Phase contrast micro-CT with adjustable in-slice spatial resolution at constant magnification.

Objective.To report on a micro computed tomography (micro-CT) system capable of x-ray phase contrast imaging and of increasing spatial resolution at constant magnification.Approach.The micro-CT system implements the edge illumination (EI) method, which relies on two absorbing masks with periodically spaced transmitting apertures in the beam path; these split the beam into an array of beamlets and provide sensitivity to the beamlets' directionality, i.e. refraction. In EI, spatial resolution depends on the width of the beamlets rather than on the source/detector point spread function (PSF), meaning that resolution can be increased by decreasing the mask apertures, without changing the source/detector PSF or the magnification.Main results.We have designed a dedicated mask featuring multiple bands with differently sized apertures and used this to demonstrate that resolution is a tuneable parameter in our system, by showing that increasingly small apertures deliver increasingly detailed images. Phase contrast images of a bar pattern-based resolution phantom and a biological sample (a mouse embryo) were obtained at multiple resolutions.Significance.The new micro-CT system could find application in areas where phase contrast is already known to provide superior image quality, while the added tuneable resolution functionality could enable more sophisticated analyses in these applications, e.g. by scanning samples at multiple scales.

Caudal Fgfr1 disruption produces localised spinal mis-patterning and a terminal myelocystocele-like phenotype in mice.

Closed spinal dysraphisms are poorly understood malformations classified as neural tube (NT) defects. Several, including terminal myelocystocele, affect the distal spine. We have previously identified a NT closure-initiating point, Closure 5, in the distal spine of mice. Here, we document equivalent morphology of the caudal-most closing posterior neuropore (PNP) in mice and humans. Closure 5 forms in a region of active FGF signalling, and pharmacological FGF receptor blockade impairs its formation in cultured mouse embryos. Conditional genetic deletion of Fgfr1 in caudal embryonic tissues with Cdx2Cre diminishes neuroepithelial proliferation, impairs Closure 5 formation and delays PNP closure. After closure, the distal NT of Fgfr1-disrupted embryos dilates to form a fluid-filled sac overlying ventrally flattened spinal cord. This phenotype resembles terminal myelocystocele. Histological analysis reveals regional and progressive loss of SHH- and FOXA2-positive ventral NT domains, resulting in OLIG2 labelling of the ventral-most NT. The OLIG2 domain is also subsequently lost, eventually producing a NT that is entirely positive for the dorsal marker PAX3. Thus, a terminal myelocystocele-like phenotype can arise after completion of NT closure with localised spinal mis-patterning caused by disruption of FGFR1 signalling.

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.

Two-Photon Cell and Tissue Level Laser Ablation Methods to Study Morphogenetic Biomechanics.

Laser ablation is routinely performed to infer mechanical tension in cells and tissues. Here we describe our method of two-photon laser ablation at the cellular and tissue level in mouse embryos. The primary outcome of these experiments is initial retraction following ablation, which correlates with, and so can be taken as a measure of, the tensile stress that structure was under before ablation. Several experimental variables can affect interpretation of ablation tests. Pre-test factors include differences in physical properties such as viscoelasticity between experimental conditions. Factors relevant during the test include viability of the cells at the point of ablation, image acquisition rate and the potential for overzealous ablations to cause air bubbles through heat dissipation. Post-test factors include intensity-biased image registration that can artificially produce apparent directionality. Applied to the closing portion of the mouse spinal neural tube, these methods have demonstrated long-range biomechanical coupling of the embryonic structure and have identified highly contractile cell populations involved in its closure process.

Hindbrain neuropore tissue geometry determines asymmetric cell-mediated closure dynamics in mouse embryos.

Gap closure is a common morphogenetic process. In mammals, failure to close the embryonic hindbrain neuropore (HNP) gap causes fatal anencephaly. We observed that surface ectoderm cells surrounding the mouse HNP assemble high-tension actomyosin purse strings at their leading edge and establish the initial contacts across the embryonic midline. Fibronectin and laminin are present, and tensin 1 accumulates in focal adhesion-like puncta at this leading edge. The HNP gap closes asymmetrically, faster from its rostral than caudal end, while maintaining an elongated aspect ratio. Cell-based physical modeling identifies two closure mechanisms sufficient to account for tissue-level HNP closure dynamics: purse-string contraction and directional cell motion implemented through active crawling. Combining both closure mechanisms hastens gap closure and produces a constant rate of gap shortening. Purse-string contraction reduces, whereas crawling increases gap aspect ratio, and their combination maintains it. Closure rate asymmetry can be explained by asymmetric embryo tissue geometry, namely a narrower rostral gap apex, whereas biomechanical tension inferred from laser ablation is equivalent at the gaps' rostral and caudal closure points. At the cellular level, the physical model predicts rearrangements of cells at the HNP rostral and caudal extremes as the gap shortens. These behaviors are reproducibly live imaged in mouse embryos. Thus, mammalian embryos coordinate cellular- and tissue-level mechanics to achieve this critical gap closure event.

Cell non-autonomy amplifies disruption of neurulation by mosaic Vangl2 deletion in mice.

Post-zygotic mutations that generate tissue mosaicism are increasingly associated with severe congenital defects, including those arising from failed neural tube closure. Here we report that neural fold elevation during mouse spinal neurulation is vulnerable to deletion of the VANGL planar cell polarity protein 2 (Vangl2) gene in as few as 16% of neuroepithelial cells. Vangl2-deleted cells are typically dispersed throughout the neuroepithelium, and each non-autonomously prevents apical constriction by an average of five Vangl2-replete neighbours. This inhibition of apical constriction involves diminished myosin-II localisation on neighbour cell borders and shortening of basally-extending microtubule tails, which are known to facilitate apical constriction. Vangl2-deleted neuroepithelial cells themselves continue to apically constrict and preferentially recruit myosin-II to their apical cell cortex rather than to apical cap localisations. Such non-autonomous effects can explain how post-zygotic mutations affecting a minority of cells can cause catastrophic failure of morphogenesis leading to clinically important birth defects.

Characterising open chromatin in chick embryos identifies cis-regulatory elements important for paraxial mesoderm formation and axis extension.

Somites arising from paraxial mesoderm are a hallmark of the segmented vertebrate body plan. They form sequentially during axis extension and generate musculoskeletal cell lineages. How paraxial mesoderm becomes regionalised along the axis and how this correlates with dynamic changes of chromatin accessibility and the transcriptome remains unknown. Here, we report a spatiotemporal series of ATAC-seq and RNA-seq along the chick embryonic axis. Footprint analysis shows differential coverage of binding sites for several key transcription factors, including CDX2, LEF1 and members of HOX clusters. Associating accessible chromatin with nearby expressed genes identifies cis-regulatory elements (CRE) for TCF15 and MEOX1. We determine their spatiotemporal activity and evolutionary conservation in Xenopus and human. Epigenome silencing of endogenous CREs disrupts TCF15 and MEOX1 gene expression and recapitulates phenotypic abnormalities of anterior-posterior axis extension. Our integrated approach allows dissection of paraxial mesoderm regulatory circuits in vivo and has implications for investigating gene regulatory networks.

Refinement of inducible gene deletion in embryos of pregnant mice.

CreERT2-mediated gene recombination is widely applied in developmental biology research. Activation of CreERT2 is typically achieved by injection of tamoxifen in an oily vehicle into the peritoneal cavity of mid-gestation pregnant mice. This can be technically challenging and adversely impacts welfare. Here we characterize three refinements to this technique: Pipette feeding (not gavage) of tamoxifen, ex vivo CreERT2 activation in whole embryo culture and injection of cell-permeable TAT-Cre into Cre-negative cultured embryos. We demonstrate that pipette feeding of tamoxifen solution to the mother on various days of gestation reliably activates embryonic CreERT2, illustrated here using ß-Actin CreERT2 , Sox2 CreERT2 , T CreERT2 , and Nkx1.2 CreERT2 . Pipette feeding of tamoxifen induces dose-dependent recombination of Rosa26 mTmG reporters when administered at E8.5. Activation of two neuromesodermal progenitor-targeting Cre drivers, T CreERT2 , and Nkx1.2 CreERT2 , produces comparable neuroepithelial lineage tracing. Dose-dependent CreERT2 activation can also be achieved by brief exposure to 4OH-tamoxifen in whole embryo culture, allowing temporal control of gene deletion and eliminating the need to treat pregnant mice. Rosa26 mTmG reporter recombination can also be achieved regionally by injecting TAT-Cre into embryonic tissues at the start of culture. This allows greater spatial control over Cre activation than can typically be achieved with endogenous CreERT2, for example by injecting TAT-Cre on one side of the midline. We hope that our description and application of these techniques will stimulate refinement of experimental methods involving CreERT2 activation for gene deletion and lineage tracing studies. Improved temporal (ex vivo treatment) and spatial (TAT-Cre injection) control of recombination will also allow previously intractable questions to be addressed.

Rho kinase-dependent apical constriction counteracts M-phase apical expansion to enable mouse neural tube closure.

Cellular generation of mechanical forces required to close the presumptive spinal neural tube, the 'posterior neuropore' (PNP), involves interkinetic nuclear migration (INM) and apical constriction. Both processes change the apical surface area of neuroepithelial cells, but how they are biomechanically integrated is unknown. Rho kinase (Rock; herein referring to both ROCK1 and ROCK2) inhibition in mouse whole embryo culture progressively widens the PNP. PNP widening is not caused by increased mechanical tension opposing closure, as evidenced by diminished recoil following laser ablation. Rather, Rock inhibition diminishes neuroepithelial apical constriction, producing increased apical areas in neuroepithelial cells despite diminished tension. Neuroepithelial apices are also dynamically related to INM progression, with the smallest dimensions achieved in cells positive for the pan-M phase marker Rb phosphorylated at S780 (pRB-S780). A brief (2 h) Rock inhibition selectively increases the apical area of pRB-S780-positive cells, but not pre-anaphase cells positive for phosphorylated histone 3 (pHH3+). Longer inhibition (8 h, more than one cell cycle) increases apical areas in pHH3+ cells, suggesting cell cycle-dependent accumulation of cells with larger apical surfaces during PNP widening. Consequently, arresting cell cycle progression with hydroxyurea prevents PNP widening following Rock inhibition. Thus, Rock-dependent apical constriction compensates for the PNP-widening effects of INM to enable progression of closure.This article has an associated First Person interview with the first authors of the paper.

miR-133-mediated regulation of the Hedgehog pathway orchestrates embryo myogenesis.

Skeletal myogenesis serves as a paradigm to investigate the molecular mechanisms underlying exquisitely regulated cell fate decisions in developing embryos. The evolutionarily conserved miR-133 family of microRNAs is expressed in the myogenic lineage, but how it acts remains incompletely understood. Here, we performed genome-wide differential transcriptomics of miR-133 knockdown (KD) embryonic somites, the source of vertebrate skeletal muscle. These analyses, performed in chick embryos, revealed extensive downregulation of Sonic hedgehog (Shh) pathway components: patched receptors, Hedgehog interacting protein and the transcriptional activator Gli1. By contrast, Gli3, a transcriptional repressor, was de-repressed and confirmed as a direct miR-133 target. Phenotypically, miR-133 KD impaired myotome formation and growth by disrupting proliferation, extracellular matrix deposition and epithelialization. Together, these observations suggest that miR-133-mediated Gli3 silencing is crucial for embryonic myogenesis. Consistent with this idea, we found that activation of Shh signalling by either purmorphamine, or KD of Gli3 by antisense morpholino, rescued the miR-133 KD phenotype. Thus, we identify a novel Shh/myogenic regulatory factor/miR-133/Gli3 axis that connects epithelial morphogenesis with myogenic fate specification.