Developmental Dynamics最新文献

Background: Early embryonic aortic arches (AA) are a dynamic vascular structures that are in the process of shaping into the great arteries of cardiovascular system. Previously, a time-lapsed mechanosensitive gene expression map was established for AA subject to altered mechanical loads in the avian embryo. To validate this map, we investigated effects on vascular microstructure and material properties following the perturbation of key genes using an in-house microvascular gene knockdown system.

Results: All siRNA vectors show a decrease in the expression intensity of desired genes with no significant differences between vectors. In TGFβ3 knockdowns, we found a reduction in expression intensities of TGFβ3 (≤76%) and its downstream targets such as ELN (≤99.6%), Fbn1 (≤60%), COL1 (≤52%) and COL3 (≤86%) and an increase of diameter in the left AA (23%). MMP2 knockdown also reduced expression levels in MMP2 (≤30%) and a 6-fold increase in its downstream target COL3 with a decrease in stiffness of the AA wall and an increase in the diameter of the AA (55%). These in vivo measurements were confirmed using immunohistochemistry, western blotting and a computational growth model of the vascular extracellular matrix (ECM).

Conclusions: Localized spatial genetic modification of the aortic arch region governs the vascular phenotype and ECM composition of the embryo and can be integrated with mechanically-induced congenital heart disease models.

Background: Meis family of transcription factors operates in Pbx-Meis-Hox regulatory network controlling development of various tissues including eye, limbs, heart, hindbrain or craniofacial skeletal elements originating from the neural crest. Although studies in mouse provide abundant information about Meis factors function in embryogenesis, little is known about their role in zebrafish.

Results: We generated zebrafish lines carrying null mutations in meis1a, meis1b, meis2a, and meis2b genes. Only meis1b mutants are lethal at larval stage around 13 dpf whereas the other mutant lines are viable and fertile. We focused on development of neural crest-derived craniofacial structures such as tendons, cranial nerves, cartilage and accompanying muscles. Meis1b mutants displayed morphogenetic abnormalities in the cartilage originating from the first and second pharyngeal arches. Meckel's cartilage was shorter and wider with fused anterior symphysis and abnormal chondrocyte organization. This resulted in impaired tendons and muscle fiber connections while tenocyte development was not largely affected.

Conclusions: Loss-of-function mutation in meis1b affects cartilage morphology in the lower jaw that leads to disrupted organization of muscles and tendons.

Background: Mouse nodal immotile cilia mechanically sense the bending direction for left-right (L-R) determination and activate the left-side-specific signaling cascade, leading to increased Nodal activity. Asymmetric distribution of Pkd2, a crucial channel for L-R determination, on immotile cilia has been reported recently. However, the causal relationship between the asymmetric Pkd2 distribution and direction-dependent flow sensing is not well understood. Furthermore, the underlying molecular mechanism directing this asymmetric Pkd2 distribution remains unclear.

Results: The effects of several recombinant proteins and inhibitors on the Pkd2 distribution were analyzed using super-resolution microscopy. Notably, bone morphogenetic protein 4 (BMP4) affected the Pkd2 distribution. Additionally, three-dimensional manipulation of nodal immotile cilia using optical tweezers revealed that excess BMP4 caused defects in the mechanosensing ability of the cilia.

Conclusions: Experimental data together with model calculations suggest that BMP4 regulates the asymmetric distribution of Pkd2 in nodal immotile cilia, thereby affecting the ability of these cilia to sense the bending direction for L-R determination. This study, for the first time, provides insight into the relationship between the asymmetric protein distribution in cilia and their function.

Primary cilia are antenna-like sensory organelles that are evolutionarily conserved in nearly all modern eukaryotes, from the single-celled green alga, Chlamydomonas reinhardtii, to vertebrates and mammals. Cilia are microtubule-based cellular projections that have adapted to perform a broad range of species-specific functions, from cell motility to detection of light and the transduction of extracellular mechanical and chemical signals. These functions render cilia essential for organismal development and survival. The high conservation of cilia has allowed for discoveries in C. reinhardtii to inform our understanding of the basic biology of mammalian primary cilia, and to provide insight into the genetic etiology of ciliopathies. Over the last two decades, a growing number of studies has revealed that multiple aspects of ciliary homeostasis are regulated by the actin cytoskeleton, including centrosome migration and positioning, vesicle transport to the basal body, ectocytosis, and ciliary-mediated signaling. Here, we review actin regulation of ciliary homeostasis, and highlight conserved and divergent mechanisms in C. reinhardtii and mammalian cells. Further, we compare the disease manifestations of patients with ciliopathies to those with mutations in actin and actin-associated genes, and propose that primary cilia defects caused by genetic alteration of the actin cytoskeleton may underlie certain birth defects.

Background: Vertebrate left-right symmetry breaking is preceded by formation of left-right organizer. In Amphibian, this structure is formed by gastrocoel roof plate, which emerges from superficial suprablastoporal cells. GRP is subdivided into medial area, which generates leftward flow by rotating monocilia and lateral Nodal1 expressing areas, which are involved in sensing of the flow. After successful symmetry breaking, medial cells are incorporated into a deep layer where they contribute to the axial mesoderm, while lateral domains join somitic mesoderm.

Results: Here, we performed detailed analysis of spatial and temporal gene expression of important markers and the corresponding morphology of emerging GRP. Endodermal marker Sox17 and markers of superficial mesoderm display complementary patterns at all studied stages. At early stages, GRP forms Tekt2 positive epithelial domain clearly separated from underlying deep layers, while at later stages, this separation disappears. Marker of early somitic mesoderm MyoD1 was absent in emerging GRP and was induced together with Nodal1 during early neurulation. Decreasing morphological separation is accompanied by lateral to medial covering of GRP by endoderm.

Conclusion: Our data supports continuous link between superficial mesoderm at the start of gastrulation and mature GRP and suggests late induction of somitic fate in lateral GRP.