Impairment of gastric acid secretion and increase of embryonic lethality in Foxq1-deficient mice

The mouse Foxq1 gene, also known as Hfh1, encodes a winged helix/forkhead transcription factor. In adult mice, Foxq1 is highly expressed in kidney and stomach. Here, we report that Foxq1 is expressed during prenatal and postnatal stomach development and the transcripts are restricted to acid secreting parietal cells. Mice homozygous for a deletion of the Foxq1 locus on a 129/Sv x C57BL/6J hybrid genetic background display variable phenotypes consistent with requirement of the gene during embryogenesis. Approximately 50% of Foxq1-/- embryos die in utero. Surviving homozygous mutants are normal and fertile, and have a silky shiny coat. Although the parietal cell development is not affected in the absence of Foxq1, there is a lack of gastric acid secretion in response to various secretagogue stimuli. Ultrastructural analysis suggests that the gastric acid secretion defect in Foxq1-deficient mice might be due to impairment in the fusion of cytoplasmic tubulovesicles to the apical membrane of secretory canaliculi.

Uniform vascular-endothelial-cell-specific gene expression in both embryonic and adult transgenic mice

TIE2 is a vascular endothelial-specific receptor tyrosine kinase essential for the regulation of vascular network formation and remodeling. Previously, we have shown that the 1.2-kb 5' flanking region of the TIE2 promoter is capable of directing beta-galactosidase reporter gene expression specifically into a subset of endothelial cells (ECs) of transgenic mouse embryos. However, transgene activity was restricted to early embryonic stages and not detectable in adult mice. Herein we describe the identification and characterization of an autonomous endothelial-specific enhancer in the first intron of the mouse TIE2 gene. Furthermore, combination of the TIE2 promoter with an intron fragment containing this enhancer allows it to target reporter gene expression specifically and uniformly to virtually all vascular ECs throughout embryogenesis and adulthood. To our knowledge, this is the first time that an in vivo expression system has been assembled by which heterologous genes can be targeted exclusively to the ECs of the entire vasculature. This should be a valuable tool to address the function of genes during physiological and pathological processes of vascular ECs in vivo. Furthermore, we were able to identify a short region critical for enhancer function in vivo that contains putative binding sites for Ets-like transcription factors. This should, therefore, allow us to determine the molecular mechanisms underlying the vascular-EC-specific expression of the TIE2 gene.

Lethal mitochondrial cardiomyopathy in a hypomorphic Med30 mouse mutant is ameliorated by ketogenic diet

Deficiencies of subunits of the transcriptional regulatory complex Mediator generally result in embryonic lethality, precluding study of its physiological function. Here we describe a missense mutation in Med30 causing progressive cardiomyopathy in homozygous mice that, although viable during lactation, show precipitous lethality 2-3 wk after weaning. Expression profiling reveals pleiotropic changes in transcription of cardiac genes required for oxidative phosphorylation and mitochondrial integrity. Weaning mice to a ketogenic diet extends viability to 8.5 wk. Thus, we establish a mechanistic connection between Mediator and induction of a metabolic program for oxidative phosphorylation and fatty acid oxidation, in which lethal cardiomyopathy is mitigated by dietary intervention.

Angiopoietin-1 induces sprouting angiogenesis in vitro

Sprouting of new capillaries from pre-existing blood vessels is a hallmark of angiogenesis during embryonic development and solid tumor growth [1]. In addition to the vascular endothelial growth factor (VEGF) and its receptors, the Tie receptors and their newly identified ligands, the angiopoietins, have been implicated in the control of blood vessel formation [2,3]. Although 'knockouts' of the gene encoding the Tie2 receptor, or its activating ligand angiopoietin-1 (Ang1), result in embryonic lethality in mice due to an absence of remodeling and sprouting of blood vessels [4,5], biological activity in vitro has not yet been described for this receptor-ligand system. In an assay in which a monolayer of endothelial cells were cultured on microcarrier beads and embedded in three-dimensional fibrin gels, recombinant Ang1 (0.5-10 nM) induced the formation of capillary sprouts in a dose-dependent manner that was completely inhibited by soluble Tie2 receptor extracellular domains. In contrast with VEGF, which also induced sprouting of capillaries, Ang1 was only very weakly mitogenic for endothelial cells. Suboptimal concentrations of VEGF and Ang1 acted synergistically to induce sprout formation. Thus, the biological activity of Ang1 in vitro is consistent with the specific phenotype of mice deficient in Tie2 or Ang1. The data suggest that, like in other developmental systems, blood vessel formation requires a hierarchy of master-control genes in which VEGF and angiopoietins, along with their receptors, are amongst the most important regulators.

VE-PTP controls blood vessel development by balancing Tie-2 activity

Vascular endothelial protein tyrosine phosphatase (VE-PTP) is an endothelial-specific receptor-type tyrosine phosphatase that associates with Tie-2 and VE-cadherin. VE-PTP gene disruption leads to embryonic lethality, vascular remodeling defects, and enlargement of vascular structures in extraembryonic tissues. We show here that antibodies against the extracellular part of VE-PTP mimic the effects of VE-PTP gene disruption exemplified by vessel enlargement in allantois explants. These effects require the presence of the angiopoietin receptor Tie-2. Analyzing the mechanism we found that anti-VE-PTP antibodies trigger endocytosis and selectively affect Tie-2-associated, but not VE-cadherin-associated VE-PTP. Dissociation of VE-PTP triggers the activation of Tie-2, leading to enhanced endothelial cell proliferation and enlargement of vascular structures through activation of Erk1/2. Importantly, the antibody effect on vessel enlargement is also observed in newborn mice. We conclude that VE-PTP is required to balance Tie-2 activity and endothelial cell proliferation, thereby controlling blood vessel development and vessel size.

Diversity and function of mutations in p450 oxidoreductase in patients with Antley-Bixler syndrome and disordered steroidogenesis

P450 oxidoreductase (POR) is the obligatory flavoprotein intermediate that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to all microsomal cytochrome P450 enzymes. Although mouse Por gene ablation causes embryonic lethality, POR missense mutations cause disordered steroidogenesis, ambiguous genitalia, and Antley-Bixler syndrome (ABS), which has also been attributed to fibroblast growth factor receptor 2 (FGFR2) mutations. We sequenced the POR gene and FGFR2 exons 8 and 10 in 32 individuals with ABS and/or hormonal findings that suggested POR deficiency. POR and FGFR2 mutations segregated completely. Fifteen patients carried POR mutations on both alleles, 4 carried mutations on only one allele, 10 carried FGFR2 or FGFR3 mutations, and 3 patients carried no mutations. The 34 affected POR alleles included 10 with A287P (all from whites) and 7 with R457H (four Japanese, one African, two whites); 17 of the 34 alleles carried 16 "private" mutations, including 9 missense and 7 frameshift mutations. These 11 missense mutations, plus 10 others found in databases or reported elsewhere, were recreated by site-directed mutagenesis and were assessed by four assays: reduction of cytochrome c, oxidation of NADPH, support of 17alpha-hydroxylase activity, and support of 17,20 lyase using human P450c17. Assays that were based on cytochrome c, which is not a physiologic substrate for POR, correlated poorly with clinical phenotype, but assays that were based on POR's support of catalysis by P450c17--the enzyme most closely associated with the hormonal phenotype--provided an excellent genotype/phenotype correlation. Our large survey of patients with ABS shows that individuals with an ABS-like phenotype and normal steroidogenesis have FGFR mutations, whereas those with ambiguous genitalia and disordered steroidogenesis should be recognized as having a distinct new disease: POR deficiency.

The channel-activating protease CAP1/Prss8 is required for placental labyrinth maturation

The serine protease CAP1/Prss8 is crucial for skin barrier function, lung alveolar fluid clearance and has been unveiled as diagnostic marker for specific cancer types. Here, we show that a constitutive knockout of CAP1/Prss8 leads to embryonic lethality. These embryos presented no specific defects, but it is during this period, and in particular at E13.5, that wildtype placentas show an increased expression of CAP1/Prss8, thus suggesting a placental defect in the knockout situation. The placentas of knockout embryos exhibited significantly reduced vascular development and incomplete cellular maturation. In contrary, epiblast-specific deletion of CAP1/Prss8 allowed development until birth. These CAP1/Prss8-deficient newborns presented abnormal epidermis, and died soon after birth due to impaired skin function. We thus conclude that a late placental insufficiency might be the primary cause of embryonic lethality in CAP1/Prss8 knockouts. This study highlights a novel and crucial role for CAP1/Prss8 in placental development and function.

Conditional and inducible transgene expression in endothelial and hematopoietic cells using Cre/loxP and tetracycline-off systems

In the present study, the tetracycline-off and Cre/loxP systems were combined to gain temporal and spatial control of transgene expression. Mice were generated that carried three transgenes: Tie2-tTA, tet-O-Cre and either the ZEG or ZAP reporter. Tie2-tTA directs expression of tetracycline-controlled transactivator (tTA) in endothelial and hematopoietic cells under the control of the Tie2 promoter. Tet-O-Cre produces Cre recombinase from a minimal promoter containing the tet-operator (tetO). ZEG or ZAP contains a strong promoter and a loxP-flanked stop sequence, followed by an enhanced green fluorescence protein (EGFP) or human placental alkaline phosphatase (hPLAP) reporter. In the presence of tetracycline, the tTA transactivator produced by Tie-2-tTA is disabled and Cre is not expressed. In the absence of tetracycline, the tTA binds tet-O-Cre to drive the expression of Cre, which recombines the loxP sites of the ZEG or ZAP transgene and results in reporter gene expression. In the present study, the expression of the ZEG or ZAP reporter genes in embryos and adult animals with and without tetracycline treatment was examined. In the presence of tetracycline, no reporter gene expression was observed. When tetracycline was withdrawn, Cre excision was activated and the reporter genes were detected in endothelial and hematopoietic cells. These results demonstrate that this system may be used to bypass embryonic lethality and access adult phenotypes.

Postnatal Notch1 activation induces T‑cell malignancy in conditional and inducible mouse models

The Notch1 signaling pathway is essential for hematopoietic development. However, the effects of postnatal activation of Notch1 signaling on hematopoietic system is not yet fully understood. We previously generated ZEG‑IC‑Notch1 transgenic mice that have a floxed β‑geo/stop signal between a CMV promoter and intracellular domain of Notch1 (IC‑Notch1). Constitutively active IC‑Notch1 is silent until the introduction of Cre recombinase. In this study, endothelial/hematopoietic specific expression of IC‑Notch1 in double transgenic ZEG‑IC‑Notch1/Tie2‑Cre embryos induced embryonic lethality at E9.5 with defects in vascular system but not in hematopoietic system. Inducible IC‑Notch1 expression in adult mice was achieved by using tetracycline regulated Cre system. The ZEG‑IC‑Notch1/Tie2‑tTA/tet‑O‑Cre triple transgenic mice survived embryonic development when maintained on tetracycline. Post‑natal withdrawal of tetracycline induced expression of IC‑Notch1 transgene in hematopoietic cells of adult mice. The triple transgenic mice displayed extensive T‑cell infiltration in multiple organs and T‑cell malignancy of lymph nodes. In addition, the protein levels of p53 and alternative reading frame (ARF) were decreased in lymphoma‑like neoplasms from the triple transgenic mice while their mRNA expression remained unchanged, suggesting that IC‑Notch1 might repress ARF‑p53 pathway by a post‑transcriptional mechanism. This study demonstrated that activation of constitutive Notch1 signaling after embryonic development alters adult hematopoiesis and induces T‑cell malignancy.

A bHLH Complex Controls Embryonic Vascular Tissue Establishment and Indeterminate Growth in Arabidopsis

Plants have a remarkable potential for sustained (indeterminate) postembryonic growth. Following their specification in the early embryo, tissue-specific precursor cells first establish tissues and later maintain them postembryonically. The mechanisms underlying these processes are largely unknown. Here we define local control of oriented, periclinal cell division as the mechanism underlying both the establishment and maintenance of vascular tissue. We identify an auxin-regulated basic helix-loop-helix (bHLH) transcription factor dimer as a critical regulator of vascular development. Due to a loss of periclinal divisions, vascular tissue gradually disappears in bHLH-deficient mutants; conversely, ectopic expression is sufficient for triggering periclinal divisions. We show that this dimer operates independently of tissue identity but is restricted to a small vascular domain by integrating overlapping transcription patterns of the interacting bHLH proteins. Our work reveals a common mechanism for tissue establishment and indeterminate vascular development and provides a conceptual framework for developmental control of local cell divisions.

Modeling Framework for the Establishment of the Apical-Basal Embryonic Axis in Plants

The apical-basal axis of the early plant embryo determines the body plan of the adult organism. To establish a polarized embryonic axis, plants evolved a unique mechanism that involves directional, cell-to-cell transport of the growth regulator auxin. Auxin transport relies on PIN auxin transporters 1], whose polar subcellular localization determines the flow directionality. PIN-mediated auxin transport mediates the spatial and temporal activity of the auxin response machinery 2-7] that contributes to embryo patterning processes, including establishment of the apical (shoot) and basal (root) embryo poles 8]. However, little is known of upstream mechanisms guiding the (re)polarization of auxin fluxes during embryogenesis 9]. Here, we developed a model of plant embryogenesis that correctly generates emergent cell polarities and auxin-mediated sequential initiation of apical-basal axis of plant embryo. The model relies on two precisely localized auxin sources and a feedback between auxin and the polar, subcellular PIN transporter localization. Simulations reproduced PIN polarity and auxin distribution, as well as previously unknown polarization events during early embryogenesis. The spectrum of validated model predictions suggests that our model corresponds to a minimal mechanistic framework for initiation and orientation of the apical-basal axis to guide both embryonic and postembryonic plant development.

The Caenorhabditis elegans histone hairpin-binding protein is required for core histone gene expression and is essential for embryonic and postembryonic cell division.

As in all metazoans, the replication-dependent histone genes of Caenorhabditis elegans lack introns and contain a short hairpin structure in the 3' untranslated region. This hairpin structure is a key element for post-transcriptional regulation of histone gene expression and determines mRNA 3' end formation, nuclear export, translation and mRNA decay. All these steps contribute to the S-phase-specific expression of the replication-dependent histone genes. The hairpin structure is the binding site for histone hairpin-binding protein that is required for hairpin-dependent regulation. Here, we demonstrate that the C. elegans histone hairpin-binding protein gene is transcribed in dividing cells during embryogenesis and postembryonic development. Depletion of histone hairpin-binding protein (HBP) function in early embryos using RNA-mediated interference leads to an embryonic-lethal phenotype brought about by defects in chromosome condensation. A similar phenotype was obtained by depleting histones H3 and H4 in early embryos, indicating that the defects in hairpin-binding protein-depleted embryos are caused by reduced histone biosynthesis. We have confirmed this by showing that HBP depletion reduces histone gene expression. Depletion of HBP during postembryonic development also results in defects in cell division during late larval development. In addition, we have observed defects in the specification of vulval cell fate in animals depleted for histone H3 and H4, which indicates that histone proteins are required for cell fate regulation during vulval development.