Knockdown Mouse Model Research Articles

FACS Array Profiling Identifies Ecto-5′ Nucleotidase as a Striatopallidal Neuron-Specific Gene Involved in Striatal-Dependent Learning

The striatopallidal (STP) and striatonigral (STN) neurons constitute the main neuronal populations of the striatum. Despite the increasing knowledge concerning their involvement in multiple tasks associated with the striatum, it is still challenging to understand the precise differential functions of these two neuronal populations and to identify and study new genes involved in these functions. Here, we describe a reliable approach, applied on adult mouse brain, to generate specific STP and STN neuron gene profiles. STP and STN neurons were identified in the same animal using the transgenic Adora2A-Cre × Z/EG mouse model combined with retrograde labeling, respectively. Gene profiling was generated from FACS-purified neurons leading to the identification of new STP and STN neuron-specific genes. Knock-down models based on Cre-dependent lentiviral vector were developed to investigate their function either in striatal or in STP neurons. Thereby, we demonstrate that ecto-5'-nucleotidase (NT5e) is specifically expressed in STP neurons and is at the origin of most of the extracellular adenosine produced in the striatum. Behavioral analysis of striatal and STP neuron knock-down mouse models as well as NT5e knock-out mice demonstrates the implication of this STP neuron enzyme in motor learning.

Dysfunctional cardiac mitochondrial bioenergetic, lipidomic, and signaling in a murine model of Barth syndrome

Barth syndrome is a complex metabolic disorder caused by mutations in the mitochondrial transacylase tafazzin. Recently, an inducible tafazzin shRNA knockdown mouse model was generated to deconvolute the complex bioenergetic phenotype of this disease. To investigate the underlying cause of hemodynamic dysfunction in Barth syndrome, we interrogated the cardiac structural and signaling lipidome of this mouse model as well as its myocardial bioenergetic phenotype. A decrease in the distribution of cardiolipin molecular species and robust increases in monolysocardiolipin and dilysocardiolipin were demonstrated. Additionally, the contents of choline and ethanolamine glycerophospholipid molecular species containing precursors for lipid signaling at the sn-2 position were altered. Lipidomic analyses revealed specific dysregulation of HETEs and prostanoids, as well as oxidized linoleic and docosahexaenoic metabolites. Bioenergetic interrogation uncovered differential substrate utilization as well as decreases in Complex III and V activities. Transgenic expression of cardiolipin synthase or iPLA2γ ablation in tafazzin-deficient mice did not rescue the observed phenotype. These results underscore the complex nature of alterations in cardiolipin metabolism mediated by tafazzin loss of function. Collectively, we identified specific lipidomic, bioenergetic, and signaling alterations in a murine model that parallel those of Barth syndrome thereby providing novel insights into the pathophysiology of this debilitating disease.

Reversible infertility in a liver receptor homologue-1 (LRH-1)-knockdown mouse model

Liver receptor homologue-1 (LRH-1) is an orphan nuclear receptor that has been implicated in steroid hormone biosynthesis and fertility. Herein we describe a transgenic inducible short hairpin (sh) RNA mouse model that was used to study the effect of transient LRH-1 knockdown in vivo. Induction of expression of the shRNA directed against LRH-1 for 2-6 weeks resulted in 80% knockdown of LRH-1 protein in the ovary and complete infertility. Gonadotropin hyperstimulation could not rescue the observed defects in ovulation and corpus luteum formation in LRH-1-knockdown mice. The infertility phenotype was fully reversible because LRH-1-knockdown females became pregnant and delivered normal size litters and healthy pups after cessation of LRH-1 shRNA expression. Timed ovarian microarray analysis showed that, in line with the observed decrease in plasma progesterone levels, key steroid biosynthesis genes, namely Star, Cyp11a1, Hsd3b and Scarb1, were downregulated in LRH-1-knockdown ovaries. In contrast with what has been described previously, no clear effect was observed on oestrogenic activity in LRH-1-knockdown mice. Only Sult1e1 and, surprisingly, Hsd17b7 expression was modulated with potentially opposite effects on oestradiol bioavailability. In conclusion, the fully reversible infertility phenotype of LRH-1-knockdown mice shows the feasibility of an LRH-1 antagonist as new contraceptive therapy with a mechanism of action that most prominently affects cholesterol availability and progesterone production.

Hepatocyte-specific deletion of hepatocyte nuclear factor-4α in adult mice results in increased hepatocyte proliferation.

Hepatocyte nuclear factor-4α (HNF4α) is known as the master regulator of hepatocyte differentiation. Recent studies indicate that HNF4α may inhibit hepatocyte proliferation via mechanisms that have yet to be identified. Using a HNF4α knockdown mouse model based on delivery of inducible Cre recombinase via an adeno-associated virus 8 viral vector, we investigated the role of HNF4α in the regulation of hepatocyte proliferation. Hepatocyte-specific deletion of HNF4α resulted in increased hepatocyte proliferation. Global gene expression analysis showed that a majority of the downregulated genes were previously known HNF4α target genes involved in hepatic differentiation. Interestingly, ≥500 upregulated genes were associated with cell proliferation and cancer. Furthermore, we identified potential negative target genes of HNF4α, many of which are involved in the stimulation of proliferation. Using chromatin immunoprecipitation analysis, we confirmed binding of HNF4α at three of these genes. Furthermore, overexpression of HNF4α in mouse hepatocellular carcinoma cells resulted in a decrease in promitogenic gene expression and cell cycle arrest. Taken together, these data indicate that, apart from its role in hepatocyte differentiation, HNF4α actively inhibits hepatocyte proliferation by repression of specific promitogenic genes.

In vivo knockdown of Brachyury results in skeletal defects and urorectal malformations resembling caudal regression syndrome

The T-box transcription factor BRACHYURY (T) is a key regulator of mesoderm formation during early development. Complete loss of T has been shown to lead to embryonic lethality around E10.0. Here we characterize an inducible miRNA-based in vivo knockdown mouse model of T, termed KD3-T, which exhibits a hypomorphic phenotype. KD3-T embryos display axial skeletal defects caused by apoptosis of paraxial mesoderm, which is accompanied by urorectal malformations resembling the murine uro-recto-caudal syndrome and human caudal regression syndrome phenotypes. We show that there is a reduction of T in the notochord of KD3-T embryos which results in impaired notochord differentiation and its subsequent loss, whereas levels of T in the tailbud are sufficient for axis extension and patterning. Furthermore, the notochord in KD3-T embryos adopts a neural character and loses its ability to act as a signaling center. Since KD3-T animals survive until birth, they are useful for examining later roles for T in the development of urorectal tissues.

Contribution of prostacyclin regulation in the vascular tone of angiopoietin like‐2 deficient mice

Vascular tone, in response to acetylcholine (ACh), is controlled by various endothelium‐derived relaxing factors, which include nitric oxide (NO), prostacyclin, and endothelium‐derived hyperpolarizing factor. In turn, contribution of each factor may be influenced by pathologies. As such, in the insulin resistant state, NO production is impaired. Angiopoietin like‐2 (Angptl2), a member of the angiopoietin‐like protein family, was shown to cause insulin resistance related to obesity (Tabata, 2009).ObjectiveAn Angptl2 knock‐down (KD) mouse model was generated to study the role of Angptl2 in endothelial function in the femoral artery.ResultsFemoral arteries of 4‐month old Angptl2 KD and wild‐type (WT) mice showed that NO was responsible for the sensitivity of ACh‐mediated vasodilation in both groups, while prostacyclin contributed to maximal dilation in KD but not in WT. Femoral artery wall thickness of KD (20±1μm) at physiological pressure was smaller than that of WT (25±1μm; P<0.01, n=7), while constriction to phenylephrine was similar in both groups. Although KD and WT mice displayed similar metabolic parameters including fasting glucose level, KD mice showed a higher fasting insulin level (P<0.05, n=7–8).ConclusionKnock‐down of Angptl2 may lead to differences in the prostacyclin signaling pathway in the femoral arteries between the two genotypes of mice. Supported by CIHR MOP14496 and NSERC.

Hepatocyte Nuclear Factor 4 alpha (HNF4α) Knockdown Stimulates Pro‐Mitogenic Gene Expression in Hepatocytes

HNF4α, the master regulator of hepatocyte differentiation, has been shown to inhibit hepatocyte proliferation via yet to be identified mechanisms. We investigated the mechanisms of HNF4α‐induced inhibition of hepatocyte proliferation using two novel HNF4α knockdown mouse models. Hepatocyte specific deletion of HNF4α resulted in increased hepatocyte proliferation. Comparative global gene expression analysis revealed a set of genes commonly regulated in both HNF4α deletion models. The majority of the down‐regulated genes are known HNF4α target genes involved in hepatic differentiation. Interestingly, many up‐regulated genes were associated with cell proliferation and cancer. HNF4α knockdown mice treated with the known hepatic carcinogen diethylnitrosamine developed a 7‐fold increase in number of tumors, as compared to controls. Furthermore, in vitro over expression of HNF4α in mouse HCC cells resulted in a decrease in cell proliferation along with a decrease in promitogenic gene expression. Taken together these data indicate that HNF4α inhibits hepatocyte proliferation by repression of specific pro‐mitogenic genes.

Requirement of Mouse BCCIP for Neural Development and Progenitor Proliferation

Multiple DNA repair pathways are involved in the orderly development of neural systems at distinct stages. The homologous recombination (HR) pathway is required to resolve stalled replication forks and critical for the proliferation of progenitor cells during neural development. BCCIP is a BRCA2 and CDKN1A interacting protein implicated in HR and inhibition of DNA replication stress. In this study, we determined the role of BCCIP in neural development using a conditional BCCIP knock-down mouse model. BCCIP deficiency impaired embryonic and postnatal neural development, causing severe ataxia, cerebral and cerebellar defects, and microcephaly. These development defects are associated with spontaneous DNA damage and subsequent cell death in the proliferative cell populations of the neural system during embryogenesis. With in vitro neural spheroid cultures, BCCIP deficiency impaired neural progenitor's self-renewal capability, and spontaneously activated p53. These data suggest that BCCIP and its anti-replication stress functions are essential for normal neural development by maintaining an orderly proliferation of neural progenitors.

TOSO-Deficient B Cells Show An Impaired NF-κB and Migration Signaling In Vivo

Abstract 1779 Background:Our group firstly demonstrated that TOSO (FAIM3) is over-expressed in CLL compared to healthy B cell subsets as well as other B cell lymphomas. Furthermore, we detected a significant correlation of high TOSO expression to high lymphocyte count, unmutated IgVH status and Binet C, which are all markers for poor prognosis. TOSO has been described as pro-survival gene also in other settings. However, its mode of action is discussed controversially.Therefore, we aimed to elucidate the role of TOSO in B-cell specific gene expression by creating a knockdown mouse model. CD40 ligation and B cell receptor (BCR) activation influences TOSO expression and the fact that transcriptional regulation of TOSO is still unknown, we were eager to determine transcriptional factors that are directly responsible for the alterable TOSO levels. Methods:Faim3-floxed C57BL/6 FLP deleter mice were crossbred with CD19 specific Cre recombinase expressing mice. B-cells from the TOSOCD19−/− (KO) mice were isolated and gene expression was analyzed via mRNA based Illumina microchip array. Convincing results were verified by flow cytometry and blood count was carried out in addition.To determine the promoter region of TOSO, three overlapping DNA fragments (containing either NF-κB, Bcl-6 or both binding sites) upstream of the transcription start site of the first TOSO exon were cloned into a luciferase reporter vector lacking a promoter. Those constructs were transfected into HeLa cells. After 24 hours luciferase assays were performed. The involvement of NF-κB in the regulation of TOSO transcription was measured by TNFα stimulation of transfected cells prior to luminescence measurement. Targeted mutagenesis of the NF-κB binding site was performed to confirm the data. In addition, Bcl-6 expression vector was co-transfected for evaluation of repressing influence on TOSO expression. Results:In order to cover the functional part of TOSO, we generated a B-cell specific TOSOCD19−/− mouse model. Downstream effects of TOSO were validated via microarray-based gene expression analysis. Results displayed a clear clustering of deregulated genes compared to control mice. Nearly 400 genes showed expression alterations; genes involved in the NF-κB pathway and migration processes were downregulated in TOSOCD19−/−. These results were confirmed by flow cytometry analysis. The TOSO KO displayed also relevant effects on the hematopoietic system. Lymphocyte (p=0,0048), neutrophil (p=0,0007) and red blood cell counts (p=0,0051) were significantly decreased in the TOSOCD19−/− mice. Most important, the B-cell count was significantly reduced in TOSO-deficient settings (n=9; p=0,032).Since TOSO level seems to be so important for such fundamental pathways, investigation of gene expression regulation is mandatory. In situ analysis of the TOSO promoter region revealed NF-κB and Bcl-6 as promising results. Luciferase reporter assays including targeted mutagenesis confirmed the positive regulation of NF-κB and the repressing influence of Bcl-6 on TOSO expression significantly. Conclusions:We reveal for the first time a TOSO-dependent expression profile. We identified TOSO-dependent deregulated genes, which were involved in NF-κB signaling and migration, suggesting that TOSO represents an important factor in these pathways. Additionally, TOSO KO caused a decrease of peripheral B-cells in vivo. Furthermore, we identified NF-κB and Bcl-6 to regulate the TOSO expression in an opposite manner. Disclosures:No relevant conflicts of interest to declare.

P2.16 Deposition of the inner limiting membrane in the eye of a mouse model for Muscle Eye Brain disease

Dystroglycan is composed of two subunits; β-dystroglycan (transmembrane) and α-dystroglycan (peripheral membrane protein), the latter of which binds to several extracellular matrix proteins including laminin, perlecan and agrin. α-Dystroglycan is extensively glycosylated and mutations in at least 8 known genes have so far been shown to be associated with defective dystroglycan processing and a phenotype now commonly referred to as a dystroglycanopathy. Mutations in one of these genes, fukutin related protein or FKRP is associated with a wide clinical spectrum, the most severe of which includes Walker Warburg Syndrome and Muscle Eye Brain disease both of which are associated with eye abnormalities. To further investigate the underlying basis for this we used a FKRP knock-down mouse model to examine the pattern of α-dystroglycan glycosylation and associated basement membrane proteins at the inner limiting membrane (ILM) from E12.5 up until birth (P0). We demonstrate that the glycosylation of α-dystroglycan is developmentally regulated and that glycosylation defects in the FKRP knock-down mouse is associated with breaches in the ILM during development.

Liver receptor homolog-1 is critical for adequate up-regulation of Cyp7a1 gene transcription and bile salt synthesis during bile salt sequestration

Liver receptor homolog-1 (LRH-1) is a nuclear receptor that controls a variety of metabolic pathways. In cultured cells, LRH-1 induces the expression of CYP7A1 and CYP8B1, key enzymes in bile salt synthesis. However, hepatic Cyp7a1 mRNA levels were not reduced upon hepatocyte-specific Lrh-1 deletion in mice. The reason for this apparent paradox has remained elusive. We describe a novel conditional whole-body Lrh-1 knockdown (LRH-1-KD) mouse model to evaluate the dependency of bile salt synthesis and composition on LRH-1. Surprisingly, Cyp7a1 expression was increased rather than decreased under chow-fed conditions in LRH-1-KD mice. This coincided with a significant reduction in expression of intestinal Fgf15, a suppressor of Cyp7a1 expression, and a 58% increase in bile salt synthesis. However, when fecal bile salt loss was stimulated by feeding the bile salt sequestrant colesevelam, Cyp7a1 expression was up-regulated in wildtype mice but not in LRH-1-KD mice (+593% in wildtype versus +9% in LRH-1-KD). This translated into an increase in bile salt synthesis of +272% in wildtype versus +21% in LRH-1-KD mice. Our data provide mechanistic insight into a missing link in the maintenance of bile salt homeostasis during enhanced fecal loss and support the view that LRH-1 controls Cyp7a1 expression from two distinct sites, i.e., liver and ileum, in the enterohepatic circulation.

Characterization of a Transgenic Short Hairpin RNA-Induced Murine Model of Tafazzin Deficiency

Barth's syndrome (BTHS) is an X-linked mitochondrial disease that is due to a mutation in the Tafazzin (TAZ) gene. Based on sequence homology, TAZ has been characterized as an acyltransferase involved in the metabolism of cardiolipin (CL), a unique phospholipid almost exclusively located in the mitochondrial inner membrane. Yeast, Drosophila, and zebrafish models have been invaluable in elucidating the role of TAZ in BTHS, but until recently a mammalian model to study the disease has been lacking. Based on in vitro evidence of RNA-mediated TAZ depletion, an inducible short hairpin RNA (shRNA)-mediated TAZ knockdown (TAZKD) mouse model has been developed (TaconicArtemis GmbH, Cologne, Germany), and herein we describe the assessment of this mouse line as a model of BTHS. Upon induction of the TAZ-specific shRNA in vivo, transgenic mouse TAZ mRNA levels were reduced by >89% in cardiac and skeletal muscle. TAZ deficiency led to the absence of tetralineoyl-CL and accumulation of monolyso-CL in cardiac muscle. Furthermore, mitochondrial morphology from cardiac and skeletal muscle was altered. Skeletal muscle mitochondria demonstrated disrupted cristae, and cardiac mitochondria were significantly enlarged and displace neighboring myofibrils. Physiological measurements demonstrated a reduction in isometric contractile strength of the soleus and a reduction in cardiac left ventricular ejection fraction of TAZKD mice compared with control animals. Therefore, the inducible TAZ-deficient model exhibits some of the molecular and clinical characteristics of BTHS patients and may ultimately help to improve our understanding of BTHS-related cardioskeletal myopathy as well as serve as an important tool in developing therapeutic strategies for BTHS.

Hepatocyte nuclear factor 4 alpha (HNF4α) knockdown stimulates pro‐mitogenic gene expression in hepatocytes

HNF4α, the master regulator of hepatocyte differentiation, has been recently shown to inhibit hepatocyte proliferation via yet to be identified mechanisms. We investigated the mechanisms of HNF4α-induced inhibition of hepatocyte proliferation using two novel HNF4α knockdown mouse models. HNF4α was deleted in adult livers using inducible cre-lox technology. Deletion of HNF4α resulted in increased hepatocyte proliferation confirmed by PCNA staining. Comparative global gene expression analysis revealed a set of genes commonly regulated in both HNF4α deletion models. Majority of the down-regulated genes were previously known HNF4α target genes involved in hepatic differentiation. Interestingly, majority of the 500+ up-regulated genes were associated with cell proliferation and cancer. Real Time PCR analysis further confirmed up-regulation of select genes including Egr-1, various Cyclins, Aurora Kinase A, BIRC5, Cdc20, Cdca3, Ect2, Eid1, and Polo-like Kinase 1. A combined gene expression-ChIP-seq analysis revealed a set of 32 HNF4α-regulated pro-mitogenic genes that are repressed in adult hepatocytes. Our data indicate that HNF4α inhibits hepatocyte proliferation by repression of specific pro-mitogenic genes. These data highlight the role of HNF4α in regulation of hepatocyte proliferation with implications in liver homeostasis and cancer pathogenesis.

Identification of a functional SNP in an asthma gene: IL1RL1

Background Our aim is to identify causal variants for the IL1RL1 gene previously associated with asthma and related phenotypes as well as perform functional assays to uncover the mechanism underlying its involvement in the disease pathogenesis. IL1RL1 has been shown to be sufficient to induce experimental allergic airway inflammation using transgenic and knockdown mouse models. Its expression has been shown to increase in murine and human asthmatic lungs; the ligand for IL1RL1 is Interleukin-33 (IL33). The signaling cascade resulting from the binding of TSLP and IL33 is crucial in eosinophilic inflammation characteristic of asthma. The IL1RL1 gene lies in chromosome 2 in the midst of a cytokine gene cluster with IL1R1, IL1RL2, IL18R1 and IL18RAP: all encoding for proteins involved in the immune response characteristic of asthma. The region is in relatively high linkage disequilibrium, thus an excellent candidate for narrowing down the asthma association signal to one or more causal SNPs.

Loss of Maternal ATRX Results in Centromere Instability and Aneuploidy in the Mammalian Oocyte and Pre-Implantation Embryo

The α-thalassemia/mental retardation X-linked protein (ATRX) is a chromatin-remodeling factor known to regulate DNA methylation at repetitive sequences of the human genome. We have previously demonstrated that ATRX binds to pericentric heterochromatin domains in mouse oocytes at the metaphase II stage where it is involved in mediating chromosome alignment at the meiotic spindle. However, the role of ATRX in the functional differentiation of chromatin structure during meiosis is not known. To test ATRX function in the germ line, we developed an oocyte-specific transgenic RNAi knockdown mouse model. Our results demonstrate that ATRX is required for heterochromatin formation and maintenance of chromosome stability during meiosis. During prophase I arrest, ATRX is necessary to recruit the transcriptional regulator DAXX (death domain associated protein) to pericentric heterochromatin. At the metaphase II stage, transgenic ATRX-RNAi oocytes exhibit abnormal chromosome morphology associated with reduced phosphorylation of histone 3 at serine 10 as well as chromosome segregation defects leading to aneuploidy and severely reduced fertility. Notably, a large proportion of ATRX-depleted oocytes and 1-cell stage embryos exhibit chromosome fragments and centromeric DNA–containing micronuclei. Our results provide novel evidence indicating that ATRX is required for centromere stability and the epigenetic control of heterochromatin function during meiosis and the transition to the first mitosis.

Knock-down of PQBP1 impairs anxiety-related cognition in mouse

PQBP1 (polyglutamine tract-binding protein 1) is a causative gene for a relatively frequent X-linked syndromic and non-syndromic mental retardation (MR). To analyze behavioral abnormalities of these patients from molecular basis, we developed a knock-down (KD) mouse model. The KD mice possess a transgene expressing 498 bp double-strand RNA that is endogenously cleaved to siRNA suppressing PQBP1 efficiently. After confirming that PQBP1 is selectively suppressed to nearly 50% of the control mice, we performed behavioral analyses of PQBP1-KD mice. The KD mice possessed normal ability in ordinary memory tests including water-maze test, whereas they showed abnormal anxiety-related behavior in light/dark exploration test and open-field test and showed obvious declines of anxiety-related cognition in the repetitive elevated plus maze or novel object recognition test. Correspondingly, we found c-fos upregulation and histone H3 acetylation after behavior tests were declined in neurons of amygdala, prefrontal cortex and hippocampus. Furthermore, we found that 4-phenylbutyric acid, an HDAC inhibitor, efficiently improved expression of these genes and rescued the abnormal phenotypes in adult PQBP1-KD mice. These results suggested that PQBP1 dysfunction in regulating gene expression might underlie the abnormal behavior and cognition of PQBP1-KD mice and that the recovery of expression of such PQBP1 target genes might improve the symptoms in adult patients.

G.P.2.10 Characterisation of the brain and eye phenotype of a FKRP knock-down mouse model of Muscle–Eye–Brain disease

G.P.2.10 Characterisation of the brain and eye phenotype of a FKRP knock-down mouse model of Muscle–Eye–Brain disease

P63 Induces Key Target Genes Required for Epidermal Morphogenesis

Epidermal morphogenesis is controlled by p63, a transcription factor that can be expressed as multiple isoforms. Of these, ΔNp63 isoforms are the predominantly expressed isoforms in late embryonic and postnatal epidermis. To determine the role of ΔNp63 proteins, we generated an epidermal‐specific inducible ΔNp63 knockdown mouse model. We found that downregulating ΔNp63 expression caused severe epidermal defects, including aberrant keratinocyte differentiation and impaired basement membrane formation, culminating in the development of severe skin erosions. Interestingly, these lesions were indistinguishable from lesions that develop in patients with AEC, a skin fragility disorder caused by mutations in ΔNp63α. We found that, during epidermal morphogenesis, ΔNp63α initially induces expression of Fras1, which is required for maintaining the integrity of the epidermal‐dermal interface at the basement membrane. Subsequently, ΔNp63α initiates epidermal terminal differentiation by inducing IKKα. Together, our data provide novel insights into the role of ΔNp63α in epidermal morphogenesis and homeostasis, and may contribute to our understanding of the pathogenic mechanisms underlying disorders caused by p63 mutations.This work was supported by grants from the NIH and the National Foundation for Ectodermal Dysplasias.

P63 induces key target genes required for epidermal morphogenesis

Mice lacking p63, a single gene that encodes a group of transcription factors that either contain (TA) or lack (DeltaN) a transactivation domain, fail to develop stratified epithelia as well as epithelial appendages and limbs. DeltaNp63 isoforms are predominantly expressed during late embryonic and postnatal epidermal development, however, the function of these proteins remains elusive. Using an epidermal-specific inducible knockdown mouse model, we demonstrate that DeltaNp63 proteins are essential for maintaining basement membrane integrity and terminal differentiation of keratinocytes. Furthermore, we have identified two DeltaNp63alpha target genes that mediate these processes. We propose that DeltaNp63alpha initially induces expression of the extracellular matrix component Fras1, which is required for maintaining the integrity of the epidermal-dermal interface at the basement membrane. Subsequently, induction of IkappaB kinase-alpha by DeltaNp63alpha initiates epidermal terminal differentiation resulting in the formation of the spinous layer. Our data provide insights into the role of DeltaNp63alpha in epidermal morphogenesis and homeostasis, and may contribute to our understanding of the pathogenic mechanisms underlying disorders caused by p63 mutations.

Interval Timing and Genomics: What Makes Mutant Mice Tick?

Mice can be shown to process temporal information as if they use an internal stopwatch that can be run, stopped, and reset on command and whose speed of “ticking” is adjustable. In addition, interval-timing behavior can be separated into clock, memory, and decision stages of information processing such that one stage can be modified without changing the others. In order to demonstrate the efficacy of interval-timing procedures in the evaluation of behavioral phenotypes, proline transporter (PROT) deficient mice (+/+, +/-, and -/-) were assessed for motor control (Rotarod beam), spatial memory (Morris water-maze), and temporal generalization (peak-interval procedure) competency. The findings demonstrate that interval-timing procedures can be profitably integrated into a behavioral battery and used to selectively diagnose the psychological abnormalities associated with transgenic, knock-out, and knock-down mouse models of human diseases.