Background: Luteinizing hormone (LH) and follicle stimulating hormone (FSH), produced in gonadotrope cells in the adenohypophysis are key regulators of vertebrate reproduction. The differential regulation of these hormones, however, is poorly understood and little is known about gonadotrope embryonic development. We developed a stable transgenic line of medaka with the LH beta subunit gene (lhb) promotor driving green fluorescent protein (gfp) expression to characterize development of LH‐producing gonadotropes in whole larvae and histological sections. Additionally, developmental and tissue‐specific gene expression was examined. Results: The lhb gene is maternally expressed during early embryogenesis. Transcript levels increase by stage 21 (36 hours post fertilization [hpf]) and then decrease during continued larval development. Examination of the expression of pituitary marker genes show that LH‐producing cells are initially localized outside the primordial pituitary, and they were localized to the developing gut tube by 32 hpf. At hatching, lhb‐GFP is clearly detected in the gut epithelium and in the anterior digestive tract. lhb‐GFP expression later consolidate in the developing pituitary by 2 weeks postfertilization. Conclusions: During embryonic development, lhb is primarily expressed outside the central nervous system and pituitary. The novel expression of lhb in the embryonic gut suggests that LH has a hitherto unidentified developmental function. Developmental Dynamics 241:1665–1677, 2012.

During the last two decades single-cell analysis (SCA) has revealed extensive phenotypic differences within homogenous cell populations. These phenotypic differences are reflected in the stochastic nature of gene regulation, which is often masked by qualitatively and quantitatively averaging in whole tissue analyses. The ability to isolate transcripts and investigate how genes are regulated at the single cell level requires highly sensitive and refined methods. This paper reviews different strategies currently used for SCA, including harvesting, reverse transcription, and amplification of the RNA, followed by methods for transcript quantification. The review provides the historical background to SCA, discusses limitations, and current and future possibilities in this exciting field of research.

Cardiac sodium (Na+)-calcium (Ca2+) exchanger 1 (NCX1) is central to the maintenance of normal Ca2+ homeostasis and contraction. Studies indicate that the Ca2+-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca2+ binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met369 antibody identified a novel calpain cleavage site at Met369. Engineering NCX1-Met369 into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met369 inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met369 cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction

Kisspeptins (Kiss1 and Kiss2) and their receptors (putatively Gpr54-1 and Gpr54-2) have emerged as key players in vertebrate reproduction owing to their stimulatory effect on the brain-pituitary-gonadal axis. Virtually nothing is known, however, about their role during embryogenesis. Using medaka (Teleostei) as a model system, we report, for the first time in vertebrates, an early developmental expression and putative function of kisspeptins. Expression analyses and knockdown experiments suggest that early actions of kisspeptins are probably mediated by binding to maternally supplied Gpr54-1 and late action by both Gpr54-1 and Gpr54-2. Knockdown of maternally provided kiss1 and gpr54-1 arrested development during gastrulation, before establishment of any germ layers, whereas knockdown of zygotically provided kiss1 and gpr54-1 disrupted brain development. A similar phenotype was observed for gpr54-2 knockdown embryos, suggesting a critical role for kiss1, gpr54-1, and gpr54-2 in neurulation. These data demonstrate that kisspeptin signaling is active both maternally and zygotically and is involved in embryonic survival and morphogenesis.