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J Appl Physiol 104: 1513-1521, 2008. First published January 24, 2008; doi:10.1152/japplphysiol.01383.2007
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INVITED REVIEW

HIGHLIGHTED TOPIC
Neural Control of Perinatal Respiration

Transcription factors and the genetic organization of brain stem respiratory neurons

Paul A. Gray

Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri


   ABSTRACT
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 ABSTRACT
PATTERNING THE HINDBRAIN
 IDENTIFYING THE A-P AND...
 GENETIC IDENTIFICATION OF...
 FUTURE DIRECTIONS FOR STUDYING...
 ACKNOWLEDGMENTS
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Breathing is a genetically determined behavior generated by neurons in the brain stem. Transcription factors, in part, determine the basic developmental identity of neurons, but the relationships between these genes and the neural populations generating and modulating respiration are unclear. The diversity of brain stem populations has been proposed to result from a combinatorial code of transcription factor expression corresponding to the anterior-posterior (A-P) and dorsal-ventral (D-V) location of a neuron's birth. I provide a schematic of transcription factor coding identifying at least 15 genetically distinct D-V subdivisions of brain stem neurons that, combined with A-P patterning, may provide a genetic organization of the brain stem in general, with the eventual goal of describing respiratory populations in particular. Using a combination of fate mapping in transgenic mouse lines and immunohistochemistry, we confirm the parabrachial nuclei are derived from a subset of Atoh1 expression progenitor neurons. I hypothesize the Kölliker-Fuse nucleus can be uniquely defined in the neonate mouse by the coexpression of the transcription factor FoxP2 in Atoh1-derived neurons of rhombomere 1.

breathing; development

THE MAMMALIAN NERVOUS SYSTEM contains hundreds of distinct neuron classes defined by anatomic location, projection pattern, gene expression, morphology, pharmacology, and firing pattern (13, 52, 76). At the same time the basic organization of the brain is highly stereotyped and evolutionarily conserved (66). This is also true for neurons involved in generating breathing. The basic organization of respiratory populations is strikingly similar between mammals and birds, strongly suggesting evolutionary conservation (5, 46, 119). The relationships between the genetic programs that generate neurons and the function of neurons that actually form the circuits underlying respiratory behaviors are unknown. One approach to elucidating these relationships is to correlate the physiological or genetic classification of neurons with their developmental origins in terms of transcription factor expression (51).

Transcription factors (TFs) are genes that control the expression of other genes. It has been proposed that the diversity of the nervous system is mediated at least in part by a "combinatorial code" of TFs expressed in a spatially and temporally defined manner (34, 71). Moreover, combinations of TFs may provide markers to uniquely identify specific subsets of neurons (24). Analysis of the nervous system using TFs as tools has been quite successful in understanding the genetic programs that specify neurons of the cortex, thalamus, spinal cord, and retina, but they have not been used in any detail to address respiratory networks (34, 71).

Breathing is a behavior generated by neurons within the hindbrain. To better understand the role development plays in organizing respiratory circuits, I will briefly describe the role TFs play in organizing the hindbrain along its two major axes, anterior-posterior (A-P) and dorsal-ventral (D-V), focusing on describing the latter. I will also describe what is known and what can be inferred about the developmental origins of respiratory populations. I will describe what is known about the gene expression patterns of medullary neurons that can be correlated with developmental origin. Last, I will briefly summarize genetic techniques that have been applied to studying neuronal populations and their possible uses in analyzing respiratory circuits.


   PATTERNING THE HINDBRAIN
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 ABSTRACT
 PATTERNING THE HINDBRAIN
IDENTIFYING THE A-P AND...
 GENETIC IDENTIFICATION OF...
 FUTURE DIRECTIONS FOR STUDYING...
 ACKNOWLEDGMENTS
 REFERENCES
 
A-P axis.   Early during embryogenesis [embryonic days 8–10 (e8–e10) in mice], the portion of the neural tube that will become the hindbrain is segmented into a series of seven to eight invaginations termed rhombomeres (R1–R8) (66). The boundaries between these rhombomeres correlate with the nested expression boundaries of highly evolutionarily conserved transcription factor proteins known as homeotic (Hox) genes. For example, during early development the zinc finger transcription factor gene Krox20/Egr2 is expressed in R3 and R5, while the bZIP gene Kreisler/MafB is expressed in R5–R6 (11, 26). Importantly, the diversity of neuron types is remarkably similar between rhombomeres. This means neurons with similar projections and functions, e.g. motoneurons, will be present in multiple rhombomeres, but each individual population may still show distinct differences in projection pattern, migration, and gene expression.

The effects of the genetic mutation on respiratory output of several transcription factors involved in A-P patterning of the hindbrain have been analyzed. The results of these experiments have been described in several excellent reviews (10, 16, 45). Briefly, the mutation of HoxA1, Krox20/Egr2, or Kreisler/MafB leads to the selective loss of neurons from different combinations of rhombomeres (9, 10, 17, 18, 23, 26). These and other data suggest the elimination of R2 and R5 has minor but not lethal effects on respiratory output, the R3–R4 region is vital for the production of an opioid-insensitive, antiapneic population, and the R7 region is necessary for respiratory rhythmogenesis and the formation of the pre-Bötzinger complex (preBötC) (9, 10, 17, 26, 60).

Importantly, mutations of genes that affect A-P patterning likely affect a large percentage of the neurons within affected rhombomeres. Thus the actual respiratory effects of a mutation may be mediated by the loss of a specific population, the expansion of a different population, or the combined effect on multiple populations. In addition, some TFs that play a role in early specification may continue to be expressed in subsets of neurons during later developmental stages. For example, MafB is a bZIP TF that is first expressed in rhombomeres 5 and 6 and is then later expressed in subsets of motoneurons and interneurons throughout the hindbrain and spinal cord (79). Kreisler is a naturally occurring mutant mouse with a deletion of a portion of the promoter region of the MafB gene that results in the ectopic expression of MafB early in development. This ectopic expression leads to the elimination of R5 and the modification of R3 (22, 35, 79). Kreisler heterozygous animals have an elevated respiratory frequency due to the malformation of R3, while homozygous mutants show a slowed, but not lethal, respiratory output and blunted chemosensitivity (17). The Kreisler mutant, however, does not eliminate MafB protein. MafB knockout mice produce no respiratory output, they lack preBötC neurokinin 1 receptor-expressing (NK1R) neurons, and they have disruptions of the nucleus ambiguus (NA) motor pool (9). These deficits are likely due to the loss of MafB's normal role in rhombomeric specification. Later in development, MafB is expressed in subsets of spinal cord and hindbrain motoneurons and interneurons. MafB-expressing interneurons are not located within the preBötC, which suggests the underlying deficit in these mutants is associated with an error in the initial specification of R7.

D-V axis.   Similar to A-P patterning, the D-V axis of the developing hindbrain and spinal cord has overlapping patterns of highly conserved genes in both the developing progenitor cells as well as in postmitotic neurons (e9–e13). In the spinal cord, at least 13 genetically distinct populations have been identified on the basis of their spatial and/or temporal combination of TF expression (57, 117). In the hindbrain, homologs of 12 of these populations have been identified on the basis of similar patterns of TF expression. At least three additional populations of neurons with no related spinal cord homologs have been identified (20, 25, 104, 122). Figure 1 shows a schematic of the pattern of D-V expression for 19 TFs in the hindbrain that can define at least 15 genetically distinct subpopulations of neurons present at or before e11.5. The terminology of dorsal populations follows that of Sieber et al. (104, 121) (Fig. 1A). The most ventral hindbrain domain [here named as V3-like (V3-L)] arises from the same Nkx2.2-expressing region that before e11.5 generates motoneurons and as such is not a true progenitor domain (91a). Figure 2 shows a schematic of the A-P distribution of the nine dorsalmost D-V domains present at e11.5 in the brain stem. Note that not every population is present in every rhombomere. For example, the Phox2b-expressing DA3 domain is present only in R4–R7. I only describe 19 relatively well-known TFs for ease of use but point out that this is only a small subset of TFs expressed in the hindbrain. A recent genome scale screen of TF expression by in situ hybridization identified well over 100 different TF genes expressed in subsets of brain stem neurons, but their expression cannot be covered here (52).


Figure 1
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  		Fig. 1. Transcription factor coding of dorsal-ventral (D-V) populations in the brain stem. A: nomenclature of transcription factor (TF)-defined neuronal populations in the e11.5 spinal cord (left) and brain stem (right) (adapted from Refs. 51, 57, 104). Domains in red are brain stem specific. D indicates a dorsal population while V indicates a ventral population. The pMN domain gives rise to motoneurons in the spinal cord and either somatic (pMNs) or visceral (pMNv) motoneurons in the brain stem. B: schematic diagram of expression for 19 TFs. Each colored block indicates a D-V region of expression in at least one rhombomere. Gene abbreviations are listed across the top. Genes expressed only in progenitor cells are in italics. Blocks outlined in black indicate brain stem-specific expression. C: table indicating the fast neurotransmitters expressed and the commissural (Comm) projection pattern of each D-V domain.

 

Figure 2
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  		Fig. 2. Rhombomeric mapping of D-V populations in the brain stem. Schematic diagram of the rhombomeric expression of the 9 dorsalmost D-V progenitor domains in the e11.5 brain stem. Rhombomeres are listed across the top with the most rostral (R1) at the left. Each row indicates the span of expression for each population not accounting for migration. SC, spinal cord.

 
The most salient aspect of D-V patterning is that each domain produces neurons that share major aspects of neural identity, such as projection pattern and transmitter identity (Fig. 1C). These features are a direct consequence of genetic origin and may differ significantly from neurons born in adjacent progenitor domains. In the cortex, developmental origin has been shown to directly relate to peptide expression as well as neuronal morphology, and this is likely to be the case in the hindbrain and spinal cord as well (13, 114). Importantly, progenitor domains can still produce a number of genetically distinct subpopulations due to divergent TF expression, time of birth, or even epigenetic mechanisms that are still unclear. For these reasons, there is reason to believe that no two neurons express exactly the same genes but that having a shared developmental origin provides a number of shared characteristics that may be fundamental to brain function (76, 114).


   IDENTIFYING THE A-P AND D-V ORIGINS OF RESPIRATORY POPULATIONS
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Mutation of a number of TFs expressed in the brain stem has been shown to affect breathing in vivo, in vitro, or both. In fact, lethality is a common consequence of mutating many D-V patterning TFs. Of the 19 TFs in Fig. 1, the genetic elimination of 13 of these genes leads to lethality either during embryogenesis or perinatally (7, 44, 54, 57, 83, 85, 93, 102, 103). Understanding the exact mechanisms of this lethality is often difficult as many of these genes are expressed in multiple organs or multiple neural populations. For example, DA1 interneurons of the hindbrain are derived from proliferating precursor neurons expressing the basic helix-loop-helix gene Atoh1 (Math1). Atoh1 mutants die at birth without taking a breath (7). Atoh1, however, is necessary for the specification of a number of different central nervous system (CNS) populations, including granule cells of the cerebellum, the parabrachial nuclei (see below), lateral reticular nuclei, proprioceptive neurons of the spinal cord, and scattered other populations throughout the brain stem (74, 117). Similarly, both Tlx3 and Lmx1b mutants die from respiratory distress shortly after birth, but their patterns of expression make it difficult to uniquely identify a single locus of action (19, 27, 29, 94). The challenge is to find ways to correlate gene expression patterns with the identity of neurons described anatomically and/or physiologically.

Neurons involved in breathing are found throughout the extent of the hindbrain. Populations that contain significant numbers of neurons phasically active during breathing are clustered in four general regions in the brain stem. The dorsolateral pons (DLP) contains both the parabrachial and Kölliker-Fuse (KF) nuclei (30, 82, 96, 107, 109). The caudal pons contains the A5 region as well as the intertrigeminal region (ITR) (14, 15, 58). The dorsal medial medulla contains the nucleus tractus solitarius (NTS) (8, 37, 98). Finally, the ventrolateral medulla contains several different respiratory populations, the Bötzinger complex (BötC), preBötC, rostral ventral respiratory group (rVRG), caudal ventral respiratory group, and, most recently described, the parafacial respiratory group (pFRG) (36, 37, 53, 81, 87, 88, 105). These last four populations together have been termed the ventral respiratory column (VRC) (1, 2). In addition, several other populations have been implicated in directly modulating breathing, including the retrotrapezoid nucleus (RTN), medullary raphe, as well as neurons distributed along the ventral medullary surface (12, 21, 55, 62, 84, 92, 97, 99). In some cases, the exact boundaries between these populations are unclear or controversial, but many researchers can agree on the general center of each of these regions (31, 38, 55, 56, 61, 65, 89).

Only a small subset of brain stem neurons migrate across rhombomere boundaries (66). Because of this, it is possible to combine the existing data of cell location with the known developmental origin of adjacent populations, such as motoneurons and catecholaminergic populations, to hypothesize the rhombomeric origin of several respiratory populations. On the basis of their location rostral to the locus ceruleus, Chatonnet et al. (18) recently provided strong data indicating the parabrachial and Kölliker-Fuse (KF) nuclei are derived from the R1 region (18). Similarly, analysis from a variety of mutant animals predicts the A5 and ITR regions are derived from some subset of R4–R6 precursors, and the NTS and VRC populations are derived from R7–R8. Recent experiments describing a differential opioid sensitivity between the two putative respiratory oscillators, preBötC and pFRG, and the ability of naloxone to promote survival in Krox20 mutant mice, suggests that at least a portion of pFRG neurons are derived from the R3 and/or R5 region and migrate toward the ventral medulla (80, 90). Fate-mapping analysis using rhombomere-specific reporter mice (see below) in the future will provide direct tests of these predictions.

Correlating respiratory populations with D-V identity is more complicated because of extensive D-V migration. One successful approach has been to identify a specific TF candidate gene by matching its expression pattern to a known or expected respiratory population by observing a knockout phenotype with a respiratory deficit. This is extremely useful in cases where TF expression is within known anatomic boundaries, is maintained into the neonatal and/or adult stage, and high-quality antibody or in situ probes are available. Variations of this approach were used by two different groups interested in respiratory modulating populations. In the first, Guyenet and colleagues (55, 63, 84, 118) identified a small population of neurons expressing the vesicular glutamate transporter 2 (VGlut2) on the ventral surface of the hindbrain, beneath and adjacent to the VII motonucleus. This region corresponds to the loosely defined RTN (86, 92). They found these neurons fired tonically, but their firing rate was directly related to extracellular pH and CO2. They further found these neurons express the transcription factor Phox2b (3, 110). Phox2b mutations are responsible for more than 90% of congenital central hypoventilation syndrome (CCHS; Ondine's curse) cases, and CCHS patients have deficits in CO2 chemosensitivity. Recently, transgenic mice expressing the most common human CCHS Phox2b mutation were generated. These animals have fatal central apneas and lack CO2 sensitivity similar to humans. Importantly, these animals show a selective loss of the RTN Phox2b neurons but not other Phox2b populations, strongly suggesting an anatomic locus mediating CCHS (28).

Second, in an elegant genetic experiment, the Lmx1b gene was deleted only from neurons that express the ETS transcription factor Pet1 (124). Pet1 is uniquely expressed in the subset of Lmx1b neurons that are serotonergic. Loss of Pet1 eliminates most but not all serotonergic neurons, while loss of Lmx1b completely eliminates both serotonergic neurons as well as other neural populations. Mice in which Lmx1b is eliminated from Pet1-expressing cells have no serotonergic neurons, yet breathe normally in normoxia. These results suggest the lethal phenotype of complete Lmx1b mutant mice is due either to the loss of a different neural population than serotonergic neurons, or the combined loss of multiple populations (124).

Determining the role in breathing of TF-expressing neurons that are either outside of clearly definable nuclei or that do not coexpress clear markers is more difficult. For example, the respiratory disruption in Tlx3 mutants was originally proposed to be due to a failure in respiratory rhythmogenesis, as a subset of Tlx3-expressing neurons are located within the ventrolateral medulla, and the rhythmic, respiratory-related output of the isolated brain stem was highly irregular (102). Analysis of a homologous Tlx3 expressing population in the spinal cord found a decrease in the number of glutamatergic neurons and an increase in the number of GABAergic neurons in Tlx3 mutant mice. This suggested a similar effect occurs in the hindbrain. Indeed, blockade of fast GABAergic transmission rescued the ataxic pattern of rhythmic output in in vitro preparations of Tlx3 mutant mice. This indicates the defect was not inherent to rhythm generation in vitro (19).

From these and similar experiments, the analysis of TF-defined populations has been extremely productive (Fig. 1). For example, experiments have identified the developmental origins of the NTS and medullary catecholaminergic neurons (DA3), as well as the raphe nuclei (V3-like) and inhibitory neurons of the SpV (104). For these populations, it is possible to begin to determine exactly which genes are necessary for each individual population. In the caudal medulla (R7), Lmx1b, Phox2b, and Tlx3 are all coexpressed in DA3 interneurons. This precursor population generates the glutamatergic neurons of the NTS and area postrema (AP), as well as the A1, A2, C1, and C2 catecholaminergic neurons, some of which are also glutamatergic (25, 95, 122). Loss of Phox2b eliminates the NTS and AP, loss of Phox2b or Tlx3 eliminates noradrenergic neurons, and Lmx1b loss eliminates the raphe and may effect the NTS (20, 25, 27, 91). Phox2b, however, is also expressed in visceral motoneurons and RTN neurons, Lmx1b is expressed in serotonergic neurons, and both Lmx1b and Tlx3 are expressed in noncatecholaminergic neurons throughout the brain stem. The necessary role in breathing for any of these other populations is unknown.


   GENETIC IDENTIFICATION OF RESPIRATORY NEURONS
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The developmental origins of the majority of respiratory neurons, especially within the VRC, are still unknown. Several approaches for identifying the genetic identity of respiratory populations are likely to be productive. As described above, the candidate gene approach based on respiratory phenotype can be very informative, but the respiratory effects of relatively few TF mutants have been analyzed in enough detail to make predictions possible. A different approach is to correlate the known genetic properties of respiratory populations with the characteristic properties of developmental populations. This is similar to a classic forward genetic screen where one looks to discover the genes responsible for specific phenotypic properties of known neurons. Conveniently, much is known about gene expression within respiratory populations. These data can be used to make predictions about the possible developmental origin of specific populations in order to identify candidate TF genes. For example, the choice of transmitter expressed within a neuron is genetically coupled to its developmental identity, as transmitter enzymes and transporter expression begins within a half day after leaving the cell cycle (19, 49). In the hindbrain, the transmitter identity of several, physiologically defined populations are known. In the NTS, pump cells receive glutamatergic input from lung stretch receptor afferents and then send an inhibitory projection (mostly GABA but also some glycine) to the VRC and RTN (67, 115). In the BötC, expiratory neurons release glycine as defined by expression of the vesicular glycine transporter (Slc6a5 or GlyT2) (39–41, 100). Spinally projecting neurons from the KF and rVRG have been shown to release glutamate or express glutamatergic marker proteins in their terminals (43, 113, 123). NK1R neurons of the preBötC express VGlut2 mRNA (111). Also, recent experiments suggesting the pFRG's role in respiratory timing during opioid-mediated inhibition strongly suggest at least some of these neurons are also glutamatergic. Combining known transmitter identity with other developmentally defined properties such as projection pattern in the forward genetic fashion may provide reasonable evidence for the role of specific developmentally defined populations in breathing. The major limitation is that many of the genetic properties of respiratory neurons are shared by a significant percentage of brain stem neurons (Fig. 1C).

One alternate to the hypothesis-based approaches described above is to use advances in genomics or genomic tools to screen large numbers of genes to identify candidates on the basis of their anatomic location. For example, microarray analysis of a number of organ systems and cell types has been extremely informative for identifying genes (101). Unfortunately, the heterogeneity of brain stem populations has limited the usefulness of these data for studying the neurons important for breathing.

A number of large-scale gene expression projects, in contrast, have provided preliminary data as to the anatomic location of a large percentage of the genome in the mouse brain stem (47, 52, 72, 75). These data can provide a starting point for the analysis of specific functional populations. Of course, this approach is hampered by the relative heterogeneity of brain stem circuits, the varying quality and resolution of analysis for different genes between different groups, and the ease of use of the large data sets. The major advantage here is that the initial large-scale effort is already complete, and it is possible to identify a relatively small number of candidates for more detailed analysis in a short time.

Last, one particularly informative approach has been to use gene promoters (TFs as well as other genes), either in transgenics or knock-in animals, not to study the genes themselves, but to drive reporter genes for the analysis of anatomy, circuitry, and behavior (13, 59, 74, 108). In this approach, also known as "fate mapping," these animals express either reporter genes such as β-galactosidase or a fluorescent protein such as green or yellow fluorescent protein (GFP/YFP) in specific subsets of neurons based on their genetic history. The stability of these reporters allows for the identification of cells even if the pattern of gene expression is only transient. Alternately, transgenic approaches using dual recombinases [reviewed in Dymecki et al. (32)], allows for a continuous or temporally specific expression of reporters, the cell type-specific elimination of target genes, or the expression of exogenous genes.

Figure 3 shows an example of the combination of several approaches specifically to determine the genetic origin of neurons of the dorsolateral pons. This region, containing the parabrachial (PB) and Kölliker-Fuse (KF) nuclei, is thought to be vitally important for the integration of sensory or state-dependent information with respiration (78). The genetic relationships between and actual boundaries of the PB and KF nuclei are controversial (31). Several groups using fate-mapping approaches found neurons of the parabrachial region are derived from precursor cells that express Atoh1 in the dorsalmost region of the rostral brain stem (33, 42, 74, 117). From our own genome-scale TF screen, we identified a number of genes expressed in subsets of DLP populations (52). Given the importance of these regions and the current lack of anatomic clarity, they make ideal candidates for genetic analysis. Using an Atoh1-cre transgenic mouse crossed with a cre-dependent YFP reporter mouse, one can label the entire population of neurons derived from the DA1 domain of the brain stem (Figs. 3 and 4) (77, 108). Combining reporter expression with immunohistochemistry for other TF genes, we found that, consistent with previous findings, the DLP is derived from Atoh1 precursors (Figs. 3 and 4). Importantly, however, we also found that a subpopulation of these neurons uniquely coexpresses the forkhead TF, FoxP2 in a location that corresponds to the functionally defined KF nucleus (Figs. 3A and 4B). FoxP2 is also expressed in a population derived from adjacent precursors expressing the Hox gene, Ptf1a (Fig. 4A) (64, 121). Several other TFs are expressed in mutually exclusive patterns in the same general region, underscoring the genetic heterogeneity of this region (Fig. 3). From these data, we can hypothesize the KF is at least partially developmentally related to the parabrachial nuclei but can be genetically distinguished from it by its coexpression of FoxP2. Whether the adjacent, Ptf1a-derived FoxP2 neurons are also within the KF and functionally similar is unclear. Interestingly, FoxP2 mutations in humans produce a profound deficit in the ability to produce speech as well as other complicated orofacial movements (6, 69, 103). Mutation in mice eliminates vocalizations (103). The expression of this gene in a region proposed to be important for gating respiratory behaviors suggests some aspects of this disorder may be related to the function of the KF nucleus (31).


Figure 3
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  		Fig. 3. Combinatorial TF expression suggests the developmental origin of subpopulations of pontine neurons. Confocal mosaic images of six 20-µm hemisections encompassing 180 µm at the level of the dorsolateral pons in a p0 double transgenic (Atoh1-cre x Rosa26 floxed YFP) (77, 108) mouse brain stem. Neurons derived from the Atoh1-expressing precursor domain (DA1) are labeled via immunohistochemistry for yellow fluorescent protein (YFP, green) in the lateral and medial parabrachial nuclei (LPB, MPB), pontine nucleus (PN), trapezoid body (TB), and reticulotegmental nucleus (RtTg) (A–F). DA1 interneurons are distinct but adjacent to monoaminergic neurons of the locus ceruleus, subceruleus (subLC), and dorsal raphe (DR) (B); scattered interneurons expressing either Phox2b (C) or Lmx1b (E); and motoneurons of the motor trigeminal nucleus (Mo5) (D). Atoh1-derived neurons coexpress FoxP2 in a region corresponding to the Kölliker-Fuse nucleus (KF) (F). Pr5, principal sensory trigeminal nucleus. Scale bar, 200 µm.

 

Figure 4
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  		Fig. 4. Fate mapping suggests a specific genetic origin of the KF nucleus. Confocal mosaic images of immunohistochemistry for FoxP2 (magenta) and YFP (green), at the level of the KF nucleus, in two different transgenic mouse lines. A: FoxP2 (magenta) is coexpressed with YFP (green) ventral to the principal sensory trigeminal nucleus (Pr5) in a p0 double transgenic mouse (Ptf1a-cre x Rosa26 floxed YFP) (64). B: FoxP2 (magenta) is coexpressed with YFP (green) in a region corresponding to the KF nucleus in a p0 double transgenic mouse (arrow; this image is the same as Fig. 2F). Scale bar, 200 µm. CB, cerebellum; MPB, medial parabrachial nucleus.

 

   FUTURE DIRECTIONS FOR STUDYING BREATHING
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Combining a fate-mapping approach with anatomic analysis of other genes in transgenic mice is relatively straightforward and will likely be remarkably insightful for understanding the genetic organization of respiratory populations. The existing literature has already identified a number of "marker genes," such as peptides, receptors, and calcium binding proteins, that have been proposed to identify specific populations of respiratory neurons (1, 73, 111–113). For example, in the adult rat, spinally projecting rVRG respiratory neurons, as well as more rostrally located noncatecholaminergic cardiovascular neurons, express mRNA for preproenkephalin. Similarly, the differential expression of calcium binding proteins has been suggested to define subsets of premotor neurons (1, 48). In addition, a number of transgenic reporter and/or recombinase mice have already been generated for many TF genes. For example, fate-mapping transgenic mouse lines have been generated for at least 12 of the 19 genes listed in Fig. 1. Combining the analysis of markers in these transgenic lines will be fairly straightforward.

The real value of identifying the developmental origins of respiratory populations is the ability to apply genetic tools to study breathing either in reduced preparations or in vivo. The inclusion of fluorescent reporters in specific neurons allows for the targeted electrophysiological recording of genetically defined neurons in reduced preparations. For example, Obata and colleagues (68) recorded from GFP-labeled neurons in the ventrolateral medulla in a GAD67-GFP knock-in mouse and found GABAergic neurons were few and only weakly respiratory. Similarly, Hinkley et al. (59) recorded from a specific subpopulation of HB9-expressing interneurons in the hemisected spinal cord that were rhythmically active during fictive locomotion.

More interesting has been the use of TF promoters and recombinases for circuit breaking. Similar to the isolated in vitro brain stem preparations, the isolated neonate lumbar spinal cord has been found to be capable of generating fictive locomotion with the application of 5-hydroxytryptamine and N-methyl-D-aspartate (106). En1 is expressed in a population of inhibitory interneurons, including Renshaw cells, in the ventral spinal cord (120). En1 mutant spinal cords produce a very slow locomotor rhythm. To test whether the locomotor effect was due to the loss of En1-expressing neurons or some modification of the network due to compensation, mice expressing Cre under the En1 promoter were crossed with two different cre-dependent lines. In the first, cre excision of a stop cassette induces the expression of diptheria toxin (DTA) (4). This kills the neurons shortly after birth. In the second, Cre excision induces the expression of a Drosophila allatostatin receptor. This is a Gi-coupled, seven-transmembrane receptor for which there is no endogenous ligand in the mouse. In the presence of allatostatin peptide, only neurons that at one time expressed the recombinase become strongly inhibited (70, 116). Crossing the En1-Cre line with either of these cre-dependent lines leads to a slowing of the locomotor rhythm (50). These experiments highlighted the functional role of a genetically defined class of neurons in generating one relatively simple behavior.

In conclusion, for at least some populations, developmental analyses allow for the generation of hypotheses about genetic identities of specific populations involved in the generation of breathing. A great deal is left to be determined. Perhaps most obviously missing in this review is that most current models of how the brain stem generates breathing are focused on the phase of firing of neurons. The relationship between the genes that specify neurons and their electrophysiological and circuit properties is completely unknown. Combinations of genetic approaches and physiological analysis may allow us to directly address many of the important questions related to breathing.


   ACKNOWLEDGMENTS
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I thank Jean-François Brunet, Frank Constantini, Christo Gordis, Tom Jessell, Randy Johnson, David Rowitch, and Christopher Wright for providing antibodies or transgenic mice and David Van Essen for reading the manuscript. I apologize to those whose work I was unable to directly cite.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. A. Gray, Dept. of Anatomy and Neurobiology, Washington Univ. School of Medicine, Box 8108, 660 S. Euclid Ave., St. Louis, MO 63110-1093 (e-mail: pgray{at}pcg.wustl.edu)


   REFERENCES
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 ABSTRACT
 PATTERNING THE HINDBRAIN
 IDENTIFYING THE A-P AND...
 GENETIC IDENTIFICATION OF...
 FUTURE DIRECTIONS FOR STUDYING...
 ACKNOWLEDGMENTS
 REFERENCES
 

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