Chapter 3 - Genetic factors determining the functional organization of neural circuits controlling rhythmic movements: the murine embryonic parafacial rhythm generator

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Abstract

In mammals, fetal movements governed by central pattern generators are essential for the development of adaptive behaviors such as breathing, walking, and chewing, which are vital after birth. Combining targeted mutations and genetic fate mapping can help to define the molecular determinants that control the development of these central pattern generators. In this chapter, recent results are presented on the embryonic parafacial (e-pF) rhythm generator, one of the two oscillators involved in controlling the breathing behavior and chemosensitive responsiveness.

Introduction

The respiratory rhythm generator is probably one of the best models to understand how genes have been selected and conserved to control development of central pattern generators responsible for adaptive behavior in vertebrates. Recent advances in the neurobiology of neonatal rodents suggest that breathing rests on two prominent rhythmogenic sites, the parafacial respiratory group and the pre-Bötzinger complex (preBötC, see Feldman et al., 2009, Feldman & Del Negro, 2006, Bouvier et al., 2010 and references herein). The complex circuitry in which central pattern generators participate, is established during development and thought to rely on spatially and temporally ordered appearance of neurons. The parafacial region is well understood with respect to the transcription factors that underlie specification of neural progenitors. Hox paralogs and Hox-regulating genes such as Egr2 (Krox-20) govern transient formation of developmental compartments, the rhombomeres, in which rhythmic neuronal networks develop (r1–r7; see Thoby-Brisson et al., 2009 and references herein). Along the dorsoventral axis of the rhombomeres, and in the spinal cord, the different neuronal types to be incorporated in synaptic circuits appear in progenitor domains, such as the “dorsal” domain broadly defined by the expression of Pax7 (dA1-dB4), then Lbx1 (dB1–dB4; see Pagliardini et al., 2008) and further subdivided by, inter alia, basic helix-loop-helix (bHLH) proneural genes (see Storm et al., 2009). In addition to these neural-type specific transcription domains, developmental gene expression might also match neuronal populations sharing the same connectivity and function: this is the case of Phox2b expression (three distinct columns (dA3, dB2, V3) in the neural tube along the DV axis, spanning different rhombomeres on the anteroposterior axis) which eventually provides molecular fingerprinting of the entire visceral nervous system from afferent chemosensory function (in carotid bodies, area postrema) to efferent branchiomotor, preganglionic, and autonomic nuclei and ganglia (see Dubreuil et al., 2009a, Dubreuil et al., 2009b). A general view is emerging on the role of developmental transcription factors allowing the coordinated integration of different neuronal types to produce motor rhythmic patterns. The present chapter concentrates on recent findings on the embryonic parafacial (e-pF) rhythm generator in the more general context of respiratory and motor development.

Section snippets

Molecular identification of central oscillators during embryonic development: the e-pF oscillator

Evidence for the developmental origin and functional nature of the respiratory rhythm generating circuits involved in fetal and neonatal breathing has been obtained using mutant mice in which developmental genes encoding transcription factors are inactivated. Inactivation may lead to abnormal breathing behavior as a result of the elimination of crucial neuronal systems. In particular, inactivation of the gene for the zinc finger transcription factor Egr2 that controls the formation of odd

Odd rhombomeric (Egr2/Krox20, Hox) patterning of the parafacial hindbrain

Comparing the postnatal behavior of several mouse models (reviewed by Champagnat et al., 2009) demonstrated that specific respiratory deficits in vivo are assignable to anteroposterior segments of the brainstem, suggesting that the respiratory neuronal network is organized according to the rhombomeric patterning by Egr2 and Hox expression. Three anteroposterior levels can be distinguished with corresponding deficits. Inactivation of e-pF activity causes life-threatening neonatal apnoeas

The paired-like homeobox 2b (Phox2b) gene, a visceral cell-type fingerprint in respiratory control: link with central chemosensitivity and the congenital central hyperventilation syndrome

There are now several mice models showing that Phox2b is a major actor during the e-pF development (Dubreuil et al., 2009a, Dubreuil et al., 2009b and references herein). Phox2b is the homeodomain transcription factor specifically expressed (since E9) and required in cells that form the phylogenetically ancient visceral reflex circuits controlling digestive, cardiovascular, and respiratory functions, thereby maintaining bodily homeostasis through branchial motor, parasympathetic, sympathetic,

The e-pF expresses the proneural mouse atonal homolog 1 (Atoh1/Math 1)

One important class of genes that regulates cellular diversity in the nervous system encodes bHLH transcription factors, which act as generic proneural factors within the Notch pathway to single out neuronal progenitors and promote their differentiation. Subsequent analysis revealed the important roles of bHLH factors and their interaction in the determination of neuronal fates, in relation to the morphogenetic gradients of bone morphogenetic proteins (dorsal) and sonic hedgehog (ventral),

Perspective: the spinal connection

Recent studies shed some light upon cell autonomous genetic network specifying the e-pF neuronal lineage and eventually endowing it with rhythm generating and chemoresponsive cellular properties. As important would be to understand how the e-pF rhythm may influence coordinated breathing movements. In vertebrates, the cell bodies of spinal motor neurons are organized in columns along the rostrocaudal extent of the brainstem and spinal cord. Neurons in a column send axons to a distinct set of

Perspective: insertion of inhibitory interneurons

There is increasing consensus to consider the rodent parafacial group as the controller of active expiration, with neurons active in late expiration (preinspiration; Onimaru et al., 2008) and with preferential action on expiratory (e.g., lumbar) motoneurons (Janczewski and Feldman, 2006). Virtually all e-pF neurons are glutamatergic, while rhythm generation and synchronization within each e-pF cell cluster does not require glutamatergic synapses (in clear contrast with the pre-BötC oscillator,

Perspective: the pre-Bötzinger complex

Because the respiratory rhythm generator includes multiple oscillators in lampreys (Martel et al., 2007), the murine preBötC might be considered a conserved homologous of e-pF's partners during gnathostome evolution. This, however, is probably not the case because anteroposterior location and function of postotic rhythm generators seem variable in different vertebrates (see Bass et al., 2008, Kinkead, 2009, Wilson et al., 2006). Isolation and imaging of nonmammal oscillators together with an

Acknowledgments

This work was supported by institutional support from the CNRS (Centre National de la Recherche Scientifique, France) and a grant ANR-07-Neuro-007-01 (Agence Nationale de la Recherche, France) to G.F. This work benefited from the facilities and expertise of the Imagif Cell Biology Unit of the CNRS Research Center of Gif-sur-Yvette.

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