The distribution and characterization of HNK-1 antigens in the developing avian heart

The heart originates from splanchnic mesoderm and to a lesser extent from neural crest cells. The HNK-1 monoclonal antibody is a marker for early migrating neural crest cells, but reacts also with structures which are not derived from the neural crest. We investigated whether heart structures are HNK-1 positive before neural crest cells colonize these target tissues. To that end, we determined the HNK-1 antigen expression in the developing avian heart on immunohistochemical sections and on Western blots. The HNK-1 immunoreactivity in the developing chick heart is compared with data from literature cm the localization of neural crest cells in chick/quail chimeras. Structures with neural crest contribution, including parts of the early outflow tract and the related endocardial cushions, the primordia of the semilunar valve leaflets and the aorticopulmonary septum were HNK-1 positive. Furthermore, other structures were HNK-1 positive, such as the atrioventricular cushions, the wall of the sinus venosus at stage HH 15 through 21, parts of the endocardium at E3, parts of the myocardium at E6, and the extracellular matrix in the myocardial base of the semilunar valves at E14. HNK-1 expression was particularly observed in morphologically dynamic regions such as the developing valves, the outflow tract cushion, the developing conduction system and the autonomie nervous system of the heart. We observed that atrioventricular endocardial cushions are HNK-1 positive. We conclude that: a HNK-1 immunoreactivity does not always coincide with the presence of neural crest cells or their derivatives; (2) the outflow tract cushions and atrioventricular endocardial cushions are HNK-1 positive before neural crest cells are expected (stage HH 19) to enter the endocardial cushions of the outflow tract; (3) the observed spatio-temporal HNK-1 patterns observed in the developing heart correspond with various HNK-1 antigens. Apart from a constant pattern of HNK-1 antigens during development, stage-dependent HNK-1 antigens were also found

The transcription factor GATA6 is essential for branching morphogenesis and epithelial cell differentiation during fetal pulmonary development

Recent loss-of-function studies in mice show that the transcription factor GATA6 is important for visceral endoderm differentiation. It is also expressed in early bronchial epithelium and the observation that this tissue does not receive any contribution from Gata6 double mutant embryonic stem (ES) cells in chimeric mice suggests that GATA6 may play a crucial role in lung development. The aim of this study was to determine the role of GATA6 in fetal pulmonary development. We show that Gata6 mRNA is expressed predominantly in the developing pulmonary endoderm and epithelium, but at E15.5 also in the pulmonary mesenchyme. Blocking or depleting GATA6 function results in diminished branching morphogenesis both in vitro and in vivo. TTF1 expression is unaltered in chimeric lungs whereas SPC and CC10 expression are attenuated in abnormally branched areas of chimeric lungs. Chimeras generated in a ROSA26 background show that endodermal cells in these abnormally branched areas are derived from Gata6 mutant ES cells, implicating that the defect is intrinsic to the endoderm. Taken together, these data demonstrate that GATA6 is not essential for endoderm specification, but is required for normal branching morphogenesis and late epithelial cell differentiation

Variants in CHEK2 other than 1100delC do not make a major contribution to breast cancer susceptibility

We recently reported that a sequence variant in the cell-cycle-checkpoint kinase CHEK2 (CHEK2 1100delC) is a low-penetrance breast cancer-susceptibility allele in noncarriers of BRCA1 or BRCA2 mutations. To investigate whether other CHEK2 variants confer susceptibility to breast cancer, we screened the full CHEK2 coding sequence in BRCA1/2-negative breast cancer cases from 89 pedigrees with three or more cases of breast cancer. We identified one novel germline variant, R117G, in two separate families. To evaluate the possible association of R117G and two germline variants repo

Positional mapping of loci in the DiGeorge critical region at chromosome 22q11 using a new marker (D22S183)

The majority of patients with DiGeorge syndrome (DGS) and velo-cardio-facial syndrome (VCFS) and a minority of patients with non-syndromic conotruncal heart defects are hemizygous for a region of chromosome 22q11. The chromosomal region that is commonly deleted is larger than 2 Mb. It has not been possible to narrow the smallest region of overlap (SRO) of the deletions to less than ca 500 kb, which suggests that DGS/VCFS might be a contiguous gene syndrome. The saturation cloning of the SRO is being carried out, and one gene (TUPLE1) has been identified. By using a cosmid probe (M51) and fluorescence in situ hybridization, we show here that the anonymous DNA marker locus D22S183 is within the SRO, between TUPLE1 and D22S75 (probe N25). A second locus with weak homology to D22S183, recognized by cosmid M56, lies immediately outside the common SRO of the DGS and VCFS deletions, but inside the SRO of the DGS deletions. D22S183 sequences are strongly conserved in primates and weaker hybridizing signals are found in DNA of other mammalian species; no transcripts are however detected in polyA+ RNA from various adult human organs. Probe M51 allows fast reliable screening for 22q11 deletions using fluorescence in situ hybridization. A deletion was found in 11 out of 12 DGS patients and in 3 out of 7 VCFS patients. Two patients inherited the deletion from a parent with mild (atypical) symptoms

Ablation of various regions within the avian vagal neural crest has differential effects on ganglion formation in the fore‐, mid‐ and hindgut

The vagal neural crest adjacent to the first seven somites gives rise to both ganglionic and ectomesenchymal derivatives. Ganglionic derivatives are the neurons and supportive cells of the enteric nervous system (ENS), cardiac, and dorsal root ganglia. Ectomesenchymal derivatives are cells in the cardiac outflow tract and the mesenchymal components of thymus and parathyroids. Ectomesenchymal derivatives are formed by a segment of the vagal neural crest, from the level of the otic vesicle down to the caudal boundary of the third somite, called the cardiac neural crest. We performed neural crest ablations to study regional differences within the avian vagal neural crest with regard to the formation of the ENS. Ablation of the entire vagal neural crest from the mid‐otic vesicle down to the seventh somite plus the nodose placode resulted in the absence of ganglia in the midgut (jejunum and ileum) and hindgut (colon). The foregut (esophagus, proventriculus, gizzard, and duodenum) was normally innervated. After ablation of the vagal neural crest adjacent to somites 3–5, ganglia were absent in the hindgut. Ablations of vagal neural crest not including this segment had no effect on the formation of the ENS. We surmise that the innervation of the hindgut in vivo depends specifically on the neural crest adjacent to somites 3–5, whereas innervation of the midgut can be accomplished by all segments within the vagal neural crest. The foregut can also be innervated by a source outside the vagal neural crest. To study intrinsic differences between various vagal neural crest segments regarding ENS formation, we performed chorioallantoic membrane cocultures of segments of quail vagal neural anlage and E4 chicken hindgut. We found that all vagal neural crest segments were able to give rise to enteric ganglia in the hindgut. When the neural crest of somites 6 and 7 was included in the segment, we also found melanocytes in the hindgut, suggesting that this segment is more related to trunk neural crest. Furthermore, we found that the vagal neural anlage from older embryos (>18 somites) showed an increased potential to form enteric ganglia. This suggests that vagal neural crest cells that have been in prolonged contact with the neural tube in vivo, because of either late emigration or delayed migration, have an increased probability to form enteric ganglia

The murine homologue of HIRA, a DiGeorge syndrome candidate gene, is expressed in embryonic structures affected in human CATCH22 patients

A wide spectrum of birth defects is caused by deletions of the DiGeorge syndrome chromosomal region at 22q11. Characteristic features include cranio-facial, cardiac and thymic malformations, which are thought to arise form disturbances in the interactions between hindbrain neural crest cells and the endoderm of the pharyngeal p

Microscopic localisation of protoporphyrin IX in normal mouse skin after topical application of 5-aminolevulinic acid or methyl 5-aminolevulinate

Light fractionation does not enhance the response to photodynamic therapy (PDT) after topical methyl-aminolevulinate (MAL) application, whereas it is after topical 5-aminolevulinic acid (ALA). The differences in biophysical and biochemical characteristics between MAL and ALA may result in differences in localisation that cause the differences in response to PDT. We therefore investigated the spatial distribution of protoporphyrin IX (PpIX) fluorescence in normal mouse skin using fluorescence microscopy and correlated that with the PDT response histologically observed at 2.5, 24 and 48 h after PDT. As expected high fluorescence intensities were observed in the epidermis and pilosebaceous units and no fluorescence in the cutaneous musculature after both MAL and ALA application. The dermis showed localised fluorescence that corresponds to the cytoplasma of dermal cells like fibroblast and mash cells. Spectral analysis showed a typical PpIX fluorescence spectrum confirming that it is PpIX fluorescence. There was no clear difference in the depth and spatial distribution of PpIX fluorescence between the two precursors in these normal mouse skin samples. This result combined with the conclusion of Moan et al. that ALA but not MAL is systemically distributed after topical application on mouse skin [Moan et al., Pharmacology of protoporphyrin IX in nude mice after application of ALA and ALA esters, Int. J. Cancer 103 (2003) 132-135] suggests that endothelial cells are involved in increased response of tissues to ALA-PDT using light fractionation. Histological analysis 2.5 h after PDT showed more edema formation after ALA-PDT compared to MAL-PDT that was not accompanied by a difference in the inflammatory response. This suggests that endothelial cells respond differently to ALA and MAL-PDT. Further investigation is needed to determine the role of endothelial cells in ALA-PDT and the underlying mechanism behind the increased effectiveness of light fractionation using a dark interval of 2 h found after ALA but not after MAL-PDT. (c) 2008 Elsevier B.V. All rights reserve

Positional mapping of loci in the DiGeorge critical region at chromosome 22q11 using a new marker (D22S183)

The majority of patients with DiGeorge syndrome (DGS) and velo-cardio-facial syndrome (VCFS) and a minority of patients with non-syndromic conotruncal heart defects are hemizygous for a region of chromosome 22q11. The chromosomal region that is commonly deleted is larger than 2 Mb. It has not been possible to narrow the smallest region of overlap (SRO) of the deletions to less than ca 500 kb, which suggests that DGS/VCFS might be a contiguous gene syndrome. The saturation cloning of the SRO is being carried out, and one gene (TUPLE1) has been identified. By using a cosmid probe (M51) and fluorescence in situ hybridization, we show here that the anonymous DNA marker locus D22S183 is within the SRO, between TUPLE1 and D22S75 (probe N25). A second locus with weak homology to D22S183, recognized by cosmid M56, lies immediately outside the common SRO of the DGS and VCFS deletions, but inside the SRO of the DGS deletions. D22S183 sequences are strongly conserved in primates and weaker hybridizing signals are found in DNA of other mammalian species; no transcripts are however detected in polyA+ RNA from various adult human organs. Probe M51 allows fast reliable screening for 22q11 deletions using fluorescence in situ hybridization. A deletion was found in 11 out of 12 DGS patients and in 3 out of 7 VCFS patients. Two patients inherited the deletion from a parent with mild (atypical) symptoms.</p

Homozygous nonsense mutations in KIAA1279 are associated with malformations of the central and enteric nervous systems

We identified, by homozygosity mapping, a novel locus on 10q21.3-q22.1 for Goldberg-Shprintzen syndrome (GOSHS) in a consanguineous Moroccan family. Phenotypic features of GOSHS in this inbred family included microcephaly and mental retardation, which are both central nervous system defects, as well as Hirschsprung disease, an enteric nervous system defect. Furthermore, since bilateral generalized polymicogyria was diagnosed in all patients in this family, this feature might also be considered a key feature of the syndrome. We demonstrate that homozygous nonsense mutations in KIAA1279 at 10q22.1, encoding a protein with two tetratrico peptide repeats, underlie this syndromic form of Hirschsprung disease and generalized polymicrogyria, establishing the importance of KIAA1279 in both enteric and central nervous system development