In vertebrates, the developing brainstem generates an enormous diversity of neuron types that control bodily homeostasis and process multiple modalities of sensory information (Nieuwenhuys, 2011; Puelles et al., 2013; Venkatraman et al., 2017). These neurons vary not only in their morphological, chemical, and electrophysiological properties, but also in their connectivity patterns that allow them to form elaborate circuits, such as those required to generate, monitor, and adjust breathing to meet various external and internal physiological demands. How this neuronal diversity emerges during development and how it contributes to functional circuits has been intensively investigated by many generations of neuroscientists, as well as by cellular, molecular, and developmental biologists.

The work of these brainstem enthusiasts has already revealed that neuronal diversity in this brain region depends on the temporal and spatial patterning of local neural progenitors (Gray, 2008, 2013; Alexander et al., 2009; Hernandez-Miranda et al., 2017a; van der Heijden and Zoghbi, 2020; Isik and Hernandez-Miranda, 2022). This patterning is first achieved by early diffusible cues that impose an anterior-posterior identity, and subsequently by other morphogens that provide a distinctive dorsal-ventral molecular signature to progenitor cells. This means that distinct progenitor cells can be distinguished primarily based on their differential expression of numerous transcription factors that commit them to generate specific neuron types. Most transcription factors normally expressed in progenitor cells are later silenced in their progeny, which allows for the postmitotic maturation of the differentiated neurons. At the same time, each neuron type can also be characterized by the expression of particular sets of transcription factors that form part of their cell physiology and identity.

In this review, we aim to summarize our current knowledge on the transcriptional programs that allow for the speciation of brainstem respiratory neurons. We will first present a general overview of the anterior-posterior and dorsal-ventral patterning of the developing brainstem as an entry point to understand its neuronal diversity. Next, we will focus on the specification of three large groups of neurons that play key roles in respiration: (i) the pontine groups, (ii) the dorsal medullary respiratory column, and (iii) the ventral medullary respiratory column. These neurons locate to the pons and the medulla oblongata where they perform a variety of functions, such as respiratory rhythm generation, respiratory modulation, and tissue gas monitoring. Lastly, we will discuss some genetic disturbances that affect breathing and respiratory neuron specification.

Brainstem development is an evolutionary conserved process that begins with the specification of the mesencephalon (midbrain) and rhombencephalon (hindbrain) by the isthmic organizer. This organizer is located at the midbrain-hindbrain border and produces diffusible morphogens (particularly Wnt1 and Fgf8 ligands) that act directly on the neighboring nervous tissue to impose midbrain and hindbrain cell fates (Zervas et al., 2004; Dworkin et al., 2012; Gibbs et al., 2017; Gutzman et al., 2018; Hidalgo-Sanchez et al., 2022). Various transcription factors are differentially expressed anterior and posterior to the isthmic organizer, such as the antagonistic homeodomain factors Otx2 (anterior) and Gbx2 (posterior), whose expression defines the rostral and caudal regions of the developing central nervous system (Figure 1A; Millet et al., 1996, 1999; Broccoli et al., 1999; Hidalgo-Sanchez et al., 1999; Joyner et al., 2000; Katahira et al., 2000; Di Giovannantonio et al., 2014). Alterations in the expression of Otx2 and Gbx2 are catastrophic for early brainstem development. For instance, the ablation of Otx2 and its closely related family member Otx1 results in the loss of midbrain tissue, which becomes re-specified into more rostral hindbrain-like regions, such as the cerebellum (Acampora et al., 1997; Suda et al., 1997; Meyers et al., 1998). Similarly, the misexpression of Gbx2 disrupts the correct positioning of the isthmic organizer and the development of rostral hindbrain (Acampora et al., 1997; Wassarman et al., 1997; Millet et al., 1999; Puelles et al., 2003; Waters and Lewandoski, 2006).

The regulation of both Otx2 and Gbx2 largely depends on a complex molecular network that centers on the instructive signals of Fgf8 (Figure 1B; Crossley et al., 1996; Meyers et al., 1998; Martinez et al., 1999; Matsunaga et al., 2002; Chi et al., 2003; Prakash et al., 2006). About embryonic (E) 7.5 in mice, Fgf8 expression is activated by the diffusible ligand Fgf4 (from the notochord) that directly induces the presumptive midbrain tissue to express the transcription factor En1, which in turns activates Fgf8 expression in the isthmic organizer (Shamim et al., 1999). Two Fgf8 isoforms appear to differentially act on the specification of the midbrain and rostral hindbrain. Fgf8a safeguards midbrain identities, while Fgf8b directs rostral hindbrain development. It is important to note that the expression of both isoforms is indispensable for the development of the midbrain and the hindbrain (Lee et al., 1997; Liu et al., 1999, 2003; Sato et al., 2001; Guo and Li, 2007). Notably, the duration of Fgf8 expression in the isthmic organizer is crucial, as available evidence shows that its sustained expression is required to restrict Otx2 rostral to the isthmic organizer, while simultaneously maintaining Gbx2 caudal to it (Liu et al., 1999; Martinez et al., 1999; Sato and Joyner, 2009). In addition to controlling the expression patterns of Otx2 and Gbx2, Fgf8 induces the expression of the homeodomain transcription factor Lmx1b, which primary function is to stabilize Wnt1 expression in the isthmic organizer. In this regard, several studies show that the concomitant ablation of Lmx1b and its family member Lmx1a severely alters the specification of the hindbrain, which adopts a “spinal cord-like” fate in Lmx1a and Lmx1b double mutant mice (Mishima et al., 2009; Su et al., 2014; Glover et al., 2018; Chizhikov et al., 2021). Thus, the establishment of the midbrain–hindbrain border, a prerequisite for brainstem development, relies on a complex molecular network of various transcription factors and signaling cascades (briefly summarized in Figure 1B).

Soon after the establishment of the isthmic organizer, the hindbrain undergoes a series of morphological changes that transiently divide it into seven or eight smaller segments called rhombomeres, from which the cerebellum, pons and medulla oblongata emerge (Bally-Cuif and Wassef, 1995; Lumsden and Krumlauf, 1996). In mice, these segments are recognizable at E8.5, whereas in humans they appear by E29 (Figure 1A; Lumsden and Krumlauf, 1996). Each of these rhombomeres develops a specific set of cellular and molecular features that distinguishes them from the adjacent nervous tissue. Although still unclear, recent anatomical studies indicate that some of rhombomeres might be further regionalized according to the expression of some patterning genes (Tomas-Roca et al., 2016; Watson et al., 2017, 2019; Hirsch et al., 2021). An interesting trait in the “rhombomerization” of the hindbrain is the differential expression of the Hox superfamily of transcription factors, whose expression creates molecular codes that coincides with the morphological borders of each rhombomere (Figure 2; Fienberg et al., 1987; Fraser et al., 1990; Krumlauf et al., 1993; Alexander et al., 2009). One should note that these transcriptional codes are not limited to the hindbrain, as some members of the Hox family are differentially expressed in the developing spinal cord, from which the characteristic cervical, thoracic, lumbar, sacral and coccygeal levels emerge (Alexander et al., 2009). In addition to Hox genes, several other transcription factors show rhombomeric specific expression patterns during early hindbrain development, such as Pax2 in rhombomere 1, Meis2 in rhombomeres 2 and 3, Egr2 (formely known as Krox20) in rhombomeres 3 and 5, and MafB (previously called Kreisler) in rhombomeres 5 and 6 (Figure 2; Seitanidou et al., 1997; Rowitch et al., 1999; Bouchard et al., 2000, 2005; Voiculescu et al., 2001; Choe et al., 2002; Manzanares et al., 2002; Giudicelli et al., 2003; Aragon et al., 2005; Stedman et al., 2009). The timely expression of hox genes, as well as the above mentioned transcription factors, is essential for the correct development of each rhombomere and significantly depends on active derivatives of vitamin A, such as retinoic acid (Gavalas and Krumlauf, 2000). Indeed, dietary deficiencies in vitamin A produce gross disturbances in hindbrain “rhombomerization” that can lead to the complete absence of caudal rhombomeres, as manifested by the loss of specific genes and neuron types normally produced in these regions (Gavalas and Krumlauf, 2000; Glover et al., 2006; Kam et al., 2012; Addison et al., 2018). Thus, early brainstem development relies on the function of diffusible ligands (i.e., Fgf8 and retinoids) that determine its anterior-posterior identity.

Once the brainstem acquires its anterior-posterior identity, a second series of diffusible cues further pattern the identity of progenitor cells along its dorsal-ventral axis. During this patterning, the ventralizing Sonic hedgehog (produced by the floor plate), as well as the dorsalizing bone morphogenetic proteins and Wnt ligands (secreted by the roof plate) create concentration gradients that differentially signal onto brainstem progenitor cells (Roelink et al., 1995; Liem et al., 1997; Lee et al., 1998, 2000; Muroyama et al., 2002; Ulloa and Marti, 2010; Hernandez-Miranda et al., 2017a). These gradients create a great diversity of molecularly distinct progenitor domains that vary depending on their spatial distance from the signaling source (Figure 3 and Table 1).

Depending on the rhombomere, six to eight progenitor domains can be distinguished in the dorsal (also known as the alar plate) aspect of the developing hindbrain, while four to five progenitor domains can be identified in its ventral (or basal) plate (Figure 3). Outstandingly, there exist great resemblance between the developing hindbrain and the spinal cord, in terms of progenitor domains that locate to their dorsal-ventral axis, which share similar gene expression patterns, illustrating common developmental programs that occur between these two nervous system regions (Gray, 2008, 2013; Di Bonito and Studer, 2017; Hernandez-Miranda et al., 2017a; van der Heijden and Zoghbi, 2020; Diek et al., 2022). In the dorsal-most part of the alar plate, the combinatorial expression of the basic Helix-loop-Helix (bHLH) transcription factor Olig3 with other bHLH genes distinguishes four progenitor domains that give rise to dorsal (d) class A neurons: dA1, dA2, dA3, and dA4 (Figure 3 and Tables 1, 2 for a list of genes expressed in these progenitors and postmitotic neurons) (Muller et al., 2005; Zechner et al., 2007; Liu et al., 2008; Storm et al., 2009). Ventral to class A progenitors, the alar plate contains four more progenitor domains that collectively generate class B neurons that express and depend on the homeodomain factor Lbx1 for their proper specification (Gross et al., 2002; Muller et al., 2002; Cheng et al., 2005; Sieber et al., 2007; Pagliardini et al., 2008). The combinatorial expression of Lbx1 with additional homeodomain transcription factors demarcates class B neurons into four types: dB1, dB2, dB3, and dB4 (Figure 3 and Tables 1, 2). Like in the alar plate, progenitor cells of the basal plate exhibit specific molecular codes of transcription factor expression that impose distinctive identities to at least five major neuron types: ventral (V) 0, V1, and V2 interneurons as well as somatic motor and vicero/branchio motor neurons (Figure 3 and Tables 1, 2; Gray, 2008, 2013; Alaynick et al., 2011; Di Bonito and Studer, 2017; van der Heijden and Zoghbi, 2020).

Thus, complex networks of signaling cues pattern the developing brainstem along its anterior-posterior and dorsal-ventral axes to form a molecular grid of longitudinally and transversely distinct progenitor domains, each displaying a particular molecular code of transcription factor expression that singles out the specification of particular neuron types. In the following sections we will discuss the current knowledge of the transcriptional codes that safeguard the specification of pontine and medullary neurons that form the central respiratory circuits in the hindbrain, which collectively generate, monitor and modulate breathing (Figure 4).

These respiratory groups locate to the pons and include two major structures: (i) the dorso-lateral parabrachial complex and its associated Kölliker-Fuse nucleus (for simplicity here shortened to parabrachial/Kölliker-Fuse complex) that surrounds the cerebellar peduncle, and (ii) the intertrigeminal (also known as the peritrigeminal) region that surrounds the trigeminal motor nucleus. The parabrachial/Kölliker-Fuse complex is key in the transition phase between inspiration and expiration. It is classically considered the major component of the pontine pneumotaxic center that controls the amount of air inspired in each breath by providing an off-switch for inspiration (Chamberlin and Saper, 1994; Alheid et al., 2004; Song et al., 2006). The function of the intertrigeminal region is yet to be defined, although available evidence suggests that this region might be an anti-apneic breathing center (Radulovacki et al., 2003, 2004a,b; Stoiljkovic et al., 2009; van der Heijden and Zoghbi, 2018).

Using conditional mutagenesis and lineage-tracing experiments, the group of Huda Y. Zoghbi has studied the development of these pontine groups to great effect. These studies show that both the parabrachial/Kölliker-Fuse complex and intertrigeminal region depend on the bHLH transcription factor Atoh1 (formerly called Math1) for their development, as ablation of Atoh1 results in the absence of these pontine groups in mice (Wang et al., 2005; Rose et al., 2009a,b). Recently, Van der Heijden and Zoghbi traced the rhombomeric origin of the parabrachial/Kölliker-Fuse complex to rhombomere 1, whereas they identified the intertrigeminal region to derive from rhombomere 2 (van der Heijden and Zoghbi, 2018). One should recall that rhombomere 1 development depends on the correct expression of En1, whereas Hoxa2 is the most rostral Hox gene expressed in the developing hindbrain and delimits the border between rhombomeres 1 and 2 (Figures 1, 2). The importance of neurons derived from En1-expressing cells to respiration has not been specifically investigated, but an early report showed that En1 null mutant mice die at birth and present with severe malformations of the cerebellum, midbrain and the parabrachial/Kölliker-Fuse complex (Wurst and Bally-Cuif, 2001). Chatonnet et al. (2007) first explored the function of Hoxa2-expressing neurons in respiration in the 2000s, whose work unveiled marked respiratory phenotypes and neonatal death in Hoxa2 null mutant mice. However, the loss of numerous brainstem structures in these mutant mice precluded the identification of the particular respiratory neurons lost by the constitutive ablations of En1 or Hoxa2.

In the more recent van der Heijden and Zoghbi study, the ablation of Atoh1 from En1-expressing cells, using En1^Cre/+;Atoh1^LacZ/Flox mice, leads to the absence of a recognizable parabrachial/Kölliker-Fuse complex (van der Heijden and Zoghbi, 2018). Physiologically, the absence of this respiratory group does not affect basal respiratory parameters, although spontaneous apneas and frequent sighing behavior is observed in En1^Cre/+;Atoh1^LacZ/Flox mice. Notably, the specific elimination of the parabrachial/Kölliker-Fuse complex impairs respiratory chemoreflexes to hypoxia (low oxygen) and hypercarbia (high carbon dioxide). Despite the fact the parabrachial/Kölliker-Fuse complex does not sense changes in blood gases by itself, it is known to form reciprocal connections with the nucleus tractus solitarius, a center known to mediate respiratory chemoreflexes (Alheid et al., 2004, 2011; Hernandez-Miranda et al., 2017b). Thus, the loss of communication between the nucleus tractus solitarius and the parabrachial/Kölliker-Fuse complex might account for the impaired chemoreflexes observed in En1^Cre/+;Atoh1^LacZ/Flox mutants. In addition, van der Heijden and Zoghbi restricted the ablation of Atoh1 to rhombomere 2 by using a transgenic mouse line that specifically expresses Cre in rhombomere 2 derived cells (Hoxa2^:CreTG;Atoh1^LacZ/Flox mice). In doing so, these scientists anatomically demonstrated the aberrant migration and defective location of intertrigeminal neurons in their conditional mutants. Physiologically, these animals show sigh-induced spontaneous apneas and smaller respiratory tidal volumes that led to a mild hypoventilation phenotype, but otherwise they are fully capable to respond to hypoxic and hypercarbic chemoreflexes. Thus, the anterior-posterior origin of the parabrachial/Kölliker-Fuse complex and intertrigeminal region has been assigned to rhombomeres 1 and 2, respectively.

During development, Atoh1 is transiently expressed (E10.5-E13.5) in progenitor cells of the dA1 domain that encompasses rhombomere 1 (also known as the rostral or upper rhombic lip) and rhombomeres 2–7/8 (known as the caudal or lower rhombic lip) (Figure 3 and Table 1; Ben-Arie et al., 1997; Helms and Johnson, 1998; Bermingham et al., 2001; Storm et al., 2009; Lowenstein et al., 2021). Therefore, the study of van der Heijden and Zoghbi (2018) indicates that the upper rhombic lip is the bona fide origin of the parabrachial/Kölliker-Fuse complex. The progenitor domain that generates the intertrigeminal region remains to be conclusively identified. Given that intertrigeminal neurons express the homeodomain factors Lbx1 and Phox2b, in addition to Atoh1, the source of intertrigeminal neurons could be: the dA1 (Atoh1 +) or the dB2 (Phox2b +) progenitor domain in rhombomere 2 (Wang et al., 2005; Dubreuil et al., 2009; Rose et al., 2009a,b; Hernandez-Miranda and Birchmeier, 2015; Ruffault et al., 2015; Hernandez-Miranda et al., 2017a,2018). Hence, either dA1-derived neurons activate Phox2b and Lbx1 expression or dB2-derived (Phox2b+/Lbx1+) neurons switch on the expression of Atoh1. Current evidence suggests that the latter option is the correct, as retrotrapezoid neurons, a sub-population of dB2 neurons produced in rhombomere 3 or 5, originates from Phox2b + (dB2) progenitors whose progeny subsequently express Lbx1 and Atoh1 (Dubreuil et al., 2009; Huang et al., 2012; Hernandez-Miranda et al., 2018).

This respiratory column contains neurons known to be critical for the regulation of inspiratory activity. Most neurons forming this respiratory column reside within the nucleus tractus solitarius (nTS, bilaterally located in the dorsal medulla oblongata), although a few neurons belonging to this column can be found in the reticular formation adjacent to the nTS. The nTS extends from the caudal level of the facial motor nucleus (at the border between rhombomeres 6/7) to the cervical spinal cord. It represents the primary entry site of peripheral viscerosensory information into the central nervous system (Figure 5A; Machado et al., 1997; Travagli, 2007; Grill and Hayes, 2009). This nucleus contains second order sensory neurons that further process and relay this information to other brain regions in the brainstem (i.e., parabrachial/Kölliker-Fuse complex), forebrain and spinal cord (Aicher et al., 1995, 1996; Alheid et al., 2011). The intermediate (at the level of the area postrema) and caudal regions of the nTS receive cardiovascular and respiratory viscerosensory afferents, while the rostral to intermediate nTS primarily receives digestive information from the vagal and glossopharyngeal nerves (Kumada et al., 1990; Vangiersbergen et al., 1992; Zoccal et al., 2014; Kawai, 2018). With respect to respiration, the intermediate nTS receives afferent information from slowly adapting pulmonary stretch receptors, whereas the caudal nTS receives afferent information from the peripheral chemoreceptors (that is the carotid bodies) and from rapidly adapting pulmonary stretch receptors (Mifflin et al., 1988; Mifflin, 1992, 1993; Machado et al., 1997; Kubin et al., 2006; Chang et al., 2015; Zhao et al., 2022). Furthermore, the nTS harbors several groups of premotor neurons that can control, for instance, the laryngeal and expiratory motor activity used in breathing-associated behaviors, such as in vocalization (Hernandez-Miranda et al., 2017b).

Histological studies traced the origin of excitatory nTS neurons to the dA3 progenitor domain (located between rhombomeres 4 and 7/8; Figure 3). This progenitor domain shares molecular traits with a progenitor domain in the spinal cord called dl3 (Muller et al., 2005; Storm et al., 2009; Hernandez-Miranda et al., 2017a). Indeed, neuronal derivatives from these progenitor domains are excitatory and seem to only vary in the expression of the transcription factors Phox2b (in dA3 neurons) and Isl1 (in dI3 neurons), but otherwise they co-express the transcription factors Pou4f1, Tlx3, Prrxl1, and Lmx1b (Figures 3, 5B; Chen et al., 2001; Qian et al., 2001, 2002; Cheng et al., 2004; Muller et al., 2005; Liu et al., 2008; D’Autreaux et al., 2011). For over a quarter century the group of Christo Goridis and Jean-François Brunet has characterized the critical roles of Phox2a and Phox2b in visceral nervous system development. These homeobox transcription factors are necessary for the development of central and peripheral noradrenergic neurons, parasympathetic and sympathetic ganglia, branchial, and visceral motor neurons, as well as primary and secondary viscerosensory neurons (Morin et al., 1997; Pattyn et al., 1997, 1999, 2000a,2000b,2006; Fode et al., 1998; Dauger et al., 2003; D’Autreaux et al., 2011; Espinosa-Medina et al., 2016). Phox2b is critical for the specification of excitatory nTS neurons, and its mutation precludes the formation of this brainstem center in mice (Pattyn et al., 1999; Dauger et al., 2003; Hernandez-Miranda et al., 2017b). In addition, most excitatory nTS neurons co-express the transcription factor Tlx3 (previously known as Rnx) that seems to stabilize and maintain the expression of Phox2b in nTS neurons (Qian et al., 2001; Dauger et al., 2003; Storm et al., 2009; Hernandez-Miranda et al., 2017b).

In the developing hindbrain, the precise identity of dA3 progenitor cells is determined by the co-expression of the bHLH transcription factors Olig3, Ascl1 and Ngn2 (Figure 5B; Pattyn et al., 2006; Liu et al., 2008; Storm et al., 2009; Hernandez-Miranda et al., 2017a). In addition to excitatory nTS neurons, dA3 progenitors also generate other excitatory neurons that include: area postrema neurons (associated with vomiting reflexes) and caudal (nor)adrenergic neurons (the baroreflex-associated A1 and A2 groups) (Qian et al., 2001; Dauger et al., 2003; Pattyn et al., 2006; Storm et al., 2009; Zhang et al., 2021). Progenitors in the dA3 domain generate these neuron types in a temporal order in which (nor)adrenergic neurons are generated first, followed by excitatory nTS neurons and lastly area postrema neurons (Hernandez-Miranda et al., 2017b). Although most nTS neurons are excitatory, a substantial amount of inhibitory neurons also reside within this nucleus. Inhibitory nTS neurons depend on Lbx1 and appear to derive from the dB1 or dB2 progenitor domains (Hernandez-Miranda et al., 2018). The rhombomeric origin of the nTS has not been directly investigated, incidental evidence, however, suggests that it primarily originates from rhombomeres 7 & 8, as the ablation of the transcription factor MafB, which severely affects the development of rhombomeres 5 and 6, does not significantly interfere with nTS development (Blanchi et al., 2003).

Several anatomical and physiological distinct groups of respiratory-related neurons have been identified in the ventral medulla oblongata, which form the ventral medullary respiratory column, these groups include: (a) the retrotrapezoid/parafacial nucleus, (b) the Bötzinger complex, (c) the preBötzinger complex, (d) the PiCo complex (dorsal to the Bötzinger and preBötzinger complexes), (e) the rostral ventral and caudal respiratory groups, as well as (f) the raphe obscurus (Figure 4). Neurons within these groups can either project onto respiratory premotor neurons or themselves act as premotor neurons to regulate the motoric behavior associated with breathing. Interestingly, three of these groups exhibit intrinsic rhythmic activity during prenatal development: (i) the parafacial nucleus, (ii) the preBötzinger complex and (iii) a newly identified PostInspiratory COmplex (PiCo), and are believed to be central for the generation of the respiratory rhythm (Smith et al., 1991; Onimaru and Homma, 2003; Anderson et al., 2016; Del Negro et al., 2018; Ramirez and Baertsch, 2018). Except for the recently identified PiCo complex, the development of all other ventral respiratory groups has been studied.

From a developmental point of view, most brainstem respiratory neurons emerge from a few molecularly defined cell-lineages and progenitor domains: (i) an Atoh1-lineage that contributes to the development of the parabrachial/Kölliker-Fuse complex (dA1, in rhombomere 1) and the intertrigeminal region (either dA1 or dB2, in rhombomere 2); (ii) an Olig3/Phox2b/Tlx3-lineage (dA3, in rhombomere 7/8) that generates the dorsal medullary respiratory column (nTS); iii) a Phox2b/Lbx1/Atoh1-lineage (dB2, in rhombomere 3 and/or 5) that generates the retrotrapezoid/parafacial nuclei; (iv) an Lbx1-lineage (presumably dB1, unknown rhombomeric origin) that produces the Bötzinger complex; v) a Dbx1-lineage (V0) that gives rise to the preBötzinger complex (in rhombomeres 6-7/8) and the rVRG (and possibly the cVRG, in rhombomere 7/8) groups of premotor neurons; as well as (vi) an Nkx2.2/Ascl1/Lmx1b-lineage that produces raphe serotonergic neurons (pMNv, across rhombomeres).

One should not forget, however, that each of these progenitor domains produce a much greater diversity of neuron types than just respiratory neurons. A good example of this is the dA1 (Atoh1 +) domain in rhombomere 1 (upper rhombic lip). This domain generates: in addition to the parabrachial/Kölliker-Fuse complex, all excitatory deep cerebellar neurons, all cerebellar granule cell progenitors, as well as all cerebellar and cochlear unipolar brush cells (Englund et al., 2006; Fink et al., 2006; Ray and Dymecki, 2009; Hernandez-Miranda et al., 2017a; van der Heijden and Zoghbi, 2018, 2020; Elliott et al., 2021; Lowenstein et al., 2021, 2022; Fritzsch et al., 2022). How these progenitors generate such a vast array of neuron types is currently being investigated and appears to depend on the temporal expression of transcription factors that act as selector genes. In this context, the co-expression of Atoh1 with Olig3 is critical for deep cerebellar neuron development, whereas the co-expression of Atoh1 with Neurod1 is essential for granule cell progenitor specification and cerebellar and cochlear unipolar brush cell development (Ben-Arie et al., 1997; Chizhikov and Millen, 2003; Gazit et al., 2004; Pan et al., 2009; Machold et al., 2011; Lowenstein et al., 2021). The selector gene for the specification of parabrachial/Kölliker-Fuse complex is presently unknown.

It might not be surprising that across rhombomeres, each progenitor domain generates different neuron types. Nonetheless, an interesting trait of these spatially segregated progenitor domains is that their shared expression of transcription factors might instruct their progeny to synaptically connect and form functional circuits. For instance, both Atoh1 + /Olig3 + (dA1) progenitors in rhombomere 7 or the pdI1 progenitors in the spinal cord (equivalent to dA1) produce second relay neurons that project to the cerebellum and synapse onto granule cells and deep cerebellar neurons that derive from rhombomere 1 dA1 (Atoh1 + /Olig3 +) progenitors (Muller et al., 2005; Liu et al., 2008; Storm et al., 2009; Hernandez-Miranda et al., 2017a; Lowenstein et al., 2021, 2022). Another example of this molecular logic could be the inferior olive-Purkinje cell circuit. Indeed, inferior olive cells that derive from Olig3 + /Ptf1a + (dA4 in rhombomere 7) progenitors send axons that synapse onto Purkinje cells that emerge from Olig3 + /Ptf1a + progenitors in the cerebellar ventricular zone in rhombomere 1 (Liu et al., 2008; Storm et al., 2009; Hernandez-Miranda et al., 2017a; Lowenstein et al., 2021, 2022). To which extent these shared transcriptional codes allow for the interconnection of the distinct neurons that form the brainstem respiratory circuit is presently unknown. However, emerging evidence shows similar developmental strategies, i.e., preBötzinger complex neurons that connect with the rVRG are both derivatives of the V0 domain (Bouvier et al., 2010; Gray et al., 2010; Wu et al., 2017). An even more conspicuous case is the Phox2b-lineage that generates virtually all neurons that form the central and peripheral visceral nervous system (Morin et al., 1997; Pattyn et al., 1997, 1999, 2000a, 2000b, 2003, 2004, 2006; Fode et al., 1998; Dauger et al., 2003; D’Autreaux et al., 2011; Espinosa-Medina et al., 2016; Isik and Hernandez-Miranda, 2022). The question of how these developmental strategies emerged during evolution remains to be explored.

The enormous knowledge gained during the last three decades of brainstem development research now allows us to further dissect the function of respiratory neurons with unprecedented detail. Several experimental and theoretical approaches have recently taken advantage of the developmental trajectories of respiratory neurons to explore the complex character of the respiratory rhythm generator or to elucidate different components of the central chemoreceptor circuit (Depuy et al., 2011; Ray et al., 2011; Brust et al., 2014; Wang et al., 2014; Vann et al., 2016, 2018).

Many aspects of how respiratory neuron diversity emerges during development remain to be elucidated. The identification of how the brainstem generates respiratory neurons is critical to understand this complex behavior and essential for the development of new therapeutic approaches for the management of respiratory diseases. In this context, recent studies on the genetic disturbances causing congenital respiratory syndromes are currently steering our views into both the development and function of respiratory neurons. A clear example is the study of congenital central hypoventilation syndrome (CCHS, also known as Ondine’s curse; OMIM 209880). Although rare (1 in 200,000 live births), this disorder is life threatening and characterized by slow, apneic and shallow breathing (hypoventilation) while awake and respiratory arrest during sleep (Weese-Mayer et al., 2008, 2017; Sivan et al., 2019; Ceccherini et al., 2022). Frequently, CCHS patients also present with blunted responses to hypercabia and have abnormal levels of PCO₂. Early genetic studies identified de novo mutations in PHOX2B as the most prevalent cause of CCHS (Amiel et al., 2003). Two types of PHOX2B mutations that cause CCHS have been identified: (i) polyalanine repeat expansions, and (ii) non-polyalanine repeat expansions that are more prevalent in severe cases of CCHS (Amiel et al., 2003; Zhou et al., 2021). CCHS patients with PHOX2B mutations frequently manifest Hirschsprung’s disease, revealing that genetic disturbances on PHOX2B can simultaneously alter the development of both the central and peripheral nervous systems (Weese-Mayer et al., 2008, 2017; Sivan et al., 2019; Ceccherini et al., 2022). PHOX2B is a central factor in the development and function of the visceral nervous system, whose mutation results in midterm fetal lethality (Dauger et al., 2003). Interestingly, the insertion of a frequent poly-alanine PHOX2B mutation (called PHOX2B^+7ala) into the murine genome has shown that this aberrant expansion only affects a subset of Phox2b functions, as only one Phox2b-dependent neuron type (the retrotrapezoid nucleus) does not develop correctly in Phox2b^+7ala mutant mice (Dubreuil et al., 2008). More recently, the characterization of a CCHS disease-causing frameshift mutation in LBX1 has further revealed that this respiratory disorder originates from the misspecification of dB2 neurons in mice, and that this is caused by a lack of cooperativity between PHOX2B and LBX1 (Hernandez-Miranda et al., 2018).

For many years, the size and location of the brainstem was a major impediment to comprehend its physiology. With the advent of new technologies such as single-cell transcriptomics, monosynaptic viral tracing as well as opto- and chemo- genetic tools, several of the long-lasting obstacles associated with the identification and modulation of specific breathing behaviors are now amenable for scientific exploration. There is no doubt that the years to come will foster and propel our understanding of this elementary and humble animal behavior in ways that we can only now imagine.

YX, KC, AA, EL, and LH-M reviewed the literature. LH-M wrote the original draft and edited it with the input from all authors. All authors contributed to the article and approved the submitted final version.