Biology subjects

Levic, D. S.

Publications and source records attributed to Levic, D. S..

4 recordsLinked to original sources

bioRxiv2024

Plxnd1-mediated mechanosensing of blood flow controls the caliber of the Dorsal Aorta via the transcription factor Klf2

The cardiovascular system generates and responds to mechanical forces. The heartbeat pumps blood through a network of vascular tubes, which adjust their caliber in response to the hemodynamic environment. However, how endothelial cells in the developing vascular system integrate inputs from circulatory forces into signaling pathways to define vessel caliber is poorly understood. Using vertebrate embryos and in vitro-assembled microvascular networks of human endothelial cells as models, flow and genetic manipulations, and custom software, we reveal that Plexin-D1, an endothelial Semaphorin receptor critical for angiogenic guidance, employs its mechanosensing activity to serve as a crucial positive regulator of the Dorsal Aortas (DA) caliber. We also uncover that the flow-responsive transcription factor KLF2 acts as a paramount mechanosensitive effector of Plexin-D1 that enlarges endothelial cells to widen the vessel. These findings illuminate the molecular and cellular mechanisms orchestrating the interplay between cardiovascular development and hemodynamic forces. HighlightsO_LIPlexin-D1 mechanosensing of blood flow tunes the caliber of the Dorsal Aorta (DA) C_LIO_LIThe DA widens without raising endothelial cell numbers, which can change separate from the caliber C_LIO_LIThe Kruppel-like transcription factor 2 (KLF2) is a key Plexin-D1 mechano-effector during development C_LIO_LIKLF2 increases endothelial cell size to expand the DA caliber C_LI

developmental biology↗

bioRxiv2023

Rediscovering the Rete Ovarii: a secreting auxiliary structure to the ovary

The rete ovarii (RO) is an appendage of the ovary that has been given little attention. Although the RO appears in drawings of the ovary in early versions of Grays Anatomy, it disappeared from recent textbooks, and is often dismissed as a functionless vestige in the adult ovary. Using PAX8 immunostaining and confocal microscopy, we characterized the fetal development of the RO in the context of the ovary. The RO consists of three distinct regions that persist in adult life, the intraovarian rete (IOR), the extraovarian rete (EOR), and the connecting rete (CR). While the cells of the IOR appear to form solid cords within the ovary, the EOR rapidly develops into a convoluted tubular epithelium ending in a distal dilated tip. Cells of the EOR are ciliated and exhibit cellular trafficking capabilities. The CR, connecting the EOR to the IOR, gradually acquires tubular epithelial characteristics by birth. Using microinjections into the distal dilated tip of the EOR, we found that luminal contents flow towards the ovary. Mass spectrometry revealed that the EOR lumen contains secreted proteins potentially important for ovarian function. We show that the cells of the EOR are closely associated with vasculature and macrophages, and are contacted by neuronal projections, consistent with a role as a sensory appendage of the ovary. The direct proximity of the RO to the ovary and its integration with the extraovarian landscape suggest that it plays an important role in ovary development and homeostasis.

developmental biology↗

bioRxiv2022

Regulated extracellular matrix trafficking shapes cell growth during cartilage morphogenesis

Craniofacial malformations are present in more than one third of all congenital syndromes, but the pathogenesis of skeletal dysmorphology is poorly understood. Here, using an unbiased forward genetics approach in zebrafish, we identified a mutation in erc1b that leads to craniofacial defects, including micrognathia and hypertelorism caused by impaired cartilage and bone growth. To date, ERC1 has not been considered a candidate gene for craniofacial syndromes. Using live in vivo imaging, genetic depletion and replacement experiments, and transgenic approaches, we interrogated erc1b function. We found that Erc1b regulates extracellular matrix (ECM) trafficking required for the highly conserved "stack of coins" organization of chondrocytes in cartilage that is essential for skeletal growth and integrity. Erc1b functions cellautonomously at the chondrocyte cell cortex to regulate traffic of ECM and plasma membrane expansion in a microtubule dependent manner during isometric cell growth. Disruption of Erc1-Rab8-Kinesin-1 axis leads to failure of cartilage maturation, endochondral bone formation and ultimately chondrocyte cell death. Our study identifies Erc1b as a candidate genetic factor for craniofacial syndromes.

developmental biology↗

bioRxiv2021

Knock-in tagging in zebrafish facilitated by insertion into non-coding regions

Zebrafish provide an excellent model for in vivo cell biology studies due to their amenability to live imaging. Protein visualization in zebrafish has traditionally relied on overexpression of fluorescently tagged proteins from heterologous promoters, making it difficult to recapitulate endogenous expression patterns and protein function. One way to circumvent this problem is to tag the proteins by modifying their endogenous genomic loci. Such an approach is not widely available to zebrafish researchers due to inefficient homologous recombination and the error-prone nature of targeted integration in zebrafish. Here, we report a simple approach for tagging proteins in zebrafish on their N- or C termini with fluorescent markers by inserting PCR-generated donor amplicons into non-coding regions of the corresponding genes. Using this approach, we generated endogenously tagged alleles for several genes critical for epithelial biology and organ development including the tight junction components ZO-1 and Cldn15la, the trafficking effector Rab11a, and the ECM receptor {beta}1 integrin. Our approach facilitates the generation of knock-in lines in zebrafish, opening the way for accurate quantitative imaging studies. Summary statementGeneration of endogenously tagged stable zebrafish knock-in lines is simplified by the integration of fluorescent protein cassettes with mRNA splicing elements into non-coding regions of genes.

developmental biology↗