John J. Greer

Loss of MeCP2 in aminergic neurons causes cell-autonomous defects in neurotransmitter synthesis and specific behavioral abnormalities

Rett syndrome (RTT) is characterized by specific motor, cognitive, and behavioral deficits. Because several of these abnormalities occur in other disease states associated with alterations in aminergic neurotransmitters, we investigated the contribution of such alterations to RTT pathogenesis. We found that both individuals with RTT and Mecp2-null mice have lower-than-normal levels of aminergic metabolites and content. Deleting Mecp2 from either TH-positive dopaminergic and noradrenergic neurons or PET1-positive serotonergic neurons in mice decreased corresponding neurotransmitter concentration and specific phenotypes, likely through MeCP2 regulation of rate-limiting enzymes involved in aminergic neurotransmitter production. These data support a cell-autonomous, MeCP2-dependent mechanism for the regulation of aminergic neurotransmitter synthesis contributing to unique behavioral phenotypes.

Fog2 Is Required for Normal Diaphragm and Lung Development in Mice and Humans

Congenital diaphragmatic hernia and other congenital diaphragmatic defects are associated with significant mortality and morbidity in neonates; however, the molecular basis of these developmental anomalies is unknown. In an analysis of E18.5 embryos derived from mice treated with N-ethyl-N-nitrosourea, we identified a mutation that causes pulmonary hypoplasia and abnormal diaphragmatic development. Fog2 (Zfpm2) maps within the recombinant interval carrying the N-ethyl-N-nitrosourea-induced mutation, and DNA sequencing of Fog2 identified a mutation in a splice donor site that generates an abnormal transcript encoding a truncated protein. Human autopsy cases with diaphragmatic defect and pulmonary hypoplasia were evaluated for mutations in FOG2. Sequence analysis revealed a de novo mutation resulting in a premature stop codon in a child who died on the first day of life secondary to severe bilateral pulmonary hypoplasia and an abnormally muscularized diaphragm. Using a phenotype-driven approach, we have established that Fog2 is required for normal diaphragm and lung development, a role that has not been previously appreciated. FOG2 is the first gene implicated in the pathogenesis of nonsyndromic human congenital diaphragmatic defects, and its necessity for pulmonary development validates the hypothesis that neonates with congenital diaphragmatic hernia may also have primary pulmonary developmental abnormalities.

Etiology of congenital diaphragmatic hernia: the retinoid hypothesis.

Congenital diaphragmatic hernia (CDH) is a major life-threatening cause of respiratory failure in the newborn. Although significant efforts have been undertaken to unravel the pathophysiology of CDH, our current understanding of the etiology remains spare. Here we outline recent evidence suggesting that abnormalities linked with the retinoid signaling pathway early in gestation may contribute to the etiology of CDH. These studies include 1) the effect of altering the retinoid system in vitamin A deficient and transgenic animals;2) disruption of the retinoid system in teratogen-induced CDH in rodents, 3) the effect of co-administration of retinoids in nitrofen-induced CDH on lung and diaphragm development, and 4) clinical evidence suggesting decreased markers of vitamin A status in human CDH. Given the substantial mortality and morbidity associated with this serious developmental anomaly, advancements in this area will be critical. We feel that there is now sufficient circumstantial and direct experimental evidence to warrant further testing of the retinoid-CDH etiology hypothesis, including examination of retinoid-regulated target genes that could be candidates for involvement in CDH.

A partial loss of function allele of Methyl-CpG-binding protein 2 predicts a human neurodevelopmental syndrome

Rett Syndrome, an X-linked dominant neurodevelopmental disorder characterized by regression of language and hand use, is primarily caused by mutations in methyl-CpG-binding protein 2 (MECP2). Loss of function mutations in MECP2 are also found in other neurodevelopmental disorders such as autism, Angelman-like syndrome and non-specific mental retardation. Furthermore, duplication of the MECP2 genomic region results in mental retardation with speech and social problems. The common features of human neurodevelopmental disorders caused by the loss or increase of MeCP2 function suggest that even modest alterations of MeCP2 protein levels result in neurodevelopmental problems. To determine whether a small reduction in MeCP2 level has phenotypic consequences, we characterized a conditional mouse allele of Mecp2 that expresses 50% of the wild-type level of MeCP2. Upon careful behavioral analysis, mice that harbor this allele display a spectrum of abnormalities such as learning and motor deficits, decreased anxiety, altered social behavior and nest building, decreased pain recognition and disrupted breathing patterns. These results indicate that precise control of MeCP2 is critical for normal behavior and predict that human neurodevelopmental disorders will result from a subtle reduction in MeCP2 expression.

Retinal Dehydrogenase-2 Is Inhibited by Compounds that Induce Congenital Diaphragmatic Hernias in Rodents

Currently, the etiology of the serious developmental anomaly congenital diaphragmatic hernia (CDH) is unknown. We have used an animal model of CDH to address this issue. We characterized four separate teratogens that produced diaphragmatic defects in embryonic rats that are similar to those in infants with CDH. We then tested the hypothesis that all these agents share the common mechanism of perturbing the retinoid-signaling pathway. Specifically, inhibition of retinal dehydrogenase-2 (RALDH2), a key enzyme necessary for the production of retinoic acid and that is expressed in the developing diaphragm, was assayed by measuring retinoic acid production in cytosolic extracts from an oligodendrocyte cell line. The following compounds all induce posterolateral defects in the rat diaphragm; nitrofen, 4-biphenyl carboxylic acid, bisdiamine, and SB-210661. Importantly, we demonstrate that they all share the common mechanism of inhibiting RALDH2. These data provide an important component of mounting evidence suggesting that the retinoid system warrants consideration in future studies of the etiology of CDH.

Serotonergic and noradrenergic effects on respiratory neural discharge in the medullary slice preparation of neonatal rats

Rhythmically active medullary slice preparations isolated from neonatal rats (postnatal days 0-3, P0-P3) were used to study the modulation of respiraory rhythmogenesis and hypoglossal (XII) nerve discharge by serotonin (5-hydroxytryptamine, 5-HT) and noradrenaline (NA). 5-HT, NA and their respective receptor agonists and antagonists were applied either to the bathing medium or focally via pressure injection into regions encompassing the pre-Bötzinger complex or XII motoneurons. The effects of endogenously released 5-HT were also studied by chemical stimulation of neurons within the raphe obscurus. The frequency of respiratory burst discharge was increased when 5-HT was applied: (1) to the bathing medium (37±16%; 30 µM; P < 0.05); (2) via pressure injection into the region of the pre-Bötzinger complex (22 ± 14%; < 25 pmol; P < 0.05); or (3) endogenously released in response to activation of neurons within the raphe obscurus via pressure injection of (R,S)-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydro-bromide (AMPA, 34 ± 15%; P < 0.05) or 5-HT (33 ± 5%; P < 0.05). All of these effects were antagonized by bath application of methysergide (30-40 µM). NA caused a reduction of respiratory burst frequency when applied to the bathing medium (40 ± 15%; 100 µM; P < 0.05) or when pressure injected into the region of the pre-Bötzinger complex (22 ± 11 %; < 25 pmol; P < 0.05). These effects were blocked by the bath application of the a2-receptor antagonist idazoxan (2 µM). 5-HT and NA both caused an augmentation of tonic discharge of XII nerves when applied either to the bathing medium or via pressure injection into the XII motoneuron pool. The 5-HT-induced XII nerve tonic discharge was mimicked by the 5-HT2 receptor agonist R(-)2-(2,5-dimethoxy-4-iodophenyl) (DOI.HC1, 5 µM) and blocked by the 5-HT2 receptor antagonist ketanser-ine tartrate (30-40 µM). The NA-induced XII nerve tonic discharge was mimicked by the α1-receptor agonist phenylephrine HC1 (500 µM) and blocked by the α1-receptor antagonist prozasin HC1 (@#@ 1 µM).

A genetic model for a central (septum transversum) congenital diaphragmatic hernia in mice lacking Slit3

Congenital diaphragmatic hernia (CDH) is a significant cause of pediatric mortality in humans with a heterogeneous and poorly understood etiology. Here we show that mice lacking Slit3 developed a central (septum transversum) CDH. Slit3 encodes a member of the Slit family of guidance molecules and is expressed predominantly in the mesothelium of the diaphragm during embryonic development. In Slit3 null mice, the central tendon region of the diaphragm fails to separate from liver tissue because of abnormalities in morphogenesis. The CDH progresses through continuous growth of the liver into the thoracic cavity. This study establishes the first genetic model for CDH and identifies a previously unsuspected role for Slit3 in regulating the development of the diaphragm.

Math1 is essential for the development of hindbrain neurons critical for perinatal breathing.

Mice lacking the proneural transcription factor Math1 (Atoh1) lack multiple neurons of the proprioceptive and arousal systems and die shortly after birth from an apparent inability to initiate respiration. We sought to determine whether Math1 was necessary for the development of hindbrain nuclei involved in respiratory rhythm generation, such as the parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN), defects in which are associated with congenital central hypoventilation syndrome (CCHS). We generated a Math1-GFP fusion allele to trace the development of Math1-expressing pFRG/RTN and paratrigeminal neurons and found that loss of Math1 did indeed disrupt their migration and differentiation. We also identified Math1-dependent neurons and their projections near the pre-Bötzinger complex, a structure critical for respiratory rhythmogenesis, and found that glutamatergic modulation reestablished a rhythm in the absence of Math1. This study identifies Math1-dependent neurons that are critical for perinatal breathing that may link proprioception and arousal with respiration.

Embryological origins and development of the rat diaphragm

Textbooks of embryology provide a standard set of drawings and text reflecting the traditional interpretation of phrenic nerve and diaphragm development based on anatomical dissections of embryonic tissue. Here, we revisit this issue, taking advantage of immunohistochemical markers for muscle precursors in conjunction with mouse mutants to perform a systematic examination of phrenic‐diaphragm embryogenesis. This includes examining the spatiotemporal relationship of phrenic axon outgrowth and muscle precursors during different stages of myogenesis. Additionally, mutant mice lacking c‐met receptors were used to visualize the mesenchymal substratum of the developing diaphragm in the absence of myogenic cells. We found no evidence for contributions to the diaphragm musculature from the lateral body wall, septum transversum, or esophageal mesenchyme, as standard dogma would state. Nor did the data support the hypothesis that the crural diaphragm is of distinct embryological origins. Rather, we found that myogenic cells and axons destined to form the neuromuscular component of the diaphragm coalesce within the pleuroperitoneal fold (PPF). It is the expansion of these components of the PPF that leads to the formation of the diaphragm. Furthermore, we extended these studies to examine the developing diaphragm in an animal model of congenital diaphragmatic hernia (CDH). We find that malformation of the PPF mesenchymal substratum leads to the defect characteristic of CDH. In summary, the data demonstrates that a significant revision of narratives describing normal and pathological development of the diaphragm is warranted. J. Comp. Neurol. 455:477–487, 2003.

Ontogeny of the pre-Bötzinger complex in perinatal rats.

The aim of this study was to provide a systematic examination of the ontogenesis of the mammalian respiratory rhythm generating center, the pre-Bötzinger complex (pre-BötC). A combination of immunohistochemical markers and electrophysiological recordings was used to determine the time of inception of the pre-BötC and the developmental changes during the perinatal period in rats spanning from embryonic day 15 (E15) to postnatal day 7. The first clear indication of neurons immunopositive for neurokinin-1 receptors (NK1Rs) and somatostatin expression, two proposed markers for pre-BötC neurons, was at ∼E17. Birth dating of neurons in the ventrolateral medulla using 5-bromo-2′-deoxyuridine demonstrated that NK1R-positive neurons populating the area of the pre-BötC during late E16-E18 are born at E12.5-E13.5, ∼2 d later than adjacent NK1R-positive neurons in the ventrolateral medulla. Extracellular recordings of neuronal populations within the pre-BötC of perinatal medullary slice preparations demonstrated that the onset of rhythmical respiratory discharge commences at ∼E17. Application of substance P, a ligand for NK1R receptors, to the media bathing E17 medullary slice and brainstem-spinal cord preparations resulted in a marked increase in respiratory frequency. These data provide insights into the ontogeny of the pre-BötC, giving fundamental information on the genesis, settlement, and inception of rhythmic activity within the group of neurons proposed to be responsible for the respiratory rhythm generation. Furthermore, this provides the foundation for further analyses of cell lineage, the transcriptional control of respiratory neuronal development, and electrophysiological and pharmacological properties of the pre-BötC during the prenatal period.