Hearing is normal without connexin30.

Gjb2 and Gjb6, two contiguous genes respectively encoding the gap junction protein connexin26 (Cx26) and connexin 30 (Cx30) display overlapping expression in the inner ear. Both have been linked to the most frequent monogenic hearing impairment, the recessive isolated deafness DFNB1. Although there is robust evidence for the direct involvement of Cx26 in cochlear functions, the contribution of Cx30 is unclear since deletion of Cx30 strongly downregulates Cx26 both in human and in mouse. Thus, it is imperative that any role of Cx30 in audition be clearly evaluated. Here, we developed a new Cx30 knock-out mouse model (Cx30(Δ/Δ)) in which half of Cx26 expression was preserved. Our results show that Cx30 and Cx26 coordinated expression is dependent on the spacing of their surrounding chromosomic region, and that Cx30(Δ/Δ) mutants display normal hearing. Thus, in deaf patients with GJB6 deletion as well as in the previous Cx30 knock-out mouse model, defective Cx26 expression is the likely cause of deafness, and in contrast to current opinion, Cx30 is dispensable for cochlear functions.

Depressed levels of prostaglandin F2α in mice lacking Akr1b7 increase basal adiposity and predispose to diet-induced obesity.

Negative regulators of white adipose tissue (WAT) expansion are poorly documented in vivo. Prostaglandin F(2α) (PGF(2α)) is a potent antiadipogenic factor in cultured preadipocytes, but evidence for its involvement in physiological context is lacking. We previously reported that Akr1b7, an aldo-keto reductase enriched in adipose stromal vascular fraction but absent from mature adipocytes, has antiadipogenic properties possibly supported by PGF(2α) synthase activity. To test whether lack of Akr1b7 could influence WAT homeostasis in vivo, we generated Akr1b7(-/-) mice in 129/Sv background. Akr1b7(-/-) mice displayed excessive basal adiposity resulting from adipocyte hyperplasia/hypertrophy and exhibited greater sensitivity to diet-induced obesity. Following adipose enlargement and irrespective of the diet, they developed liver steatosis and progressive insulin resistance. Akr1b7 loss was associated with decreased PGF(2α) WAT contents. Cloprostenol (PGF(2α) agonist) administration to Akr1b7(-/-) mice normalized WAT expansion by affecting both de novo adipocyte differentiation and size. Treatment of 3T3-L1 adipocytes and Akr1b7(-/-) mice with cloprostenol suggested that decreased adipocyte size resulted from inhibition of lipogenic gene expression. Hence, Akr1b7 is a major regulator of WAT development through at least two PGF(2α)-dependent mechanisms: inhibition of adipogenesis and lipogenesis. These findings provide molecular rationale to explore the status of aldo-keto reductases in dysregulations of adipose tissue homeostasis.

The AMPKγ1 subunit plays an essential role in erythrocyte membrane elasticity, and its genetic inactivation induces splenomegaly and anemia.

AMP-activated protein kinase (AMPK) is an αßγ heterotrimer conserved throughout evolution and important for energy sensing in all eukaryote cells. AMPK controls metabolism and various cellular events in response to both hormones and changes in cellular energy status. The γ subunit senses intracellular energy status through the competitive binding of AMP and ATP. We show here that targeted disruption of the mouse AMPKγ1 gene (Prkag1) causes regenerative hemolytic anemia by increasing the sequestration of abnormal erythrocytes. Prkag1(-/-) mice displayed splenomegaly and iron accumulation due to compensatory splenic erythropoiesis and erythrophagocytosis. Moreover, AMPKγ1-deficient erythrocytes were highly resistant to osmotic hemolysis and poorly deformable in response to increasing shear stress, consistent with greater membrane rigidity. No change in cytoskeletal protein composition was observed; however, the phosphorylation level of adducin, a protein promoting the binding of spectrin to actin, was higher in AMPKγ1-deficient erythrocytes. Together, these results demonstrate that AMPKγ1 subunit is required for the maintenance of erythrocyte membrane elasticity.

Sumoylation by Ubc9 regulates the stem cell compartment and structure and function of the intestinal epithelium in mice.

BACKGROUND & AIMS: Small ubiquitin-like modifiers (SUMOs) are attached to other proteins to regulate their function (sumoylation). We investigated the role of Ubc9, which covalently attaches SUMOs to proteins, in the gastrointestinal tract of adult mice. METHODS: We investigated the effects of decreased sumoylation in adult mammals by generating mice with an inducible knockout (by injection of 4-hydroxytamoxifen) of the E2 enzyme Ubc9 (Ubc9fl/-/ROSA26-CreERT2 mice). We analyzed the phenotypes using a range of histologic techniques. RESULTS: Loss of Ubc9 from adult mice primarily affected the small intestine. Ubc9fl/-/ROSA26-CreERT2 mice died within 6 days of 4-hydroxytamoxifen injection, losing 20% or less of their body weight and developing severe diarrhea on the second day after injection. Surprisingly, other epithelial tissues appeared to be unaffected at that stage. Decreased sumoylation led to the depletion of the intestinal proliferative compartment and to the rapid disappearance of stem cells. Sumoylation was required to separate the proliferative and differentiated compartments from the crypt and control differentiation and function of the secretory lineage. Sumoylation was required for nucleus positioning and polarized organization of actin in the enterocytes. Loss of sumoylation caused detachment of the enterocytes from the basal lamina, as observed in tissue fragility diseases. We identified the intermediate filament keratin 8 as a SUMO substrate in epithelial cells. CONCLUSIONS: Sumoylation maintains intestinal stem cells and the architecture, mechanical stability, and function of the intestinal epithelium of mice.

Cubilin is essential for albumin reabsorption in the renal proximal tubule.

Receptor-mediated endocytosis is responsible for protein reabsorption in the proximal tubule. This process involves two interacting receptors, megalin and cubilin, which form a complex with amnionless. Whether these proteins function in parallel or as part of an integrated system is not well understood. Here, we report the renal effects of genetic ablation of cubilin, with or without concomitant ablation of megalin, using a conditional Cre-loxP system. We observed that proximal tubule cells did not localize amnionless to the plasma membrane in the absence of cubilin, indicating a mutual dependency of cubilin and amnionless to form a functional membrane receptor complex. The cubilin-amnionless complex mediated internalization of intrinsic factor-vitamin B12 complexes, but megalin considerably increased the uptake. Furthermore, cubilin-deficient mice exhibited markedly decreased uptake of albumin by proximal tubule cells and resultant albuminuria. Inactivation of both megalin and cubilin did not increase albuminuria, indicating that the main role of megalin in albumin reabsorption is to drive the internalization of cubilin-albumin complexes. In contrast, cubulin deficiency did not affect urinary tubular uptake or excretion of vitamin D-binding protein (DBP), which binds cubilin and megalin. In addition, we observed cubilin-independent reabsorption of the "specific" cubilin ligands transferrin, CC16, and apoA-I, suggesting a role for megalin and perhaps other receptors in their reabsorption. In summary, with regard to albumin, cubilin is essential for its reabsorption by proximal tubule cells, and megalin drives internalization of cubilin-albumin complexes. These genetic models will allow further analysis of protein trafficking in the progression of proteinuric renal diseases.

A murine model of Denys-Drash syndrome reveals novel transcriptional targets of WT1 in podocytes.

The Wilms tumor-suppressor gene WT1, a key player in renal development, also has a crucial role in maintenance of the glomerulus in the mature kidney. However, molecular pathways orchestrated by WT1 in podocytes, where it is highly expressed, remain unknown. Their defects are thought to modify the cross-talk between podocytes and other glomerular cells and ultimately lead to glomerular sclerosis, as observed in diffuse mesangial sclerosis (DMS) a nephropathy associated with WT1 mutations. To identify podocyte WT1 targets, we generated a novel DMS mouse line, performed gene expression profiling in isolated glomeruli and identified excellent candidates that may modify podocyte differentiation and growth factor signaling in glomeruli. Scel, encoding sciellin, a protein of the cornified envelope in the skin, and Sulf1, encoding a 6-O endosulfatase, are shown to be expressed in wild-type podocytes and to be strongly down-regulated in mutants. Co-expression of Wt1, Scel and Sulf1 was also found in a mesonephric cell line, and siRNA-mediated knockdown of WT1 decreased Scel and Sulf1 mRNAs and proteins. By ChIP we show that Scel and Sulf1 are direct WT1 targets. Cyp26a1, encoding an enzyme involved in the degradation of retinoic acid, is shown to be up-regulated in mutant podocytes. Cyp26a1 may play a role in the development of glomerular lesions but does not seem to be regulated by WT1. These results provide novel clues in our understanding of normal glomerular function and early events involved in glomerulosclerosis.

Stereocilin-deficient mice reveal the origin of cochlear waveform distortions.

Although the cochlea is an amplifier and a remarkably sensitive and finely tuned detector of sounds, it also produces conspicuous mechanical and electrical waveform distortions. These distortions reflect nonlinear mechanical interactions within the cochlea. By allowing one tone to suppress another (masking effect), they contribute to speech intelligibility. Tones can also combine to produce sounds with frequencies not present in the acoustic stimulus. These sounds compose the otoacoustic emissions that are extensively used to screen hearing in newborns. Because both cochlear amplification and distortion originate from the outer hair cells-one of the two types of sensory receptor cells-it has been speculated that they stem from a common mechanism. Here we show that the nonlinearity underlying cochlear waveform distortions relies on the presence of stereocilin, a protein defective in a recessive form of human deafness. Stereocilin was detected in association with horizontal top connectors, lateral links that join adjacent stereocilia within the outer hair cell's hair bundle. These links were absent in stereocilin-null mutant mice, which became progressively deaf. At the onset of hearing, however, their cochlear sensitivity and frequency tuning were almost normal, although masking was much reduced and both acoustic and electrical waveform distortions were completely lacking. From this unique functional situation, we conclude that the main source of cochlear waveform distortions is a deflection-dependent hair bundle stiffness resulting from constraints imposed by the horizontal top connectors, and not from the intrinsic nonlinear behaviour of the mechanoelectrical transducer channel.

Gain-of-function mutant of angiotensin II receptor, type 1A, causes hypertension and cardiovascular fibrosis in mice.

The role of the renin-angiotensin system has been investigated by overexpression or inactivation of its different genes in animals. However, there is no data concerning the effect of the constitutive activation of any component of the system. A knockin mouse model has been constructed with a gain-of-function mutant of the Ang II receptor, type 1A (AT(1A)), associating a constitutively activating mutation (N111S) with a C-terminal deletion, which impairs receptor internalization and desensitization. In vivo consequences of this mutant receptor expression in homozygous mice recapitulate its in vitro characteristics: the pressor response is more sensitive to Ang II and longer lasting. These mice present with a moderate (~20 mmHg) and stable increase in BP. They also develop early and progressive renal fibrosis and cardiac fibrosis and diastolic dysfunction. However, there was no overt cardiac hypertrophy. The hormonal parameters (low-renin and inappropriately normal aldosterone productions) mimic those of low-renin human hypertension. This new model reveals that a constitutive activation of AT(1A) leads to cardiac and renal fibrosis in spite of a modest effect on BP and will be useful for investigating the role of Ang II in target organs in a model similar to some forms of human hypertension.

Otoferlin, defective in a human deafness form, is essential for exocytosis at the auditory ribbon synapse.

The auditory inner hair cell (IHC) ribbon synapse operates with an exceptional temporal precision and maintains a high level of neurotransmitter release. However, the molecular mechanisms underlying IHC synaptic exocytosis are largely unknown. We studied otoferlin, a predicted C2-domain transmembrane protein, which is defective in a recessive form of human deafness. We show that otoferlin expression in the hair cells correlates with afferent synaptogenesis and find that otoferlin localizes to ribbon-associated synaptic vesicles. Otoferlin binds Ca(2+) and displays Ca(2+)-dependent interactions with the SNARE proteins syntaxin1 and SNAP25. Otoferlin deficient mice (Otof(-/-)) are profoundly deaf. Exocytosis in Otof(-/-) IHCs is almost completely abolished, despite normal ribbon synapse morphogenesis and Ca(2+) current. Thus, otoferlin is essential for a late step of synaptic vesicle exocytosis and may act as the major Ca(2+) sensor triggering membrane fusion at the IHC ribbon synapse.

Targeted disruption of the hepcidin 1 gene results in severe hemochromatosis.

We previously reported that mice made deficient for the transcriptional factor USF2 fail to express hepcidin 1 and hepcidin 2 genes as a consequence of targeted disruption of the Usf2 gene lying just upstream in the locus. These mice developed an iron overload phenotype with excess iron deposition in parenchymal cells and decreased reticuloendothelial iron. At that time, although the role of USF2 was still confounding, we proposed for the first time the role of hepcidin as a negative regulator of iron absorption and iron release from macrophages. Accordingly, we subsequently demonstrated that hyperexpression of hepcidin 1, but not hepcidin 2, resulted in a profound hyposideremic anemia. To analyze the consequences of hepcidin 1 deletion on iron metabolism without any disturbance due to USF2 deficiency, we disrupted the hepcidin 1 gene by targeting almost all the coding region. Confirming our prior results, Hepc1(-/-) mice developed early and severe multivisceral iron overload, with sparing of the spleen macrophages, and demonstrated increased serum iron and ferritin levels as compared with their controls.

Branching and nucleokinesis defects in migrating interneurons derived from doublecortin knockout mice.

Type I lissencephaly results from mutations in the doublecortin (DCX) and LIS1 genes. We generated Dcx knockout mice to further understand the pathophysiological mechanisms associated with this cortical malformation. Dcx is expressed in migrating interneurons in developing human and mouse brains. Video microscopy analyses of such tangentially migrating neuron populations derived from the medial ganglionic eminence show defects in migratory dynamics. Specifically, the formation and division of growth cones, leading to the production of new branches, are more frequent in knockout cells, although branches are less stable. Dcx-deficient cells thus migrate in a disorganized manner, extending and retracting short branches and making less long-distant movements of the nucleus. Despite these differences, migratory speeds and distances remain similar to wild-type cells. These novel data thus highlight a role for Dcx, a microtubule-associated protein enriched at the leading edge in the branching and nucleokinesis of migrating interneurons.

Lactadherin promotes VEGF-dependent neovascularization.

Vascular endothelial growth factor (VEGF)-induced blood vessel growth is involved in both physiological and pathological angiogenesis and requires integrin-mediated signaling. We now show that an integrin-binding protein initially described in milk-fat globule, MFG-E8 (also known as lactadherin), is expressed in and around blood vessels and has a crucial role in VEGF-dependent neovascularization in the adult mouse. Using neutralizing antibodies and lactadherin-deficient animals, we show that lactadherin interacts with alphavbeta3 and alphavbeta5 integrins and alters both VEGF-dependent Akt phosphorylation and neovascularization. In the absence of VEGF, lactadherin administration induced alphavbeta3- and alphavbeta5-dependent Akt phosphorylation in endothelial cells in vitro and strongly improved postischemic neovascularization in vivo. These results show a crucial role for lactadherin in VEGF-dependent neovascularization and identify lactadherin as an important target for the modulation of neovascularization.

Six1 and Six4 homeoproteins are required for Pax3 and Mrf expression during myogenesis in the mouse embryo.

In mammals, Six5, Six4 and Six1 genes are co-expressed during mouse myogenesis. Six4 and Six5 single knockout (KO) mice have no developmental defects, while Six1 KO mice die at birth and show multiple organ developmental defects. We have generated Six1Six4 double KO mice and show an aggravation of the phenotype previously reported for the single Six1 KO. Six1Six4 double KO mice are characterized by severe craniofacial and rib defects, and general muscle hypoplasia. At the limb bud level, Six1 and Six4 homeogenes control early steps of myogenic cell delamination and migration from the somite through the control of Pax3 gene expression. Impaired in their migratory pathway, cells of the somitic ventrolateral dermomyotome are rerouted, lose their identity and die by apoptosis. At the interlimb level, epaxial Met expression is abolished, while it is preserved in Pax3-deficient embryos. Within the myotome, absence of Six1 and Six4 impairs the expression of the myogenic regulatory factors myogenin and Myod1, and Mrf4 expression becomes undetectable. Myf5 expression is correctly initiated but becomes restricted to the caudal region of each somite. Early syndetomal expression of scleraxis is reduced in the Six1Six4 embryo, while the myotomal expression of Fgfr4 and Fgf8 but not Fgf4 and Fgf6 is maintained. These results highlight the different roles played by Six proteins during skeletal myogenesis.

Colorectal cancers in a new mouse model of familial adenomatous polyposis: influence of genetic and environmental modifiers.

Murine models of familial adenomatous polyposis harbor a germinal heterozygous mutation on Apc tumor suppressor gene. They are valuable tools for studying intestinal carcinogenesis, as most human sporadic cancers contain inactivating mutations of APC. However, Apc(+/-) mice, such as the well-characterized Apc(Min/+) model, develop cancers principally in the small intestine, while humans develop mainly colorectal cancers. We used a Cre-loxP strategy to achieve a new model of germline Apc invalidation in which exon 14 is deleted. We compared the phenotype of these Apc(Delta14/+) mice to that of the classical Apc(Min/+). The main phenotypic difference is the shift of the tumors in the distal colon and rectum, often associated with a rectal prolapse. Thus, the severity of the colorectal phenotype is partly due to the particular mutation Delta14, but also to environmental parameters, as mice raised in conventional conditions developed more colon cancers than those raised in pathogen-free conditions. All lesions, including early lesions, revealed Apc LOH and loss of Apc gene expression. They accumulated beta-catenin, overexpressed the beta-catenin target genes cyclin D1 and c-Myc, and the distribution pattern of glutamine synthetase, a beta-catenin target gene recently identified in the liver, was mosaic in intestinal adenomas. The Apc(Delta14/+) model is thus a useful new tool for studies on the molecular mechanisms of colorectal tumorigenesis.

Targeted inactivation of serum response factor in the developing heart results in myocardial defects and embryonic lethality.

Serum response factor (SRF) is at the confluence of multiple signaling pathways controlling the transcription of immediate-early response genes and muscle-specific genes. There are active SRF target sequences in more than 50 genes expressed in the three muscle lineages including normal and diseased hearts. However, the role of SRF in heart formation has not been addressed in vivo thus far due to the early requirement of SRF for mesoderm formation. We have generated a conditional mutant of SRF by using Cre-LoxP strategy that will be extremely useful to study the role of SRF in embryonic and postnatal cardiac functions, as well as in other tissues. This report shows that heart-specific deletion of SRF in the embryo by using a new beta MHC-Cre transgenic mouse line results in lethal cardiac defects between embryonic day 10.5 (E10.5) and E13.5, as evidenced by abnormally thin myocardium, dilated cardiac chambers, poor trabeculation, and a disorganized interventricular septum. At E9.5, we found a marked reduction in the expression of essential regulators of heart development, including Nkx2.5, GATA4, myocardin, and the SRF target gene c-fos prior to overt maldevelopment. We conclude that SRF is crucial for cardiac differentiation and maturation, acting as a global regulator of multiple developmental genes.

Altered myogenesis in Six1-deficient mice.

Six homeoproteins are expressed in several tissues, including muscle, during vertebrate embryogenesis, suggesting that they may be involved in diverse differentiation processes. To determine the functions of the Six1 gene during myogenesis, we constructed Six1-deficient mice by replacing its first exon with the lacZ gene. Mice lacking Six1 die at birth because of severe rib malformations and show extensive muscle hypoplasia affecting most of the body muscles in particular certain hypaxial muscles. Six1(-/-) embryos have impaired primary myogenesis, characterized, at E13.5, by a severe reduction and disorganisation of primary myofibers in most body muscles. While Myf5, MyoD and myogenin are correctly expressed in the somitic compartment in early Six1(-/-) embryos, by E11.5 MyoD and myogenin gene activation is reduced and delayed in limb buds. However, this is not the consequence of a reduced ability of myogenic precursor cells to migrate into the limb buds or of an abnormal apoptosis of myoblasts lacking Six1. It appears therefore that Six1 plays a specific role in hypaxial muscle differentiation, distinct from those of other hypaxial determinants such as Pax3, cMet, Lbx1 or Mox2.

Cre-mediated germline mosaicism: a new transgenic mouse for the selective removal of residual markers from tri-lox conditional alleles.

The binary Cre-lox conditional knockout system requires an essential part of the target gene to be flanked by loxP sites, enabling excision in vivo upon Cre expression. LoxP sites are introduced by homologous recombination, together with a selectable marker. However, this marker can disturb gene expression and should be removed. The marker is therefore often prepared with a third, flanking loxP site (tri-lox construct), facilitating its selective removal by partial Cre-lox recombination. We have shown that this excision can be achieved in vivo in the germline using EIIaCre transgenic mice, and have described the advantages of in vivo over in vitro removal. We show here that MeuCre40, a new transgenic mouse, more reliably and reproducibly generates an optimal partial mosaic Cre-lox recombination pattern in the early embryo. This mosaicism was transmitted to the germline and to many other tissues. Alleles with partial deletions, in particular floxed alleles from which the selectable marker was removed, were readily recovered in the next generation, after segregation from the transgene. Segregation via paternal or maternal transmission led to successful recovery of the alleles of interest. We also obtained total deletion of the floxed regions in the same experiment, making this transgene a polyvalent Cre-lox tool. We rigorously tested the ability of MeuCre40 to solve tri-lox problems, by using it for the in vivo removal of neo(R)- and hprt-expression cassettes from three different tri-lox mutants.

Intralysosomal cystine accumulation in mice lacking cystinosin, the protein defective in cystinosis.

Cystinosis is an autosomal recessive disorder characterized by an accumulation of intralysosomal cystine. The causative gene, CTNS, encodes cystinosin, a seven-transmembrane-domain protein, which we recently showed to be a lysosomal cystine transporter. The most severe and frequent form of cystinosis, the infantile form, appears around 6 to 12 months, with a proximal tubulopathy (de Toni-Debré-Fanconi syndrome) and ocular damage. End-stage renal failure is reached by 10 years of age. Accumulation of cystine in all tissues eventually leads to multisystemic disease. Treatment with cysteamine, which reduces the concentration of intracellular cystine, delays disease progression but has undesirable side effects. We report the first Ctns knockout mouse model generated using a promoter trap approach. We replaced the last four Ctns exons by an internal ribosome entry site-betagal-neo cassette and showed that the truncated protein was mislocalized and nonfunctional. Ctns(-/-) mice accumulated cystine in all organs tested, and cystine crystals, pathognomonic of cystinosis, were observed. Ctns(-/-) mice developed ocular changes similar to those observed in affected individuals, bone defects and behavioral anomalies. Interestingly, Ctns(-/-) mice did not develop signs of a proximal tubulopathy, or renal failure. A preliminary therapeutic trial using an oral administration of cysteamine was carried out and demonstrated the efficiency of this treatment for cystine clearance in Ctns(-/-) mice. This animal model will prove an invaluable and unique tool for testing emerging therapeutics for cystinosis.