intraflagellar transport (IFT) is essential for construction and maintenance of cilia. IFT proteins concentrate at the basal body, where they are thought to assemble into trains and bind cargoes for transport. To study the mechanisms of IFT recruitment to this peri-basal body pool, we quantified protein dynamics of eight IFT proteins, as well as five other basal body localizing proteins, using fluorescence recovery after photobleaching in vertebrate multiciliated cells. We found that members of the IFT-A and IFT-B protein complexes show distinct turnover kinetics from other basal body components. Additionally, known IFT sub-complexes displayed shared dynamics, and these dynamics were not altered during cilia regeneration as compared to homeostasis. Finally, we evaluated the mechanisms of basal body recruitment by depolymerizing cytosolic MTs, which suggested that IFT proteins are recruited to basal bodies through a diffusion-to-capture mechanism. Our survey of IFT protein dynamics provides new insights into IFT recruitment to basal bodies, a crucial step in ciliogenesis.

Developmental disorders, often called ‘birth defects’, represent the number one cause of infant mortality in the USA and are nearly as lethal in the UK and Australia. The incidence of birth defects is highest in countries with the lowest income per capita. Owing to the highly heterogeneous

Summary: This Spotlight summarizes conclusions from The Company of Biologists Workshop ‘Understanding Human Birth Defects in the Genomic Age’ held in the UK in November 2019, identifying key hurdles in the field, as well as priorities for overcoming them.

Explanted tissues from vertebrate embryos reliably develop in culture and have provided essential paradigms for understanding embryogenesis, from early embryological investigations of induction, to the extensive study ofXenopusanimal caps, to the current studies of mammalian gastruloids. Cultured explants of theXenopusdorsal marginal zone (“Keller” explants) serve as a central paradigm for studies of convergent extension cell movements, yet we know little about the global patterns of gene expression in these explants. In an effort to more thoroughly develop this important model system, we provide here a time-resolved bulk transcriptome for developing Keller explants.

Thiabendazole (TBZ) is an FDA-approved benzimidazole widely used for its antifungal and antihelminthic properties. We showed previously that TBZ is also a potent vascular disrupting agent and inhibits angiogenesis at the tissue level by dissociating vascular endothelial cells in newly formed blood vessels. Here, we uncover TBZ’s molecular target and mechanism of action. Using human cell culture, molecular modeling, and humanized yeast, we find that TBZ selectively targets only 1 of 9 human β-tubulin isotypes (TUBB8) to specifically disrupt endothelial cell microtubules. By leveraging epidemiological pesticide resistance data and mining chemical features of commercially used benzimidazoles, we discover that a broader class of benzimidazole compounds, in extensive use for 50 years, also potently disrupt immature blood vessels and inhibit angiogenesis. Thus, besides identifying the molecular mechanism of benzimidazole-mediated vascular disruption, this study presents evidence relevant to the widespread use of these compounds while offering potential new clinical applications.