Newborn Mutant Mice Research Articles

Gene therapy of Dent disease type 1 in newborn ClC-5 null mice for sustained transgene expression and gene therapy effects.

Dent disease type 1 is caused by changes in the chloride voltage-gated channel 5 (CLCN5) gene on chromosome X, resulting in the lack or dysfunction of chloride channel ClC-5. Individuals affected by Dent disease type 1 show proteinuria and hypercalciuria. Previously we found that lentiviral vector-mediated hCLCN5 cDNA supplementary therapy in ClC-5 null mice was effective only for three months following gene delivery, and the therapeutic effects disappeared four months after treatment, most likely due to immune responses to the ClC-5 proteins expressed in the treated cells. Here we tried two strategies to reduce possible immune responses: 1) confining the expression of ClC-5 expression to the tubular cells with tubule-specific Npt2a and Sglt2 promoters, and 2) performing gene therapy in newborn mutant mice whose immune system has not fully developed. We found that although Npt2a and Sglt2 promoters successfully drove ClC-5 expression in the kidneys of the mutant mice, the treatment did not ameliorate the phenotypes. However, gene delivery to the kidneys of newborn Clcn5 mutant mice enabled long-term transgene expression and phenotype improvement. Our data suggest that performing gene therapy on Dent disease affected subjects soon after birth could be a promising strategy to attenuate immune responses in Dent disease type 1 gene therapy.

Analysis of Postnatal Mutant Phenotypes in Mice.

Viable homozygous mutant newborn mice may show effects of a mutation at any time during their development by exhibiting abnormal structure, function, or lethality. This overview guides the analysis of postnatal mice through gross anatomical assessment and the detection of visible phenotypes prior to weaning such as altered growth patterns, neurological problems, or abnormalities in movement or coordination. Advice on marking pups for identification purposes and providing adequate nutrition in the event of eating problems is given. After weaning and at the onset of puberty, different phenotypes may become manifest, including compromised growth and vigor and reproductive problems in males and/or females. Assessing infertility in each sex is addressed.

A Carboxy-terminal Smarcb1 Point Mutation Induces Hydrocephalus Formation and Affects AP-1 and Neuronal Signalling Pathways in Mice

The BAF (BRG1/BRM-associated factor) chromatin remodelling complex is essential for the regulation of DNA accessibility and gene expression during neuronal differentiation. Mutations of its core subunit SMARCB1 result in a broad spectrum of pathologies, including aggressive rhabdoid tumours or neurodevelopmental disorders. Other mouse models have addressed the influence of a homo- or heterozygous loss of Smarcb1, yet the impact of specific non-truncating mutations remains poorly understood. Here, we have established a new mouse model for the carboxy-terminal Smarcb1 c.1148del point mutation, which leads to the synthesis of elongated SMARCB1 proteins. We have investigated its impact on brain development in mice using magnetic resonance imaging, histology, and single-cell RNA sequencing. During adolescence, Smarcb11148del/1148del mice demonstrated rather slow weight gain and frequently developed hydrocephalus including enlarged lateral ventricles. In embryonic and neonatal stages, mutant brains did not differ anatomically and histologically from wild-type controls. Single-cell RNA sequencing of brains from newborn mutant mice revealed that a complete brain including all cell types of a physiologic mouse brain is formed despite the SMARCB1 mutation. However, neuronal signalling appeared disturbed in newborn mice, since genes of the AP-1 transcription factor family and neurite outgrowth-related transcripts were downregulated. These findings support the important role of SMARCB1 in neurodevelopment and extend the knowledge of different Smarcb1 mutations and their associated phenotypes.

Assessment of the atrial adenosinergic system in the onset of atrial fibrillation: A pre-clinical study in a mouse model with genetic susceptibility to atrial fibrillation

Atrial fibrillation (AF) is a multifactorial sustained cardiac arrhythmia with genetic predisposition. Genome-wide association studies (GWAS) have identified loci near key cardiac transcription factors encoding genes (TBX5, GATA4, NKX2.5) associated with AF risk. Consistently, adult-specific Tbx5 knockout mice showed primary spontaneous and sustained AF, induced by calcium handling abnormalities. The adenosinergic system may be implicated in the onset of AF. We previously showed that adenosine plasma levels increase in persistent AF, and atrial adenosine receptor overexpression is associated with reentry mechanisms. While calcium signaling proteins and potassium channels are major effectors of the adenosinergic system, and also triggers of AF, the link between TBX5, AF and the adenosinergic system has not yet been studied. To assess how the intra-atrial adenosinergic system is associated with a higher susceptibility to atrial fibrillation in a mouse model. Transgenic Cx40-CreERT2::Tbx5 floxed/floxed mice allow conditional deletion of Tbx5 in atria and the ventricular conduction system. Cre-mediated recombination was induced by intraperitoneal injection of tamoxifen in control (Cx40-CreERT2::Tbx5++) and mutant newborn mice. AF was investigated by surface ECGs between 2 weeks and 3 months. The adenosinergic system was assessed by adenosine plasma measurement, in vivo pharmacological tests, immunofluorescence and western blot analyses. At 2 weeks, prolonged P wave and PR interval durations ( P < 0.001; P = 0.03, respectively) and reduced P wave amplitude ( P = 0.01) were measured in transgenic mice ( n = 9) compared to controls ( n = 8). AF episodes were more frequently observed in conditional mutant mice (33% vs. 0%; P = 0.21). Tbx5 deletion specifically in atria increases susceptibility for atrial fibrillation confirming our model as a good one to study the role of the adenosinergic system in AF.

Mutation of Murine Rpl5 reveals a New Model for Diamond Blackfan Anemia Characterized By Defective Erythropoiesis

Background Ribosome dysfunction is implicated in several abnormal developmental conditions and diseases in humans. Heterozygous germline ribosomal protein (RP) variants are found in the majority of individuals with Diamond Blackfan anemia (DBA) with RPL5 (uL18) being the second most frequently mutated gene. Patients with RPL5 mutations are for unknown reasons at increased risk of congenital defects when compared to other genotypes. There is currently no available Rpl5 mouse model with anemia and therefore hematopoiesis in this genotype has not been well studied. Methods We characterized a novel mutant mouse with a growth and kinky tail defect, and increased mortality. The mouse was generated in a chromosome 11 balancer ENU mutagenesis screen. Following genetic mapping and whole exome sequencing, we identified a heterozygous intronic Rpl5 missense variant, which segregated with all affected mice. We first performed molecular analyses to validate the impact of this variant on RPL5 and ribosome levels, and then examined the development and physiology of multiple tissues by histology, micro-CT, echocardiogram and measurement of erythroid differentiation to determine the etiology of the observed increased mortality in newborn Rpl5 mutant mice. Results This variant led to decreased Rpl5 mRNA and protein, and consequential polysome defects (decreased 60S and 80S ribosome subunits) consistent with RPL5 haploinsufficiency. All Rpl5+/- neonates exhibited deficient terminal erythroid differentiation. A fraction of the mutants progressed to severe anemia leading to increased mortality while others appeared to undergo spontaneous remission. Surviving adult/aged mice exhibited no anemia although preliminary studies indicate reduced numbers of hematopoietic progenitors. Most newborn mutant mice had large ventricular septal defects (VSD) while surviving adult mice had small VSDs on echocardiogram. Rpl5+/- mice were significantly smaller without craniofacial abnormalities as assessed by micro-CT or histology. Bone and cartilage staining of newborn mice revealed that the kinky tail defect resulted from delayed endochondral ossification. Conclusion This model recapitulates the major developmental and erythroid differentiation defects of DBA in an animal model. We propose that this novel Rpl5 mutant provides an opportunity to study the mechanism of the variable penetrance of anemia and the congenital defects normally encountered in DBA. Disclosures Singh: Emmaus Medical, Inc: Consultancy.

Abstract 627: Primary Cilia of the Cardiac Neural Crest Orchestrate Critical Aspects of Ventricular Maturation and Postnatal Cardiac Function: A Role for Hedgehog Signaling

Primary cilia are tiny, plasma-membrane bound organelles that function to modulate intra- and extracellular signaling, in particular Hedgehog signaling, implicated in both normal development as well as in various disease pathologies, including congenital heart disease (CHD). Cardiac neural crest cells (CNCC) display primary cilia and function as a major progenitor cell population contributing to the developing heart. We hypothesized that damage to or loss of primary cilia in CNCC would impair normal heart development, leading to CHD. We present 1) a precise description of CHD’s resulting from loss of primary cilia of CNCC in vivo ; 2) characterization of the functional, electrophysiological aspects of the perinatal phenotype; and 3) a preliminary investigation of the molecular mechanisms leading to this phenotype. Using a Wnt1:Cre-2, Ift88-targeted conditional elimination of primary cilia, and a Td-tomato reporter to track CNCC, we observed loss of cilia in CNCC at embryonic day E9.5, with notable CNCC contribution to the ventricular myocardium and pronounced disorganization of the endocardium by E10.5.The phenotype resulting from cilia loss in CNCC was characterized by a variety predicted CHDs in addition to disorganization of the ventricular endocardium, pronounced noncompaction of the ventricular myocardium, and perinatal lethality. To investigate potential changes in Hedgehog signaling resulting from cilia loss, we implemented an in vivo conditional knockout in which Wnt1:Cre-2 drives expression of a mutant, constitutively-active Hedgehog receptor. Noncompaction observed in all Wnt1:Cre-2/Ift88 mutants became exacerbated in a primary-cilia dependent fashion with the introduction of forced Hedgehog signaling in CNCC. These results support a critical role for both primary cilia and Hedgehog signaling in the development of the ventricular myocardium. Further electrophysiological assessments of Wnt1:Cre-2/Ift88 newborn mutant mice revealed bradycardia, conduction defects, and ECG tracings consistent with ventricular hypertrophy. These data collectively support a role for Hedgehog signaling via primary cilia of CNCC in the pathogenesis of outflow tract defects, VSD, and most notably, ventricular maturation CHDs.

Cux1 regulation of the cyclin kinase inhibitor p27kip1 in polycystic kidney disease is attenuated by HDAC inhibitors.

Cux1 is a homeodomain protein involved in cell cycle regulation and kidney development. Cux1 represses the cyclin kinase inhibitor p27 during early kidney development, promoting cell proliferation in the nephrogenic zone. Promoter reporter analysis of p27 revealed that Cux1 represses p27 in a concentration dependent manner, and immunoprecipitation showed that Cux1 interacts with the co-repressor Grg4 and the histone deacetylases HDAC1 and HDAC3. Chromatin immunoprecipitation (ChIP) identified the interaction of Cux1, Grg4, HDAC1, and HDAC3 at two different sites in the p27 promoter. To determine whether there was an interaction between these two loci in the developing kidney, we performed chromatin conformation capture (3C) assay. Analysis of newborn kidney tissue with 3C and ChIP-loop showed that the p27 promoter forms a loop intersecting at these two loci and that Cux1 bridges these two sites. To determine whether HDACs are required for Cux1 repression of p27 we analyzed p27 promoter activity in the presence of the HDAC inhibitor trichostatin A (TSA). TSA treatment completely relieved the repression of p27 by Cux1 and Grg4, demonstrating that Cux1 represses p27 in an HDAC dependent manner. To begin to test whether HDAC inhibitors could be used to target Cux1 repression of p27 for the treatment of PKD, we treated Pkd1 targeted pregnant mice with TSA or vehicle beginning at embryonic day 10.5 until embryonic day 18.5. Newborn Pkd1 mutant mice that received vehicle exhibited extensive collecting duct cysts, while newborn Pkd1 mutant mice that received TSA showed a significant reduction in cysts. Moreover, p27 expression was upregulated in TSA treated Pkd1 mice. Taken together, these results suggest that HDACs are required for cyst growth, and further support studies indicating that HDAC inhibitors may be an effective treatment for PKD.

The microRNA-183/96/182 Cluster is Essential for Stereociliary Bundle Formation and Function of Cochlear Sensory Hair Cells

The microRNA (miR)-183/96/182 cluster plays important roles in the development and functions of sensory organs, including the inner ear. Point-mutations in the seed sequence of miR-96 result in non-syndromic hearing loss in both mice and humans. However, the lack of a functionally null mutant has hampered the evaluation of the cluster’s physiological functions. Here we have characterized a loss-of-function mutant mouse model (miR-183CGT/GT), in which the miR-183/96/182 cluster gene is inactivated by a gene-trap (GT) construct. The homozygous mutant mice show profound congenital hearing loss with severe defects in cochlear hair cell (HC) maturation, alignment, hair bundle formation and the checkboard-like pattern of the cochlear sensory epithelia. The stereociliary bundles retain an immature appearance throughout the cochlea at postnatal day (P) 3 and degenerate soon after. The organ of Corti of mutant newborn mice has no functional mechanoelectrical transduction. Several predicted target genes of the miR-183/96/182 cluster that are known to play important roles in HC development and function, including Clic5, Rdx, Ezr, Rac1, Myo1c, Pvrl3 and Sox2, are upregulated in the cochlea. These results suggest that the miR-183/96/182 cluster is essential for stereociliary bundle formation, morphogenesis and function of the cochlear HCs.

Local gene therapy durably restores vestibular function in a mouse model of Usher syndrome type 1G

Our understanding of the mechanisms underlying inherited forms of inner ear deficits has considerably improved during the past 20 y, but we are still far from curative treatments. We investigated gene replacement as a strategy for restoring inner ear functions in a mouse model of Usher syndrome type 1G, characterized by congenital profound deafness and balance disorders. These mice lack the scaffold protein sans, which is involved both in the morphogenesis of the stereociliary bundle, the sensory antenna of inner ear hair cells, and in the mechanoelectrical transduction process. We show that a single delivery of the sans cDNA by the adenoassociated virus 8 to the inner ear of newborn mutant mice reestablishes the expression and targeting of the protein to the tips of stereocilia. The therapeutic gene restores the architecture and mechanosensitivity of stereociliary bundles, improves hearing thresholds, and durably rescues these mice from the balance defects. Our results open up new perspectives for efficient gene therapy of cochlear and vestibular disorders by showing that even severe dysmorphogenesis of stereociliary bundles can be corrected.

Knock-In of the Recurrent R368X Mutation of PRKAR1A that Represses cAMP-Dependent Protein Kinase A Activation: A Model of Type 1 Acrodysostosis.

In humans, activating mutations in the PRKAR1A gene cause acrodysostosis 1 (ACRDYS1). These mutations result in a reduction in PKA activation caused by an impaired ability of cAMP to dissociate mutant PRKAR1A from catalytic PKA subunits. Two striking features of this rare developmental disease are renal resistance to PTH and chondrodysplasia resulting from the constitutive inhibition of PTHR1/Gsa/AC/cAMP/PKA signaling. We developed a knock-in of the recurrent ACRDYS1 R368X PRKAR1A mutation in the mouse. No litters were obtained from [R368X]/[+] females (thus no homozygous [R368X]/[R368X] mice). In [R368X]/[+] mice, Western blot analysis confirmed mutant allele heterozygous expression. Growth retardation, peripheral acrodysostosis (including brachydactyly affecting all digits), and facial dysostosis were shown in [R368X]/[+] mice by weight curves and skeletal measurements (μCT scan) as a function of time. [R368X]/[+] male and female mice were similarly affected. Unexpected, however, whole-mount skeletal preparations revealed a striking delay in mineralization in newborn mutant mice, accompanied by a decrease in the height of terminal hypertrophic chondrocyte layer, an increase in the height of columnar proliferative prehypertrophic chondrocyte layer, and changes in the number and spatial arrangement of proliferating cell nuclear antigen (PCNA)-positive chondrocytes. Plasma PTH and basal urinary cAMP were significantly higher in [R368X]/[+] compared to WT mice. PTH injection increased urinary cAMP similarly in [R368X]/[+] and WT mice. PRKACA expression was regulated in a tissue (kidney not bone and liver) manner. This model, the first describing the germline expression of a PRKAR1A mutation causing dominant repression of cAMP-dependent PKA, reproduced the main features of ACRDYS1 in humans. It should help decipher the specificity of the cAMP/PKA signaling pathway, crucial for numerous stimuli. In addition, our results indicate that PRKAR1A, by tempering intracellular cAMP levels, is a molecular switch at the crossroads of signaling pathways regulating chondrocyte proliferation and differentiation. © 2016 American Society for Bone and Mineral Research.

Partitioning-Defective 1a/b Depletion Impairs Glomerular and Proximal Tubule Development.

The kidney is a highly polarized epithelial organ that develops from undifferentiated mesenchyme, although the mechanisms that regulate the development of renal epithelial polarity are incompletely understood. Partitioning-defective 1 (Par1) proteins have been implicated in cell polarity and epithelial morphogenesis; however, the role of these proteins in the developing kidney has not been established. Therefore, we studied the contribution of Par1a/b to renal epithelial development. We examined the renal phenotype of newborn compound mutant mice carrying only one allele of Par1a or Par1b. Loss of three out of four Par1a/b alleles resulted in severe renal hypoplasia, associated with impaired ureteric bud branching. Compared with kidneys of newborn control littermates, kidneys of newborn mutant mice exhibited dilated proximal tubules and immature glomeruli, and the renal proximal tubular epithelia lacked proper localization of adhesion complexes. Furthermore, Par1a/b mutants expressed low levels of renal Notch ligand Jag1, activated Notch2, and Notch effecter Hes1. Together, these data demonstrate that Par1a/b has a key role in glomerular and proximal tubule development, likely via modulation of Notch signaling.

Dullard/Ctdnep1 regulates endochondral ossification via suppression of TGF-β signaling.

Transforming growth factor (TGF)-β signaling plays critical roles during skeletal development and its excessive signaling causes genetic diseases of connective tissues including Marfan syndrome and acromelic dysplasia. However, the mechanisms underlying prevention of excessive TGF-β signaling in skeletogenesis remain unclear. We previously reported that Dullard/Ctdnep1 encoding a small phosphatase is required for nephron maintenance after birth through suppression of bone morphogenetic protein (BMP) signaling. Unexpectedly, we found that Dullard is involved in suppression of TGF-β signaling during endochondral ossification. Conditional Dullard-deficient mice in the limb and sternum mesenchyme by Prx1-Cre displayed the impaired growth and ossification of skeletal elements leading to postnatal lethality. Dullard was expressed in early cartilage condensations and later in growth plate chondrocytes. The tibia growth plate of newborn Dullard mutant mice showed reduction of the proliferative and hypertrophic chondrocyte layers. The sternum showed deformity of cartilage primordia and delayed hypertrophy. Micromass culture experiments revealed that Dullard deficiency enhanced early cartilage condensation and differentiation, but suppressed mineralized hypertrophic chondrocyte differentiation, which was reversed by treatment with TGF-β type I receptor kinase blocker LY-364947. Dullard deficiency induced upregulation of protein levels of both phospho-Smad2/3 and total Smad2/3 in micromass cultures without increase of Smad2/3 mRNA levels, suggesting that Dullard may affect Smad2/3 protein stability. The phospho-Smad2/3 level was also upregulated in perichondrium and hypertrophic chondrocytes in Dullard-deficient embryos. Response to TGF-β signaling was enhanced in Dullard-deficient primary chondrocyte cultures at late, but not early, time point. Moreover, perinatal administration of LY-364947 ameliorated the sternum deformity in vivo. Thus, we identified Dullard as a new negative regulator of TGF-β signaling in endochondral ossification.

The Candidate Splicing Factor Sfswap Regulates Growth and Patterning of Inner Ear Sensory Organs

The Notch signaling pathway is thought to regulate multiple stages of inner ear development. Mutations in the Notch signaling pathway cause disruptions in the number and arrangement of hair cells and supporting cells in sensory regions of the ear. In this study we identify an insertional mutation in the mouse Sfswap gene, a putative splicing factor, that results in mice with vestibular and cochlear defects that are consistent with disrupted Notch signaling. Homozygous Sfswap mutants display hyperactivity and circling behavior consistent with vestibular defects, and significantly impaired hearing. The cochlea of newborn Sfswap mutant mice shows a significant reduction in outer hair cells and supporting cells and ectopic inner hair cells. This phenotype most closely resembles that seen in hypomorphic alleles of the Notch ligand Jagged1 (Jag1). We show that Jag1; Sfswap compound mutants have inner ear defects that are more severe than expected from simple additive effects of the single mutants, indicating a genetic interaction between Sfswap and Jag1. In addition, expression of genes involved in Notch signaling in the inner ear are reduced in Sfswap mutants. There is increased interest in how splicing affects inner ear development and function. Our work is one of the first studies to suggest that a putative splicing factor has specific effects on Notch signaling pathway members and inner ear development.

In vivo function and evolution of the eutherian-specific pluripotency marker UTF1.

Embryogenesis in placental mammals is sustained by exquisite interplay between the embryo proper and placenta. UTF1 is a developmentally regulated gene expressed in both cell lineages. Here, we analyzed the consequence of loss of the UTF1 gene during mouse development. We found that homozygous UTF1 mutant newborn mice were significantly smaller than wild-type or heterozygous mutant mice, suggesting that placental insufficiency caused by the loss of UTF1 expression in extra-embryonic ectodermal cells at least in part contributed to this phenotype. We also found that the effects of loss of UTF1 expression in embryonic stem cells on their pluripotency were very subtle. Genome structure and sequence comparisons revealed that the UTF1 gene exists only in placental mammals. Our analyses of a family of genes with homology to UTF1 revealed a possible mechanism by which placental mammals have evolved the UTF1 genes.

TCF/Lef1-Mediated Control of Lipid Metabolism Regulates Skin Barrier Function

Defects in the function of the skin barrier are associated with a wide variety of skin diseases, many of which are not well characterized at the molecular level. Using Lef1 (lymphoid enhancer-binding factor 1) dominant-negative mutant mice, we demonstrate here that altered epidermal TCF (T cell factor)/Lef1 signaling results in severe impairment of the stratum corneum skin barrier and early postnatal death. Barrier defects were accompanied by major changes in lipid composition and ultrastructural abnormalities in assembly and extrusion of lipid lamellae of the interfollicular epidermis, as well as abnormal processing of profilaggrin. In contrast, tight-junction formation and stratified organization of the interfollicular epidermis was not obviously disturbed in Lef1 mutant mice. Molecular analysis revealed that TCF/Lef1 signaling regulates expression of lipid-modifying enzymes, such as Elovl3 and stearoyl coenzyme A desaturase 1 (SCD1), which are key regulators of barrier function. Promoter analysis and chromatin immunoprecipitation experiments indeed showed that SCD1 is a direct target of Lef1. Together, our data demonstrate that functional TCF/Lef1 signaling governs important aspects of epidermal differentiation and lipid metabolism, thereby regulating skin barrier function.

Loss of ceramide synthase 3 causes lethal skin barrier disruption

The stratum corneum as the outermost epidermal layer protects against exsiccation and infection. Both the underlying cornified envelope (CE) and the intercellular lipid matrix contribute essentially to these two main protective barriers. Epidermis-unique ceramides with ultra-long-chain acyl moities (ULC-Cers) are key components of extracellular lipid lamellae (ELL) and are bound to CE proteins, thereby contributing to the cornified lipid envelope (CLE). Here, we identified human and mouse ceramide synthase 3 (CerS3), among CerS1-6, to be exclusively required for the ULC-Cer synthesis in vitro and of mouse CerS3 in vivo. Deficiency of CerS3 in mice results in complete loss of ULC-Cers (≥C26), lack of continuous ELL and a non-functional CLE. Consequently, newborn mutant mice die shortly after birth from transepidermal water loss. Mutant skin is prone to Candida albicans infection highlighting ULC-Cers to be pivotal for both barrier functions. Persistent periderm, hyperkeratosis and deficient cornification are hallmarks of mutant skin demonstrating loss of Cers to trigger a keratinocyte maturation arrest at an embryonic pre-barrier stage.

Control of Exocytosis by Synaptotagmins and Otoferlin in Auditory Hair Cells

In pre-hearing mice, vesicle exocytosis at cochlear inner hair cell (IHC) ribbon synapses is triggered by spontaneous Ca(2+) spikes. At the onset of hearing, IHC exocytosis is then exclusively driven by graded potentials, and is characterized by higher Ca(2+) efficiency and improved synchronization of vesicular release. The molecular players involved in this transition are still unknown. Here we addressed the involvement of synaptotagmins and otoferlin as putative Ca(2+) sensors in IHC exocytosis during postnatal maturation of the cochlea. Using cell capacitance measurements, we showed that Ca(2+)-evoked exocytosis in mouse IHCs switches from an otoferlin-independent to an otoferlin-dependent mechanism at postnatal day 4. During this early exocytotic period, several synaptotagmins (Syts), including Syt1, Syt2 and Syt7, were detected in IHCs. The exocytotic response as well as the release of the readily releasable vesicle pool (RRP) was, however, unchanged in newborn mutant mice lacking Syt1, Syt2 or Syt7 (Syt1(-/-), Syt2(-/-) and Syt7(-/-) mice). We only found a defect in RRP recovery in Syt1(-/-) mice which was apparent as a strongly reduced response to repetitive stimulations. In post-hearing Syt2(-/-) and Syt7(-/-) mutant mice, IHC synaptic exocytosis was unaffected. The transient expression of Syt1 and Syt2, which were no longer detected in IHCs after the onset of hearing, indicates that these two most common Ca(2+)-sensors in CNS synapses are not involved in mature IHCs. We suggest that otoferlin underlies highly efficient Ca(2+)-dependent membrane-membrane fusion, a process likely essential to increase the probability and synchrony of vesicle fusion events at the mature IHC ribbon synapse.

Mesenchymal stem cells rescue Purkinje cells and improve motor functions in a mouse model of cerebellar ataxia

Mesenchymal stem cells have been proven to be potentially effective in the treatment of a large variety of diseases, including neurodegenerative disorders. Of these, cerebellar ataxia is a group of disorders characterized by the degeneration of the cerebellum, particularly the Purkinje cells, responsible for motor coordination and control of the motor functions. To analyze the possibility of using bone marrow-derived mesenchymal stem cells in treating ataxia, we transplanted these cells in the cerebellum of newborn Lurcher mutant mice, a very aggressive mouse model characterized by the selective early post-natal death of Purkinje cells in the cerebellum. Two months after the surgical procedure, the treated mice presented significant improvements in the motor behavior tests performed. Histological analysis of the cerebellum indicated that the donor cells had migrated throughout the cerebellum, as well as a significant increase in the number of Purkinje cells. Many grafted stem cells were located adjacent to the Purkinje cell layer, and expressed BDNF, NT-3 or GDNF, neurotrophic factors implicated in Purkinje cell survival. Also, a small percentage of the grafted stem cells had fused with Purkinje cells. Thus, we have shown that mesenchymal stem cells are capable of integrating into the central nervous system, migrate towards the areas where neurodegenerative processes are occurring, and rescue the degenerating cells through cell trophic effects. This is an adequate and feasible model that could be translated into a therapeutic approach for clinical assays in neurodegenerative diseases.

Expression of neuropeptide Y and agouti-related peptide in the hypothalamic arcuate nucleus of newborn neurogenin3 null mutant mice

Mice deficient in neurogenin 3 (Ngn3) fail to generate pancreatic endocrine cells and intestinal endocrine cells. Hypothalamic neuropeptides implicated in the control of energy homeostasis might also be affected in Ngn3 homozygous null mutant mice. We investigated the expression of two prominent orexigenic neuropeptides, neuropeptide Y (NPY) and agouti-related protein (AgRP), in the hypothalamic arcuate nucleus of newborn wild-type and Ngn3 null mutant mice. Immunohistochemical analysis demonstrated that, in Ngn3 null mutants, the number of NPY-immunoreactive neurons and nerve fibers was markedly increased in the arcuate nucleus, and the nerve fibers were widely distributed in the hypothalamic area, including the paraventricular and dorsomedial nuclei. Little increase of AgRP immunoreactivity was detected in the arcuate nucleus of mutant mice. In situ hybridization analysis confirmed the increased population of the NPY neurons in the arcuate nucleus of the mutants. The NPY mRNA level, as estimated by laser capture microdissection and real-time quantitative polymerase chain reaction, was 371% higher in Ngn3 null mutants than in wild-type mice. AgRP mRNA levels did not differ significantly between the null mutants and wild-type mice. Thus, up-regulation of the hypothalamic NPY system is probably a feature characteristic of Ngn3 null mice.

Canonical WNT/β-catenin signaling is required for ureteric branching

WNT/β-catenin signaling has an established role in nephron formation during kidney development. Yet, the role of β-catenin during ureteric morphogenesis in vivo is undefined. We generated a murine genetic model of β-catenin deficiency targeted to the ureteric bud cell lineage. Newborn mutant mice demonstrated bilateral renal aplasia or renal dysplasia. Analysis of the embryologic events leading to this phenotype revealed that abnormal ureteric branching at E12.5 precedes histologic abnormalities at E13.5. Microarray analysis of E12.5 kidney tissue identified decreased Emx2 and Lim1 expression among a small subset of renal patterning genes disrupted at the stage of abnormal branching. These alterations are followed by decreased expression of genes downstream of Emx2, including Lim1, Pax2, and the ureteric tip markers, c-ret and Wnt 11. Together, these data demonstrate that β-catenin performs essential functions during renal branching morphogenesis via control of a hierarchy of genes that control ureteric branching.