Background Patients with congenital heart disease (CHD) and heterotaxy show high postsurgical morbidity/mortality, with some developing respiratory complications. Although this finding is often attributed to the CHD, airway clearance and left-right patterning both require motile cilia function. Thus, airway ciliary dysfunction (CD) similar to that of primary ciliary dyskinesia (PCD) may contribute to increased respiratory complications in heterotaxy patients. Methods and Results We assessed 43 CHD patients with heterotaxy for airway CD. Videomicrocopy was used to examine ciliary motion in nasal tissue, and nasal nitric oxide (nNO) was measured; nNO level is typically low with PCD. Eighteen patients exhibited CD characterized by abnormal ciliary motion and nNO levels below or near the PCD cutoff values. Patients with CD aged >6 years show increased respiratory symptoms similar to those seen in PCD. Sequencing of all 14 known PCD genes in 13 heterotaxy patients with CD, 12 without CD, 10 PCD disease controls, and 13 healthy controls yielded 0.769, 0.417, 1.0, and 0.077 novel variants per patient, respectively. One heterotaxy patient with CD had the PCD causing DNAI1 founder mutation. Another with hyperkinetic ciliary beat had 2 mutations in DNAH11, the only PCD gene known to cause hyperkinetic beat. Among PCD patients, 2 had known PCD causing CCDC39 and CCDC40 mutations. Conclusions Our studies show that CHD patients with heterotaxy have substantial risk for CD and increased respiratory disease. Heterotaxy patients with CD were enriched for mutations in PCD genes. Future studies are needed to assess the potential benefit of prescreening and prophylactically treating heterotaxy patients for CD.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder associated with ciliary defects and situs inversus totalis, the complete mirror image reversal of internal organ situs (positioning). A variable incidence of heterotaxy, or irregular organ situs, also has been reported in PCD patients, but it is not known whether this is elicited by the PCD-causing genetic lesion. We studied a mouse model of PCD with a recessive mutation in Dnahc5, a dynein gene commonly mutated in PCD. Analysis of homozygous mutant embryos from 18 litters yielded 25% with normal organ situs, 35% with situs inversus totalis, and 40% with heterotaxy. Embryos with heterotaxy had complex structural heart defects that included discordant atrioventricular and ventricular outflow situs and atrial/pulmonary isomerisms. Variable combinations of a distinct set of cardiovascular anomalies were observed, including superior-inferior ventricles, great artery alignment defects, and interrupted inferior vena cava with azygos continuation. The surprisingly high incidence of heterotaxy led us to evaluate the diagnosis of PCD. PCD was confirmed by EM, which revealed missing outer dynein arms in the respiratory cilia. Ciliary dyskinesia was observed by videomicroscopy. These findings show that Dnahc5 is required for the specification of left-right asymmetry and suggest that the PCD-causing Dnahc5 mutation may also be associated with heterotaxy.

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Forward genetic screens with ENU (N-ethyl-N-nitrosourea) mutagenesis can facilitate gene discovery, but mutation identification is often difficult. We present the first study in which an ENUinduced mutation was identified by massively parallel DNA sequencing. This mutation causes heterotaxy and complex congenital heart defects and was mapped to a 2.2-Mb interval on mouse chromosome 7. Massively parallel sequencing of the entire 2.2-Mb interval identified 2 single-base substitutions, one in an intergenic region and a second causing replacement of a highly conserved cysteine with arginine (C193R) in the gene Megf8. Megf8 is evolutionarily conserved from human to fruit fly, and is observed to be ubiquitously expressed. Morpholino knockdown of Megf8 in zebrafish embryos resulted in a high incidence of heterotaxy, indicating a conserved role in laterality specification. Megf8 C193R mouse mutants show normal breaking of symmetry at the node, but Nodal signaling failed to be propagated to the left lateral plate mesoderm. Videomicroscopy showed nodal cilia motility, which is required for left-right patterning, is unaffected. Although this protein is predicted to have receptor function based on its amino acid sequence, surprisingly confocal imaging showed it is translocated into the nucleus, where it is colocalized with Gfi1b and Baf60C, two proteins involved in chromatin remodeling. Overall, through the recovery of an ENU-induced mutation, we uncovered Megf8 as an essential regulator of left-right patterning.cardiogenesis ͉ left-right ͉ nodal

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The electrophysiological properties of the mouse anterior pituitary cell line AtT-20/D16-16 were investigated with intracellular and patch-clamp techniques. Clonal AtT-20/D16-16 cells were found to be electrically excitable, with most cells exhibiting spontaneous bursting action potentials. The mean burst rates varied from 1.4 Hz at -55 mV to 8.2 Hz at -25 mV, showing an approximately linear frequency-current relationship in the low current range. The bursts consisted of one to several fast Na4 spikes superimposed on a slow pacemaker potential, followed by a Ca2+ spike and a Ca2+-sensitive afterhyperpolarization. Removal of either Na+ or Ca2+ from the bathing medium led to cessation of spontaneous activity and the appearance of arrhythmic firing patterns. Single channel recordings revealed the presence of Ca2+-dependent K+ channels with unitary conductances of =130 pS in physiological medium. These channels were activated by both intracellular Ca2+ and membrane depolarization. Addition of norepinephrine (10 ,LM) led to increases in burst frequency and f8-endorphin secretion mediated by activation of f3-adrenergic receptors. Our results, in conjunction with previous work, suggest that the Ca2+ that enters the cell during the burst may be involved in hormone secretion.Clonal AtT-20/D16v mouse anterior pituitary tumor cells synthesize, store, and secrete corticotropin (ACTH), f3-lipotropin, ,3-endorphin, and other peptides derived from the corticotropin/p3-endorphin common precursor protein (1-5). The coordinate secretion of corticotropin and P3-endorphin by AtT-20/ D16v cells, or by cells of other clones, is stimulated up to 30-fold by depolarization with 50-80 mM KCI (4, 6-8) and by 10 ,uM norepinephrine (5). Secretion stimulated by these compounds is dependent on extracellular Ca2" (5-8), indicating a requirement for Ca2" influx in the release process.Secretions from a number of endocrine systems have been reported to involve Ca2" entry through voltage-sensitive channels. Thus, action potentials in which Ca2" was identified as the principal charge carrier have been recorded from cells of the ,B-pancreatic islets (9, 10), the anterior pituitary gland (11) Unless stated otherwise, the cells were treated with 1 mM dibutyryl cAMP (Boehringer Mannheim) for 1-3 wk prior to recording. This procedure doubled the average cell diameter, facilitating microelectrode impalement, but did not appreciably alter the excitability of the cells (8).Patch-clamp recordings were made at room temperature (22-24°C) by the method of Hamill et al. (18). The patch electrode was fabricated on a microforge and had a resistance of 3-5 MfQ when filled with physiological saline.The control solution had the following composition (mM):NaCI, 145; KCI, 5.4; MgCl2, 0.8; CaCl2, 1.8; glucose,25; Hepes/ NaOH, 10. Na+-free solutions were prepared by isosmotic substitution of sucrose for NaCl. Ca2+-free solutions were prepared by omitting CaCl2 from the control solution and adding EGTA to 0.5 mM and additional MgCl2 to 2.8 mM. All solutions we...

During spermatogenesis, several genes are expressed in a germ cell-specific manner. Previous studies have demonstrated that rat and mouse spermatogenic cells produce a 1,700-nucleotide proenkephalin RNA, while somatic cells that express the proenkephalin gene contain a 1,450-nucleotide transcript. Using cDNA cloning, RNA protection, and primer extension analyses, we showed that transcription of the rat and mouse spermatogenic-cell RNAs is initiated downstream from the proenkephalin somatic promoter in the first somatic intron (intron As). In both species, the germ cell cap site region consists of multiple start sites distributed over a length of approximately 30 base pairs. Within rat and mouse intron As, the region upstream of the germ cell cap sites is GC rich and lacks TATA sequences. A consensus binding site for the transcription factor SP1 was identified in intron As downstream of the proenkephalin germ cell cap site region. These features are characteristic of several previously described promoters that lack TATA sequences. Homologies were also identified between the proenkephalin and rat cytochrome c spermatogenic-cell promoters, including the absence of a TATA box, a multiple start site region, and several common sequences. This promoter motif thus may be shared with other genes expressed in male germ cells.Spermatogenesis is a complex program of cellular differentiation that results in the formation of haploid spermatozoa. While this developmental sequence of events has been well characterized morphologically, a description of the molecular mechanisms regulating spermatogenic cell differentiation has only recently been initiated (18). Germ cell gene expression is highly stage specific, with different gene products being expressed at distinct phases of development. Both stage-specific transcriptional and translational regulation of early-transcribed mRNAs contribute to these differentiational changes (18,19). Another characteristic of spermatogenic-cell gene expression is the presence of germ cell-specific transcripts not produced in somatic cells. These unique RNAs may be generated by multiple mechanisms, including transcription of genes selectively expressed in germ cells (5,37,49), utilization of distinct transcriptional initiation or termination sites (12, 39), and alternative RNA splicing.The gene for the opioid precursor proenkephalin is expressed in both spermatogenic and somatic cells (13,(28)(29)(30) MATERIALS AND METHODSIsolation of proenkephalin cDNA from mouse testis. A mouse testis cDNA library constructed in the EcoRI site of AgtlO (kindly provided by Ken Kleene, Biology Department, University of Massachusetts, Boston) was screened for proenkephalin-containing phage by hybridization to a PvuII fragment from rat brain proenkephalin cDNA [pRPE-1(165-600) (21)]. The largest insert from the positive bacteriophage clones was isolated by EcoRI digestion and subcloned into the EcoRI site of pBluescript SK (Stratagene, San Diego, Calif.). Standard procedures were used throughout for the growth and...

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