Mitochondrial dynamics regulate cell morphology in the developing cochlea.

In multicellular tissues, the size and shape of cells are intricately linked with their physiological functions. In the vertebrate auditory organ, the neurosensory epithelium develops as a mosaic of sensory hair cells (HCs), and their glial-like supporting cells, which have distinct morphologies and functional properties at different frequency positions along its tonotopic long axis. In the chick cochlea, the basilar papilla (BP), proximal (high-frequency) HCs, are larger than their distal (low-frequency) counterparts, a morphological feature essential for sound perception. Mitochondrial dynamics, which constitute the equilibrium between fusion and fission, regulate differentiation and functional refinement across a variety of cell types. We investigate this as a potential mechanism for regulating the shape of developing HCs. Using live imaging in intact BP explants, we identify distinct remodelling of mitochondrial networks in proximal compared with distal HCs. Manipulating mitochondrial dynamics in developing HCs alters their normal morphology along the proximal-distal (tonotopic) axis. Inhibition of the mitochondrial fusion machinery decreased proximal HC surface area, whereas promotion of fusion increased the distal HC surface area. We identify mitochondrial dynamics as a key regulator of HC morphology in developing inner ear epithelia.

Notch signalling influences cell fate decisions and HOX gene induction in axial progenitors.

The generation of the post-cranial embryonic body relies on the coordinated production of spinal cord neurectoderm and presomitic mesoderm cells from neuromesodermal progenitors (NMPs). This process is orchestrated by pro-neural and pro-mesodermal transcription factors that are co-expressed in NMPs together with Hox genes, which are essential for axial allocation of NMP derivatives. NMPs reside in a posterior growth region, which is marked by the expression of Wnt, FGF and Notch signalling components. Although the importance of Wnt and FGF in influencing the induction and differentiation of NMPs is well established, the precise role of Notch remains unclear. Here, we show that the Wnt/FGF-driven induction of NMPs from human embryonic stem cells (hESCs) relies on Notch signalling. Using hESC-derived NMPs and chick embryo grafting, we demonstrate that Notch directs a pro-mesodermal character at the expense of neural fate. We show that Notch also contributes to activation of HOX gene expression in human NMPs, partly in a non-cell-autonomous manner. Finally, we provide evidence that Notch exerts its effects via the establishment of a negative-feedback loop with FGF signalling.

A chemo-mechanical model of endoderm movements driving elongation of the amniote hindgut.

Although mechanical and biochemical descriptions of development are each essential, integration of upstream morphogenic cues with downstream tissue mechanics remains understudied during vertebrate morphogenesis. Here, we developed a two-dimensional chemo-mechanical model to investigate how mechanical properties of the endoderm and transport properties of fibroblast growth factor (FGF) regulate avian hindgut morphogenesis in a coordinated manner. Posterior endoderm cells convert a gradient of FGF ligands into a contractile force gradient, leading to a force imbalance that drives collective cell movements that elongate the forming hindgut tube. We formulated a 2D reaction-diffusion-advection model describing the formation of an FGF protein gradient as a result of posterior displacement of cells transcribing unstable Fgf8 mRNA during axis elongation, coupled with translation, diffusion and degradation of FGF protein. The endoderm was modeled as an active viscous fluid that generates contractile stresses in proportion to FGF concentration. With parameter values constrained by experimental data, the model replicates key aspects of hindgut morphogenesis, suggests that graded isotropic contraction is sufficient to generate large anisotropic cell movements, and provides new insight into how chemo-mechanical coupling across the mesoderm and endoderm coordinates hindgut elongation with axis elongation.

CNKSR2, a downstream mediator of retinoic acid signaling, modulates the Ras/Raf/MEK pathway to regulate patterning and invagination of the chick forebrain roof plate.

During embryonic development, the forebrain roof plate undergoes invagination, leading to separation of the cerebral hemispheres. Any defects in this process, in humans, lead to middle interhemispheric holoprosencephaly (MIH-HPE). In this study, we have identified a previously unreported downstream mediator of retinoic acid (RA) signaling, CNKSR2, which is expressed in the forebrain roof plate in the chick embryo. Knockdown of CNKSR2 affects invagination, cell proliferation and patterning of the roof plate, similar to the phenotypes observed upon inhibition of RA signaling. We further demonstrate that CNKSR2 functions by modulating the Ras/Raf/MEK signaling. This appears to be crucial for patterning of the forebrain roof plate and its subsequent invagination, leading to the formation of the cerebral hemispheres. Thus, a set of novel molecular players have been identified that regulate the morphogenesis of the avian forebrain.

Accurate staging of chick embryonic tissues via deep learning of salient features.

Recent work shows that the developmental potential of progenitor cells in the HH10 chick brain changes rapidly, accompanied by subtle changes in morphology. This demands increased temporal resolution for studies of the brain at this stage, necessitating precise and unbiased staging. Here, we investigated whether we could train a deep convolutional neural network to sub-stage HH10 chick brains using a small dataset of 151 expertly labelled images. By augmenting our images with biologically informed transformations and data-driven preprocessing steps, we successfully trained a classifier to sub-stage HH10 brains to 87.1% test accuracy. To determine whether our classifier could be generally applied, we re-trained it using images (269) of randomised control and experimental chick wings, and obtained similarly high test accuracy (86.1%). Saliency analyses revealed that biologically relevant features are used for classification. Our strategy enables training of image classifiers for various applications in developmental biology with limited microscopy data.

Aquaporin regulates cell rounding through vacuole formation during endothelial-to-hematopoietic transition.

Endothelial-to-hematopoietic transition (EHT) is crucial for hematopoietic stem cell (HSC) generation. During EHT, the morphology of hemogenic endothelial cells (HECs) changes from flat and adherent to spherical hematopoietic cells, which detach from the dorsal aorta. HECs attain a rounded shape in a mitosis-independent manner before cell adhesion termination, suggesting an atypical cell-rounding mechanism. However, the direct mechanisms underlying this change in cell morphology during EHT remain unclear. Here, we show that large vacuoles were transiently formed in avian HECs, and that aquaporin 1 (AQP1) was localized in the vacuole and plasma membranes. Overexpression of AQP1 in non-HECs induced ectopic vacuole expansion, cell rounding and subsequent cell detachment from the endothelium into the bloodstream, mimicking EHT. Loss of redundant AQP functions by CRISPR/Cas9 gene editing in HECs impeded the morphological EHT. Our findings provide the first evidence to indicate that morphological segregation of hematopoietic cells from endothelial cells is regulated by water influx into vacuoles. These findings provide important insights for further exploration of the mechanisms underlying cell/tissue morphogenesis through water-adoptive cellular responses.

Neurogenesis redirects ß-catenin from adherens junctions to the nucleus to promote axonal growth.

Here, we show that, in the developing spinal cord, after the early Wnt-mediated Tcf transcription activation that confers dorsal identity to neural stem cells, neurogenesis redirects ß-catenin from the adherens junctions to the nucleus to stimulate Tcf-dependent transcription in a Wnt-independent manner. This new ß-catenin activity regulates genes implicated in several aspects of contralateral axon growth, including axon guidance and adhesion. Using live imaging of ex-vivo chick neural tube, we showed that the nuclear accumulation of ß-catenin and the rise in Tcf-dependent transcription both initiate before the dismantling of the adherens junctions and remain during the axon elongation process. Notably, we demonstrated that ß-catenin activity in post-mitotic cells depends on TCF7L2 and is central to spinal commissural axon growth. Together, our results reveal Wnt-independent Tcf/ß-catenin regulation of genes that control the growth and guidance of commissural axons in chick spinal cord.

Tlx3 mediates neuronal differentiation and proper condensation of the developing trigeminal ganglion.

The trigeminal ganglion, the largest of the vertebrate cranial ganglia, is comprised of sensory neurons that relay sensations of pain, touch, and temperature to the brain. These neurons are derived from two embryonic cell types, the neural crest and ectodermal placodes, whose interactions are critical for proper ganglion formation. While the T-cell leukemia homeobox 3 (Tlx3) gene is known to be expressed in placodally-derived sensory neurons and necessary for their differentiation, little was known about Tlx3 expression and/or function in the neural crest-derived component of the developing trigeminal ganglion. By combining lineage labeling with in situ hybridization in the chick embryo, we show that neural crest-derived cells that contribute to the cranial trigeminal ganglion express Tlx3 at a time point that coincides with the onset of ganglion condensation. Importantly, loss of Tlx3 function in vivo diminishes the overall size and abundance of neurons within the trigeminal ganglion. Conversely, ectopic expression of Tlx3 in migrating cranial neural crest results in their premature neuronal differentiation. Taken together, our results demonstrate a critical role for Tlx3 in neural crest-derived cells during chick trigeminal gangliogenesis.

Voltage-gated ion channel's gene expression in the myocardium of embryo and adult chickens.

The functioning of the cardiovascular system is critical for embryo survival. Cardiac contractions depend on the sequential activation of different classes of voltage-gated ion channels. Understanding the fundamental features of these interactions is important for identifying the mechanisms of pathologies development in the myocardium. However, at present there is no consensus on which ion channels are involved in the formation of automaticity in the early embryonic stages. The aim of this study was to elucidate the expression of genes encoding various types of ion channels that are involved in the generation of electrical activity chicken heart at different stages of ontogenesis. We analyzed the expression of 14 genes from different families of ion channels. It was revealed that the expression profiles of ion channel genes change depending on the stages of ontogenesis. The HCN4, CACNA1D, SCN1A, SCN5A, KCNA1 genes have maximum expression at the tubular heart stage. In adult, a switch occurs to the higher expression of CACNA1C, KCNH6, RYR and SLC8A1 genes. This data correlated with the results obtained by the microelectrode method. It can be assumed that the automaticity of the tubular heart is mainly due to the mechanism of the «membrane-clock¼ (hyperpolarization-activated current (If), Ca2+-current L-type (ICaL), Na+-current (INa) and the slow component of the delayed rectifier K+-current (IKs)). Whereas in adult birds, the mechanism for generating electrical impulses is determined by both « membrane- clock¼ and «Ca2+-clock¼.

Transcriptomic Perspectives on Neocortical Structure, Development, Evolution, and Disease.

The cerebral cortex is the source of our most complex cognitive capabilities and a vulnerable target of many neurological and neuropsychiatric disorders. Transcriptomics offers a new approach to understanding the cortex at the level of its underlying genetic code, and rapid technological advances have propelled this field to the high-throughput study of the complete set of transcribed genes at increasingly fine resolution to the level of individual cells. These tools have revealed features of the genetic architecture of adult cortical areas, layers, and cell types, as well as spatiotemporal patterning during development. This has allowed a fresh look at comparative anatomy as well, illustrating surprisingly large differences between mammals while at the same time revealing conservation of some features from avians to mammals. Finally, transcriptomics is fueling progress in understanding the causes of neurodevelopmental diseases such as autism, linking genetic association studies to specific molecular pathways and affected brain regions.

Rectified random cell motility as a mechanism for embryo elongation.

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.

Fatty acid-binding proteins and fatty acid synthase influence glial reactivity and promote the formation of Müller glia-derived progenitor cells in the chick retina.

A recent comparative transcriptomic study of Müller glia (MG) in vertebrate retinas revealed that fatty acid binding proteins (FABPs) are among the most highly expressed genes in chick ( Hoang et al., 2020). Here, we investigate how FABPs and fatty acid synthase (FASN) influence glial cells in the chick retina. During development, FABP7 is highly expressed by retinal progenitor cells and maturing MG, whereas FABP5 is upregulated in maturing MG. PMP2 (FABP8) is expressed by oligodendrocytes and FABP5 is expressed by non-astrocytic inner retinal glial cells, and both of these FABPs are upregulated by activated MG. In addition to suppressing the formation of Müller glia-derived progenitor cells (MGPCs), we find that FABP-inhibition suppresses the proliferation of microglia. FABP-inhibition induces distinct changes in single cell transcriptomic profiles, indicating transitions of MG from resting to reactive states and suppressed MGPC formation, with upregulation of gene modules for gliogenesis and decreases in neurogenesis. FASN-inhibition increases the proliferation of microglia and suppresses the formation of MGPCs. We conclude that fatty acid metabolism and cell signaling involving fatty acids are important in regulating the reactivity and dedifferentiation of MG, and the proliferation of microglia and MGPCs.

'Neighbourhood watch' model: embryonic epiblast cells assess positional information in relation to their neighbours.

In many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret these. Using experimental manipulation of early chick embryos, including misexpression of an inducer (VG1 or ACTIVIN) and an inhibitor (BMP4), we test two alternative models for their ability to explain how the site of primitive streak formation is positioned relative to the rest of the embryo. In one model, cells read morphogen concentrations cell-autonomously. In the other, cells sense changes in morphogen status relative to their neighbourhood. We find that only the latter model can account for the experimental results, including some counter-intuitive predictions. This mechanism (which we name the 'neighbourhood watch' model) illuminates the classic 'French Flag Problem' and how positional information is interpreted by a sheet of cells in a large developing system.

High-resolution ultrasound and speckle tracking: a non-invasive approach to assess in vivo gastrointestinal motility during development.

Gastrointestinal motor activity has been extensively studied in adults; however, only few studies have investigated fetal motor skills. It is unknown when the gastrointestinal tract starts to contract during the embryonic period and how this function evolves during development. Here, we adapted a non-invasive high-resolution echography technique combined with speckle tracking analysis to examine the gastrointestinal tract motor activity dynamics during chick embryo development. We provided the first recordings of fetal gastrointestinal motility in living embryos without anesthesia. We found that, although gastrointestinal contractions appear very early during development, they become synchronized only at the end of the fetal period. To validate this approach, we used various pharmacological inhibitors and BAPX1 gene overexpression in vivo. We found that the enteric nervous system determines the onset of the synchronized contractions in the stomach. Moreover, alteration of smooth muscle fiber organization led to an impairment of this functional activity. Altogether, our findings show that non-invasive high-resolution echography and speckle tracking analysis allows visualization and quantification of gastrointestinal motility during development and highlight the progressive acquisition of functional and coordinated gastrointestinal motility before birth.

Single-cell imaging of the cell cycle reveals CDC25B-induced heterogeneity of G1 phase length in neural progenitor cells.

Although lengthening of the cell cycle and G1 phase is a generic feature of tissue maturation during development, the underlying mechanism remains poorly understood. Here, we develop a time-lapse imaging strategy to measure the four cell cycle phases in single chick neural progenitor cells in their endogenous environment. We show that neural progenitors are widely heterogeneous with respect to cell cycle length. This variability in duration is distributed over all phases of the cell cycle, with the G1 phase contributing the most. Within one cell cycle, each phase duration appears stochastic and independent except for a correlation between S and M phase duration. Lineage analysis indicates that the majority of daughter cells may have a longer G1 phase than mother cells, suggesting that, at each cell cycle, a mechanism lengthens the G1 phase. We identify that the CDC25B phosphatase known to regulate the G2/M transition indirectly increases the duration of the G1 phase, partly through delaying passage through the restriction point. We propose that CDC25B increases the heterogeneity of G1 phase length, revealing a previously undescribed mechanism of G1 lengthening that is associated with tissue development.

Mirk/Dyrk1B controls ventral spinal cord development via Shh pathway.

Cross-talk between Mirk/Dyrk1B kinase and Sonic hedgehog (Shh)/Gli pathway affects physiology and pathology. Here, we reveal a novel role for Dyrk1B in regulating ventral progenitor and neuron subtypes in the embryonic chick spinal cord (SC) via the Shh pathway. Using in ovo gain-and-loss-of-function approaches at E2, we report that Dyrk1B affects the proliferation and differentiation of neuronal progenitors at E4 and impacts on apoptosis specifically in the motor neuron (MN) domain. Especially, Dyrk1B overexpression decreases the numbers of ventral progenitors, MNs, and V2a interneurons, while the pharmacological inhibition of endogenous Dyrk1B kinase activity by AZ191 administration increases the numbers of ventral progenitors and MNs. Mechanistically, Dyrk1B overexpression suppresses Shh, Gli2 and Gli3 mRNA levels, while conversely, Shh, Gli2 and Gli3 transcription is increased in the presence of Dyrk1B inhibitor AZ191 or Smoothened agonist SAG. Most importantly, in phenotype rescue experiments, SAG restores the Dyrk1B-mediated dysregulation of ventral progenitors. Further at E6, Dyrk1B affects selectively the medial lateral motor neuron column (LMCm), consistent with the expression of Shh in this region. Collectively, these observations reveal a novel regulatory function of Dyrk1B kinase in suppressing the Shh/Gli pathway and thus affecting ventral subtypes in the developing spinal cord. These data render Dyrk1B a possible therapeutic target for motor neuron diseases.

DLX gene expression in the developing chick pharyngeal arches and relationship to endothelin signaling and avian jaw patterning.

BACKGROUND: A hinged jaw that articulates with the skull base is a striking feature of the vertebrate head and has been greatly modified between, and within, vertebrate classes. Genes belonging to the DLX homeobox family are conserved mediators of local signaling pathways that distinguish the dorsal and ventral aspects of the first pharyngeal arch. Specifically, a subset of DLX genes are expressed in the cranial neural crest-derived mandibular ectomesenchyme in response to ventral endothelin signaling, an important step that confers the first arch with maxillary and mandibular identities. Downstream targets of DLX genes then execute the morphogenetic processes that lead to functional jaws. Identifying lineage-specific variations in DLX gene expression and the regulatory networks downstream of DLX action is necessary to understand how different kinds of jaws evolved. RESULTS: Here, we describe and compare the expression of all six DLX genes in the chick pharyngeal arches, focusing on the period of active patterning in the first arch. Disruption of endothelin signaling results in the down-regulation of ventral-specific DLX genes and confirms their functional role in avian jaw patterning. CONCLUSIONS: This expression resource will be important for comparative embryology and for identifying synexpression groups of DLX-regulated genes in the chick.

Synchronisation of apical constriction and cell cycle progression is a conserved behaviour of pseudostratified neuroepithelia informed by their tissue geometry.

Neuroepithelial cells balance tissue growth requirement with the morphogenetic imperative of closing the neural tube. They apically constrict to generate mechanical forces which elevate the neural folds, but are thought to apically dilate during mitosis. However, we previously reported that mitotic neuroepithelial cells in the mouse posterior neuropore have smaller apical surfaces than non-mitotic cells. Here, we document progressive apical enrichment of non-muscle myosin-II in mitotic, but not non-mitotic, neuroepithelial cells with smaller apical areas. Live-imaging of the chick posterior neuropore confirms apical constriction synchronised with mitosis, reaching maximal constriction by anaphase, before division and re-dilation. Mitotic apical constriction amplitude is significantly greater than interphase constrictions. To investigate conservation in humans, we characterised early stages of iPSC differentiation through dual SMAD-inhibition to robustly produce pseudostratified neuroepithelia with apically enriched actomyosin. These cultured neuroepithelial cells achieve an equivalent apical area to those in mouse embryos. iPSC-derived neuroepithelial cells have large apical areas in G2 which constrict in M phase and retain this constriction in G1/S. Given that this differentiation method produces anterior neural identities, we studied the anterior neuroepithelium of the elevating mouse mid-brain neural tube. Instead of constricting, mid-brain mitotic neuroepithelial cells have larger apical areas than interphase cells. Tissue geometry differs between the apically convex early midbrain and flat posterior neuropore. Culturing human neuroepithelia on equivalently convex surfaces prevents mitotic apical constriction. Thus, neuroepithelial cells undergo high-amplitude apical constriction synchronised with cell cycle progression but the timing of their constriction if influenced by tissue geometry.

Regulation of aortic morphogenesis and VE-cadherin dynamics by VEGF.

In amniote vertebrates, the definitive dorsal aorta is formed by the fusion of two primordial aortic endothelial tubes. Formation of the definitive dorsal aorta requires extensive cellular migrations and rearrangements of the primordial tubes in order to generate a single vessel located at the embryonic ventral midline. This study examines the role of VEGF signaling in the generation of the definitive dorsal aorta. Through gain- and loss-of-function studies in vivo in the chick embryo, we document a requirement for VEGF signaling in growth and remodeling of the paired primordia. We find that regions of the aorta are differentially sensitive to levels of VEGF signaling, and present evidence that areas of low blood flow are more sensitive to the loss of VEGF signaling. We also find that VEGF signaling regulates the intracellular distribution between membrane and cytoplasm of the cell-cell adhesion molecule VE-cadherin in aortic endothelial cells in vivo. Together, these finding identify mechanisms that likely contribute to the dynamic behavior of endothelial cells during aorta morphogenesis.

Single-cell reconstruction with spatial context of migrating neural crest cells and their microenvironments during vertebrate head and neck formation.

The dynamics of multipotent neural crest cell differentiation and invasion as cells travel throughout the vertebrate embryo remain unclear. Here, we preserve spatial information to derive the transcriptional states of migrating neural crest cells and the cellular landscape of the first four chick cranial to cardiac branchial arches (BA1-4) using label-free, unsorted single-cell RNA sequencing. The faithful capture of branchial arch-specific genes led to identification of novel markers of migrating neural crest cells and 266 invasion genes common to all BA1-4 streams. Perturbation analysis of a small subset of invasion genes and time-lapse imaging identified their functional role to regulate neural crest cell behaviors. Comparison of the neural crest invasion signature to other cell invasion phenomena revealed a shared set of 45 genes, a subset of which showed direct relevance to human neuroblastoma cell lines analyzed after exposure to the in vivo chick embryonic neural crest microenvironment. Our data define an important spatio-temporal reference resource to address patterning of the vertebrate head and neck, and previously unidentified cell invasion genes with the potential for broad impact.