Hypoxia induces a transcriptional early primitive streak signature in pluripotent cells enhancing spontaneous elongation and lineage representation in gastruloids

The cellular microenvironment, together with intrinsic regulators, shapes stem cell identity and differentiation capacity. Mammalian early embryos are exposed to hypoxia in vivo and appear to benefit from hypoxic culture in vitro. Yet, how hypoxia influences stem cell transcriptional networks and lineage choices remain poorly understood. Here, we investigated the molecular effects of acute and prolonged hypoxia on embryonic and extra-embryonic stem cells as well as the functional impact on differentiation potential. We find a temporal and cell type-specific transcriptional response including an early primitive streak signature in hypoxic embryonic stem cells mediated by HIF1α. Using a 3D gastruloid differentiation model, we show that hypoxia-induced T expression enables symmetry breaking and axial elongation in the absence of exogenous WNT activation. When combined with exogenous WNT activation, hypoxia enhances lineage representation in gastruloids, as demonstrated by highly enriched signatures of gut endoderm, notochord, neuromesodermal progenitors and somites. Our findings directly link the microenvironment to stem cell function and provide a rationale supportive of applying physiological conditions in models of embryo development

Morphogenesis of Developmental Stages of Dirofilaria immitis (Nematoda) in the Dog

ABSTRACT: Morphogenesis of the dog heartworm, Dirofilaria immitis, through the third and fourth molts to the fifth stage is described. Specimens were collected from mosquitoes 14 or 15 days after infection (DAI) or from dogs 3-79 DAI. Infective larvae from mosquitoes were 0.7-1.0 mm long with a tapered blunt anterior end, bilayered cuticle, refractile granules in the anterior % of the glandular esophagus, and a pair of small, broadly based, subventral bumps near the larger conical tail tip. Sexes were distinguished by the position of the oval-shaped genital primordium (GP) (near the anterior part of the glandular esophagus in females and just anterior to midbody in males), and by the presence of spicular primordia in males. When larvae were first collected from dogs 3 DAI most had completed the third molt. Fourth-stage larvae (L-4) at 3-6 DAI were 1.0-1.5 mm long, had untapered anterior ends, and had sharply defined, angular submedian papillae near a buttonlike tail tip. Posterior growth of the male GP was seen first in L-4's 9 DAI and it reached the rectum 30 DAI. Genital papillae and developing spicules of the fifth stage were evident 41 DAI. Formation of the vagina was initiated 9 DAI by hypodermal invasion of the GP at its attachment point. By 12 DAI the female GP was enlarged and swollen posteriorly with two large nuclei near the posterior tip. By 21 DAI two branches of the GP developed, each with a large nucleus at its posterior tip; by 30 DAI each branch extended beyond the junction of the esophagus and intestine (E-I). The vulva was located near but anterior to the E-I 41-58 DAI. The fourth molt occurred 50-58 DAI. Some specimens of both sexes completed ecdysis by 58 DAI. Early fifthstage specimens at 58 DAI were 12-14.8 mm long with a thick, finely striated cuticle. Spicules were almost completely sclerotized and as large as in mature males by 79 DAI. Spermatozoa were present in the testis but not in the vas deferens 79 DAI. The development of the dog heartworm, Dirofilaria immitis, in the dog has been described by Materials and Methods Specimens Specimens of Dirofilaria immitis for study were collected in a previous project on development in dogs reported by Infective larvae (third stage) were allowed to emerge from the mouthparts of cold-inactivated mosquitoes, Aedes aegypti (Liverpool strain), into Hanks' balanced salt solution 14 or 15 DA

Effect of Salicylic Acid on the Early Development of the Chick Limb-Bud

Transplanted hind limb-buds of chick embryos were treated with salicylic acid. Treatment with this chemical caused reduction in length, weight and volume of the developing limb-buds. A side effect observed was an apparent loosening of the basal membrane and examined with regard to length, weight, and volume. The above observations are discussed in relation to the different hypotheses of the ectoderm-mesoderm interaction. The physiological role of the basal membrane, as well as the possible implications of the basal findings to the question of limb development and morphogenesis, are also discussed

MicroRNA-277 Modulates the Neurodegeneration Caused by Fragile X Premutation rCGG Repeats

Fragile X-associated tremor/ataxia syndrome (FXTAS), a late-onset neurodegenerative disorder, has been recognized in older male fragile X premutation carriers and is uncoupled from fragile X syndrome. Using a Drosophila model of FXTAS, we previously showed that transcribed premutation repeats alone are sufficient to cause neurodegeneration. MiRNAs are sequence-specific regulators of post-transcriptional gene expression. To determine the role of miRNAs in rCGG repeat-mediated neurodegeneration, we profiled miRNA expression and identified selective miRNAs, including miR-277, that are altered specifically in Drosophila brains expressing rCGG repeats. We tested their genetic interactions with rCGG repeats and found that miR-277 can modulate rCGG repeat-mediated neurodegeneration. Furthermore, we identified Drep-2 and Vimar as functional targets of miR-277 that could modulate rCGG repeat-mediated neurodegeneration. Finally, we found that hnRNP A2/B1, an rCGG repeat-binding protein, can directly regulate the expression of miR-277. These results suggest that sequestration of specific rCGG repeat-binding proteins could lead to aberrant expression of selective miRNAs, which may modulate the pathogenesis of FXTAS by post-transcriptionally regulating the expression of specific mRNAs involved in FXTAS

Extracardiac septum transversum/proepicardial endothelial cells pattern embryonic coronary arterio–venous connections

Recent reports suggest that mammalian embryonic coronary endothelium (CoE) originates from the sinus venosus and ventricular endocardium. However, the contribution of extracardiac cells to CoE is thought to be minor and nonsignificant for coronary formation. Using classic (Wt1(Cre)) and previously undescribed (G2-Gata4(Cre)) transgenic mouse models for the study of coronary vascular development, we show that extracardiac septum transversum/proepicardium (ST/PE)-derived endothelial cells are required for the formation of ventricular coronary arterio-venous vascular connections. Our results indicate that at least 20% of embryonic coronary arterial and capillary endothelial cells derive from the ST/PE compartment. Moreover, we show that conditional deletion of the ST/PE lineage-specific Wilms' tumor suppressor gene (Wt1) in the ST/PE of G2-Gata4(Cre) mice and in the endothelium of Tie2(Cre) mice disrupts embryonic coronary transmural patterning, leading to embryonic death. Taken together, our results demonstrate that ST/PE-derived endothelial cells contribute significantly to and are required for proper coronary vascular morphogenesi

Specific expression of a TRIM-containing factor in ectoderm cells affects the skeletal morphogenetic program of the sea urchin embryo

In the indirect developing sea urchin embryo, the primary mesenchyme cells (PMCs) acquire most of the positional and temporal information from the overlying ectoderm for skeletal initiation and growth. In this study, we characterize the function of the novel gene strim1, which encodes a tripartite motif-containing (TRIM) protein, that adds to the list of genes constituting the epithelial-mesenchymal signaling network. We report that strim1 is expressed in ectoderm regions adjacent to the bilateral clusters of PMCs and that its misexpression leads to severe skeletal abnormalities. Reciprocally, knock down of strim1 function abrogates PMC positioning and blocks skeletogenesis. Blastomere transplantation experiments establish that the defects in PMC patterning, number and skeletal growth depend upon strim1 misexpression in ectoderm cells. Furthermore, clonal expression of strim1 into knocked down embryos locally restores skeletogenesis. We also provide evidence that the Otp and Pax2/5/8 regulators, as well as FGFA, but not VEGF, ligand act downstream to strim1 in ectoderm cells, and that strim1 triggers the expression of the PMC marker sm30, an ectoderm-signaling dependent gene. We conclude that the strim1 function elicits specific gene expression both in ectoderm cells and PMCs to guide the skeletal biomineralization during morphogenesis

Enhanced muscarinic M1 receptor gene expression in the corpus striatum of streptozotocin-induced diabetic rats

Acetylcholine (ACh), the first neurotransmitter to be identified, regulate the activities of central and peripheral functions through interactions with muscarinic receptors. Changes in muscarinic acetylcholine receptor (mAChR) have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). Previous reports from our laboratory on streptozotocin (STZ) induced diabetic rats showed down regulation of muscarinic M1 receptors in the brainstem, hypothalamus, cerebral cortex and pancreatic islets. In this study, we have investigated the changes of acetylcholine esterase (AChE) enzyme activity, total muscarinic and muscarinic M1 receptor binding and gene expression in the corpus striatum of STZ – diabetic rats and the insulin treated diabetic rats. The striatum, a neuronal nucleus intimately involved in motor behaviour, is one of the brain regions with the highest acetylcholine content. ACh has complex and clinically important actions in the striatum that are mediated predominantly by muscarinic receptors. We observed that insulin treatment brought back the decreased maximal velocity (Vmax) of acetylcholine esterase in the corpus striatum during diabetes to near control state. In diabetic rats there was a decrease in maximal number (Bmax) and affinity (Kd) of total muscarinic receptors whereas muscarinic M1 receptors were increased with decrease in affinity in diabetic rats. We observed that, in all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR experiment confirmed the increase in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. These results suggest the diabetes-induced changes of the cholinergic activity in the corpus striatum and the regulatory role of insulin on binding parameters and gene expression of total and muscarinic M1 receptors

Dynamics of Spatial Pattern Formation: Cases of Spikes and Droplets

This thesis studies the gradient system that forms spatial patterns such that the minimum distances of pairs among various points are maximized in the end. As this problem innately involves singularity issues, an extended system of the gradient system is proposed. Motivated by the spatial pattern suggested by a numerical example, this extended system is applied to a three-point problem and then to a two-point problem in a quotient space of R2 modulo a lattice