Whole embryo culture, transcriptomics and RNA interference identify TBX1 and FGF11 as novel regulators of limb development in the mouse

Gautier Tejedor,Patricia Luz-Crawford,Paul Chuchana,Marc Mathieu,Karima Kissa,Béryl Laplace-Builhé,Christian Jorgensen,Jérôme Collignon,Said Assou,Farida Djouad,Audrey Barthelaix

doi:10.1038/s41598-020-60217-w

Abstract

Identifying genes involved in vertebrate developmental processes and characterizing this involvement are daunting tasks, especially in the mouse where viviparity complicates investigations. Attempting to devise a streamlined approach for this type of study we focused on limb development. We cultured E10.5 and E12.5 embryos and performed transcriptional profiling to track molecular changes in the forelimb bud over a 6-hour time-window. The expression of certain genes was found to diverge rapidly from its normal path, possibly reflecting the activation of a stress-induced response. Others, however, maintained for up to 3 hours dynamic expression profiles similar to those seen in utero. Some of these resilient genes were known regulators of limb development. The implication of the others in this process was either unsuspected or unsubstantiated. The localized knockdown of two such genes, Fgf11 and Tbx1, hampered forelimb bud development, providing evidence of their implication. These results show that combining embryo culture, transcriptome analysis and RNA interference could speed up the identification of genes involved in a variety of developmental processes, and the validation of their implication.

Highlights

A number of studies have attempted to integrate what is known of the genetic and transcriptional control of vertebrate limb formation into temporal gene networks[1,2,3,4]
Tbx[1] and Fgf[11], were tested for their implication in limb bud development and found to be involved. These results show that gene expression profiles of cultured embryos quickly diverge from their normal course, their analysis can deliver valuable information and lead to a better understanding of actual developmental processes
Analysis of the transcriptomes of forelimb buds (FB) from cultured E10.5 and E12.5 embryos, and comparison with those from their freshly dissected counterparts, allowed us to distinguish over a short time-window changes in gene expression that still reflected ongoing developmental process as they normally take place in utero, from changes in gene expression that were dependent on a stress-induced response triggered by the adaptation of the embryos to culture conditions

Summary

Introduction

A number of studies have attempted to integrate what is known of the genetic and transcriptional control of vertebrate limb formation into temporal gene networks[1,2,3,4] These efforts were based in part on transcriptome data obtained from mouse embryos collected 0.5 or 1 day apart from E9.5 to E13.53,5. Tbx[1] and Fgf[11], were tested for their implication in limb bud development and found to be involved These results show that gene expression profiles of cultured embryos quickly diverge from their normal course, their analysis can deliver valuable information and lead to a better understanding of actual developmental processes

Objectives

Methods

Results

Conclusion