Biomedical subjects

S Steven Potter

Publications and source records attributed to S Steven Potter.

At least 19 recordsLinked to original sources

PubMed2006

Comprehensive microarray analysis of Hoxa11/Hoxd11 mutant kidney development.

The Hox11 paralogous genes play critical roles in kidney development. They are expressed in the early metanephric mesenchyme and are required for the induction of ureteric bud formation and its subsequent branching morphogenesis. They are also required for the normal nephrogenesis response of the metanephric mesenchyme to inductive signals from the ureteric bud. In this report, we use microarrays to perform a comprehensive gene expression analysis of the Hoxa11/Hoxd11 mutant kidney phenotype. We examined E11.5, E12.5, E13.5 and E16.5 developmental time points. A novel high throughput strategy for validation of microarray data is described, using additional biological replicates and an independent microarray platform. The results identified 13 genes with greater than 3-fold change in expression in early mutant kidneys, including Hoxa11s, GATA6, TGFbeta2, chemokine ligand 12, angiotensin receptor like 1, cytochrome P450, cadherin5, and Lymphocyte antigen 6 complex, Iroquois 3, EST A930038C07Rik, Meox2, Prkcn, and Slc40a1. Of interest, many of these genes, and others showing lower fold expression changes, have been connected to processes that make sense in terms of the mutant phenotype, including TGFbeta signaling, iron transport, protein kinase C function, growth arrest and GDNF regulation. These results identify the multiple molecular pathways downstream of Hox11 function in the developing kidney.

Animals↗

PubMed2006

The zinc finger transcription factor Sp8 regulates the generation and diversity of olfactory bulb interneurons.

The molecular mechanisms that regulate the production and diversity of olfactory bulb interneurons remain poorly understood. With the exception of the GABAergic/dopaminergic subtype in the glomerular layer, no information exists concerning the generation of the other subtypes. Here we show that the recently identified zinc finger transcription factor Sp8 is expressed in neurogenic regions, which give rise to olfactory bulb interneurons at embryonic and postnatal time points and remains expressed in the calretinin-expressing and GABAergic/nondopaminergic interneurons of the glomerular layer. Conditional inactivation of Sp8 in the embryonic ventral telencephalon reveals a requirement for the normal generation of these interneuron subtypes. Sp8 conditional mutants exhibit an increase in cell death within the lateral ganglionic eminence and rostral migratory stream. Moreover, mutant neuroblasts/interneurons are misspecified and display abnormal migration patterns in the olfactory bulb, indicating that Sp8 contributes to olfactory bulb interneuron diversity by regulating the survival, migration, and molecular specification of neuroblasts/interneurons.

Animals↗

PubMed2005

Abnormal neurodevelopment, neurosignaling and behaviour in Npas3-deficient mice.

Npas3 is a member of the bHLH-PAS superfamily of transcription factors that is expressed broadly in the developing neuroepithelium. To study the function of this gene, mice deficient in Npas3 were generated and characterized. Npas3-/- mice were growth-retarded and exhibited developmental brain abnormalities that included a reduction in size of the anterior hippocampus, hypoplasia of the corpus callosum and enlargement of the ventricles. A number of behavioural abnormalities were identified in Npas3-/- mice including locomotor hyperactivity, subtle gait defects, impairment of prepulse inhibition of acoustic startle, deficit in recognition memory and altered anxiety-related responses. Characterization of neurosignaling pathways using several pharmacological agents revealed dysfunctional glutamate, dopamine and serotonin neurotransmitter signaling. Consistent with these findings, we identified a significant alteration in cortical PSD-95 expression, a PDZ-containing protein that has been shown to be involved in postsynaptic signal transduction. Together, our observations indicate an important role for Npas3 in controlling normal brain development and neurosignaling pathways.

Animals↗

PubMed2004

Large-scale reprogramming of cranial neural crest gene expression by retinoic acid exposure.

Although retinoic acid (RA), the active form of vitamin A, is required for normal embryonic growth and development, it is also a powerful teratogen. Infants born to mothers exposed to retinoids during pregnancy have a 25-fold increased risk for malformations, nearly exclusively of cranial neural crest-derived tissues. To characterize neural crest cell responses to RA, we exposed murine crest cultures to teratogenic levels of RA and subjected their RNA to microarray-based gene expression profile analysis using Affymetrix MG-U74Av2 GeneChips. RNAs were isolated from independent cultures treated with 10(-6) M RA for 6, 12, 24, or 48 h. Statistical analyses of gene expression profile data facilitated identification of the 205 top-ranked differentially regulated genes whose expression was reproducibly changed by RA over time. Cluster analyses of these genes across the independently treated sample series revealed distinctive kinetic patterns of altered gene expression. The largest group was transiently affected within the first 6 h of exposure, representing early responding genes. Group 2 showed sustained induction by RA over all times, whereas group 3 was characterized by the suppression of a time-dependent expression increase normally seen in untreated cells. Additional patterns demonstrated time-dependent increased or decreased expression among genes not normally regulated to a significant extent. Gene function analysis revealed that more than one-third of all RA-regulated genes were associated with developmental regulation, including both canonical and noncanonical Wnt signaling pathways. Multiple genes associated with cell adhesion and cell cycle regulation, recognized targets for the biological effects of RA, were also affected. Taken together, these results support the hypothesis that the teratogenic effects of RA derive from reprogramming gene expression of a host of genes, which play critical roles during embryonic development regulating pathways that determine subsequent differentiation of cranial neural crest cells.

Animals↗

PubMed2004

Behavioral and regulatory abnormalities in mice deficient in the NPAS1 and NPAS3 transcription factors.

Laboratory mice bearing inactivating mutations in the genes encoding the NPAS1 and NPAS3 transcription factors have been shown to exhibit a spectrum of behavioral and neurochemical abnormalities. Behavioral abnormalities included diminished startle response, as measured by prepulse inhibition, and impaired social recognition. NPAS1/NPAS3-deficient mice also exhibited stereotypic darting behavior at weaning and increased locomotor activity. Immunohistochemical staining assays showed that the NPAS1 and NPAS3 proteins are expressed in inhibitory interneurons and that the viability and anatomical distribution of these neurons are unaffected by the absence of either transcription factor. Adult brain tissues from NPAS3- and NPAS1/NPAS3-deficient mice exhibited a distinct reduction in reelin, a large, secreted protein whose expression has been reported to be attenuated in the postmortem brain tissue of patients with schizophrenia. These observations raise the possibility that a regulatory program controlled in inhibitory interneurons by the NPAS1 and NPAS3 transcription factors may be either substantively or tangentially relevant to psychosis.

Aging↗

PubMed2004

Microarray analysis of focal segmental glomerulosclerosis.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a leading cause of chronic renal failure in children. Recent studies have begun to define the molecular pathogenesis of this heterogeneous condition. Here we use oligonucleotide microarrays to obtain a global gene expression profile of kidney biopsy specimens from patients with FSGS in order to better understand the pathogenesis of this disease. METHODS: We extracted RNA from renal biopsy samples of 10 patients with the diagnosis of FSGS and from 5 control kidney samples, and produced labeled cRNA for hybridization to Affymetrix human U133A microarrays. RESULTS: We identified a gene expression fingerprint for FSGS that contained 429 of 22,283 possible genes, each with a p < 0.01, using RMA normalization, Welch t test, and at least a 1.8-fold change in 5 of the 10 patients examined. We also found gene expression differences in samples from subsets of patients who had either nephrotic syndrome or renal insufficiency. This screen identified many genes and genetic pathways that have already been implicated in the pathogenesis of FSGS. In addition, we found changes in gene expression in genetic pathways that have not been studied in FSGS. CONCLUSIONS: Oligonucleotide DNA microarray analysis of renal biopsy specimens identified a gene expression fingerprint in samples from a heterogeneous population of patients with FSGS. The genes and genetic pathways identified in this study can be compared to results of similar studies of other diseases to examine specificity and used to study the pathogenesis of FSGS.

Adolescent↗

PubMed2004

Atlas of Hox gene expression in the developing kidney.

Hox genes often play important roles in segment identity determination and organogenesis. To better understand the roles of Hox genes during kidney development, we performed an extensive analysis of their expression patterns. Section in situ hybridizations were used to define the expression of 37 Hox genes at embryonic day (E) 12.5, E13.5, E15.5, and E17.5 of kidney development. Several interesting principles emerged. First, the concept of colinearity was preserved. Hox genes from the more 3' positions in clusters were more often expressed in the ureteric bud, which is derived from the anterior of the intermediate mesoderm. Second, Hox genes were expressed throughout the ureteric bud without any segment specificity. Third, in the different segments of the forming nephron we did observe overlapping domains of Hox gene expression, which initiated distally at the junction between the nephron and ureteric bud, and extended proximally variable distances. Finally, we observed that paralogous Hox genes often showed surprisingly diverse expression patterns. Indeed, contiguous genes on a single cluster more often showed similar expression patterns than paralogs. In summary, the resulting atlas of Hox gene expression provides a foundation for further study of the overlapping functions Hox genes in the developing kidney.

Animals↗

PubMed2004

Profiling gene expression in kidney development.

Rapid determination of the expression levels of all genes can be achieved with microarrays. This technology is beginning to revolutionize the study of kidney development. Genome-wide expression studies of the developing rat, mouse and human kidneys have found thousands of expressed genes, with many hundreds showing changes in expression as a function of developmental time. The resulting gene expression profiles provide an important discovery function, identifying new genes and pathways not previously implicated in kidney organogenesis. In combination with microdissection techniques, microarrays further extend global gene expression analysis to discrete components or cell types within the kidney. The resulting detailed molecular portrait of normal kidney development provides a baseline for studies of the many mouse mutants available with abnormal kidney development.

Animals↗

PubMed2003

Sp8 is crucial for limb outgrowth and neuropore closure.

In this report we describe the developmental expression and function of Sp8, a member of the Sp family of zinc finger transcription factors, and provide evidence that the legless transgene insertional mutant is a hypomorphic allele of the Sp8 gene. Sp8 is expressed during embryogenesis in the forming apical ectodermal ridge (AER), restricted regions of the central nervous system, and tail bud. Targeted deletion of the Sp8 gene gives a striking phenotype, with severe truncation of both forelimbs and hindlimbs, absent tail, as well as defects in anterior and posterior neuropore closure leading to exencephaly and spina bifida. Outgrowth of the limb depends on formation of the AER, a signaling center that forms at the limb bud apex. In Sp8 mutants, the AER precursor cells are induced and initially express multiple appropriate marker genes, but expression of these genes is not maintained and progression to a mature AER is blocked. These observations indicate that Sp8 functions downstream of Wnt3, Fgf10, and Bmpr1a in the signaling cascade that mediates AER formation.

Amino Acid Sequence↗

PubMed2003

Gene expression in early ischemic renal injury: clues towards pathogenesis, biomarker discovery, and novel therapeutics.

Acute renal failure (ARF) represents a common and serious problem in clinical medicine. Renal ischemia-reperfusion injury (IRI) is the major cause of ARF in the native and transplanted kidney. Several decades of research have provided successful therapeutic approaches in animal models, but translational efforts in humans have yielded disappointing results. The major reasons for this include a lack of early markers for ARF (and hence a delay in initiating therapy), and the multi-factorial nature of the disease. This review focuses on the use of cDNA microarrays to elucidate the molecular genetic mechanisms underlying tubule cell apoptosis, and to identify novel biomarkers for early renal IRI. Also presented is a comparative temporal analysis of cDNA microarray results from mature kidneys following IRI and during normal nephrogenesis. Molecular genetic evidence for the notion that regeneration recapitulates development in the kidney, and that injured tubule cells possess the capacity to de-differentiate to the earliest stages of development, is presented. The implications of these findings to the ability of the kidney to repair itself and potential strategies for accelerating recovery are briefly discussed.

Acute Kidney Injury↗

PubMed2003

A catalogue of gene expression in the developing kidney.

BACKGROUND: Although many genes with important function in kidney morphogenesis have been described, it is clear that many more remain to be discovered. Microarrays allow a more global analysis of the genetic basis of kidney organogenesis. METHODS: In this study, Affymetrix U74Av2 microarrays, with over 12,000 genes represented, were used in conjunction with robust target microamplification techniques to define the gene expression profiles of the developing mouse kidney. RESULTS: Microdissected murine ureteric bud and metanephric mesenchyme as well as total kidneys at embryonic day E11.5, E12.5, E13.5, E16.5, and adult were examined. This work identified, for example, 3847 genes expressed in the E12.5 kidney. Stringent comparison of the E12.5 versus adult recognized 428 genes with significantly elevated expression in the embryonic kidney. These genes fell into several functional categories, including transcription factor, growth factor, signal transduction, cell cycle, and others. In contrast, surprisingly few differences were found in the gene expression profiles of the ureteric bud and metanephric mesenchyme, with many of the differences clearly associated with the more epithelial character of the bud. In situ hybridizations were used to confirm and extend microarray-predicted expression patterns in the developing kidney. For three genes, Cdrap, Tgfbi, and Col15a1, we observed strikingly similar expression in the developing kidneys and lungs, which both undergo branching morphogenesis. CONCLUSION: The results provide a gene discovery function, identifying large numbers of genes not previously associated with kidney development. This study extends developing kidney microarray analysis to the powerful genetic system of the mouse and establishes a baseline for future examination of the many available mutants. This work creates a catalogue of the gene expression states of the developing mouse kidney and its microdissected subcomponents.

Animals↗

PubMed2003

GeneChip microarrays facilitate identification of Protease Nexin-1 as a target gene of the Prx2 (S8) homeoprotein.

The paired-related homeobox genes, Prx1 and Prx2, are important for normal skeletal and cardiovascular development as well as adult vascular remodeling. The identification and characterization of Prx downstream targets is crucial to understanding their function in normal developmental processes and congenital malformations. To identify Prx2 regulated genes, stably transfected NIH3T3 clones expressing Prx2 sense or antisense transcripts were generated. Expression profiles initially were established for two of the clones using Affymetrix GeneChip arrays. Over 6,400 genes were screened by the microarray approach, and approximately 500 genes differed in expression by a factor of two or more. Fifteen genes were chosen for further analysis. RT-PCR of the two transfectants used in the GeneChip analysis demonstrated that five out of the 15 genes were differentially expressed. However, after screening additional stable transfectant clones only one of the 15 genes, Protease Nexin-1 (PN-1), was differentially expressed. Subsequent Northern blot, RT-PCR, and further GeneChip analysis of additional stable transfectants confirmed that PN-1 expression is increased at least fivefold when Prx2 is overexpressed. It was demonstrated that Prx2 directly regulates PN-1 because (1) Prx2 binds to a cis element in the PN-1 promoter in vitro, and (2) Prx2 regulates the PN-1 promoter in transient transfection assays. The GeneChip analysis generated a prioritized list of other potential targets. The utility and limitations of cell culture models combined with microarray analysis for elucidating complex regulatory cascades are discussed.

3T3 Cells↗

PubMed2003

Hox genes and kidney patterning.

PURPOSE OF REVIEW: Hox gene activity is essential for proper organization or pattern of the vertebrate body plan and is necessary for organogenesis. Sequence conservation within this family of genes is high yet they are involved in very diverse developmental processes. How this family functions in these processes is a challenging question, but is important for the understanding of renal organogenesis. Multiple Hox genes are expressed in the kidney and mutation in at least one group of paralogous genes results in severe renal defects. RECENT FINDINGS: Recent studies in mice with targeted Hox gene mutations and in kidney cell lines demonstrate that these genes have evolved to control tissue specific functions through their ability to regulate the expression of renal morphogens. The studies also demonstrate that Hox gene activity is not only restricted by the domain of expression but also by the specificity of the DNA binding homeodomain. Interestingly, these conserved homeodomains are not wholly interchangeable for normal renal organogenesis while they do appear to be interchangeable for axial skeleton development. SUMMARY: It is clear that Hox genes regulate important interactions between the ureteric bud and metanephric mesenchyme. Nevertheless, much work remains to define the expression patterns of multiple Hox genes during kidney development, to better determine the functional relationships of the encoded proteins, and to identify additional Hox downstream targets.

Animals↗

PubMed2003

Early orchiopexy restores fertility in the Hoxa 11 gene knockout mouse.

PURPOSE: We investigated whether infertility could be reversed in cryptorchid mice (with disrupted expression of the homeobox gene Hoxa 11) by orchiopexy and mating such animals with females of proven fertility. MATERIALS AND METHODS: Hoxa 11 mutant and WT male mice were genotyped by polymerase chain reaction. Surgery (orchiopexy or sham operation) was performed at age 18 days and fertility was assessed at ages 6 to 8 weeks. Animals were sacrificed at ages 6 to 9 months and computer assisted semen analysis was performed on fluid obtained by epididymal puncture. RESULTS: Five of 28 mutant mice proved fertile following orchiopexy versus 0 of 22 after sham operation (p <0.05). Values in WT mice were 18 of 35 and 25 of 33, respectively (p <0.01). Mean spermatozoa counts +/- SEM were 21.7 +/- 5.9 x 106/ml in 8 mutant mice with orchiopexy, 2.78 +/- 1.59 x 106/ml in 8 sham operated mutant mice (p <0.002), 15.6 +/- 4.9 x 106/ml in 7 WT mice with orchiopexy and 36.3 +/- 10.5 x 106/ml in 9 sham operated WT mice (p <0.02). CONCLUSIONS: Testicular position following orchiopexy is important to achieve fertility but the surgical procedure was associated with a degree of damage. Since mutant animals did not attain the fertility rates observed in WT animals following orchiopexy, other factors (possibly vaso-epididymal) may be necessary for normal spermatogenesis. Further studies of this model may allow the identification of such factors.

Animals↗

PubMed2002

Hoxa 11 is upstream of Integrin alpha8 expression in the developing kidney.

Mutation of the functionally redundant Hoxa 11/Hoxd 11 genes gives absent or rudimentary kidneys resulting from a dramatic reduction of the growth and branching of the ureteric bud. To understand better the molecular mechanisms of Hoxa 11/Hoxd 11 function in kidney development, it is necessary to identify the downstream target genes regulated by their encoded transcription factors. To this end, we conducted a screen for Hoxa 11-responsive genes in two kidney cell lines. HEK293 cells, which usually do not express Hoxa 11, were modified to allow inducible Hoxa 11 expression. The mK10 cells, derived specifically for this study from Hoxa 11/Hoxd 11 double-mutant mice, were also modified to give cell populations with and without Hoxa 11 expression. Differential display, Gene Discovery Arrays, and Affymetrix genechip probe arrays were used to screen for genes up- or down-regulated by Hoxa 11. Nine genes, PDGF A, Cathepsin L, annexin A1, Mm.112139, Est2 repressor factor, NrCAM, ZNF192, integrin-associated protein, and GCM1, showed reproducible 3-fold or smaller changes in gene expression in response to Hoxa 11. One gene, the Integrin alpha8, was up-regulated approximately 20-fold after Hoxa 11 expression. The Integrin alpha8 gene is expressed together with Hoxa 11 in metanephric mesenchyme cells, and mutation of Integrin alpha8 gives a bud-branching morphogenesis defect very similar to that observed in Hoxa 11/Hoxd 11 mutant mice. In situ hybridizations showed a dramatic regional reduction in Integrin alpha8 expression in the developing kidneys of Hoxa 11/Hoxd 11 mutant mice. This work suggests that the Integrin alpha8 gene may be a major effector of Hoxa 11/Hoxd 11 function in the developing kidney.

Animals↗

PubMed2002

Functional comparison of the Hoxa 4, Hoxa 10, and Hoxa 11 homeoboxes.

A number of models attempt to explain the functional relationships of Hox genes. The functional equivalence model states that mammalian Hox-encoded proteins are largely functionally equivalent, and that Hox quantity is more important than Hox quality. In this report, we describe the results of two homeobox swaps. In one case, the homeobox of Hoxa 11 was replaced with that of the very closely related Hoxa 10. Developmental function was assayed by analyzing the phenotypes of all possible allele combinations, including the swapped allele, and null alleles for Hoxa 11 and Hoxd 11. This chimeric gene provided wild-type function in the development of the axial skeleton and male reproductive tract, but served as a hypomorph allele in the development of the appendicular skeleton, kidneys, and female reproductive tract. In the other case, the Hoxa 11 homeobox was replaced with that of the divergent Hoxa 4 gene. This chimeric gene provided near recessive null function in all tissues except the axial skeleton, which developed normally. These results demonstrate that even the most conserved regions of Hox genes, the homeoboxes, are not functionally interchangeable in the development of most tissues. In some cases, developmental function tracked with the homeobox, as previously seen in simpler organisms. Homeoboxes with more 5' cluster positions were generally dominant over more 3' homeoboxes, consistent with phenotypic suppression seen in Drosophila. Surprisingly, however, all Hox homeoboxes tested did appear functionally equivalent in the formation of the axial skeleton. The determination of segment identity is one of the most evolutionarily ancient functions of Hox genes. It is interesting that Hox homeoboxes are interchangeable in this process, but are functionally distinct in other aspects of development.

Alleles↗

PubMed2002

Microarray analysis of novel cell lines representing two stages of metanephric mesenchyme differentiation.

Clonal cell lines representing different developmental stages of the metanephric mesenchyme were made from transgenic mice with the Simian Virus 40 T-antigen (SV40 Tag) gene driven by the Hoxa 11 promoter. The resulting mK3 cell line represented early metanephric mesenchyme, prior to induction by the ureteric bud. These cells showed a spindle-shaped, fibroblast morphology. They expressed genes characteristic of early mesenchyme, including Hoxa 11, Hoxd 11, collagen I, and vimentin. Moreover, the mK3 cells displayed early metanephric mesenchyme biological function. In organ co-culture experiments they were able to induce growth and branching of the ureteric bud. Another cell line, mK4, represented later, induced metanephric mesenchyme undergoing epithelial conversion. These cells were more polygonal, or epithelial in shape, and expressed genes diagnostic of late mesenchyme, including Pax-2, Pax-8, Wnt-4, Cadherin-6, Collagen IV, and LFB3. To better define the gene expression patterns of kidney metanephric mesenchyme cells at these two stages of development, RNAs from the mK3 and mK4 cells were hybridized to Affymetrix GeneChip probe arrays. Over 4000 expressed genes were identified and thereby implicated in kidney formation. Comparison of the mK3 and mK4 gene expression profiles revealed 121 genes showing greater than a ten-fold difference in expression level. Several are known to be expressed during metanephric mesenchyme differentiation, but most had not been previously associated with this process. In situ hybridizations were used to confirm that selected novel genes were expressed in the developing kidney.

Animals↗

PubMed2002

Microarray analysis of novel cell lines representing two stages of metanephric mesenchyme differentiation.

Animals↗