Connexinplexity: The spatial and temporal expression of connexin genes during vertebrate organogenesis

Animal development requires coordinated communication between cells. The Connexin family of proteins is a major contributor to intercellular communication in vertebrates by forming gap junction channels that facilitate the movement of ions, small molecules, and metabolites between cells. Additionally, individual hemichannels can provide a conduit to the extracellular space for paracrine and autocrine signaling. Connexin-mediated communication is well appreciated in epithelial, neural, and vascular development and homeostasis, and most tissues likely use this form of communication. In fact, Connexin disruptions are of major clinical significance contributing to disorders developing from all major germ layers. Despite the fact that Connexins serve as an essential mode of cellular communication, the temporal and cell-type specific expression patterns of connexin genes remain unknown in vertebrates. A major challenge is the large and complex connexin gene family. To overcome this barrier, we probed the expression of all connexins in zebrafish using single-cell RNA-sequencing of entire animals across several stages of organogenesis. Our analysis of expression patterns has revealed that few connexins are broadly expressed, but rather, most are expressed in tissue- or cell-type-specific patterns. Additionally, most tissues possess a unique combinatorial signature of connexin expression with dynamic temporal changes across the organism, tissue, and cell. Our analysis has identified new patterns for well-known connexins and assigned spatial and temporal expression to genes with no-existing information. We provide a field guide relating zebrafish and human connexin genes as a critical step towards understanding how Connexins contribute to cellular communication and development throughout vertebrate organogenesis.

Neural cells and their progenitors in regenerating zebrafish spinal cord

The zebrafish (Danio rerio), among all amniotes is emerging as a powerful model to study vertebrate organogenesis and regeneration. In contrast to mammals, the adult zebrafish is capable of regenerating damaged axonal tracts; it can replace neurons and glia lost after spinal cord injury (SCI) and functionally recover. In the present paper, we report ultrastructural and cell biological analyses of regeneration processes after SCI. We have focused on event specific analyses of spinal cord regeneration involving different neuronal and glial cell progenitors, such as radial glia, oligodendrocyte progenitors (OPC), and Schwann cells. While comparing the different events, we frequently refer to previous ultrastructural analyses of central nervous system (CNS) injury in higher vertebrates. Our data show (a) the cellular events following injury, such as cell death and proliferation; (b) demyelination and remyelination followed by target innervation and regeneration of synaptic junctions and c) the existence of different progenitors and their roles during regeneration. The present ultrastructural analysis corroborates the cellular basis of regeneration in the zebrafish spinal cord and confirms the presence of both neuronal and different glial progenitors.

Ubc9 Regulates Mitosis and Cell Survival during Zebrafish Development

Many proteins are modified by conjugation with Sumo, a gene-encoded, ubiquitin-related peptide, which is transferred to its target proteins via an enzymatic cascade. A central component of this cascade is the E2-conjugating enzyme Ubc9, which is highly conserved across species. Loss-of-function studies in yeast, nematode, fruit fly, and mouse blastocystes point to multiple roles of Ubc9 during cell cycle regulation, maintenance of nuclear architecture, chromosome segregation, and viability. Here we show that in zebrafish embryos, reduction of Ubc9 activity by expression of a dominant negative version causes widespread apoptosis, similar to the effect described in Ubc9-deficient mice. However, antisense-based knock down of zygotic ubc9 leads to much more specific defects in late proliferating tissues, such as cranial cartilage and eyes. Affected cartilaginous elements are of relatively normal size and shape, but consist of fewer and larger cells. Stainings with mitotic markers and 5-Bromo-2′-deoxyuridine incorporation studies indicate that fewer chondrocyte precursors are in mitosis, whereas the proportion of cells in S-phase is unaltered. Consistently, FACS analyses reveal an increase in the number of cells with a DNA content of 4n or even 8n. Our data indicate an in vivo requirement of Ubc9 for G2/M transition and/or progression through mitosis during vertebrate organogenesis. Failed mitosis in the absence of Ubc9 is not necessarily coupled with cell death. Rather, cells can continue to replicate their DNA, grow to a larger size, and finish their normal developmental program.