A spindle matrix has been proposed to help organize and stabilize the microtubule spindle during mitosis, though molecular evidence corroborating its existence has been elusive. In Drosophila, we have cloned and characterized a novel nuclear protein, skeletor, that we propose is part of a macromolecular complex forming such a spindle matrix. Skeletor antibody staining shows that skeletor is associated with the chromosomes at interphase, but redistributes into a true fusiform spindle structure at prophase, which precedes microtubule spindle formation. During metaphase, the spindle, defined by skeletor antibody labeling, and the microtubule spindles are coaligned. We find that the skeletor-defined spindle maintains its fusiform spindle structure from end to end across the metaphase plate during anaphase when the chromosomes segregate. Consequently, the properties of the skeletor-defined spindle make it an ideal substrate for providing structural support stabilizing microtubules and counterbalancing force production. Furthermore, skeletor metaphase spindles persist in the absence of microtubule spindles, strongly implying that the existence of the skeletor-defined spindle does not require polymerized microtubules. Thus, the identification and characterization of skeletor represents the first direct molecular evidence for the existence of a complete spindle matrix that forms within the nucleus before microtubule spindle formation.
One of the overarching goals in developmental biology is the elucidation of mechanisms that elaborate form and function. To this end, an accurate morphological description of embryonic development is essential. However, visualizing dynamic changes in the three-dimensional (3D) structure of the developing embryo has been a "holy grail" in the field of developmental biology. The fundamental difficulties that have hindered all efforts in 3D reconstruction using two-dimensional (2D) image stacks revolve around the seemingly intractable problems of section registration and distortion. A remarkably simple solution has come about with the development of a new technique referred to as episcopic fluorescence image capture (EFIC). With EFIC imaging, tissue autofluorescence is used to image the block face prior to cutting each section. The 2D resolution obtained is close to that achieved by histology, and such 2D image stacks can be readily reconstructed in 3D. The 3D models generated provide fine structural details with resolution unmatched by 3D reconstructions obtained with any other imaging modalities. Given the perfect registration of EFIC image stacks, another important capability provided by EFIC is digital resectioning in any plane. This provides complete flexibility in the selection of optimal virtual sectioning planes for viewing different features in a specimen, and is invaluable for analyzing dynamic changes in tissue structure in the developing embryo. The capabilities provided by EFIC for rapid high resolution 3D reconstruction together with digital resectioning make this an unparalleled tool for characterizing morphogenetic events in the developing embryo. Although our review is focused on using EFIC for studying embryonic development, it is important to note that there is no intrinsic limitation on the size of the specimen that can be analyzed by EFIC imaging. Overall, EFIC should serve as an important imaging technique that will complement other 3D imaging modalities such as MRI and optical tomography. Given the feasibility of generating EFIC image stacks using cryoembedded or polyethylene glycol (PEG)-embedded specimens, there is the possibility that EFIC may be combined with 3D RNA or protein expression profiling. Together, such studies may help further elucidate the relationship between form and function.
We have cloned and characterized JIL-1, a novel tandem kinase in Drosophila that associates with the chromosomes throughout the cell cycle. Antibody staining and live imaging of JIL-1-GFP transgenic flies show that JIL-1 localizes to the gene-rich interband regions of larval polytene chromosomes and is upregulated almost 2-fold on the hypertranscribed male X chromosome compared to autosomes. Phylogenetic analysis suggests that JIL-1 together with human MSKs defines a separate family of tandem kinases. That JIL-1 is a functional kinase was demonstrated by autophosphorylation and phosphorylation of histone H3 in vitro. Based on these findings, we propose that JIL-1 may play a role in transcriptional control potentially by regulating chromatin structure.
The connexin43 knockout (Cx43alpha1 KO) mouse dies at birth from outflow obstruction associated with infundibular pouches. To elucidate the origin of the infundibular pouches, we used microarray analysis to investigate gene expression changes in the pouch tissue. We found elevated expression of many genes encoding markers for vascular smooth muscle (VSM), endothelial cells, and fibroblasts, cell types that are epicardially derived and essential for coronary vasculogenesis. This was accompanied by increased expression of VEGF and genes in the TGFbeta and VEGF/Notch/Eph cell-signaling pathways known to regulate vasculogenesis/angiogenesis. Using immunohistochemistry and a VSM lacZ reporter gene, we confirmed an abundance of ectopic VSM and endothelial cells in the infundibular pouch and in some regions of the right ventricle forming secondary pouches. This was associated with distinct thinning of the compact myocardium. TUNEL labeling showed increased apoptosis in the pouch tissue, in agreement with the finding of altered expression of many apoptotic genes. Defects in vascular remodeling were indicated by a marked reduction in the branching complexity of the distal coronary arteries. In the near term KO mouse, we also observed a profusion of large coronary vascular plexuses subepicardially. This was associated with elevated epicardial expression of VEGF and abnormal epicardial cell morphology. Together, these observations indicate that dysregulated coronary vasculogenesis plays a pivotal role in formation of the infundibular pouches and suggests an essential role for Cx43alpha1 gap junctions in coronary vasculogenesis and vascular remodeling.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.