Expression Studies In Mice Research Articles

The mouse Gene Expression Database (GXD): 2021 update

The Gene Expression Database (GXD; www.informatics.jax.org/expression.shtml) is an extensive and well-curated community resource of mouse developmental gene expression information. For many years, GXD has collected and integrated data from RNA in situ hybridization, immunohistochemistry, RT-PCR, northern blot, and western blot experiments through curation of the scientific literature and by collaborations with large-scale expression projects. Since our last report in 2019, we have continued to acquire these classical types of expression data; developed a searchable index of RNA-Seq and microarray experiments that allows users to quickly and reliably find specific mouse expression studies in ArrayExpress (https://www.ebi.ac.uk/arrayexpress/) and GEO (https://www.ncbi.nlm.nih.gov/geo/); and expanded GXD to include RNA-Seq data. Uniformly processed RNA-Seq data are imported from the EBI Expression Atlas and then integrated with the other types of expression data in GXD, and with the genetic, functional, phenotypic and disease-related information in Mouse Genome Informatics (MGI). This integration has made the RNA-Seq data accessible via GXD’s enhanced searching and filtering capabilities. Further, we have embedded the Morpheus heat map utility into the GXD user interface to provide additional tools for display and analysis of RNA-Seq data, including heat map visualization, sorting, filtering, hierarchical clustering, nearest neighbors analysis and visual enrichment.

GXD's RNA-Seq and Microarray Experiment Search: using curated metadata to reliably find mouse expression studies of interest.

The Gene Expression Database (GXD), an extensive community resource of curated expression information for the mouse, has developed an RNA-Seq and Microarray Experiment Search (http://www.informatics.jax.org/gxd/htexp_index). This tool allows users to quickly and reliably find specific experiments in ArrayExpress and the Gene Expression Omnibus (GEO) that study endogenous gene expression in wild-type and mutant mice. Standardized metadata annotations, curated by GXD, allow users to specify the anatomical structure, developmental stage, mutated gene, strain and sex of samples of interest, as well as the study type and key parameters of the experiment. These searches, powered by controlled vocabularies and ontologies, can be combined with free text searching of experiment titles and descriptions. Search result summaries include link-outs to ArrayExpress and GEO, providing easy access to the expression data itself. Links to the PubMed entries for accompanying publications are also included. More information about this tool and GXD can be found at the GXD home page (http://www.informatics.jax.org/expression.shtml). Database URL: http://www.informatics.jax.org/expression.shtml

Genetic Variants in HSD17B3, SMAD3, and IPO11 Impact Circulating Lipids in Response to Fenofibrate in Individuals With Type 2 Diabetes.

Individuals with type 2 diabetes (T2D) and dyslipidemia are at an increased risk of cardiovascular disease. Fibrates are a class of drugs prescribed to treat dyslipidemia, but variation in response has been observed. To evaluate common and rare genetic variants that impact lipid responses to fenofibrate in statin-treated patients with T2D, we examined lipid changes in response to fenofibrate therapy using a genomewide association study (GWAS). Associations were followed-up using gene expression studies in mice. Common variants in SMAD3 and IPO11 were marginally associated with lipid changes in black subjects (P < 5 × 10-6 ). Rare variant and gene expression changes were assessed using a false discovery rate approach. AKR7A3 and HSD17B13 were associated with lipid changes in white subjects (q < 0.2). Mice fed fenofibrate displayed reductions in Hsd17b13 gene expression (q < 0.1). Associations of variants in SMAD3, IPO11, and HSD17B13, with gene expression changes in mice indicate that transforming growth factor-beta (TGF-β) and NRF2 signaling pathways may influence fenofibrate effects on dyslipidemia in patients with T2D.

Epidermal growth factor receptor restoration rescues the fatty liver regeneration in mice.

Hepatic steatosis is a common histological finding in obese patients. Even mild steatosis is associated with delayed hepatic regeneration and poor outcomes following liver resection or transplantation. We sought to identify and target molecular pathways that mediate this dysfunction. Lean mice and mice made obese through feeding of a high-fat, hypercaloric diet underwent 70 or 80% hepatectomy. After 70% resection, obese mice demonstrated 100% survival but experienced increased liver injury, reduced energy stores, reduced mitoses, increased necroapoptosis, and delayed recovery of liver mass. Increasing liver resection to 80% was associated with mortality of 40% in lean and 80% in obese mice (P < 0.05). Gene expression profiling showed decreased epidermal growth factor receptor (EGFR) in fatty liver. Meta-analysis of expression studies in mice, rats, and patients also demonstrated reduction of EGFR in fatty liver. In mice, both EGFR and phosphorylated EGFR decreased with increasing percent body fat. Hydrodynamic transfection of EGFR plasmids in mice corrected fatty liver regeneration, reducing liver injury, increasing proliferation, and improving survival after 80% resection. Loss of EGFR expression is rate limiting for liver regeneration in obesity. Therapies directed at increasing EGFR in steatosis might promote liver regeneration and survival following hepatic resection or transplantation.

The Beta Subunit of Hemoglobin (HBB2/HBB) Suppresses Neuroblastoma Growth and Metastasis.

Soluble pulmonary factors have been reported to be capable of inhibiting the viability of cancer cells that metastasize to the lung, but the molecular identity was obscure. Here we report the isolation and characterization of the beta subunit of hemoglobin as a lung-derived antimetastatic factor. Peptide mapping in the beta subunit of human hemoglobin (HBB) defined a short C-terminal region (termed Metox) as responsible for activity. In tissue culture, both HBB and murine HBB2 mediated growth arrest and apoptosis of lung-metastasizing neuroblastoma cells, along with a variety of other human cancer cell lines. Metox acted similarly and its administration in human tumor xenograft models limited the development of adrenal neuroblastoma tumors as well as spontaneous lung and bone marrow metastases. Expression studies in mice indicated that HBB2 is produced by alveolar epithelial and endothelial cells and is upregulated in mice bearing undetectable metastasis. Our work suggested a novel function for HBB as a theranostic molecule: an innate antimetastasis factor with potential utility as an anticancer drug and a biomarker signaling the presence of clinically undetectable metastasis. Cancer Res; 77(1); 14-26. ©2016 AACR.

Genetics of Congenital Heart Defects: The NKX2-5 Gene, a Key Player.

Congenital heart defects (CHDs) represent the biggest fraction of morbid congenital anomalies worldwide. Owing to their complex inheritance patterns and multifactorial etiologies, these defects are difficult to identify before complete manifestation. Research over the past two decades has established firmly the role of genetics in the development of these congenital defects. While syndromic CHDs are more straightforward, non-syndromic CHDs are usually characterized by multiple mutations that affect intricate inter-connected developmental pathways. Knock-out and gene expression studies in mice and other genetic models have been performed to elucidate the roles of these implicated genes. Functional analysis has not been able to resolve the complete picture, as increasingly more downstream effects are continuously being assigned to CHD mutant factors. NKX2-5, a cardiac transcription factor, has received much attention for its role in cardiac dysmorphogenesis. Approximately 50 different mutations in this gene have been identified to date, and only a few have been functionally characterized. The mutant NKX2-5 factor can regulate a number of off-targets downstream to facilitate CHD development. This review summarizes the genetic etiology of congenital heart defects and emphasizes the need for NKX2-5 mutation screening.

Deletion of the miR-143/145 Cluster Leads to Hydronephrosis in Mice

Obstructive nephropathy, the leading cause of kidney failure in children, can be anatomic or functional. The underlying causes of functional hydronephrosis are not well understood. miRNAs, which are small noncoding RNAs, regulate gene expression at the post-transcriptional level. We found that miR-145-5p, a member of the miR-143/145 cluster that is highly expressed in smooth muscle cells of the renal vasculature, was present in the pelvicalyceal system and the ureter. To evaluate whether the miR-143/145 cluster is involved in urinary tract function we performed morphologic, functional, and gene expression studies in mice carrying a whole-body deletion of miR-143/145. miR-143/145-deficient mice developed hydronephrosis, characterized by severe papillary atrophy and dilatation of the pelvicalyceal system without obvious physical obstruction. Moreover, mutant mice showed abnormal ureteral peristalsis. The number of ureter contractions was significantly higher in miR-143/145-deficient mice. Peristalsis was replaced by incomplete, short, and more frequent contractions that failed to completely propagate in a proximal-distal direction. Microarray analysis showed 108 differentially expressed genes in ureters of miR-143/145-deficient mice. Ninety genes were up-regulated and 18 genes were down-regulated, including genes with potential regulatory roles in smooth muscle contraction and extracellular matrix-receptor interaction. We show that miR-143/145 are important for the normal peristalsis of the ureter and report an association between the expression of these miRNAs and hydronephrosis.

Genome-wide mapping of copy number variations in patients with both anorectal malformations and central nervous system abnormalities.

Anorectal malformations (ARM) have a prevalence of around 1 in 2500 live births. In around 50% of patients, the malformation is isolated, while in the remainder it arises within the context of complex genetic abnormalities or a defined genetic syndrome. Recent studies have implicated rare copy number variations (CNVs) in both isolated and nonisolated ARM, and identified plausible candidate genes. In the present study, array-based molecular karyotyping was performed to identify causative CNVs in 32 sporadic ARM patients with comorbid abnormalities of the central nervous system (CNS). This phenotype was selected to enrich for rare CNVs, since previous research has implicated rare CNVs in both CNS abnormalities and ARM. In five patients, a probable disease-causing CNV was identified (del6q14.3q16.3, del14q32.2, del17q12q21.2, and two patients with del22q11.21). In three of these patients, the CNVs were de novo. For the remaining two patients, no parental DNA was available. Deletions at 22q11.21 and 6q14.3 have been associated with both CNS abnormalities and ARM. In contrast, deletions at 14q32.2 have only been described in patients with CNS abnormalities, and the del17q12q21.2 is a novel CNV. Expression studies in mice suggest that NEUROD2 and RARA, which reside within the newly identified del17q12q21.2 region, are candidate genes for the formation of microcephaly and ARM. The present data suggest that CNVs are a frequent cause of the ARM with CNS abnormalities phenotype, and that array-analysis is indicated in such patients.

De novo microduplications at 1q41, 2q37.3, and 8q24.3 in patients with VATER/VACTERL association

The acronym VATER/VACTERL association describes the combination of at least three of the following congenital anomalies: vertebral defects (V), anorectal malformations (A), cardiac defects (C), tracheoesophageal fistula with or without esophageal atresia (TE), renal malformations (R), and limb defects (L). We aimed to identify highly penetrant de novo copy number variations (CNVs) that contribute to VATER/VACTERL association. Array-based molecular karyotyping was performed in a cohort of 41 patients with VATER/VACTERL association and 6 patients with VATER/VACTERL-like phenotype including all of the patients' parents. Three de novo CNVs were identified involving chromosomal regions 1q41, 2q37.3, and 8q24.3 comprising one (SPATA17), two (CAPN10, GPR35), and three (EPPK1, PLEC, PARP10) genes, respectively. Pre-existing data from the literature prompted us to choose GPR35 and EPPK1 for mouse expression studies. Based on these studies, we prioritized GPR35 for sequencing analysis in an extended cohort of 192 patients with VATER/VACTERL association and VATER/VACTERL-like phenotype. Although no disease-causing mutation was identified, our mouse expression studies suggest GPR35 to be involved in the development of the VATER/VACTERL phenotype. Follow-up of GPR35 and the other genes comprising the identified duplications is warranted.

Abstract 2607: A GWAS From The Vitamin Intervention For Stroke Prevention (VISP) Clinical Trial Identifies Variants That Contribute To Recurrent Stroke And Combined Vascular Endpoints

We have performed a genome-wide association study (GWAS), using the Illumina Omni 1M SNP platform, in 2,100 stroke patients from the Vitamin Intervention for Stroke Prevention (VISP) trial. VISP was a controlled, double blinded clinical trial, designed to determine whether the daily intake of high dose folic acid, vitamins B6 and B12 reduce recurrent cerebral infarction. Extensive quality control (QC) measures were performed, resulting in a total of 737,081 SNPs for analysis. Principal Component Analysis (PCA), implemented in KING, was utilized to address confounders due to population substructure. We performed genome wide association (survival) analyses for recurrent stroke and combined vascular endpoints (stroke, MI, and CHD death), using stratified Cox model. Clinic sites were treated as strata and age, sex, treatment arm, and the top 10 principal components were used as covariates for these analyses. There is no inflation in our GWAS scan results, with GC lambda values of 1.008 and 0.995 for recurrent stroke and combined vascular endpoints, respectively. For recurrent stroke, we observed association with two SNPs that reached genome wide significance (≤10 -8 ). Both SNPs were located in a gene sparse region of chromosome 1p31 (P=1.73×10 -8 ; beta=-0.64; P=3.43×10 -8 ; beta=-0.62). Two additional SNPs approached genome wide significance, one located in the same 1p31 region (P=7.60×10 -7 ) and the second located in the GMPR gene (P=8.69×10 -7 ). Variants in the GMPR gene region have been associated with LDL-cholesterol measures in previous GWAS. While no SNPs reached genome wide significance for combined vascular endpoints, six SNPs approached genome wide significance (P values ranging from 9.51×10 -6 to 2.56×10 -7 ). These variants are located in genes that have implications in vascular disease. In particular, GPR37L1 has been identified as a potential target for heart failure in mouse expression studies while NSMAF may play a role in regulating TNF-induced cellular responses (e.g. inflammation). Additionally, ILDR2 is located in a region that has shown previous linkage for hypertension, suggesting potential biological implications for these variants in relation to stroke and vascular disease. Our GWAS scans represent the first large scale genetic association study of recurrent stroke and combined vascular events. Our findings have identified novel associations for genes that contribute to recurrent stroke and composite vascular endpoints.

Mutations in KAT6B, Encoding a Histone Acetyltransferase, Cause Genitopatellar Syndrome

Genitopatellar syndrome (GPS) is a skeletal dysplasia with cerebral and genital anomalies for which the molecular basis has not yet been determined. By exome sequencing, we found de novo heterozygous truncating mutations in KAT6B (lysine acetyltransferase 6B, formerly known as MYST4 and MORF) in three subjects; then by Sanger sequencing of KAT6B, we found similar mutations in three additional subjects. The mutant transcripts do not undergo nonsense-mediated decay in cells from subjects with GPS. In addition, human pathological analyses and mouse expression studies point to systemic roles of KAT6B in controlling organismal growth and development. Myst4 (the mouse orthologous gene) is expressed in mouse tissues corresponding to those affected by GPS. Phenotypic differences and similarities between GPS, the Say-Barber-Biesecker variant of Ohdo syndrome (caused by different mutations of KAT6B), and Rubinstein-Taybi syndrome (caused by mutations in other histone acetyltransferases) are discussed. Together, the data support an epigenetic dysregulation of the limb, brain, and genital developmental programs.

Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells

The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.

Gellan Gum-PEI Nanocomposites as Efficient Gene Delivery Agents

Of the non-viral vectors, a cationic polymer like PEI is an attractive candidate which however, has been negatively impacted due to its marked toxicity. An anionic sugar polymer gelan gum (GG) has been introduced into PEI system to increase transfection efficiency with minimal toxicity. We showed that one of the synthesized (GP1-GP6) GG-PEI nanocomposites (NCs), GP3, exhibited negligible toxicity in in vitro (primary keratinocytes, HEK293, HeLa and HepG2 cells) and in vivo (Drosophila melanogaster) as compared to PEI or lipofectamin. GP3-pDNA complex was found to be transfected efficiently in the above cells as confirmed by FACS analysis (72.0 + 5.5%) while lipofectamine showed only 12.4 + 3.5% efficiency. GP3 mediated GFP specific siRNA delivery resulted in the knockdown of the GFP expression by approximately 77% and JNK (60%). In vivo gene expression studies in mice revealed reporter gene expression in spleen. The study demonstrates that GG blended PEI NCs hold promise for future applications in gene delivery both in vitro and in vivo.

Secreted frizzled related protein 1 is a paracrine modulator of epithelial branching morphogenesis, proliferation, and secretory gene expression in the prostate: Joesting MS, Cheever TR, Volzing KG, Yamaguchi TP, Wolf V, Naf D, Rubin JS, Marker PC, Department of Genetics, Cellular Biology, and Development, University of Minnesota, Minneapolis, MN

Previous in vitro studies identified secreted frizzled related protein 1 (SFRP1) as a candidate pro-proliferative signal during prostatic development and cancer progression. This study determined the in vivo roles of SFRP1 in the prostate using expression studies in mice and by creating loss- and gain-of-function mouse genetic models. Expression studies using an Sfrp1(lacZ) knock-in allele showed that Sfrp1 is expressed in the developing mesenchyme/stroma of the prostate. Nevertheless, Sfrp1 null prostates exhibited multiple prostatic developmental defects in the epithelium, including reduced branching morphogenesis, delayed proliferation, and increased expression of genes encoding prostate-specific secretory proteins. Interestingly, over-expression of SFRP1 in the adult prostates of transgenic mice yielded opposite effects, including prolonged epithelial proliferation and decreased expression of genes encoding secretory proteins. These data demonstrated a previously unrecognized role for Sfrp1 as a stromal-to-epithelial paracrine modulator of epithelial growth, branching morphogenesis, and epithelial gene expression. To clarify the mechanism of SFRP1 action in the prostate, the response of Wnt signaling pathways to SFRP1 was examined. Forced expression of SFRP1 in prostatic epithelial cells did not alter canonical Wnt/β-catenin signaling or the activation of CamKII. However, forced expression of SFRP1 led to sustained activation of JNK, and inhibition of JNK activity blocked the SFRP1-induced proliferation of prostatic epithelial cells, suggesting that SFRP1 acts through the noncanonical Wnt/JNK pathway in the prostate.

Alterations in Fos-Related Antigen 2 and σ1 Receptor Gene and Protein Expression Are Associated with the Development of Cocaine-Induced Behavioral Sensitization: Time Course and Regional Distribution Studies

Repeated exposure to cocaine results in neuroadaptations that can alter the way the brain responds to subsequent stimuli. Earlier studies demonstrated that acute administration of cocaine up-regulates the immediate-early gene fos-related antigen 2 (fra-2) followed by a later up-regulation of sigma(1) receptor gene and protein levels in brain regions involved in addiction and reward. To test whether such alterations could have long-term consequences on behavior, the present study was undertaken. Using a cocaine-induced behavioral sensitization model coupled with gene and protein expression studies in mice, the results show that cocaine induces the expression of fra-2, which leads to a progressive increase in sigma(1) receptor gene and protein expression over a period of days. This progressive increase in sigma(1) expression corresponds to the steady increase in the locomotor response to repeated cocaine administration in mice. The cocaine-induced changes in fra-2 and sigma(1) receptor gene and protein expression occur in brain regions that subserve drug abuse, such as the cortex, striatum, and hippocampus, but not the cerebellum. Moreover, the prototypic sigma(1) receptor antagonist 1-[2-(3,4-dichloropheny)ethyl]-4-methylpiperazine (BD1063) significantly attenuates both the molecular adaptations and behavioral sensitization induced by cocaine. These data suggest that repeated exposure to cocaine elicits alterations in fra-2 and sigma(1) receptor-mediated mechanisms, which ultimately manifest as altered behavioral responses to cocaine.

Secreted frizzled related protein 1 is a paracrine modulator of epithelial branching morphogenesis, proliferation, and secretory gene expression in the prostate

Previous in vitro studies identified secreted frizzled related protein 1 (SFRP1) as a candidate pro-proliferative signal during prostatic development and cancer progression. This study determined the in vivo roles of SFRP1 in the prostate using expression studies in mice and by creating loss- and gain-of-function mouse genetic models. Expression studies using an Sfrp1lacZ knock-in allele showed that Sfrp1 is expressed in the developing mesenchyme/stroma of the prostate. Nevertheless, Sfrp1 null prostates exhibited multiple prostatic developmental defects in the epithelium including reduced branching morphogenesis, delayed proliferation, and increased expression of genes encoding prostate-specific secretory proteins. Interestingly, over-expression of SFRP1 in the adult prostates of transgenic mice yielded opposite effects including prolonged epithelial proliferation and decreased expression of genes encoding secretory proteins. These data demonstrated a previously unrecognized role for Sfrp1 as a stromal-to-epithelial paracrine modulator of epithelial growth, branching morphogenesis, and epithelial gene expression. To clarify the mechanism of SFRP1 action in the prostate, the response of WNT signaling pathways to SFRP1 was examined. Forced expression of SFRP1 in prostatic epithelial cells did not alter canonical WNT/β-catenin signaling or the activation of CamKII. However, forced expression of SFRP1 led to sustained activation of JNK, and inhibition of JNK activity blocked the SFRP1-induced proliferation of prostatic epithelial cells, suggesting that SFRP1 acts through the non-canonical WNT/JNK pathway in the prostate.

166. Modulation of Wnt Pathway Signaling Determines Ectopic Craniofacial Fusion and Orofacial Clefting

Introduction: The etiology of facial clefting is a complex synthesis of multiple environmental and genetic factors. Therefore few inroads have been made into the design or development of targeted preventions or treatments for these birth defects. The identification of a developmental signaling pathway that definitively regulates facial fusion would be a crucial first step. Previous gene expression studies in mice have demonstrated that Wnt genes are expressed in the right time and place to be critical for facial fusion. Studies of mouse strains prone to facial clefting have identified causative mutations in the Wnt9b locus. This study examines for the first time how the experimental manipulation of Wnt signaling at the pathway level influences the normal events of craniofacial morphogenesis. Here we use two animal models in which the early events of facial development are remarkably conserved, combining mouse transgenetics with in ovo viral-mediated gene transfer in developing chick embryos. Methods: Wild type (CD1) or Wnt reporter (BATGAL) mouse embryos and normal chicken embryos representing the normal course of craniofacial outgrowth and fusion were collected at E10.5-E13.5 (mouse) and St. 25-28 (E5-E7; chick). Gene expression was detected by non-radioactive in situ hybridization or X-gal detection of beta-galactosidase transgene activity in either whole-mount or sectioned embryos. To achieve viral-mediated gene transfer in chick embryos, adenovirus encoding the soluble Wnt inhibitor Dkk1 or retrovirus encoding Wnt3a ligand were delivered in ovo to the maxillary prominence (MXP) by microinjection. Results: in mouse and chick embryos, Wnt7b and Wnt9b ligand gene expression was found to localize to the surface ectoderm of fusing facial prominences. In BATGAL mice, Wnt-responsive cells were found in the surface ectoderm of the fusing lateral and frontonasal promences, as well as in the underlying mesenchyme of the developing facial prominences. To determine whether repression of Wnt pathway activity prevented normal facial fusion, Ad-Dkk1 or Ad-Fc (as a control) was injected into the developing maxillary prominence at St. 25 (E5.5). After 36 hours of incubation, facial clefts were visible in embryos treated with Ad-Dkk1, whereas the face had fused normally in controls. To assess whether exogenous Wnt pathway activation might induce ectopic fusion of craniofacial tissues, RCAS-Wnt3a or RCAS-AP (alkaline phosphatase) was injected into the developing maxillary facial prominence at St. 23 (E4). After 36 hours of incubation, the injected maxillary prominences of RCAS-Wnt3a treated embryos had grown out and fused to the frontonasal prominence, whereas the contralateral, uninjected maxillary facial prominence and the RCAS-AP injected MXP had not yet fused. Conclusions: Our gain- and loss-of-function data provide strong evidence that normal Wnt pathway activity is necessary for physiologic facial fusion, and that exogenous activation of the Wnt pathway by administration of the ligand is sufficient to induce ectopic fusion events within the craniofacial complex. Further studies using this model will determine how regulation of the Wnt pathway influence epithelial seam cell fate decisions during facial fusion, such as proliferation rates, apoptosis, migration, and epithelial-mesenchymal transformations. Finally, our model suggests that therapeutic modulation of a cell signaling pathway may be a viable strategy for in utero treatment of facial clefts.

Markers of prostate region-specific epithelial identity define anatomical locations in the mouse prostate that are molecularly similar to human prostate cancers

Although the basic functions of the prostate gland are conserved among mammals, its morphology varies greatly among species. Comparative studies between mouse and human are important because mice are widely used to study prostate cancer, a disease that occurs in a region-restricted manner within the human prostate. An informatics-based approach was used to identify prostate-specific human genes as candidate markers of region-specific identity that might distinguish prostatic ducts prone to prostate cancer from ducts that rarely give rise to cancer. Subsequent analysis of normal and cancerous human prostates demonstrated that the genes microseminoprotein-β ( MSMB) and transglutaminase 4 ( TGM4) were expressed in distinct groups of ducts in the normal human prostate, and only MSMB was detected in areas of prostate cancer. The mouse orthologs of MSMB and TGM4 were then used for expression studies in mice along with the mouse ventrally expressed gene spermine binding protein ( SBP). All three genes were informative markers of region-specific epithelial identity with distinct expression patterns that collectively accounted for all ducts in the mouse prostate. Together with the human data, this suggested that MSMB expression defines an anatomical domain in the mouse prostate that is molecularly most similar to human prostate cancers. Computer-assisted serial section reconstruction was used to visualize the complete expression domains for MSMB, SBP, and TGM4 in the mouse prostate. This showed that MSMB is expressed in prostatic ducts that comprise 21% of the mouse dorso-lateral prostate. Finally, the expression of MSMB, SBP, and TGM4 was evaluated in a mouse prostate cancer model created by the prostate epithelium-specific deletion of the tumor suppressor PTEN. MSMB and TGM4 were rapidly and dramatically down-regulated in response to PTEN deletion suggesting that this model of prostate cancer includes a more rapid de-differentiation of the prostatic epithelium than is observed in organ-confined human prostate cancers.

MSX1 and Orofacial Clefting with and without Tooth Agenesis

MSX1 has been considered a strong candidate for orofacial clefting, based on mouse expression studies and knockout models, as well as association and linkage studies in humans. MSX1 mutations are also causal for hereditary tooth agenesis. We tested the hypothesis that individuals with orofacial clefting with or without tooth agenesis have MSX1 coding mutations by screening 33 individuals with cleft lip with or without cleft palate (CL/P) and 19 individuals with both orofacial clefting and tooth agenesis. Although no MSX1 coding mutations were identified, the known 101C > G variant occurred more often in subjects with both CL/P and tooth agenesis (p = 0.0008), while the *6C-T variant was found more often in CL/P subjects (p = 0.001). Coding mutations in MSX1 are not the cause of orofacial clefting with or without tooth agenesis in this study population. However, the significant association of MSX1 with both phenotypes implies that MSX1 regulatory elements may be mutated.

Human artificial chromosomes containing chromosome 17 alphoid DNA maintain an active centromere in murine cells but are not stable

Human artificial chromosomes (HACs) are autonomous molecules that can function and segregate as normal chromosomes in human cells. De novo HACs have successfully been used as gene expression vectors to complement genetic deficiencies in human cultured cells. HACs now offer the possibility of studying the regulation and expression of large genes in a variety of cell types from different tissues and correcting gene deficiencies caused by human inherited diseases. Complementary gene expression studies in mice, especially in mouse models of human genetic diseases, are also important in determining if large human transgenes can be expressed appropriately from artificial chromosomes. Toward this aim we are establishing artificial chromosomes in murine cells as novel gene expression vectors. Initially we transferred HAC vectors into murine cells, but were unable to generate de novo HACs at a reasonable frequency. We then transferred HACs previously established in human HT1080 cells to three different murine cell types by microcell fusion, followed by positive selection. We observed that the HACs in murine cells bound centromere protein C (CENP-C), a marker of active centromeres, and were detected under selection but rapidly lost when selection was removed. These results suggest that the HACs maintain at least a partially functional centromere complex in murine cells, but other factors are required for stability and segregation. Artificial chromosomes containing mouse centromeric sequences may be required for better stability and maintenance in murine cells.