Understanding the role of the shha-expressing cells during zebrafish caudal fin regeneration

Abstract

Zebrafish is able to regenerate its fins following amputation. We are interested in elucidating the cellular and molecular events that are underlying regeneration of the bones of the fin rays. Previous studies from our laboratory showed that genes involved in the Hedgehog Hh pathway are re-expressed during fin regeneration. In addition, gain and loss of function experiments previously showed that Hedgehog signalling might be involved in regenerate outgrowth and osteoblasts differentiation and patterning. Two members of the hedgehog gene family are expressed during fin regeneration: ihha (Indian Hedgehog) is expressed in the newly differentiated osteoblasts, while shha (Sonic Hedgehog) is expressed in a small domain of the basal epidermal layer in the differentiation zone. To understand the specific role of the shha-expressing cells, we precisely monitored these cells during the process of branching morphogenesis, using the transgenic line 2.4shha:GFPABC#15. We subsequently defined 3 stages that are successively taking place during the formation of the branches in the regenerating fin ray. We also assessed the expression profile of genes that might be involved in potential cross-talks between HH/FGF signalling during fin ray regeneration. Shh has been shown to play a role during branching morphogenesis in different organs that branche, through inducing cell proliferation. We analysed the cell proliferation profile during branching morphogenesis in the regenerating fin rays, and we suggest that shha-expressing cells might regulate the proliferation rate during branching morphogenesis. Previous results obtained from laser ablation experiments, where the transgenic line 2.4shha:gfp:ABC#15 was used to specifically but transiently ablate the shha-expressing cells using a laser beam showed that the transient absence of the shha --expressing cells from the regenerating ray results in a branching delay. Based on this finding, along with our results from cell proliferation analysis, and the time course analysis of the shha --expressing cells during branching morphogenesis, we propose a model for branches formation during fin regeneration, in which the splitting of shha ---expressing cells govern branches formation. We also elaborated a new approach based on the generation of a transgenic line expressing the nitroreductase in the shha-expressing cells, through which we aim to conditionally ablate these cells in the fin regenerate by addition of the prodrug, metronidazole. This will allow us to maintain the absence the shh-expressing cells long enough to observe the long term ablation effects and to better understand the role of these cells during caudal fin regeneration.