Molecular Characterization of the Gastrula in the Turtle Emys orbicularis: An Evolutionary Perspective on Gastrulation

Due to the presence of a blastopore as in amphibians, the turtle has been suggested to exemplify a transition form from an amphibian- to an avian-type gastrulation pattern. In order to test this hypothesis and gain insight into the emergence of the unique characteristics of amniotes during gastrulation, we have performed the first molecular characterization of the gastrula in a reptile, the turtle Emys orbicularis. The study of Brachyury, Lim1, Otx2 and Otx5 expression patterns points to a highly conserved dynamic of expression with amniote model organisms and makes it possible to identify the site of mesoderm internalization, which is a long-standing issue in reptiles. Analysis of Brachyury expression also highlights the presence of two distinct phases, less easily recognizable in model organisms and respectively characterized by an early ring-shaped and a later bilateral symmetrical territory. Systematic comparisons with tetrapod model organisms lead to new insights into the relationships of the blastopore/blastoporal plate system shared by all reptiles, with the blastopore of amphibians and the primitive streak of birds and mammals. The biphasic Brachyury expression pattern is also consistent with recent models of emergence of bilateral symmetry, which raises the question of its evolutionary significance

Taspase1-dependent TFIIA cleavage coordinates head morphogenesis by limiting Cdkn2a locus transcription

Head morphogenesis requires complex signal relays to enable precisely coordinated proliferation, migration, and patterning. Here, we demonstrate that, during mouse head formation, taspase1-mediated (TASP1-mediated) cleavage of the general transcription factor TFIIA ensures proper coordination of rapid cell proliferation and morphogenesis by maintaining limited transcription of the negative cell cycle regulators p16Ink4a and p19Arf from the Cdkn2a locus. In mice, loss of TASP1 function led to catastrophic craniofacial malformations that were associated with inadequate cell proliferation. Compound deficiency of Cdkn2a, especially p16Ink4a deficiency, markedly reduced the craniofacial anomalies of TASP1-deficent mice. Furthermore, evaluation of mice expressing noncleavable TASP1 targets revealed that TFIIA is the principal TASP1 substrate that orchestrates craniofacial morphogenesis. ChIP analyses determined that noncleaved TFIIA accumulates at the p16Ink4a and p19Arf promoters to drive transcription of these negative regulators. In summary, our study elucidates a regulatory circuit comprising proteolysis, transcription, and proliferation that is pivotal for construction of the mammalian head

Live Imaging of Xwnt5A-ROR2 Complexes

Secreted molecules of the Wnt family regulate key decisions in embryogenesis and adult tissue homeostasis by activating a complex network of Wnt signaling pathways. Although the different branches of Wnt signaling have been studied for more than 25 years, fluorophore tagged constructs for live cell imaging of Wnt molecules activating the Wnt/β-catenin pathway have become available only recently. We have generated a fluorophore tagged Wnt construct of the Xenopus Wnt5a protein (Xwnt5A) with the enhanced green fluorescent protein (EGFP), Xwnt5A-EGFP. This construct activates non-canonical Wnt pathways in an endocytosis dependent manner and is capable of compensating for the loss of endogenous Xwnt5A in Xenopus embryos. Strikingly, non-canonical Wnt pathway activation was restricted to short-range signaling while an inhibitory effect was observed in transwell cell cultures taken as long-range signaling model sytem. We used our Xwnt5A-EGFP construct to analyze in vivo binding of Wnt5A to its co-receptor ROR2 on the microscopic and on the molecular level. On the microscopic level, Xwnt5A-EGFP clusters in the membrane and recruits ROR2-mCherry to these clusters. Applying dual-colour dual-focus line-scanning fluorescence correlation spectroscopy on dorsal marginal zone explants, we identified membrane tethered Xwnt5A-EGFP molecules binding to ROR2-mCherry molecules. Our data favour a model, in which membrane-tethered Wnt-5A recruits ROR2 to form large ligand/receptor clusters and signals in an endocytosis-dependent manner

Cell Cycle Regulation and Cytoskeletal Remodelling Are Critical Processes in the Nutritional Programming of Embryonic Development

Many mechanisms purport to explain how nutritional signals during early development are manifested as disease in the adult offspring. While these describe processes leading from nutritional insult to development of the actual pathology, the initial underlying cause of the programming effect remains elusive. To establish the primary drivers of programming, this study aimed to capture embryonic gene and protein changes in the whole embryo at the time of nutritional insult rather than downstream phenotypic effects. By using a cross-over design of two well established models of maternal protein and iron restriction we aimed to identify putative common “gatekeepers” which may drive nutritional programming

Dental cell type atlas reveals stem and differentiated cell types in mouse and human teeth

Understanding cell types and mechanisms of dental growth is essential for reconstruction and engineering of teeth. Therefore, we investigated cellular composition of growing and non-growing mouse and human teeth. As a result, we report an unappreciated cellular complexity of the continuously-growing mouse incisor, which suggests a coherent model of cell dynamics enabling unarrested growth. This model relies on spatially-restricted stem, progenitor and differentiated populations in the epithelial and mesenchymal compartments underlying the coordinated expansion of two major branches of pulpal cells and diverse epithelial subtypes. Further comparisons of human and mouse teeth yield both parallelisms and differences in tissue heterogeneity and highlight the specifics behind growing and non-growing modes. Despite being similar at a coarse level, mouse and human teeth reveal molecular differences and species-specific cell subtypes suggesting possible evolutionary divergence. Overall, here we provide an atlas of human and mouse teeth with a focus on growth and differentiation. Unlike human teeth, mouse incisors grow throughout life, based on stem and progenitor cell activity. Here the authors generate single cell RNA-seq comparative maps of continuously-growing mouse incisor, non-growing mouse molar and human teeth, combined with lineage tracing to reveal dental cell complexity.Peer reviewe

Cell-Sorting at the A/P Boundary in the Drosophila Wing Primordium: A Computational Model to Consolidate Observed Non-Local Effects of Hh Signaling

Non-intermingling, adjacent populations of cells define compartment boundaries; such boundaries are often essential for the positioning and the maintenance of tissue-organizers during growth. In the developing wing primordium of Drosophila melanogaster, signaling by the secreted protein Hedgehog (Hh) is required for compartment boundary maintenance. However, the precise mechanism of Hh input remains poorly understood. Here, we combine experimental observations of perturbed Hh signaling with computer simulations of cellular behavior, and connect physical properties of cells to their Hh signaling status. We find that experimental disruption of Hh signaling has observable effects on cell sorting surprisingly far from the compartment boundary, which is in contrast to a previous model that confines Hh influence to the compartment boundary itself. We have recapitulated our experimental observations by simulations of Hh diffusion and transduction coupled to mechanical tension along cell-to-cell contact surfaces. Intriguingly, the best results were obtained under the assumption that Hh signaling cannot alter the overall tension force of the cell, but will merely re-distribute it locally inside the cell, relative to the signaling status of neighboring cells. Our results suggest a scenario in which homotypic interactions of a putative Hh target molecule at the cell surface are converted into a mechanical force. Such a scenario could explain why the mechanical output of Hh signaling appears to be confined to the compartment boundary, despite the longer range of the Hh molecule itself. Our study is the first to couple a cellular vertex model describing mechanical properties of cells in a growing tissue, to an explicit model of an entire signaling pathway, including a freely diffusible component. We discuss potential applications and challenges of such an approach

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) and serine biosynthetic pathway genes are co-ordinately increased during anabolic agent-induced skeletal muscle growth

We aimed to identify novel molecular mechanisms for muscle growth during administration of anabolic agents. Growing pigs (Duroc/(Landrace/Large-White)) were administered Ractopamine (a beta-adrenergic agonist; BA; 20ppm in feed) or Reporcin (recombinant growth hormone; GH; 10mg/48hours injected) and compared to a control cohort (feed only; no injections) over a 27-day time course (1, 3, 7, 13 or 27-days). Longissimus Dorsi muscle gene expression was analyzed using Agilent porcine transcriptome microarrays and clusters of genes displaying similar expression profiles were identified using a modified maSigPro clustering algorithm. Anabolic agents increased carcass (p=0.002) and muscle weights (Vastus Lateralis: p<0.001; Semitendinosus: p=0.075). Skeletal muscle mRNA expression of serine/one-carbon/glycine biosynthesis pathway genes (Phgdh, Psat1 and Psph) and the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase-M (Pck2/PEPCK-M), increased during treatment with BA, and to a lesser extent GH (p<0.001, treatment x time interaction). Treatment with BA, but not GH, caused a 2-fold increase in phosphoglycerate dehydrogenase (PHGDH) protein expression at days 3 (p<0.05) and 7 (p<0.01), and a 2-fold increase in PEPCK-M protein expression at day 7 (p<0.01). BA treated pigs exhibit a profound increase in expression of PHGDH and PEPCK-M in skeletal muscle, implicating a role for biosynthetic metabolic pathways in muscle growth

Travelling and splitting of a wave of hedgehog expression involved in spider-head segmentation

During development segmentation is a process that generates a spatial periodic pattern. Peak splitting of waves of gene expression is a mathematically predicted, simple strategy accounting for this type of process, but it has not been well characterized biologically. Here we show temporally repeated splitting of gene expression into stripes that is associated with head axis growth in the spider Achaearanea embryo. Preceding segmentation, a wave of hedgehog homologue gene expression is observed to travel posteriorly during development stage 6. This stripe, co-expressing an orthodenticle homologue, undergoes two cycles of splitting and shifting accompanied by convergent extension, serving as a generative zone for the head segments. The two orthodenticle and odd-paired homologues are identified as targets of Hedgehog signalling, and evidence suggests that their activities mediate feedback to maintain the head generative zone and to promote stripe splitting in this zone. We propose that the 'stripe-splitting' strategy employs genetic components shared with Drosophila blastoderm subdivision, which are required for participation in an autoregulatory signalling network