Hemato-vascular specification requires arnt1 and arnt2 genes in zebrafish embryos.

During embryonic development, a subset of cells in the mesoderm germ layer are specified as hemato-vascular progenitor cells, which then differentiate into endothelial cells and hematopoietic stem and progenitor cells. In zebrafish, the transcription factor npas4l (cloche) is required for the specification of hemato-vascular progenitor cells. However, it is unclear whether npas4l is the sole factor at the top of the hemato-vascular specification cascade. Here, we show that arnt1 and arnt2 genes are required for hemato-vascular specification. We found that arnt1;arnt2 double mutant zebrafish embryos, but not arnt1 or arnt2 single mutants, lack blood cells and most endothelial cells. arnt1/2 mutants have reduced or absent expression of etsrp and tal1, the earliest known endothelial and hematopoietic transcription factor genes. We found that Npas4l binds both Arnt1 and Arnt2 proteins in vitro, consistent with the idea that PAS domain-containing bHLH transcription factors act in a multimeric complex to regulate gene expression. Our results demonstrate that npas4l, arnt1 and arnt2 act together to regulate endothelial and hematopoietic cell fate, where each gene is necessary, but not sufficient, to drive hemato-vascular specification.

G protein-coupled estrogen receptor regulates embryonic heart rate in zebrafish.

Estrogens act by binding to estrogen receptors alpha and beta (ERα, ERß), ligand-dependent transcription factors that play crucial roles in sex differentiation, tumor growth and cardiovascular physiology. Estrogens also activate the G protein-coupled estrogen receptor (GPER), however the function of GPER in vivo is less well understood. Here we find that GPER is required for normal heart rate in zebrafish embryos. Acute exposure to estrogens increased heart rate in wildtype and in ERα and ERß mutant embryos but not in GPER mutants. GPER mutant embryos exhibited reduced basal heart rate, while heart rate was normal in ERα and ERß mutants. We detected gper transcript in discrete regions of the brain and pituitary but not in the heart, suggesting that GPER acts centrally to regulate heart rate. In the pituitary, we observed gper expression in cells that regulate levels of thyroid hormone triiodothyronine (T3), a hormone known to increase heart rate. Compared to wild type, GPER mutants had reduced levels of T3 and estrogens, suggesting pituitary abnormalities. Exposure to exogenous T3, but not estradiol, rescued the reduced heart rate phenotype in gper mutant embryos, demonstrating that T3 acts downstream of GPER to regulate heart rate. Using genetic and mass spectrometry approaches, we find that GPER regulates maternal estrogen levels, which are required for normal embryonic heart rate. Our results demonstrate that estradiol plays a previously unappreciated role in the acute modulation of heart rate during zebrafish embryonic development and suggest that GPER regulates embryonic heart rate by altering maternal estrogen levels and embryonic T3 levels.

Gray platelet syndrome: natural history of a large patient cohort and locus assignment to chromosome 3p.

Gray platelet syndrome (GPS) is an inherited bleeding disorder characterized by macrothrombocytopenia and absence of platelet α-granules resulting in typical gray platelets on peripheral smears. GPS is associated with a bleeding tendency, myelofibrosis, and splenomegaly. Reports on GPS are limited to case presentations. The causative gene and underlying pathophysiology are largely unknown. We present the results of molecular genetic analysis of 116 individuals including 25 GPS patients from 14 independent families as well as novel clinical data on the natural history of the disease. The mode of inheritance was autosomal recessive (AR) in 11 and indeterminate in 3 families. Using genome-wide linkage analysis, we mapped the AR-GPS gene to a 9.4-Mb interval on 3p21.1-3p22.1, containing 197 protein-coding genes. Sequencing of 1423 (69%) of the 2075 exons in the interval did not identify the GPS gene. Long-term follow-up data demonstrated the progressive nature of the thrombocytopenia and myelofibrosis of GPS resulting in fatal hemorrhages in some patients. We identified high serum vitamin B(12) as a consistent, novel finding in GPS. Chromosome 3p21.1-3p22.1 has not been previously linked to a platelet disorder; identification of the GPS gene will likely lead to the discovery of novel components of platelet organelle biogenesis. This study is registered at www.clinicaltrials.gov as NCT00069680 and NCT00369421.

Correlation of kidney function, volume and imaging findings, and PKHD1 mutations in 73 patients with autosomal recessive polycystic kidney disease.

BACKGROUND AND OBJECTIVES: Renal function and imaging findings have not been comprehensively and prospectively characterized in a broad age range of patients with molecularly confirmed autosomal recessive polycystic kidney disease (ARPKD). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Ninety potential ARPKD patients were examined at the National Institutes of Health Clinical Center. Seventy-three fulfilled clinical diagnostic criteria, had at least one PKHD1 mutation, and were prospectively evaluated using magnetic resonance imaging (MRI), high-resolution ultrasonography (HR-USG), and measures of glomerular and tubular function. RESULTS: Among 31 perinatally symptomatic patients, 25% required renal replacement therapy by age 11 years; among 42 patients who became symptomatic beyond 1 month (nonperinatal), 25% required kidney transplantation by age 32 years. Creatinine clearance (CrCl) for nonperinatal patients (103 +/- 54 ml/min/1.73 m(2)) was greater than for perinatal patients (62 +/- 33) (P = 0.002). Corticomedullary involvement on HR-USG was associated with a significantly worse mean CrCl (61 +/- 32) in comparison with medullary involvement only (131 +/- 46) (P < 0.0001). Among children with enlarged kidneys, volume correlated inversely with function, although with wide variability. Severity of PKHD1 mutations did not determine kidney size or function. In 35% of patients with medullary-only abnormalities, standard ultrasound was normal and the pathology was detectable with HR-USG. CONCLUSIONS: In ARPKD, perinatal presentation and corticomedullary involvement are associated with faster progression of kidney disease. Mild ARPKD is best detected by HR-USG. Considerable variability occurs that is not explained by the type of PKHD1 mutation.

PKHD1 sequence variations in 78 children and adults with autosomal recessive polycystic kidney disease and congenital hepatic fibrosis.

PKHD1, the gene mutated in autosomal recessive polycystic kidney disease (ARPKD)/congenital hepatic fibrosis (CHF), is an exceptionally large and complicated gene that consists of 86 exons and has a number of alternatively spliced transcripts. Its longest open reading frame contains 67 exons that encode a 4074 amino acid protein called fibrocystin or polyductin. The phenotypes caused by PKHD1 mutations are similarly complicated, ranging from perinatally-fatal PKD to CHF presenting in adulthood with mild kidney disease. To date, more than 300 mutations have been described throughout PKHD1. Most reported cohorts include a large proportion of perinatal-onset ARPKD patients; mutation detection rates vary between 42% and 87%. Here we report PKHD1 sequencing results on 78 ARPKD/CHF patients from 68 families. Differing from previous investigations, our study required survival beyond 6 months and included many adults with a CHF-predominant phenotype. We identified 77 PKHD1 variants (41 novel) including 19 truncating, 55 missense, 2 splice, and 1 small in-frame deletion. Using computer-based prediction tools (GVGD, PolyPhen, SNAP), we achieved a mutation detection rate of 79%, ranging from 63% in the CHF-predominant group to 82% in the remaining families. Prediction of the pathogenicity of missense variants will remain challenging until a functional assay is available. In the meantime, use of PKHD1 sequencing data for clinical decisions requires caution, especially when only novel or rare missense variants are identified.