Showing papers in "Development in 2022"

Cell trajectory modeling identifies a primitive trophoblast state defined by BCAM enrichment.

Abstract: In early placental development, progenitor cytotrophoblasts (CTB) differentiate along one of two cellular trajectories: the villous or extravillous pathways. CTB committed to the villous pathway fuse with neighboring CTB to form the outer multinucleated syncytiotrophoblast (SCT), whereas CTB committed to the extravillous pathway differentiate into invasive extravillous trophoblasts (EVT). Unfortunately, little is known about the processes controlling human CTB progenitor maintenance and differentiation. To address this, we established a single cell RNA sequencing (scRNA-seq) dataset from first trimester placentas to identify cell states important in trophoblast progenitor establishment, renewal and differentiation. Multiple distinct trophoblast states were identified, representing progenitor CTB, column CTB, SCT precursors and EVT. Lineage trajectory analysis identified a progenitor origin that was reproduced in human trophoblast stem cell organoids. Heightened expression of basal cell adhesion molecule (BCAM) defined this primitive state, where BCAM enrichment or gene silencing resulted in enhanced or diminished organoid growth, respectively. Together, this work describes at high-resolution trophoblast heterogeneity within the first trimester, resolves gene networks within human CTB progenitors and identifies BCAM as a primitive progenitor marker and possible regulator.

<i>Arabidopsis</i> root responses to salinity depend on pectin modification and cell wall sensing

Abstract: ABSTRACT Owing to its detrimental effect on plant growth, salinity is an increasing worldwide problem for agriculture. To understand the molecular mechanisms activated in response to salt in Arabidopsis thaliana, we investigated the Catharanthus roseus receptor-like kinase 1-like family, which contains sensors that were previously shown to be involved in sensing the structural integrity of the cell walls. We found that herk1 the1-4 double mutants, lacking the function of HERKULES1 (HERK1) and combined with a gain-of-function allele of THESEUS1 (THE1), strongly respond to salt application, resulting in an intense activation of stress responses, similarly to plants lacking FERONIA (FER) function. We report that salt triggers pectin methyl esterase (PME) activation and show its requirement for the activation of several salt-dependent responses. Because chemical inhibition of PMEs alleviates these salt-induced responses, we hypothesize a model in which salt directly leads to cell wall modifications through the activation of PMEs. Responses to salt partly require the functionality of FER alone or HERK1/THE1 to attenuate salt effects, highlighting the complexity of the salt-sensing mechanisms that rely on cell wall integrity.

The origins and roles of osteoclasts in bone development, homeostasis and repair.

Abstract: The mechanisms underlying bone development, repair and regeneration are reliant on the interplay and communication between osteoclasts and other surrounding cells. Osteoclasts are multinucleated monocyte lineage cells with resorptive abilities, forming the bone marrow cavity during development. This marrow cavity, essential to hematopoiesis and osteoclast-osteoblast interactions, provides a setting to investigate the origin of osteoclasts and their multi-faceted roles. This Review examines recent developments in the embryonic understanding of osteoclast origin, as well as interactions within the immune environment to regulate normal and pathological bone development, homeostasis and repair.

Microglia in brain development and regeneration.

Abstract: It has recently emerged that microglia, the tissue-resident macrophages of the central nervous system, play significant non-innate immune roles to support the development, maintenance, homeostasis and repair of the brain. Apart from being highly specialized brain phagocytes, microglia modulate the development and functions of neurons and glial cells through both direct and indirect interactions. Thus, recognizing the elements that influence the homeostasis and heterogeneity of microglia in normal brain development is crucial to understanding the mechanisms that lead to early disease pathogenesis of neurodevelopmental disorders. In this Review, we discuss recent studies that have elucidated the physiological development of microglia and summarize our knowledge of their non-innate immune functions in brain development and tissue repair.

Dynamic profiling and functional interpretation of histone Kcr and Kla during neural development.

Abstract: Metabolites such as crotonyl-CoA and lactyl-CoA influence gene expression by covalently modifying histones, known as histone lysine crotonylation (Kcr) and lysine lactylation (Kla). However, their existence patterns, dynamic changes, biological functions，and associations with histone lysine acetylation and gene expression during mammalian development remain largely unknown. Here, we find that histone Kcr and Kla are widely distributed in the brain and undergo global changes during neural development. By profiling genome-wide dynamics of H3K9ac, H3K9cr and H3K18la in combination with ATAC and RNA sequencing, we reveal that these marks are tightly correlated with chromatin state and gene expression, and extensively involved in transcriptome remodeling to promote cell-fate transitions in the developing telencephalon. Importantly, we demonstrate that global Kcr and Kla levels are not the consequence of transcription and identify histone deacetylase (HDAC)1-3 as novel "erasers" of H3K18la. Using P19 cells as induced neural differentiation system, we find that HDAC1-3 inhibition by MS-275 pre-activates neuronal transcriptional programs through stimulating multiple histone lysine acylations simultaneously. These findings suggest histone Kcr and Kla play critical roles in the epigenetic regulation of neural development.

Normal Table of Xenopus development: a new graphical resource

Abstract: ABSTRACT Normal tables of development are essential for studies of embryogenesis, serving as an important resource for model organisms, including the frog Xenopus laevis. Xenopus has long been used to study developmental and cell biology, and is an increasingly important model for human birth defects and disease, genomics, proteomics and toxicology. Scientists utilize Nieuwkoop and Faber's classic ‘Normal Table of Xenopus laevis (Daudin)’ and accompanying illustrations to enable experimental reproducibility and reuse the illustrations in new publications and teaching. However, it is no longer possible to obtain permission for these copyrighted illustrations. We present 133 new, high-quality illustrations of X. laevis development from fertilization to metamorphosis, with additional views that were not available in the original collection. All the images are available on Xenbase, the Xenopus knowledgebase (http://www.xenbase.org/entry/zahn.do), for download and reuse under an attributable, non-commercial creative commons license. Additionally, we have compiled a ‘Landmarks Table’ of key morphological features and marker gene expression that can be used to distinguish stages quickly and reliably (https://www.xenbase.org/entry/landmarks-table.do). This new open-access resource will facilitate Xenopus research and teaching in the decades to come.

Gynoecium and fruit development in Arabidopsis.

Abstract: Flowering plants produce flowers and one of the most complex floral structures is the pistil or the gynoecium. All the floral organs differentiate from the floral meristem. Various reviews exist on molecular mechanisms controlling reproductive development, but most focus on a short time window and there has been no recent review on the complete developmental time frame of gynoecium and fruit formation. Here, we highlight recent discoveries, including the players, interactions and mechanisms that govern gynoecium and fruit development in Arabidopsis. We also present the currently known gene regulatory networks from gynoecium initiation until fruit maturation.

Distinct contributions of ECM proteins to basement membrane mechanical properties in Drosophila.

Abstract: The basement membrane is a specialized extracellular matrix (ECM) that is crucial for the development of epithelial tissues and organs. In Drosophila, the mechanical properties of the basement membrane play an important role in the proper elongation of the developing egg chamber; however, the molecular mechanisms contributing to basement membrane mechanical properties are not fully understood. Here, we systematically analyze the contributions of individual ECM components towards the molecular composition and mechanical properties of the basement membrane underlying the follicle epithelium of Drosophila egg chambers. We find that the Laminin and Collagen IV networks largely persist in the absence of the other components. Moreover, we show that Perlecan and Collagen IV, but not Laminin or Nidogen, contribute greatly towards egg chamber elongation. Similarly, Perlecan and Collagen, but not Laminin or Nidogen, contribute towards the resistance of egg chambers against osmotic stress. Finally, using atomic force microscopy we show that basement membrane stiffness mainly depends on Collagen IV. Our analysis reveals how single ECM components contribute to the mechanical properties of the basement membrane controlling tissue and organ shape.

Biology of resident tissue macrophages.

Abstract: Although best known for their phagocytic and immunological functions, macrophages have increasingly been recognised as key players in the development, homeostasis and regeneration of their host tissues. Early during development, macrophages infiltrate and colonise all tissues within the body, developing symbiotically with their host tissues and acquiring unique functional adaptations based on the tissue microenvironment. These embryonic resident tissue macrophages (RTMs) are ontogenically distinct from the later adult bone marrow-derived monocytes, and in some tissues are self-maintained independently of general circulation at a steady state. In this article, we briefly discuss the ontogeny, maintenance and unique tissue adaptions of RTMs focusing on microglia, Kupffer cells, Langerhans cells, intestinal macrophages, cardiac macrophages and tumour-associated macrophages, and highlight their role in development, homeostasis and dysfunction.

Human assembloids.

Abstract: Deconstructing and then reconstructing developmental processes ex vivo is crucial to understanding how organs assemble and how physiology can be disrupted in disease. Human 3D stem cell-derived systems, such as organoids, have facilitated this pursuit; however, they often do not capture inter-tissue or inter-lineage cellular interactions that give rise to emergent tissue properties during development. Assembloids are self-organizing 3D cellular systems that result from the integration of multiple organoids or the combination of organoids with missing cell types or primary tissue explants. Here, we outline the concept and types of assembloids and present their applications for studying the nervous system and other tissues. We describe tools that are used to probe and manipulate assembloids and delineate current challenges and the potential for this new approach to interrogate development and disease.

Development of the mammalian main olfactory bulb

Abstract: ABSTRACT The mammalian main olfactory bulb is a crucial processing centre for the sense of smell. The olfactory bulb forms early during development and is functional from birth. However, the olfactory system continues to mature and change throughout life as a target of constitutive adult neurogenesis. Our Review synthesises current knowledge of prenatal, postnatal and adult olfactory bulb development, focusing on the maturation, morphology, functions and interactions of its diverse constituent glutamatergic and GABAergic cell types. We highlight not only the great advances in the understanding of olfactory bulb development made in recent years, but also the gaps in our present knowledge that most urgently require addressing.

Msl3 promotes germline stem cell differentiation in female <i>Drosophila</i>

Abstract: Gamete formation from germline stem cells (GSCs) is essential for sexual reproduction. However, the regulation of GSC differentiation is incompletely understood. Set2, which deposits H3K36me3 modifications, is required for GSC differentiation during Drosophila oogenesis. We discovered that the H3K36me3 reader Male-specific lethal 3 (Msl3) and histone acetyltransferase complex Ada2a-containing (ATAC) cooperate with Set2 to regulate GSC differentiation in female Drosophila. Msl3, acting independently of the rest of the male-specific lethal complex, promotes transcription of genes, including a germline-enriched ribosomal protein S19 paralog RpS19b. RpS19b upregulation is required for translation of RNA-binding Fox protein 1 (Rbfox1), a known meiotic cell cycle entry factor. Thus, Msl3 regulates GSC differentiation by modulating translation of a key factor that promotes transition to an oocyte fate.

SCARECROW is deployed in distinct contexts during rice and maize leaf development

Abstract: ABSTRACT The flexible deployment of developmental regulators is an increasingly appreciated aspect of plant development and evolution. The GRAS transcription factor SCARECROW (SCR) regulates the development of the endodermis in Arabidopsis and maize roots, but during leaf development it regulates the development of distinct cell types; bundle-sheath in Arabidopsis and mesophyll in maize. In rice, SCR is implicated in stomatal patterning, but it is unknown whether this function is additional to a role in inner leaf patterning. Here, we demonstrate that two duplicated SCR genes function redundantly in rice. Contrary to previous reports, we show that these genes are necessary for stomatal development, with stomata virtually absent from leaves that are initiated after germination of mutants. The stomatal regulator OsMUTE is downregulated in Osscr1;Osscr2 mutants, indicating that OsSCR acts early in stomatal development. Notably, Osscr1;Osscr2 mutants do not exhibit the inner leaf patterning perturbations seen in Zmscr1;Zmscr1h mutants, and Zmscr1;Zmscr1h mutants do not exhibit major perturbations in stomatal patterning. Taken together, these results indicate that SCR was deployed in different developmental contexts after the divergence of rice and maize around 50 million years ago.

Single-cell sequencing reveals PDFGRα+ stromal cell subpopulations that promote proacinar differentiation in embryonic salivary gland organoids.

Abstract: Stromal cells can direct differentiation of epithelial progenitor cells during organ development. FGF signaling is essential for submandibular salivary gland development. Through stromal fibroblast cells, FGF2 can indirectly regulate proacinar cell differentiation in organoids but the mechanisms are not understood. We performed scRNA-Seq and identified multiple stromal cell subsets, including Pdgfrα+ stromal subsets expressing both Fgf2 and Fgf10. When combined with epithelial progenitor cells in organoids, MACS-sorted PDGFRα+ cells promoted proacinar differentiation similarly to total stroma. Gene expression analysis revealed that FGF2 increases multiple stromal genes, including Bmp2 and Bmp7. Both BMP2 and BMP7 synergized with FGF2 stimulating proacinar differentiation, but not branching. However, stromal cells grown without FGF2 did not support proacinar organoid differentiation and instead differentiated into myofibroblasts. In organoids, TGFβ1 treatment stimulate myofibroblast differentiation and inhibited the epithelial progenitor cell's proacinar differentiation. Conversely, FGF2 reversed the TGFβ1's effects. We also demonstrated that adult salivary stromal cells were FGF2 responsive and could promote proacinar differentiation. These FGF2 signaling pathways may have applications in future regenerative therapies.

Intermittent ERK oscillations downstream of FGF in mouse embryonic stem cells

Abstract: Signal transduction networks generate characteristic dynamic activities to process extracellular signals and guide cell fate decisions such as to divide or differentiate. The differentiation of pluripotent cells is controlled by FGF/ERK signaling. However, only a few studies have addressed the dynamic activity of the FGF/ERK signaling network in pluripotent cells at high time resolution. Here, we use live cell sensors in wild-type and Fgf4-mutant mouse embryonic stem cells to measure dynamic ERK activity in single cells, for defined ligand concentrations and differentiation states. These sensors reveal pulses of ERK activity. Pulsing patterns are heterogeneous between individual cells. Consecutive pulse sequences occur more frequently than expected from simple stochastic models. Sequences become more prevalent with higher ligand concentration, but are rarer in more differentiated cells. Our results suggest that FGF/ERK signaling operates in the vicinity of a transition point between oscillatory and non-oscillatory dynamics in embryonic stem cells. The resulting heterogeneous dynamic signaling activities add a new dimension to cellular heterogeneity that may be linked to divergent fate decisions in stem cell cultures.

Human pluripotent stem cell-derived kidney organoids for personalized congenital and idiopathic nephrotic syndrome modeling

Abstract: Nephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a prerequisite to resolve NS pathogenesis. The detailed characterization of organoid podocytes resulting from a hybrid culture protocol showed a podocyte population that resembles adult podocytes and was superior compared with 2D counterparts, based on single-cell RNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling, as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate, puromycin aminonucleoside treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies.

LONP1-mediated mitochondrial quality control safeguards metabolic shifts in heart development.

Abstract: The mitochondrial matrix AAA+ Lon protease (LONP1) degrades misfolded or unassembled proteins, which play a pivotal role in mitochondrial quality control. During heart development, a metabolic shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation takes place, and this process relies highly on functional mitochondria. However, the relationship between mitochondrial quality control machinery and metabolic shifts is elusive. Here, we interfered with mitochondrial quality control by inactivating Lonp1 in embryonic cardiac tissue and found severely impaired heart development, leading to embryonic lethality. Mitochondrial swelling, cristae loss and abnormal protein aggregates were evident in the mitochondria of Lonp1-deficient cardiomyocytes. Accordingly, the p-eIF2α-ATF4 pathway was triggered, and nuclear translocation of ATF4 was observed. We further demonstrated that ATF4 negatively regulates the expression of Tfam while promoting that of Glut1, which was responsible for the disruption of the metabolic shift to oxidative phosphorylation. Meanwhile, elevated levels of reactive oxygen species were observed in Lonp1 mutant cardiomyocytes. This study revealed that LONP1 safeguards metabolic shifts in the developing heart by controlling mitochondrial protein quality and implies that disrupted mitochondrial quality control may cause prenatal cardiomyopathy.

High temperature perception in leaves promotes vascular regeneration and graft formation in distant tissues

Abstract: ABSTRACT Cellular regeneration in response to wounding is fundamental to maintain tissue integrity. Various internal factors including hormones and transcription factors mediate healing, but little is known about the role of external factors. To understand how the environment affects regeneration, we investigated the effects of temperature upon the horticulturally relevant process of plant grafting. We found that elevated temperatures accelerated vascular regeneration in Arabidopsis thaliana and tomato grafts. Leaves were crucial for this effect, as blocking auxin transport or mutating PHYTOCHROME INTERACTING FACTOR 4 (PIF4) or YUCCA2/5/8/9 in the cotyledons abolished the temperature enhancement. However, these perturbations did not affect grafting at ambient temperatures, and temperature enhancement of callus formation and tissue adhesion did not require PIF4, suggesting leaf-derived auxin specifically enhanced vascular regeneration in response to elevated temperatures. We also found that elevated temperatures accelerated the formation of inter-plant vascular connections between the parasitic plant Phtheirospermum japonicum and host Arabidopsis, and this effect required shoot-derived auxin from the parasite. Taken together, our results identify a pathway whereby local temperature perception mediates long distance auxin signaling to modify regeneration, grafting and parasitism. This article has an associated ‘The people behind the papers’ interview.

Stable iPSC-derived NKX2-1+ lung bud tip progenitor organoids give rise to airway and alveolar cell types

Abstract: Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. The current work aimed to develop an iPSC organoid model enriched with stable NKX2-1+ BTP-like cells. Building on prior work, we optimized a directed differentiation paradigm to generate spheroids with robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs can be purified and expanded as induced bud tip organoids (iBTO), which maintain an enriched population of bud tip progenitors. When iBTOs are directed to differentiate into airway or alveolar cell types using well-established methods, they give rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model to study human lung development and differentiation. SUMMARY STATEMENT iPSC-derived lung bud tip progenitors emerge in organoid culture, can be isolated and expanded, are transcriptionally similar to primary bud tip progenitors, and can differentiate into airway or alveolar organoids.

Imaging the onset of oscillatory signaling dynamics during mouse embryo gastrulation

Abstract: ABSTRACT A fundamental requirement for embryonic development is the coordination of signaling activities in space and time. A notable example in vertebrate embryos is found during somitogenesis, where gene expression oscillations linked to the segmentation clock are synchronized across cells in the presomitic mesoderm (PSM) and result in tissue-level wave patterns. To examine their onset during mouse embryo development, we studied the dynamics of the segmentation clock gene Lfng during gastrulation. To this end, we established an imaging setup using selective plane illumination microscopy (SPIM) that enables culture and simultaneous imaging of up to four embryos (‘SPIM- for-4’). Using SPIM-for-4, combined with genetically encoded signaling reporters, we detected the onset of Lfng oscillations within newly formed mesoderm at presomite stages. Functionally, we found that initial synchrony and the first ∼6-8 oscillation cycles occurred even when Notch signaling was impaired, revealing similarities to previous findings made in zebrafish embryos. Finally, we show that a spatial period gradient is present at the onset of oscillatory activity, providing a potential mechanism accounting for our observation that wave patterns build up gradually over the first oscillation cycles.

Identification of enhancer regulatory elements that direct epicardial gene expression during zebrafish heart regeneration.

Abstract: The epicardium is a mesothelial tissue layer that envelops the heart. Cardiac injury activates dynamic gene expression programs in epicardial tissue, which in zebrafish enables subsequent regeneration through paracrine and vascularizing effects. To identify tissue regeneration enhancer elements (TREEs) that control injury-induced epicardial gene expression during heart regeneration, we profiled transcriptomes and chromatin accessibility in epicardial cells purified from regenerating zebrafish hearts. We identified hundreds of candidate TREEs, which are defined by increased chromatin accessibility of non-coding elements near genes with increased expression during regeneration. Several of these candidate TREEs were incorporated into stable transgenic lines, with five out of six elements directing injury-induced epicardial expression but not ontogenetic epicardial expression in larval hearts. Whereas two independent TREEs linked to the gene gnai3 showed similar functional features of gene regulation in transgenic lines, two independent ncam1a-linked TREEs directed distinct spatiotemporal domains of epicardial gene expression. Thus, multiple TREEs linked to a regeneration gene can possess either matching or complementary regulatory controls. Our study provides a new resource and principles for understanding the regulation of epicardial genetic programs during heart regeneration. This article has an associated 'The people behind the papers' interview.

Immune cells in cardiac repair and regeneration

Abstract: ABSTRACT The immune system is fundamental to tissue homeostasis and is the first line of defense following infection, injury or disease. In the damaged heart, large numbers of immune cells are recruited to the site of injury. These cells play an integral part in both repair by scar formation and the initiation of tissue regeneration. They initially assume inflammatory phenotypes, releasing pro-inflammatory cytokines and removing dead and dying tissue, before entering a reparative stage, replacing dead muscle tissue with a non-contractile scar. In this Review, we present an overview of the innate and adaptive immune response to heart injury. We explore the kinetics of immune cell mobilization following cardiac injury and how the different innate and adaptive immune cells interact with one another and with the damaged tissue. We draw on key findings from regenerative models, providing insight into how to support a robust immune response permissible for cardiac regeneration. Finally, we consider how the latest technological developments can offer opportunities for a deeper and unbiased functional understanding of the immune response to heart disease, highlighting the importance of such knowledge as the basis for promoting regeneration following cardiac injury in human patients.

ABI transcription factors and PROTEIN L-ISOASPARTYL METHYLTRANSFERASE module mediate seed desiccation tolerance and longevity in Oryza sativa.

Abstract: In contrast to desiccation-tolerant orthodox seeds, recalcitrant seeds are desiccation sensitive and are unable to survive for a prolonged time. Here, our analyses of Oryza species with contrasting seed desiccation tolerance reveals that PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT), an enzyme that repairs abnormal isoaspartyl (isoAsp) residues in proteins, acts as a key player that governs seed desiccation tolerance to orthodox seeds but is ineffective in recalcitrant seeds. We observe that, unlike the orthodox seed of Oryza sativa, desiccation intolerance of the recalcitrant seeds of Oryza coarctata are linked to reduced PIMT activity and increased isoAsp accumulation due to the lack of coordinated action of ABA and ABI transcription factors to upregulate PIMT during maturation. We show that suppression of PIMT reduces, and its overexpression increases, seed desiccation tolerance and seed longevity in O. sativa. Our analyses further reveal that the ABI transcription factors undergo isoAsp formation that affect their functional competence; however, PIMT interacts with and repairs isoAsp residues and facilitates their functions. Our results thus illustrate a new insight into the mechanisms of acquisition of seed desiccation tolerance and longevity by ABI transcription factors and the PIMT module.

PRDM paralogs antagonistically balance Wnt/β-catenin activity during craniofacial chondrocyte differentiation.

Abstract: Cranial neural crest (NCC)-derived chondrocyte precursors undergo a dynamic differentiation and maturation process to establish a scaffold for subsequent bone formation, alterations in which contribute to congenital birth defects. Here, we demonstrate that transcription factor and histone methyltransferase proteins Prdm3 and Prdm16 control the differentiation switch of cranial NCCs to craniofacial cartilage. Loss of either paralog results in hypoplastic and unorganized chondrocytes due to impaired cellular orientation and polarity. We show that PRDMs regulate cartilage differentiation by controlling the timing of Wnt/β-catenin activity in strikingly different ways: Prdm3 represses while Prdm16 activates global gene expression, though both by regulating Wnt enhanceosome activity and chromatin accessibility. Finally, we show that manipulating Wnt/β-catenin signaling pharmacologically or generating prdm3-/-;prdm16-/- double mutants rescues craniofacial cartilage defects. Our findings reveal upstream regulatory roles for Prdm3 and Prdm16 in cranial NCCs to control Wnt/β-catenin transcriptional activity during chondrocyte differentiation to ensure proper development of the craniofacial skeleton.

Lmo7 recruits myosin II heavy chain to regulate actomyosin contractility and apical domain size in Xenopus ectoderm.

Abstract: Apical constriction, or reduction of the apical domain, underlies many morphogenetic events during development. Actomyosin complexes play an essential role in apical constriction, however the detailed analysis of molecular mechanisms is still pending. Here we show that Lim domain only protein 7 (Lmo7), a multidomain adaptor at apical junctions, promotes apical constriction in the Xenopus superficial ectoderm, whereas apical domain size increases in Lmo7-depleted cells. Lmo7 is primarily localized at apical junctions and promotes the formation of the dense circumferential actomyosin belt. Strikingly, Lmo7 binds non-muscle myosin II (NMII) and recruits it to apical junctions and the apical cortex. This NMII recruitment is essential for Lmo7-mediated apical constriction. Lmo7 knockdown decreases NMIIA localization at apical junctions and delays neural tube closure in Xenopus embryos. Our findings suggest that Lmo7 serves as a scaffold regulating actomyosin contractility and apical domain size.

C2CD6 regulates targeting and organization of the CatSper calcium channel complex in sperm flagella

Abstract: The CatSper cation channel is essential for sperm capacitation and male fertility. The multi-subunit CatSper complexes form highly organized calcium signaling nanodomains on flagellar membranes. Here, we report identification of an uncharacterized protein, C2CD6, as a subunit of the mouse CatSper complex. C2CD6 contains a calcium-dependent, membrane-targeting C2 domain. C2CD6 associates with the CatSper calcium-selective, core-forming subunits. Deficiency of C2CD6 depletes the CatSper nanodomains from the flagellum and results in male sterility. C2CD6-deficient sperm are defective in hyperactivation and fail to fertilize oocytes both in vitro and in vivo. CatSper currents are present but at a significantly lower level in C2CD6-deficient sperm. Transient treatments with either Ca2+ ionophore, starvation, or a combination of both restore the fertilization capacity of C2CD6-deficient sperm. C2CD6 interacts with EFCAB9, a pH-dependent calcium sensor in the CatSper complex. We postulate that C2CD6 facilitates incorporation of the CatSper complex into the flagellar plasma membrane and may function as a calcium sensor. The identification of C2CD6 may enable the long-sought reconstitution of the CatSper ion channel complex in a heterologous system for male contraceptive development.

Zebrafish vascular quantification: a tool for quantification of three-dimensional zebrafish cerebrovascular architecture by automated image analysis

Abstract: ABSTRACT Zebrafish transgenic lines and light sheet fluorescence microscopy allow in-depth insights into three-dimensional vascular development in vivo. However, quantification of the zebrafish cerebral vasculature in 3D remains highly challenging. Here, we describe and test an image analysis workflow for 3D quantification of the total or regional zebrafish brain vasculature, called zebrafish vasculature quantification (ZVQ). It provides the first landmark- or object-based vascular inter-sample registration of the zebrafish cerebral vasculature, producing population average maps allowing rapid assessment of intra- and inter-group vascular anatomy. ZVQ also extracts a range of quantitative vascular parameters from a user-specified region of interest, including volume, surface area, density, branching points, length, radius and complexity. Application of ZVQ to 13 experimental conditions, including embryonic development, pharmacological manipulations and morpholino-induced gene knockdown, shows that ZVQ is robust, allows extraction of biologically relevant information and quantification of vascular alteration, and can provide novel insights into vascular biology. To allow dissemination, the code for quantification, a graphical user interface and workflow documentation are provided. Together, ZVQ provides the first open-source quantitative approach to assess the 3D cerebrovascular architecture in zebrafish.

Rectified random cell motility as a mechanism for embryo elongation.

Abstract: The body of vertebrate embryos forms by posterior elongation from a terminal growth zone called the tail bud. The tail bud is a source of highly motile cells that eventually constitute the presomitic mesoderm (PSM), a tissue that plays an important role in elongation movements. PSM cells establish an anterior-posterior cell motility gradient that parallels a gradient associated with the degradation of a specific cellular signal (FGF) known to be implicated in cell motility. Here, we combine the electroporation of fluorescent reporters in the PSM with time-lapse imaging in the chicken embryo to quantify cell diffusive movements along the motility gradient. We show that a simple microscopic model for random cell motility induced by FGF activity along with geometric confinement leads to rectified tissue elongation consistent with our observations. A continuum analog of the microscopic model leads to a macroscopic mechano-chemical model for tissue extension that couples FGF activity-induced cell motility and tissue rheology, and is consistent with the experimentally observed speed and extent of elongation. Together, our experimental observations and theoretical models explain how the continuous addition of cells at the tail bud combined with lateral confinement can be converted into oriented movement and drive body elongation.

Reprogramming cellular identity in vivo.

Abstract: Cellular identity is established through complex layers of genetic regulation, forged over a developmental lifetime. An expanding molecular toolbox is allowing us to manipulate these gene regulatory networks in specific cell types in vivo. In principle, if we found the right molecular tricks, we could rewrite cell identity and harness the rich repertoire of possible cellular functions and attributes. Recent work suggests that this rewriting of cell identity is not only possible, but that newly induced cells can mitigate disease phenotypes in animal models of major human diseases. So, is the sky the limit, or do we need to keep our feet on the ground? This Spotlight synthesises key concepts emerging from recent efforts to reprogramme cellular identity in vivo. We provide our perspectives on recent controversies in the field of glia-to-neuron reprogramming and identify important gaps in our understanding that present barriers to progress.

Development of a 3D atlas of the embryonic pancreas for topological and quantitative analysis of heterologous cell interactions

Abstract: Generating comprehensive image maps, while preserving spatial three-dimensional (3D) context, is essential in order to locate and assess quantitatively specific cellular features and cell-cell interactions during organ development. Despite recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on two-dimensional histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in three dimensions and map tissue interactions at key time points in the mouse embryo. We demonstrate the utility of the approach by providing volumetric data, 3D distribution of three main cellular components (epithelial, mesenchymal and endothelial cells) within the developing pancreas, and quantification of their relative cellular abundance within the tissue. Interestingly, our 3D images show that endocrine cells are constantly and increasingly in contact with endothelial cells forming small vessels, whereas the interactions with mesenchymal cells decrease over time. These findings suggest distinct cell-cell interaction requirements for early endocrine cell specification and late differentiation. Lastly, we combine our image data in an open-source online repository (referred to as the Pancreas Embryonic Cell Atlas).