Brainstem Respiratory Oscillators Develop Independently of Neuronal Migration Defects in the Wnt/PCP Mouse Mutant looptail

Muriel Thoby-Brisson,Charlotte Dean,Derrick M Glasco,Michelle E Stewart,Jennifer N Murdoch,Anand Chandrasekhar,Julien Bouvier,Gilles Fortin,Jean Champagnat

doi:10.1371/journal.pone.0031140

Abstract

The proper development and maturation of neuronal circuits require precise migration of component neurons from their birthplace (germinal zone) to their final positions. Little is known about the effects of aberrant neuronal position on the functioning of organized neuronal groups, especially in mammals. Here, we investigated the formation and properties of brainstem respiratory neurons in looptail (Lp) mutant mice in which facial motor neurons closely apposed to some respiratory neurons fail to migrate due to loss of function of the Wnt/Planar Cell Polarity (PCP) protein Vangl2. Using calcium imaging and immunostaining on embryonic hindbrain preparations, we found that respiratory neurons constituting the embryonic parafacial oscillator (e-pF) settled at the ventral surface of the medulla in Vangl2Lp/+ and Vangl2Lp/Lp embryos despite the failure of tangential migration of its normally adjacent facial motor nucleus. Anatomically, the e-pF neurons were displaced medially in Lp/+ embryos and rostro-medially Lp/Lp embryos. Pharmacological treatments showed that the e-pF oscillator exhibited characteristic network properties in both Lp/+ and Lp/Lp embryos. Furthermore, using hindbrain slices, we found that the other respiratory oscillator, the preBötzinger complex, was also anatomically and functionally established in Lp mutants. Importantly, the displaced e-pF oscillator established functional connections with the preBötC oscillator in Lp/+ mutants. Our data highlight the robustness of the developmental processes that assemble the neuronal networks mediating an essential physiological function.

Highlights

The development of functional neuronal circuits requires appropriate migration of neurons from the germinal zone where they are born to their final position in the nervous tissue
Given that embryonic parafacial oscillator (e-pF) neurons are located normally in close proximity to the facial motor nucleus (FMN) [7,20], and that the FMN is misplaced in Lp/+ and Lp/Lp embryos [15,16], we used various e-pF molecular markers to examine its location in Lp mutants
Wholemount in situ hybridization for Tbx20, a T-box transcription factor expressed by migratory viscero- and branchio-motor neurons of the hindbrain [23,24], was performed in E14.5 embryos, a stage at which the FMN has completely formed at the ventro-lateral surface of a brainstem area mostly derived from rhombomere 6 (r6; Fig. 1D)

Summary

Introduction

The development of functional neuronal circuits requires appropriate migration of neurons from the germinal zone where they are born to their final position in the nervous tissue. Abnormal neuronal migration during development can cause neurological and cognitive impairments varying between mild to severe deficits [1,2,3]. Respiratory rhythmogenesis relies on the activity of a brainstem respiratory rhythm generator located in the ventral medulla, and composed of two interacting oscillators: the preBotzinger complex (preBotC) that drives inspiration [4] and the parafacial respiratory group (pFRG) controlling pre-inspiratory and expiratory activities [5,6]. It has been shown recently in rodents that respiratory oscillators emerge sequentially during development. At embryonic day (E) 14.5 in the mouse, rhythmic activity and chemosensitivity can be detected in the embryonic parafacial oscillator (e-pF) [7], while the preBotC oscillator activity appears one day later at E15.5 [8]

Methods

Results

Conclusion