Fujimori, C. | bioRxiv Science

bioRxiv2023

Enhancer evolution as a driving force for lineage-specific paralog usage in the central nervous system.

Expression patterns of paralogous genes in the functionally homologous cells sometimes show differences across species. However, no reasonable explanation for the mechanism underlying such phenomena has been discovered. To understand this mechanism, the present study focused on the hypophysiotropic GnRH neurons in vertebrates as a model. These neurons express either gnrh1 or gnrh3 paralogs depending on species, and apparent switching of the expressed paralogs in them occurred at least four times in vertebrate evolution. First, we found redundant expressions of gnrh1 and gnrh3 in a single neuron in piranha and hypothesized that this situation may indicate an ancestral condition. We tested this hypothesis by examining the activity of piranha gnrh1/gnrh3 enhancers in zebrafish and medaka, in which the two gnrh paralogs are not co-expressed. Here, the gnrh1/gnrh3 enhancer of piranha induced reporter RFP/GFP co-expressions in a single hypophysiotropic GnRH neuron in both zebrafish and medaka. From these results, we propose that long-lasting ([~]550 My) redundancy after gnrh1/3 duplication in 1R/2R WGD may be the key to apparent switching of the paralog usage among the present-day species. Moreover, interspecies analyses of enhancers indicated that the loss of enhancers rather than changes in trans-regulatory elements drove the role-division of these paralogs.

evolutionary biology↗

bioRxiv2022

In vitro and in vivo gene introduction in the cloudy catshark (Scyliorhinus torazame), a cartilaginous fish

Cartilaginous fishes have various unique physiological features such as cartilaginous skeletons and a urea-based osmoregulation strategy for adaptation to their marine environment. Also, because they are considered a sister group of bony vertebrates, understanding their unique features is important from an evolutionary perspective. However, experimental approaches are limited in cartilaginous fishes. Particularly, genetic engineering, which can analyze gene functions as well as cellular behavior, has not been effectively utilized in cartilaginous fishes. This is partly because their reproductive strategy involves internal fertilization, which results in difficulty in microinjection into fertilized eggs at the early developmental stage. Trials of gene transfer have also been limited both in in vitro cultured cells and in vivo. Here, to identify efficient gene transfer methods in cartilaginous fishes, we examined the effects of various methods both in vitro and in vivo using the cloudy catshark, a candidate model cartilaginous fish species. In all methods, green fluorescent protein (GFP) expression was used to evaluate exogenous gene introduction. First, we established a primary cell culture containing fibroblast-like and epithelial-like cells from cloudy catshark embryos. Using these primary cultured cells, we attempted gene transfection by lipofection, polyethylenimine (PEI), adenovirus, baculovirus and electroporation. Among the methods tested, lipofection, electroporation and baculovirus infection enabled the successful introduction of exogenous genes into primary cultured cells, allowing us to study physiological mechanisms at a single-cell level in culture conditions close to those in a living cartilaginous fish. We also attempted in vivo transfection into cloudy catshark embryos by electroporation and baculovirus infection. Although baculovirus-injected groups did not show GFP fluorescence, electroporation successfully introduced GFP into various tissues including muscle cells. Furthermore, we succeeded in GFP introduction into adult testis by electroporation. The in vitro and in vivo gene introduction methods that worked in this study may identify paths for future genetic manipulation including knockout experiments and cellular linage analysis in cartilaginous fishes.

molecular biology↗