The Molecular Neurobiology of Breathing...

All land vertebrates, including humans, use lungs to breathe air. The inspiratory and expiratory movements of the lungs are driven by a complex neural circuitry that consists of a central network in the brainstem that generates breathing rhythms and an output layer of motor neurons (MNs) which connect to respiratory muscles.

These respiratory circuits develop prenatally and have to become functional immediately after birth. While significant progress has been made in understanding the central pattern generator itself, very little is known about the formation of neural circuits that turn breathing rhythms into coordinated motor output.

Understanding the genetic program of respiratory MN differentiation is desirable for two reasons: First, respiratory MNs evolved after the basic vertebrate body plan was established and became more diverse during the evolution from ancestral tetrapods to mammals. Hence, understanding the logic of their genetic program might shed light on how neuronal developmental programs diversify, as organisms grow more complex. Second, in humans, loss of respiratory motor function is a leading cause of death in MN diseases such as spinal muscular atrophy and amyotrophic lateral sclerosis.

Reconstitution of respiratory muscle innervation through cell replacement therapy is therefore of considerable medical interest and will require the generation of authentic respiratory MNs in vitro from embryonic stem cells or induced pluripotent cells. Elucidating how respiratory MN identity and connectivity is established during normal embryogenesis would greatly facilitate the development of such a therapeutic approach.

The cell bodies of MNs are organized in columns along the rostro-caudal extent of the brainstem and spinal cord. Neurons in a column send axons to a distinct set of muscles during early embryonic development (Figure 1A). The columnar identity of MNs is genetically predetermined, and several elements of the transcription factor code that controls MN specification have been identified (Figure 1B). For example, the 'lateral motor column' (LMC), which projects to limb muscle, is specified by the coexpression of Hox6 (forelimb) or Hox10 (hindlimb) paralogues and Foxp1. In contrast, the identity of the 'medial motor colum' (MMC), which innervates epaxial muscle, is determined by the expression of Lhx3/Lhx4.

Respiratory muscles are controlled by two different MN subsets, both of which are part of the 'hypaxial motor column' (HMC): 1) The phrenic nucleus, which is a derivative of the cervical HMC, connects to the diaphragm muscle. The diaphragm, which is unique to mammals, forms the boundary between thoracic and abdominal cavity and contracts during inspiration. 2) Respiratory thoracic HMC neurons fall into two functional categories: MNs innervating the external intercostal muscle are inspiratory, whereas MN projecting to internal intercostal muscle and some abdominal muscles are expiratory. Both the phrenic nucleus and respiratory MNs of the thoracic HMC receive input from respiratory centers in the brainstem, which generate breathing rhythms. HMC populations that control air breathing emerged relatively late in evolution. While ancestral vertebrates such as lampreys do have MMC- and HMC-like neurons, the function of both MN subtypes in these animals is to control myomeric axial muscle and generate undulatory swimming behavior.

In contrast, HMC neurons in higher vertebrates, such as mammals, have evolved to mediate additional motor functions, in particular breathing, and show a far less stereotypic pattern of nerve-muscle connectivity. Therefore, it seems plausible that respiratory HMC identity in mammals is determined by common transcriptional element(s) that allow the anatomically diverse, yet functionally linked subsets to execute segment-specific developmental programs, as Foxp1 does in LMC neurons. Using a combination of in vitro differentiation of MNs from mouse ES cells, mouse genetics, imaging and systematic analysis of gene expression, I aim at defining the transcriptional program of respiratory MN identity (Figure 2).

In the long run, the expertise gained from this investigation will be applied to the differentiation of MNs from human ES cells. Human ES cell-derived respiratory MNs could serve as a therapeutic tool to restore the ability to breathe in individuals suffering from neuromuscular disorders or spinal cord injury.

Figure 1: Hierarchical organization of MN identity and axonal target choice. (A) Most spinal MNs belong to the vMN population that sends axons into the ventral mesenchyme. Spinal vMNs are further divided into four columnar subtypes that have distinct muscle targets. HMC: 'hypaxial motor column'; MMC: 'medial motor column'; LMC: 'lateral motor column'; PGC: 'pregangionic column'; PHR: 'phrenic nucleus'; HMCic: 'HMC innervating intercostal muscle'. (B) Columnar identity is determined by the combinatorial expression of transcription factors, in particular Hox genes. The two principal populations of respiratory MNs, cervical phrenic neurons and the thoracic HMC, are marked with circles.

Figure 2: Sorting of phrenic neurons and subsequent microarray analysis allowed the identification of phrenic nucleus-specific genes. (A )The Hb9::GFP transgene marks all spinal MNs. Phrenic neurons in E11.5 Hb9::GFP embryos were retrogradely labeled with dextran-tetramethylrhodamine (white arrow) (B) FACS purification of GFP+ TMR+ phrenic neurons from dissociated spinal cords.

(C-E) Transverse section of E11.5 ventral cervical spinal cord: In situ hybridization shows phrenic nucleus-specific expression of candidate genes identified in an Affymetrix microarray screen (red arrows).