s2fieldsofstudy: Biology Medicine

text:
Carditis After COVID-19 Vaccination With a Messenger RNA Vaccine and an Inactivated Virus Vaccine

Cases of carditis after the use of a messenger RNA (mRNA) COVID-19 vaccine have been reported in various populations worldwide. However, few studies have directly evaluated this association. Furthermore, it is unclear whether this potential risk involves vaccine platforms other than mRNA vaccines. This case–control study from Hong Kong aimed to examine the association between COVID-19 vaccination with an mRNA vaccine or an inactivated virus vaccine and the subsequent risk for carditis. The results of this study could be useful for public health policymakers to weigh the risk and benefit of population-wide COVID-19 vaccination programs.

Results: A total of 160 case patients and 1533 control participants were included. Incidence of carditis per 100 000 doses of CoronaVac and BNT162b2 administered was estimated to be 0.31 (95% CI, 0.13 to 0.66) and 0.57 (CI, 0.36 to 0.90), respectively. Multivariable analyses showed that recipients of the BNT162b2 vaccine had higher odds of carditis (adjusted odds ratio [OR], 3.57 [CI, 1.93 to 6.60]) than unvaccinated persons. Stratified by sex, the OR was 4.68 (CI, 2.25 to 9.71) for males and 2.22 (CI, 0.57 to 8.69) for females receiving the BNT162b2 vaccine. The ORs for adults and adolescents receiving the BNT162b2 vaccine were 2.41 (CI, 1.18 to 4.90) and 13.79 (CI, 2.86 to 110.38), respectively. Subanalysis showed an OR of 9.29 (CI, 3.94 to 21.91) for myocarditis and 1.06 (CI, 0.35 to 3.22) for pericarditis associated with BNT162b2. The risk was mainly seen after the second dose of BNT162b2 rather than the first. No association between CoronaVac and carditis with a magnitude similar to that for BNT162b2 was seen.
Limitation: Limited sample size, absence of electrocardiography and other clinical investigative data, and unrecorded overseas vaccination exposure.
Conclusion: Despite a low absolute risk, there is an increased risk for carditis associated with BNT162b2 vaccination. This elevated risk should be weighed against the benefits of vaccination.

Primary Funding Source: Health and Medical Research
Fund.
Ann Intern Med. doi:10.7326/M21-3700 Annals.org For author, article, and disclosure information, see end of text. This article was published at Annals.org on 25 January 2022. * Drs. Lai and Li contributed equally to this work and should be considered co-first authors.
C arditis is an acute condition presenting with inflammation of the heart. Common subtypes include myocarditis (heart muscle) (1) and pericarditis (outer lining of the heart) (2). Viral, bacterial, or parasitic infections are often causes of this condition (1)(2)(3). Noninfectious causes include autoimmune, neoplastic, and metabolic risk factors as well as various specific drugs (4). Carditis is rare and mostly self-limiting-the incidence rate of myocarditis is estimated at approximately 20 cases per 100 000 person-years globally (5,6).
Case reports of carditis after the emergency use of the messenger RNA (mRNA) COVID-19 vaccine BNT162b2 (Pfizer-BioNTech) are rapidly accruing in various populations worldwide (7)(8)(9)(10), despite the lack of an observed elevated risk from clinical trial data (phases 1 to 3), which may be limited by a relatively small sample size (11). As shown in a recent study comparing risk differences of carditis after the first and second doses of BNT162b2 (12), a predominant proportion of the cases involved younger male persons presenting with myocarditis after receiving the second dose (13,14). On 23 August 2021, the U.S. Food and Drug Administration approved the regular use of BNT162b2 for COVID-19 prevention among persons aged 16 years or older. The approval stipulates a population-based postmarketing assessment of the association between carditis and BNT162b2 in both the short and the longer term with regular data updates (15). A recent Israeli retrospective cohort study of nearly 1 million persons reported an elevated risk for myocarditis (identified using routine health care diagnostic codes) associated with receiving BNT162b2 (16), with a risk ratio of 3.24 (95% CI, 1.55 to 12.44) and an additional 2.7 events (CI, 1.0 to 4.6 events) per 100 000 vaccinated persons. Nevertheless, analytic research on this widely speculated association remains scant (14,17 (18). Since the rollout of the public-funded mass vaccination program, more than 7 million doses of the COVID-19 vaccines have been administered, alongside the implementation of a surveillance program for safety monitoring (19). As of 2 August 2021, about 3.29 million (49%) of the population aged 12 years or older (6.7 million) have been vaccinated. In this casecontrol study, we analyzed a territory-wide, routine, public health care database with linkage to population-based vaccination records to examine the association of BNT162b2 and CoronaVac vaccination with carditis.

Study Design and Data Source
We did a case-control study following the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guideline (20). Routine health care records provided by the Hospital Authority (HA) of Hong Kong were linked with population-based vaccination records at the Department of Health to allow case and control identification and exposure (vaccination status) ascertainment. Matching between inpatient and vaccination records for selected case patients and control participants was based on the Hong Kong Identity Card number or other personal identification numbers, such as foreign passports, and was done by the HA.
The HA is the sole provider of public inpatient services and a major provider of public outpatient services in Hong Kong, with a comprehensive electronic health record (EHR) system for clinical management. Each Hong Kong resident has a unique Hong Kong Identity Card number that allows the HA to create a unique EHR for each patient to link attendances to all health care facilities. Data from the HA EHR are deidentified, and pseudoanonymized data are transferred daily to the Clinical Data Analysis and Reporting System (CDARS), a nonclaims-based clinical management database with the EHRs of all patients who used HA health care services. The EHRs in CDARS include demographic characteristics, diagnoses, medication dispensing records, outpatient and primary care clinics, emergency department attendances, laboratory tests, and hospitalization details -all comprehensively recorded for research or auditing purposes (21). The database has frequently been used for high-quality pharmacovigilance studies to evaluate the safety of medicines and vaccines at population level (21)(22)(23). A previous study showed high coding accuracy for cardiovascular diagnosis in CDARS, with positive predictive values estimated at 85% to 91% (24).

Case and Control Selection
Using the definition from the ACCESS (vACCine covid-19 monitoring readinESS) project funded by the European Medicines Agency (25), we defined case patients as patients first diagnosed with carditis (acute myocarditis or pericarditis; International Classification of Diseases, Ninth Revision, Clinical Modification [ICD-9-CM] codes 420.9, 422.x, 423.9, and 429.0) in an inpatient setting from 23 February to 2 August 2021, identified in CDARS. Patients younger than 12 years and those with any history of carditis were excluded from the analysis. To reduce potential misdiagnosis, case patients with a subsequent diagnosis of myocardial infarction (ICD-9-CM codes 410.x to 411.x) after carditis diagnosis, case patients without laboratory test results of troponin levels, or case patients without an elevated troponin level during admission were excluded to ensure they were "probable cases" according to the Brighton Collaboration's definition (26). We adopted the reference thresholds used for the different testing kits of individual hospitals, which were calibrated independently, to indicate the elevation of troponin levels (Supplement Table 1, available at Annals.org). All other hospitalized patients during the same period without a diagnosis of carditis were treated as control participants. Ten control participants were randomly matched with each of the case patients according to age (within the same year), sex, and date of admission (within 1 calendar day).

Exposure
Receipt of any dose of BNT162b2 or CoronaVac before the index date (date of hospital admission) was categorized as an exposure (versus the unvaccinated). In Hong Kong, the recommended dosing intervals (number of days between doses) for BNT162b2 and CoronaVac are 21 and 28 days, respectively (27).

Covariates
Clinical history (since the launch of the EHR system in 1993) of diabetes, hypertension, coronary heart disease, stroke, heart failure, myositis, and encephalitis was included in the main analysis for multivariable adjustment. In addition, we adjusted a binary indicator for any cardiovascular medication prescribed within the past year before admission, including anticoagulants, antiplatelet medications, angiotensin-converting enzyme inhibitors, angiotensin-receptor blockers, b -adrenoceptor blocking drugs, calcium-channel blockers, digoxin, and statins. The corresponding ICD-9-CM codes, British National Formulary codes, and drug names are tabulated in Supplement Tables 2 and 3 (available at Annals.org).

Statistical Analysis
Conditional logistic regression was used to examine the association of receiving BNT162b2 and CoronaVac ORIGINAL RESEARCH Carditis Risk After Messenger RNA and Inactivated Virus Vaccination vaccination with carditis. Adjusted odds ratios (ORs) of carditis in vaccine recipients compared with unvaccinated persons were estimated for BNT162b2 and CoronaVac. Stratified analyses were also done by age group (adults aged ≥18 years and adolescents aged 12 to 17 years) and sex.
A series of sensitivity analyses were done. First, to differentiate the subtypes of carditis, we stratified the cases as pericarditis (ICD-9-CM codes 420.9 and 423.9) and myocarditis (ICD-9-CM codes 422.x and 429.0) and replicated the main analysis. A small number of patients with both diagnoses within the same hospital admission were included in both replications. Second, to compare the risk difference between the first and second doses, we replicated the analysis with vaccination status categorized into the following categories to generate specific ORs for each of these vaccine groups: unvaccinated, received 1 dose of CoronaVac, received both doses of CoronaVac, received 1 dose of BNT162b2, and received both doses of BNT162b2. Third, in addition to myocardial infarction, we removed cases with a subsequent diagnosis of heart failure to minimize the risk for misclassification of cases. Fourth, although Hong Kong had a low COVID-19 infection rate (<12 500 cumulative confirmed cases out of a population of more than 7 million) and the proportion of COVID-19-related hospitalizations in the pool of hospitalizations for the selection of control participants was extremely low (0.15%) (28), we replicated the analysis with 4 COVID-19-related hospitalizations removed from the control group. Fifth, we specified a risk window of 14 days after vaccination and conducted another sensitivity analysis with the most recent dose of vaccine (either CoronaVac or BNT162b2) administered more than 14 days before the index admission recoded as unvaccinated (nonexposed). Sixth, we included those diagnosed with carditis but without a troponin test result and replicated the analysis. Finally, in addition to the covariate adjustment in the main analysis, we further adjusted for covariates, including health care use history, additional preexisting chronic diseases, and other longterm medications, to minimize the residual confounding effects on the results.
As recommended in previous methodological research (29,30), we applied unconditional exact logistic regression in scenarios when conditional logistic regression returned extreme estimates in some of the subgroup analyses owing to a small sample size. All analyses were done using R, version 4.1.1 (R Foundation for Statistical Computing), and results were cross-checked by 2 independent researchers (K.P. and L.H.) as quality control.

Role of the Funding Source
This is a regulatory pharmacovigilance study initiated by the Department of Health and funded by the Food and Health Bureau of the Government of Hong Kong Special Administrative Region, China. The Department of Health has facilitated data extraction but has played no other roles in the study's design, conduct, or reporting.

Incidence of Carditis
In Hong Kong, 2 291 444 doses of CoronaVac and 3 496 629 doses of BNT162b2 were administered as of 2 August 2021 (27). We identified 433 patients with a diagnosis of carditis from CDARS according to ICD-9-CM codes. After applying the exclusion criteria to cases, we selected 160 case patients and 1533 control participants   Figure 2 shows the onset time distribution of included cases of carditis among vaccine recipients. After BNT162b2 vaccination, the onset time of carditis was concentrated during the first week (75.0% of all cases), and 5 out of all 7 case patients (71.4%) who received CoronaVac had carditis occur more than 30 days after vaccination. Subsequent intensive care unit (ICU) admission and death were rare among the carditis cases identified. Of 160 carditis cases, 14 patients without vaccination, 2 after CoronaVac vaccination, and none after BNT162b2 vaccination were admitted to the ICU. Only 1 case patient who received CoronaVac and 12 case patients without vaccination died within the observation period. Table 1 shows the sample characteristics by case patients and control participants. There were no marked differences between the groups in chronic disease history, with approximately half of both groups prescribed cardiovascular medications in the past year. There was a higher proportion of case patients with a preexisting heart failure diagnosis than control participants (12.9% vs. 8.0%), whereas a higher proportion of control participants had diabetes compared with case patients (20.7% vs. 11.9%). A similar proportion of around 4% in both groups had received CoronaVac but a much higher proportion of patients having received BNT162b2 was seen among case patients (12.5%) than among control participants (4.2%). Table 2 shows the characteristics of control participants by vaccination status. Although the CoronaVac and unvaccinated groups had about the same mean age of 59 years, the BNT162b2 group was only 37 years on average. All chronic conditions were more prevalent among the unvaccinated group than among the 2 vaccinated groups. The proportion of patients having received the second dose was similar between the 2 vaccinated groups (around 60%). Supplement Table 4 (available at Annals. org) shows the characteristics of case patients by vaccination status.

Case-Control Analysis
Multivariable conditional logistic regression showed that the adjusted OR of carditis was 3.57 (CI, 1.93 to 6.60) among those receiving BNT162b2 compared with unvaccinated persons (

DISCUSSION
To the best of our knowledge, this is the first casecontrol study analyzing carditis risk associated with 2 different COVID-19 vaccine technologies. We found an elevated risk for carditis after the use of the mRNA BNT162b2 vaccine but not the inactivated virus vaccine CoronaVac. This risk increase associated with BNT162b2 was predominant in males. Among both adults and adolescents, there was an increased risk for carditis, with a larger OR estimated for adolescents. Sensitivity analysis suggested the association was driven by myocarditis, not pericarditis. In addition, this risk increase was more likely to be after the second dose of BNT162b2 rather than the first. A stronger association was seen with BNT162b2, with the specification of a 14-day risk window after vaccination. Nevertheless, the absolute risk for carditis after BNT162b2 vaccination remains very low-with only approximately 0.25 cases per 100 000 first doses identified from the territory-wide routine health care database and approximately 1 case per 100 000 second doses administered according to our operational case definition. Moreover, none of the 20 case patients with carditis after BNT162b2 vaccination were admitted to the ICU or died within the observation period, compared with 14 of 133 unvaccinated patients admitted to the ICU and 12 deaths. Our observations and published studies have shown that, to date, most reported carditis cases after BNT162b2 are self-limiting with good prognosis.
Given the rare incidence of carditis, it is difficult to capture cases in clinical trials with typically no more than a few tens of thousands of participants. To our knowledge, no clinical trials have reported the occurrence of carditis after COVID-19 vaccination (11,(31)(32)(33). In the literature, aside from a recent Israeli retrospective cohort study (16), only case series or case reports examining the association between BNT162b2 and carditis were available (7-10). Our findings are consistent with these reports in that the distribution of carditis occurrence is concentrated in young male persons after the second dose (13,14,34), which is also supported by a recent meta-analysis of these cases. Earlier research before the pandemic has shown a similar marked sex difference in carditis incidence (35). The Israeli study identified a 3-fold elevated risk for myocarditis and, like our findings, did not identify a significant elevated risk for pericarditis (16). Owing to the wide variety of outcomes investigated, they relied on the validity of the diagnosis codes without considering further criteria on the validity of the diagnosis codes or further criteria on the definition of cases (16,36). In the current case-control study, we applied a more stringent case definition for the operationalization of the study to ensure case validity. Our findings have important scientific and health policy implications. An indication bias of "healthy vaccinerecipient effect"-those who received the vaccines are, in general, healthier than those who are not vaccinated-is widely reported in COVID-19 vaccine safety investigations (37,38). Analytic studies are often challenged by such bias, and the risk comparison between vaccine recipients and nonvaccinated persons can be biased as a result (16). However, recipients of CoronaVac should have reasonably similar health statuses as recipients of BNT162b2, but we did not find notably increased or reduced carditis risk after CoronaVac. Such findings further support the validity of observed increased risk for carditis associated with BNT162b2 vaccination.
Moreover, the differences in the risk after immunization with the 2 vaccines imply the presence of potential mechanisms specific to the mRNA platform, which is illustrated in the onset distribution ( Figure 2), with carditis cases typically occurring within a short period after BNT162b2 vaccination. For cases after CoronaVac vaccination, it was more evenly distributed. The potential mechanism underlying this observed association between BNT162b2 and carditis is unclear. Of note, a recent animal experiment using a mouse model suggested in vivo evidence that inadvertent intravenous injection of COVID-19 mRNA vaccines may induce carditis (39). Although the extrapolation of animal research to humans warrants great caution, this finding plausibly suggests the possibility that unintended intravenous injection may be 1 potential mechanism to explain the association. This is because no histopathologic changes of carditis were seen in mice randomly allocated to receive intramuscular BNT162b2 injection (39). Further research is warranted to identify the physiologic response triggered by COVID-19 vaccination in relation to carditis and inform further hypothesis-driven studies.
Postvaccination carditis should be particularly closely monitored in persons receiving BNT162b2. From the observed onset distribution, more than 1 month after either dose of vaccine, vaccine recipients and physicians should be aware of symptoms related to probable carditis. The symptoms include chest pain, palpitation, shortness of breath, and fatigue and the subsequent elevated risk for carditis, especially among the young and males. As some countries have already begun the rollout of the third dose, the associated risk for carditis and long-term safety profile of the vaccines should continue to be closely monitored. Regarding further vaccination of adolescents and children, the risks and benefits should be considered thoughtfully in accordance with the local epidemic situation. Nonetheless, although the estimated OR is large, the absolute risk for carditis is low, with the number of carditis cases per 100 000 doses of administered BNT162b2 estimated at 0.57. If we assume the carditis cases after CoronaVac vaccination is the background incidence-that is, 0.31 per 100 000 doses, the approximate absolute risk increase is 0.26 per 100 000 doses overall and 0.17 per 100 000 doses and 0.4 per 100 000 doses for the first and second doses, respectively. On the other hand, the risk for carditis and other serious complications after SARS-CoV-2 infection (16), including an approximate 16-times increased risk for myocarditis shown in a retrospective cohort study of 36 million persons in the United States, are also concerning (36). The risk seen in this study for carditis after BNT162b2 vaccination should be weighed against the benefits of vaccination.
Despite the strengths of a stringent case definition as well as the availability of accurate information on vaccination statuses before the onset of the outcome, there are limitations to this study. First, we did not have a sufficient sample size for the application of conditional logistic regression to the adolescent group because of the lack of variation in vaccination status within matched sets. The modest sample size also resulted in relatively wide CIs, limiting the precision of the estimates and conclusions, especially regarding CoronaVac. Hence, the interpretation of the results should be cautious. Second, in common with other large-scale epidemiologic studies using EHR databases, we were dependent on ICD-9-CM codes to identify cases. We used troponin levels as a means  (16). Fifth, long-term postdischarge outcomes were also not investigated because only a short observation period was allowed for this urgent investigation of the association of vaccines with carditis. The Hong Kong government obtained consent from patients and guardians, and a further study on the long-term outcomes is under planning. Sixth, overseas vaccine exposure was not recorded in the database, although this should be rare given various restrictions on international travel and unlikely to have a substantial effect on our results. Finally, the hospitalized control participants selected for this study may not be sufficiently representative of the risk set of the underlying cohort, and the validity of the study results largely depend on the key assumption that the selection of control participants was independent of exposure. There is no evidence that such an assumption was violated because our results are consistent with previously published data in other populations.
In conclusion, we observed an increased risk for carditis associated with the use of BNT162b2, particularly in young male persons receiving the second dose. Although the absolute risk is very low, this elevated risk should be made known to vaccine recipients and physicians and be weighed against the benefits of vaccination.


Transcription factors regulating the specification of brainstem respiratory neurons

Breathing (or respiration) is an unconscious and complex motor behavior which neuronal drive emerges from the brainstem. In simplistic terms, respiratory motor activity comprises two phases, inspiration (uptake of oxygen, O2) and expiration (release of carbon dioxide, CO2). Breathing is not rigid, but instead highly adaptable to external and internal physiological demands of the organism. The neurons that generate, monitor, and adjust breathing patterns locate to two major brainstem structures, the pons and medulla oblongata. Extensive research over the last three decades has begun to identify the developmental origins of most brainstem neurons that control different aspects of breathing. This research has also elucidated the transcriptional control that secures the specification of brainstem respiratory neurons. In this review, we aim to summarize our current knowledge on the transcriptional regulation that operates during the specification of respiratory neurons, and we will highlight the cell lineages that contribute to the central respiratory circuit. Lastly, we will discuss on genetic disturbances altering transcription factor regulation and their impact in hypoventilation disorders in humans.

Introduction
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(Gray, , 2013Alexander 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.
Anterior to posterior patterning of the developing brainstem 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., 1996Millet et al., , 1999Broccoli 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., 1999Liu et al., , 2003Sato 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 cordlike" 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 Development of the midbrain-hindbrain border. (A) Schema illustrating the molecular and anatomical establishment of the anterior and posterior brain regions by the expression of Otx2 and Gbx2, respectively, in a developing mouse embryo (between E8-E8.5). The forebrain (fb), midbrain (mb), rhomboencephalon (rb), and spinal cord (sc) are indicated. The transient morphological segments of the rhombencephalon (rhombomeres, r) are also illustrated. (B) Molecular networks acting in the midbrain-hindbrain border for the establishment of the isthmic organizer (see the text). This figure is adapted from our previous publication Lowenstein et al. (2022) that was published under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution, provided that the original author and source are credited (https://creativecommons.org/licenses/by/4.0/). 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., 2017Watson et al., , 2019Hirsch 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., 2000Bouchard et al., , 2005Voiculescu 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 anteriorposterior identity.

Dorsal to ventral patterning of the developing brainstem
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., 1998Lee et al., , 2000Muroyama et al., 2002;Ulloa and Marti, 2010;Hernandez-Miranda et al., 2017a). These gradients create a great Anterior to posterior patterning of the developing brainstem. (A) Schema illustrating the segmentation of the brainstem into rhombomeres (r) and selected transcription factors differentially express within these rhombomeres. For more details on Hox gene expression in the developing hindbrain please see Alexander et al. (2009). (B) A sagittal section of the mouse brainstem stained with antibodies against mCherry and counterstained with DAPI. The section is taken from Egr2 Cre/+ ;H2B mCherry/+ mice at birth (P0). In these mice, only r3 and r5 neural derivatives express the nuclear mCherry protein after Cre mediated recombination. This figure is adapted from our previous publication Isik and Hernandez-Miranda (2022) in Handbook of Clinical Neurology, Chapter 5, entitled Early development of the breathing network, published by Elsevier Books. The license number 5392080585902 between Hernandez-Miranda, Charite Universitätsmedizin Berlin and Elsevier allows us to reuse it in a journal/magazine. 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(Gray, , 2013Di 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, 2008Gray, , 2013Alaynick 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 dorsalventral 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).

Development of the anterior respiratory groups
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 Dorsal to ventral patterning of the developing brainstem. Left, schema illustrating concentration gradients formed by the diffusion of bone morphogenic proteins (BMPs)/WNTs and Sonic hedgehog (SHH) morphogens produced by the roof and floor plate, respectively. Right, schema illustrating a transverse section of the developing hindbrain and the patterning of 13 progenitor domains along its dorsal-ventral axis. These progenitors emerge from the differential action of BMP/WNT/SHH concentration gradients. The rhombomeric distribution of each progenitor domain and contribution to brainstem respiratory centers are indicated. Of note, the origin of the intertrigeminal region (ITR), Bötzinger complex (BötC) and caudal ventral respiratory group (cVRG) has not been conclusively determined (see text). nTS, nucleus tractus solitarius; RTN/pF, retrotrapezoid/parafacial nucleus; preBötC, preBötzinger complex; rVRG, rostral ventral respiratory group; Bmm, branchial motor neurons; Vmn, visceral motor neurons; nRaphe, Raphe nuclei. This figure is adapted from our previous publication Isik and Hernandez-Miranda (2022) in Handbook of Clinical Neurology, Chapter 5, entitled Early development of the breathing network, published by Elsevier Books. The license number 5392080585902 between Hernandez-Miranda, Charite Universitätsmedizin Berlin and Elsevier allows us the reuse of it in a journal/magazine. 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(Radulovacki et al., , 2004aStoiljkovic 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(Alheid et al., , 2011Hernandez-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.

Development of the dorsal medullary respiratory column
This respiratory column contains neurons known to be critical for the regulation of inspiratory activity. Most neurons forming this respiratory column reside within the Brainstem respiratory groups. Scheme representing the sagittal view of a mouse brainstem. This scheme illustrates the location of respiratory neurons belonging to the intertrigeminal region (ITR), parabrachial/Kölliker-Fuse complex (PB/KF), nucleus tractus solitarius (nTS), retrotrapezoid/parafacial nucleus (RTN/pF), Bötzinger complex (BötC), preBötzinger complex (preBötC), postInspiratory COmplex (PiCO), rostral and caudal ventral respiratory groups (rVRG and cVRG). The cerebellum, midbrain (mb), as well as the facial (nVII) motor nucleus and the nucleus ambiguus (na) are illustrated for anatomical orientation. This figure is adapted from our previous publication Isik and Hernandez-Miranda (2022) in Handbook of Clinical Neurology, Chapter 5, entitled Early development of the breathing network, published by Elsevier Books. The license number 5392080585902 between Hernandez-Miranda, Charite Universitätsmedizin Berlin and Elsevier allows us the reuse of it in a journal/magazine. 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(Aicher et al., , 1996Alheid 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, 1992Mifflin, , 1993Machado 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., 2001Qian et al., , 2002Cheng 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 Pattyn et al., 1997Pattyn et al., , 1999Pattyn et al., , 2000aPattyn et al., ,b, 2006Fode 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).

Development of the ventral medullary respiratory column
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.

Development of the retrotrapezoid/parafacial nucleus
The retrotrapezoid and parafacial nuclei are two small groups of excitatory neurons that are located ventro-laterally to the facial motor nucleus. Whether these groups are genuinely distinct remains to be conclusively determined. However, recent evidence seems to suggest that they are indeed physiologically distinct neuronal populations (Huckstepp et al., 2015(Huckstepp et al., , 2016Korsak et al., 2018;Zoccal et al., 2018). From an anatomical point of view, retrotrapezoid neurons locate ventrally to the facial motor nucleus and are long known to contain central respiratory chemoreceptor neurons; that is, acid-activated neurons that maintain constant levels of arterial PCO 2 (Figures 6A,B,F). For more details on the physiology of retrotrapezoid neurons and other central respiratory chemoreceptor neurons, we refer to the excellent work of Nattie and Li (2012), Bayliss (2015, 2022), Guyenet et al. (2019) and Lopez-Barneo (2022). On the other hand, neurons of the parafacial nucleus are located lateral to the facial motor nucleus. They are thought to control active expiration, a particular type of expiration that is produced when high metabolic demands induce an increase in respiration Thoby-Brisson et al., 2009;Bayliss et al., 2015;Huckstepp et al., 2015Huckstepp et al., , 2018Korsak et al., 2018;Pisanski and Pagliardini, 2019). In 2009, Dubreuil et al. (2009 identified that virtually all retrotrapezoid and parafacial neurons originate from Egr2expressing cells (that is from rhombomeres 3 and/or 5; see Figure 2). Furthermore, they identified their distinctive molecular signature, namely their co-expression of Phox2b, Lbx1, and Atoh1 ( Figure 6B). Ablation of each of these genes leads to the anatomical absence (Phox2b and Lbx1 mutant mice) or aberrant location (Atoh1 mutants) of retrotrapezoid and parafacial neurons, as well as to the loss of the hypercarbic reflex (the natural acceleration of breathing in response to increasing PCO 2 levels) and neonatal death (Wang et al., 2005;Pagliardini et al., 2008;Dubreuil et al., 2009;Rose et al., 2009a,b;Hernandez-Miranda et al., 2018). The dB2 progenitor domain generates retrotrapezoid and parafacial neurons and is exclusively located between rhombomeres 2 and 6 (Figures 3, 6C). dB2 progenitor cells express Phox2b and their postmitotic progeny co-express Lbx1 in addition to Phox2b (Figure 6C). A small number of dB2 (Lbx1 + /Phox2b +) neurons migrate ventrally toward the facial motor nucleus and activate the expression of Atoh1 during their migration (Figures 6D,E). The expression of Atoh1 seems to be essential for the migration and maturation of retrotrapezoid and parafacial nucleus neurons Development of the retrotrapezoid/parafacial nucleus. (A) Schema representing the location and function of retrotrapezoid/parafacial nucleus (RTN/pF) in mice. Under normal levels of oxygen (normoxia), the activity of the preBötzinger complex (preBötC) controls the firing rate of neurons in the phrenic motor nucleus (Phrenic) by sending signals via premotor neurons located in the rostral ventral respiratory group (rVRG). Phrenic motor neurons control diaphragm activity. Under hypercarbia (high levels of PCO 2 ) RTN/pF neurons increase their firing rate: this adjusts the activity of preBötzinger complex and eventually the firing rate of the phrenic motor neurons, which in turn increases respiration (B) Histological characterization of RTN/pF in newborn mice (P0). RTN/pF neurons co-express Phox2b and Lbx1 but not choline-acetyltransferase (ChAT), which distinguishes them from facial motor neurons (nVII) that express Phox2b and ChAT but not Lbx1. (C) Histological characterization of dB2 neurons across the indicated rhombomeres (r). dB2 neurons co-express Phox2b and Lbx1 at E11.5 in mice. This molecular signature distinguishes them from the dorsal (dA3 neurons) and ventral (pMNv) cells that express Phox2b but not Lbx1. Note that dB2 neurons emerge from r2 to r6, whereas dA3 neurons originate in r4 to r7/8 and pMNv cells can be found from r2-r7/8 (see text). (D) Transverse section of a E12.5 mouse embryo stained with antibodies against Phox2b, Atoh1, and Lbx1. Note that a subset of dB2 neurons (magenta cells) activate the expression of Atoh1 as they reach the facial (nVII) motor nucleus (boxed area). The boxed area is illustrated at the right with different combinations of the fluorescent signals. (E) Schema depicting the development of RTN/pF neurons. Phox2b + dB2 progenitor cells differentiate an initiate the expression of Lbx1 in neurons. A subset of Phox2b + /Lbx1 + (dB2) neurons ventrally migrates and activates the expression of Atoh1 as they reach the facial (nVII) motor nucleus. It is the co-expression of Phox2b + /Lbx1 + /Atoh1 + what defines the molecular signature of RTN/pF neurons. (F) Upper panels, by E14.5 in mice, RTN/pF neurons (arrowheads) settle underneath the facial motor (nVII) nucleus and express additional marker, such as neurokinin 1 receptor (Nk1R). Lower panels, ventral hindbrain view of a E14.5 wholemount brainstem preparation centered on the facial (nVII) motor nucleus showing Ca2 + green-1AM fluorescence changes ( F/F) of parifacial nucleus (pF) activity in physiological (7.4) and low (7.2) pH. This figure is adapted from our previous publication Isik and Hernandez-Miranda (2022) in Handbook of Clinical Neurology, Chapter 5, entitled Early development of the breathing network, published by Elsevier Books. The license number 5392080585902 between Hernandez-Miranda, Charite Universitätsmedizin Berlin and Elsevier allows us to reuse it in a journal/magazine. The primary data used in this figure was published in Hernandez-Miranda and Birchmeier (2015) and Hernandez-Miranda et al. (2018) under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution, provided that the original author and source are credited (https://creativecommons.org/licenses/by/4.0/). (Huang et al., 2012;Ruffault et al., 2015;Hernandez-Miranda et al., 2018). As retrotrapezoid and parafacial neurons mature throughout postnatal life, and for reasons yet to be identified, these neurons silence Lbx1 and Atoh1, but retain Phox2b. Several studies show that interfering with the specification of retrotrapezoid and parafacial neurons does not compromise neonatal or postnatal survival in mice. However, the loss of these neurons leads to a range of hypoventilation behaviors and the loss of the hypercarbic reflex in neonatal mice (Ramanantsoa et al., 2011;Huang et al., 2012;Ruffault et al., 2015;Hernandez-Miranda et al., 2018). Notably, transgenic mice lacking retrotrapezoid and parafacial neurons are able to recover some of the ventilatory responses to hypercarbia in adult life (Ramanantsoa et al., 2011;Huang et al., 2012;Ruffault et al., 2015;Hernandez-Miranda et al., 2018). How these animals gain the ability to respond to hypercarbia in adulthood is currently unknown, but this indicates that other chemoreceptor cells, either in the central or peripheral nervous system, can compensate for the loss of retrotrapezoid and parafacial neurons in the adult life.

Development of the Bötzinger complex
The major inhibitory component of the ventral respiratory column is the Bötzinger complex that uses GABA and glycine as its primary neurotransmitters (Shao and Feldman, 1997;Schreihofer et al., 1999;Song et al., 2001). Classic studies showed that the electrophysiological and pharmacological activation of Bötzinger neurons strongly inhibits inspiration and that Bötzinger neurons display decrementing postinspiratory or augmenting firing patterns during expiration (Bongianni et al., 1988;Gang and Lei, 1996). These neurons mutually interact with the preBötzinger complex to regulate the respiratory rhythm (Bryant et al., 1993;Tian et al., 1998;Ezure et al., 2003a,b,c). The Bötzinger complex innervates all other brainstem respiratory neurons and projects to spinal premotor and motor phrenic neurons (Otake et al., 1988;Jiang and Lipski, 1990;Douse and Duffin, 1992;Tian et al., 1998;Smith et al., 2007).
The precise rhombomeric origin and progenitor domain from which the Bötzinger complex develops has not yet been fully investigated. However, circumstantial evidence might indicate that this respiratory group develops from the Lbx1-lineage. Indeed, a lineage tracing study by Pagliardini et al. (2008) revealed that virtually all GABAergic and glycinergic neurons found in the anatomical region where the Bötzinger complex resides have a history of Lbx1 expression, ablation of which results in the absence of GABAergic and glycinergic neurons in this area (Pagliardini et al., 2008). Mature "Bötzinger" neurons lose the expression of Lbx1 but can be recognized by the expression of Pax2, GABA and other glycinergic markers (Pagliardini et al., 2008). As abovementioned, Lbx1 is key for the specification of four distinct neuron types, two of which express Pax2 and are GABAergic and glycinergic in nature: dB1 and dB4 neurons (Figure 3 and Table 2). In Lbx1 null mutant mice, Pax2 expression seems to be uniquely lost from the dB1 domain (that extends from rhombomeres 2-7/8), which might suggest this region as the possible source of Bötzinger neurons (Pagliardini et al., 2008). However, more research is necessary to clearly define the developmental origin of the Bötzinger complex.

Development of the preBötzinger complex
In the early 1990's, the work of Jeffrey C. Smith and Jack L. Feldman identified the preBötzinger complex as essential for generating the respiratory rhythm in mammals (Figures 7A,B; Smith et al., 1991). Later studies uncover some molecular markers such as the neurokinin 1 receptor and somatostatin to be expressed by preBötzinger neurons (Smith et al., 1991(Smith et al., , 2007Gray et al., 1999Gray et al., , 2001. How the preBötzinger complex generates the respiratory rhythm is currently the subject of intense investigation and outside the scope of this review, but we recommend the reader the excellent reviews by the groups of Anderson and Ramirez (2017), Ramirez and Baertsch (2018), Rubin and Smith (2019), Ramirez et al. (2022) and Smith (2022).
In 2010, two independent groups defined the V0 progenitor domain (in the basal plate) as the source of the preBötzinger complex (Bouvier et al., 2010;Gray et al., 2010). V0 progenitor cells share molecular traits with the p0 progenitor domain in the ventral spinal cord and are characterized by the expression of Dbx1 and Dbx2, the former is, however, unique for V0 and p0 progenitor cells ( Figure 7C and Table 1; Pierani et al., 1999Pierani et al., , 2001. Both Bouvier et al. (2010) and Gray et al. (2010) used Dbx1 LacZ mice, which express the reporter protein betagalactosidase under the control of the Dbx1 promoter, to lineage trace the preBötzinger complex and demonstrated that all the excitatory and rhythmically active neurons in this region derived from the V0 progenitor domain. Neurons emanating from this domain express the transcription factor Evx1, which distinguishes them from the surrounding dB4 and V1 neurons that express Lbx1 and En1, respectively ( Figure 7C and Table 2; Pierani et al., 1999Pierani et al., , 2001. Interestingly, V0 neuronal derivatives destined to populate the preBötzinger complex are correctly generated and reach the preBötzinger complex area in Dbx1 null mutant (Dbx1 LacZ/LacZ ) mice, but these cells do not exhibit intrinsic rhythmicity and fail to adopt their mature molecular identity, as determined by the lack of neurokinin 1 receptor or somatostatin expression in these cells (Bouvier et al., 2010).
Expression of the transcription factor Pax7 molecularly distinguishes two V0 progenitor subdomains: V0 dorsal (V0 D ; Pax7 +) and V0 ventral (V0 V ; Pax7-) ( Figure 7C). In Bouvier et al. (2010) the authors walked an extra mile and ablated Dbx1 from the V0 D domain, using Pax7 Cre ;Dbx1 LacZ/Flox mice, to elucidate which of these two subdomains generate Development of the preBötzinger complex. (A) The neurons of the preBötzinger complex locate ventral to the nucleus ambiguus (na) and can be distinguished by the co-expression of somatotatin (SST) and neurokinin 1 receptor (Nk1R) in mice at birth (P0). Please note that preBötzinger neurons co-express SST and Nk1R but not Phox2b, while motor neurons of the nucleus ambiguus co-express Nk1R and Phox2b but not SST. (B) Left, transverse section of a mouse hindbrain at E14.5 showing Ca2 + green-1AM fluorescence changes ( F/F) of preBötzinger complex (boxed area) intrinsic activity. Right, traces illustrating bursts of preBötzinger complex activity in physiological pH or after substance p (SP) treatment, which accelerates their firing. (C) Left, transverse section of a E11.5 mouse brainstem at rhombomere 7. The section was stained with antibodies against Dbx1 (green) Olig3 (red) and Bhlhe22 (blue). Note that the V0 progenitor domain can be distinguished by the expression of Dbx1. Right, histological characterization of the V0 progenitor domain and the neurons that emerge from it, using antibodies against Dbx1, Dbx2, Evx1, Pax7, Lhx1/5, Pax2 and Lbx1. Note the Dbx1 + (V0) progenitor domain generates Evx1 + neurons that co-express in addition Lhx1/5 and Pax2 but not Lbx1 (see also Tables 1, 2 and text for more details). The expression of Bhlhe22 allows the distinction of the dB4 and V1/V2 domains that sandwich the V0 progenitor domain. A fraction of Dbx1-derived neurons will differentiate into the preBötzinger complex (see text). Insets in the main photographs illustrate the co-expression of Evx1 + cells with the indicated markers. This figure is adapted from our previous publication Isik and Hernandez-Miranda (2022)  the preBötzinger complex. This elegant experiment found an anatomically, molecularly and physiologically intact preBötzinger complex, demonstrating that this respiratory center emerges from the V0 V subdomain. More recent studies have exploited this developmental knowledge to selectively activate, silence, or ablate Dbx1-derived preBötzinger neurons, resulting in respiratory changes that enhance, depress, or halt breathing in adult mice, respectively (Wang et al., 2014;Vann et al., 2016Vann et al., , 2018. Even though the anterior-posterior origin of the preBötzinger complex has not yet been identified, an early study indicates that it emerges from the most posterior rhombomeres and partly from rhombomere 6 (MafB +). In this context, a study by Blanchi et al. (2003) showed that MafB null mutant mice rarely breathe at birth and display limited phrenic nerve activity (Blanchi et al., 2003). Histologically, MafB null mutants have a significant, but not complete, loss of preBötzinger neurons (Blanchi et al., 2003). Since Egr2 null mutant mice display breathing rhythmicity and can survive for about a day after birth (Chatonnet et al., 2007), it is likely that V0 progenitors in rhombomere 6 generate a fraction of preBötzinger neurons that is complemented by V0 progenitors in rhombomeres 7/8.

Development of the rostral and caudal ventral respiratory groups
The preBötzinger complex controls breathing by activating premotor neurons that in turn regulate multiple brainstem (e.g., hypoglossal or vagal) and spinal cord (phrenic) motor neurons. Caudal to the preBötzinger complex, two sets of respiratory premotor neurons are associated with inspiratory and expiatory motor activity: the rostral (rVRG) and caudal (cVRG) ventral respiratory groups, respectively (Smith et al., 2013). A recent study from the group of Gilles Fortin identified that excitatory rVRG neurons have a history of Dbx1 expression and, as such, to originate from the V0 progenitor domain (Wu et al., 2017). Even though both preBötzinger neurons and rVRG neurons express Slc17a6 (vGlut2) and Pax2, only the former expresses neurokinin 1 receptor and somatostatin (Bouvier et al., 2010;Gray et al., 2010;Wu et al., 2017). These molecular differences could be explained by their distinct rhombomeric origins and/or by their generation in distinct V0 progenitor subdomains. The development of cVRG neurons has not been yet addressed, but some Dbx1-derivaties neurons can be observed caudal to the rVRG, suggesting that cVRG neurons are also generated from the V0 progenitor domain (Gray, 2013;Wu et al., 2017). An alternative source of cVRG neurons could be the dB4 domain, whose derivatives co-express Lbx1 and Wt1 (Table 2). Indeed, a recent study reported the presence of GABAergic (Wt1 +) neurons in the cVRG region (Schnerwitzki et al., 2020).

Development of respiratory serotonergic neurons
The monoamine neurotransmitter serotonin has long been implicated in the control of respiration. Brain serotonin is produced by nine distinct groups of cells (called raphe nuclei), all of which are in the brainstem. Among the distinct raphe nuclei, several lines of research indicate that the midline located raphe obscurus in the medulla oblongata is an important component of the central respiratory chemoreceptor circuit (Jacobs et al., 2002). First, the en masse inhibition of serotonergic neurons (Ray et al., 2011), or the targeted inhibition of raphe obscurus neurons (Brust et al., 2014), significantly impairs the chemoreflex to hypercarbia in mice. Second, the optogenetic activation of these raphe neurons accelerates respiration in conscious and anesthetized rodents (Depuy et al., 2011). Lastly, mice genetically engineered to lack raphe neurons display dulled chemoreflexes to hypercarbia (Hodges et al., 2008(Hodges et al., , 2009Buchanan and Richerson, 2010).
The ventral-most progenitor domain (termed as pMN; Figure 3) in the developing hindbrain initially generates motor neurons, and later all brainstem serotonergic neurons. Due to its proximity to the floor plate, the pMN domain is under the direct influence of Sonic Hedgehog signaling (Marti et al., 1995;Chiang et al., 1996). Molecularly, this progenitor domain is subdivided into a dorsal subdomain (pMNs; expressing the transcription factors Pax6 and Olig2) and a ventral subdomain (pMNv; expressing the transcription factors Nkx2.2, Nkx2.9, and Phox2b) (Figure 3 and Table 1). Detailed histological and genetic analyses showed that the pMNs subdomain generates somatic motor neurons (i.e., hypoglossal motor neurons), whereas the pMNv subdomain generates branchial (e.g., facial motor neurons) and visceral (i.e., nucleus ambiguous) motor neurons (Briscoe et al., 1999;Pattyn et al., 2003). Pioneer studies by Briscoe and colleagues showed that pMNv progenitors first generate branchio/viscero motor neurons before E11.5 in mice, and then serotonergic neurons (Briscoe et al., 1999;Pattyn et al., 2003). One should note that except for rhombomere 4, the pMNv progenitor domain in all other rhombomeres contributes to raphe neurons. pMNv progenitor cells of rhombomere 4 are known to generate a large group of branchial (facial) motor neurons and to have an unusual prolonged expression of Phox2b between E9.5 to E12.5, which seems to be attributable to its incapacity to generate serotonergic neurons . Indeed, the silencing of Phox2b expression seems to be a molecular switch in the transition of pMNv progenitor cells from first generating branchio/visceromotor neurons to later generating serotonergic neurons in the other rhombomeres (Briscoe et al., 1999;Pattyn et al., 2003Pattyn et al., , 2004. In support of this, analysis of Nkx2.2 null mutant mice revealed an unusual extended expression of Phox2b within the pMNv domain, which leads to the overproduction of branchio/visceromotor neurons and the absence of serotonergic neurons (Briscoe et al., 1999;Pattyn et al., 2003Pattyn et al., , 2004. Conversely, the ablation of Phox2b results in the early generation of raphe neurons at the expense of branchio/visceromotor cells (Pattyn et al., 2004).
The bHLH transcription factor Ascl1 is critical for development of peripheral (i.e., enteric nervous system) and central (raphe) serotonergic cells (Blaugrund et al., 1996;Pattyn et al., 2004). In the pMNv domain, Ascl1 is co-expressed with Phox2b during the genesis of branchio/visceromotor neurons and is retained by these progenitors throughout the specification of raphe cells (Pattyn et al., 2004). Mutation of Ascl1 does not affect Phox2b expression nor the development of branchio/visceromotor cells, but severely interferes with the specification of raphe neurons, which are completely absent in Ascl1 null mutant mice (Pattyn et al., 2004). The maturation of raphe neurons is regulated by several other transcription factors, such as Gata2, Gata3, Lmx1b and Pet1, of which the null mutation of Lmx1b or Pet1 results in the total loss or a severe Frontiers in Molecular Neuroscience 13 frontiersin.org decrease (> 70%) of raphe cells, respectively (Cheng et al., 2003;Ding et al., 2003;Hendricks et al., 2003;Dosumu-Johnson et al., 2018;Okaty et al., 2020).
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 andZoghbi, 2018, 2020;Elliott et al., 2021;Lowenstein et al., 2021Lowenstein et al., , 2022Fritzsch 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 coexpression 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., 2021Lowenstein et al., , 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., 2021Lowenstein et al., , 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 Pattyn et al., 1997Pattyn et al., , 1999Pattyn et al., , 2000aPattyn et al., ,b, 2003Pattyn et al., , 2004Pattyn et al., , 2006Fode 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.

Conclusion
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., 2016Vann et al., , 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., 2008Sivan et al., 2019;Ceccherini et al., 2022). Frequently, CCHS patients also present with blunted responses to hypercabia and have abnormal levels of PCO 2 . 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., 2008Sivan 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.