Hes1 Is Expressed in the Second Heart Field and Is Required for Outflow Tract Development

Background: Rapid growth of the embryonic heart occurs by addition of progenitor cells of the second heart field to the poles of the elongating heart tube. Failure or perturbation of this process leads to congenital heart defects. In order to provide further insight into second heart field development we characterized the insertion site of a transgene expressed in the second heart field and outflow tract as the result of an integration site position effect. Results: Here we show that the integration site of the A17-Myf5-nlacZ-T55 transgene lies upstream of Hes1, encoding a basic helix-loop-helix containing transcriptional repressor required for the maintenance of diverse progenitor cell populations during embryonic development. Transgene expression in a subset of Hes1 expression sites, including the CNS, pharyngeal epithelia, pericardium, limb bud and lung endoderm suggests that Hes1 is the endogenous target of regulatory elements trapped by the transgene. Hes1 is expressed in pharyngeal endoderm and mesoderm including the second heart field. Analysis of Hes1 mutant hearts at embryonic day 15.5 reveals outflow tract alignment defects including ventricular septal defects and overriding aorta. At earlier developmental stages, Hes1 mutant embryos display defects in second heart field proliferation, a reduction in cardiac neural crest cells and failure to completely extend the outflow tract. Conclusions: Hes1 is expressed in cardiac progenitor cells in the early embryo and is required for development of the arteria

Wnt4 is required for proper male as well as female sexual development

AbstractGenes previously implicated in mammalian sexual development have either a male- or female-specific role. The signaling molecule WNT4 has been shown to be important in female sexual development. Lack of Wnt4 gives rise to masculinization of the XX gonad and we showed previously that the role of WNT4 was to inhibit endothelial and steroidogenic cell migration into the developing ovary. Here we show that Wnt4 also has a function in the male gonad. We find that Sertoli cell differentiation is compromised in Wnt4 mutant testes and that this defect occurs downstream of the testis-determining gene Sry but upstream of Sox9 and Dhh, two early Sertoli cell markers. Genetic analysis shows that this phenotype is primarily due to the action of WNT4 within the early genital ridge. Analysis of different markers identifies the most striking difference in the genital ridge at early stages of its development between wild-type and Wnt4 mutant embryos to be a significant increase of steroidogenic cells in the Wnt4 −/− gonad. These results identify WNT4 as a new factor involved in the mammalian testis determination pathway and show that genes can have a specific but distinct role in both male and female gonad development

Kv1.1 Channels Act as Mechanical Brake in the Senses of Touch and Pain

SummaryMolecular determinants of threshold sensitivity of mammalian mechanoreceptors are unknown. Here, we identify a mechanosensitive (MS) K+ current (IKmech) that governs mechanical threshold and adaptation of distinct populations of mechanoreceptors. Toxin profiling and transgenic mouse studies indicate that IKmech is carried by Kv1.1-Kv1.2 heteromers. Mechanosensitivity is attributed to Kv1.1 subunits, through facilitation of voltage-dependent open probability. IKmech is expressed in high-threshold C-mechano-nociceptors (C-HTMRs) and Aβ-mechanoreceptors, but not in low-threshold C-mechanoreceptors. IKmech opposes depolarization induced by slow/ultraslow MS cation currents in C-HTMRs, thereby shifting mechanical threshold for firing to higher values. However, due to kinetics mismatch with rapidly-adapting MS cation currents, IKmech tunes firing adaptation but not mechanical threshold in Aβ-mechanoreceptors. Expression of Kv1.1 dominant negative or inhibition of Kv1.1/IKmech caused severe mechanical allodynia but not heat hyperalgesia. By balancing the activity of excitatory mechanotransducers, Kv1.1 acts as a mechanosensitive brake that regulates mechanical sensitivity of fibers associated with mechanical perception

Myocardium at the base of the aorta and pulmonary trunk is prefigured in the outflow tract of the heart and in subdomains of the second heart field

AbstractOutflow tract myocardium in the mouse heart is derived from the anterior heart field, a subdomain of the second heart field. We have recently characterized a transgene (y96-Myf5-nlacZ-16), which is expressed in the inferior wall of the outflow tract and then predominantly in myocardium at the base of the pulmonary trunk. Transgene A17-Myf5-nlacZ-T55 is expressed in the developing heart in a complementary pattern to y96-Myf5-nlacZ-16, in the superior wall of the outflow tract at E10.5 and in myocardium at the base of the aorta at E14.5. At E9.5, the two transgenes are transcribed in different subdomains of the anterior heart field. A clonal analysis of cardiomyocytes in the outflow tract, at E10.5 and E14.5, provides insight into the behaviour of myocardial cells and their progenitors. At E14.5, most clones are located at the base of either the pulmonary trunk or the aorta, indicating that these derive from distinct myocardial domains. At E10.5, clones are observed in subdomains of the outflow tract. The distribution of small clones indicates proliferative differences, whereas regionalisation of large clones, that derive from an early myocardial progenitor cell, reflect coherent cell growth in the heart field as well as in the myocardium. Our results suggest that myocardial differences at the base of the great arteries are prefigured in distinct progenitor cell populations in the anterior heart field, with important implications for understanding the etiology of congenital heart defects affecting the arterial pole of the heart

Molecular characterization of the <i>T55</i> transgene integration site.

<p>(A) Fluorescent in situ hybridization to metaphase chromosomes prepared from <i>T55</i> splenocytes showing transgene localization to chromosome 16 B2–B4. (B) Map of the <i>T55</i> integration site (top) and endogenous locus (bottom) showing the structure of the 3′ end of the transgene array (black box, <i>A17 Myf5</i> enhancer; blue box, <i>nlacZ</i> reporter gene) and the position of the inverse PCR primers (red arrowheads); B, <i>Bam</i>HI; Bg, <i>Bgl</i>II. Also shown are the flanking sequence isolated by inverse PCR (red) and the Southern blot probe (grey) used to identify the predicted 5.5 kb <i>BamHI</i> fragment in transgenic DNA in addition to a 3.8 kb wildtype fragment (C). (D) Ensembl map of the genomic region surrounding the integration site (red arrow) on chromosome 16, showing the position of the flanking genes <i>Opa1</i> and <i>Hes1</i> and intermediate gene predictions based on EST alignments.</p

Congenital heart defects in <i>Hes1</i> mutant mice.

<p>(A) E15.5 hearts in ventral wholemount views (left) and cryostat sections at the levels indicated showing a normal configuration of the ascending aorta (ao) and pulmonary trunk (pt) in <i>Hes1</i>^+/− hearts (top) and a dextraposed aorta overriding a ventricular septal defect (arrowhead) in <i>Hes1</i>^−/− hearts (middle (mild) and bottom (severe)). Note that the aorta in the bottom heart symmetrically overrides the ventricular septal defect. (B) Paraffin sections of E18.5 hearts in a frontal plane showing a ventricular septal defect (arrowhead) in a <i>Hes1</i>^−/− (middle and bottom) but not <i>Hes1</i>^+/+ heart (top). lv, left ventricle; rv, right ventricle. Scale bar: (A) 200 µm; (B) 500 µm.</p

<i>Hes1</i> is expressed in the second heart field.

<p>(A) Comparison of transgene and <i>Hes1</i> expression in right lateral views of hearts after X-gal staining and in situ hybridization at E10.5 (left three panels). X-gal positive cells are observed in the distal OFT wall (arrowhead) and pulmonary endoderm (arrow); <i>nlacZ</i> transcripts are observed in pulmonary endoderm but not the OFT. Low-level Hes1 transcript accumulation is observed in the distal OFT (black asterisk). Transverse sections (right two panels) show β-galactosidase and low-level <i>Hes1</i> transcript accumulation in the pericardial region (white asterisks), superior OFT wall (arrow) and mesenchymal cells in the OFT (arrowhead). (B) At E8.5 X-gal and <i>Hes1</i> positive cells are observed in ventral and right views in the pericardial wall (black arrowhead) and pharyngeal mesoderm (white arrowheads) where <i>Fgf10</i> transcripts also accumulate. (C) E8.5 sections at the levels indicated in (B), showing <i>Hes1</i> transcripts in neuroepithelium (ne), endoderm (e) and mesoderm (m) lateral and ventral to the pharynx, including the SHF (arrowheads), compared with <i>Fgf10</i> expression. Note that caudally (level e) <i>Hes1</i> and <i>Fgf10</i> transcripts are observed in endoderm and mesoderm respectively. ht, heart tube. Scale bars: (A, C) 100 µm.</p

Comparison of transgene and integration site gene expression profiles.

<p>(A) E9.5 embryos after X-gal staining (<i>T55</i> embryo, left) or in situ hybridization with a lacZ (<i>T55</i> embryo, middle) or <i>Hes1</i> riboprobe (right). Overlapping expression sites include the ventral pharyngeal region (ph), forelimb (fl), ventral neural tube (cns), segmental intersomitic region (s), midbrain (mb), tail region (t) and nasal ectoderm (ne). Asterisk indicates <i>lacZ</i> riboprobe trapping in the otic vesicle. (B) Expression profiles of <i>Opa1</i> and two predicted genes mapping to the intergenic region at E9.5.</p

Loss of <i>Hes1</i> impairs neural crest development.

<p>(A) In situ hybridization at E9.5 showing decreased <i>Crabp1</i> transcript accumulation in the caudal pharyngeal region of <i>Hes1</i>^−/− compared to <i>Hes1</i>^+/+ embryos (arrowheads). (B) Immunochemistry with anti-AP-2α (red) and anti-β-galactosidase (green) antibodies in a transverse section through the caudal pharynx of an E9.5 embryo carrying the <i>Mlc1v-nlacZ-24</i> transgene. (C) Histograms showing the percentage of β-galactosidase positive nuclei and the percentage of AP-2α positive nuclei in the pharyngeal region of <i>Hes1</i>^+/+ and <i>Hes1</i>^−/− embryos (left) and the numbers of total, β-galactosidase positive and AP-2α positive nuclei (right). Note the decrease in the number of AP-2α positive cells in <i>Hes1</i>^−/− embryos (p<0.001, Student's t-test). (D) In situ hybridization at E11.5 showing decreased <i>PlexinA2</i> transcript accumulation in the OFT of <i>Hes1</i>^−/− compared to <i>Hes1</i>^+/+ hearts (black arrowheads) in wholemount (top) and transverse sections (middle). Histological analysis reveals normal OFT cushion morphology in <i>Hes1</i>^−/− embryos at E11.5 (bottom). Scale bars (B): 50 µm; (D): 200 µm.</p

Impact of loss of <i>Hes1</i> on differentiation and proliferation in the second heart field.

<p>(A) Immunochemistry with anti-MHC (left), anti-α-actinin (right) and anti-β-galactosidase antibodies in transverse sections through the caudal pharyngeal region of an E9.5 embryo carrying the <i>Mlc1v-nlacZ-24</i> transgene. Note the β-galactosidase positive α-actinin and MF20 negative cells in the dorsal pericardial wall (arrowheads). Nuclei are labeled with Hoechst (blue). (B) Immunochemistry with anti-phospho-Histone H3 and anti-β-galactosidase antibodies in paraffin sections of E8.5 <i>Hes1</i>^+/+ and <i>Hes1</i>^−/− embryos carrying the <i>Mlc1v-nlacZ-24</i> transgene. (C) Histogram comparing the percentage of phospho-Histone H3 positive nuclei in pharyngeal endoderm, myocardium and SHF of <i>Hes1</i>^+/+ and <i>Hes1</i>^−/− embryos. A decrease in phospho-Histone H3 positive nuclei is observed in <i>Hes1</i>^−/− SHF and endoderm (p<0.001, Student's t-test). (D) Immunochemistry with an anti-p27^kip1 antibody in paraffin sections at E8.5. Note that p27^kip1 is expanded in the SHF of <i>Hes1</i>^−/− hearts (arrowheads). Nuclei are labeled with Hoechst (blue). (E) Western blot of microdissected heart and ventral pharyngeal regions of <i>Hes1</i>^+/+ and <i>Hes1</i>^−/− embryos showing elevated p27^kip1 protein levels in the absence of Hes1. Scale bars (A): 50 µm; (B): 100 µm; (D) 50 µm.</p