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Recent studies in our lab identified a mutant mouse model of obstructive nephropathy designated mgb for megabladder. Homozygotic mgb mice (mgb−/−) develop lower urinary tract obstruction in utero due to a lack of bladder smooth muscle differentiation. This defect is the result of a random transgene insertion/translocation into chromosomes 11 and 16. Transcriptional profiling identified a significantly over-expressed cluster of gene products located on the translocated fragment of chromosome 16 including urotensin II-related peptide (Urp), which was shown to be preferentially over-expressed in developing mgb−/− bladders. Pathway analysis of mgb microarray data indicated dysregulation of at least 60 gene products associated with smooth muscle development. In conclusion, the results of this study indicate that the molecular pathways controlling normal smooth muscle development are severely altered in mgb−/− bladders, and provide the first evidence that Urp may play a critical role in bladder smooth muscle development. Developmental Dynamics 237:170–186, 2008. © 2007 Wiley-Liss, Inc.

Background Bed bugs (Cimex lectularius) are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance. Results We performed a next-generation RNA sequencing (RNA-Seq) experiment to find differentially expressed genes between pesticide-resistant (PR) and pesticide-susceptible (PS) strains of C. lectularius. A reference transcriptome database of 51,492 expressed sequence tags (ESTs) was created by combining the databases derived from de novo assembled mRNA-Seq tags (30,404 ESTs) and our previous 454 pyrosequenced database (21,088 ESTs). The two-way GLMseq analysis revealed ~15,000 highly significant differentially expressed ESTs between the PR and PS strains. Among the top 5,000 differentially expressed ESTs, 109 putative defense genes (cuticular proteins, cytochrome P450s, antioxidant genes, ABC transporters, glutathione S-transferases, carboxylesterases and acetyl cholinesterase) involved in penetration resistance and metabolic resistance were identified. Tissue and development-specific expression of P450 CYP3 clan members showed high mRNA levels in the cuticle, Malpighian tubules, and midgut; and in early instar nymphs, respectively. Lastly, molecular modeling and docking of a candidate cytochrome P450 (CYP397A1V2) revealed the flexibility of the deduced protein to metabolize a broad range of insecticide substrates including DDT, deltamethrin, permethrin, and imidacloprid. Conclusions We developed significant molecular resources for C. lectularius putatively involved in metabolic resistance as well as those participating in other modes of insecticide resistance. RNA-Seq profiles of PR strains combined with tissue-specific profiles and molecular docking revealed multi-level insecticide resistance in C. lectularius. Future research that is targeted towards RNA interference (RNAi) on the identified metabolic targets such as cytochrome P450s and cuticular proteins could lay the foundation for a better understanding of the genetic basis of insecticide resistance in C. lectularius.

Background: Expression profiling, the measurement of all transcripts of a cell or tissue type, is currently the most comprehensive method to describe their physiological states. Given that accurate profiling methods currently available require RNA amounts found in thousands to millions of cells, many fields of biology working with specialized cell types cannot use these techniques because available cell numbers are limited. Currently available alternative methods for expression profiling from nanograms of RNA or from very small cell populations lack a broad validation of results to provide accurate information about the measured transcripts. Methods and Findings: We provide evidence that currently available methods for expression profiling of very small cell populations are prone to technical noise and therefore cannot be used efficiently as discovery tools. Furthermore, we present Pico Profiling, a new expression profiling method from as few as ten cells, and we show that this approach is as informative as standard techniques from thousands to millions of cells. The central component of Pico Profiling is Whole Transcriptome Amplification (WTA), which generates expression profiles that are highly comparable to those produced by others, at different times, by standard protocols or by Real-time PCR. We provide a complete workflow from RNA isolation to analysis of expression profiles. Conclusions: Pico Profiling, as presented here, allows generating an accurate expression profile from cell populations as small as ten cells.

Retinitis pigmentosa is an incurable retinal disease that leads to blindness. One puzzling aspect concerns the progression of the disease. Although most mutations that cause retinitis pigmentosa are in rod photoreceptor–specific genes, cone photoreceptors also die as a result of such mutations. To understand the mechanism of non-autonomous cone death, we analyzed four mouse models harboring mutations in rod-specific genes. We found changes in the insulin/mammalian target of rapamycin pathway that coincided with the activation of autophagy during the period of cone death. We increased or decreased the insulin level and measured the survival of cones in one of the models. Mice that were treated systemically with insulin had prolonged cone survival, whereas depletion of endogenous insulin had the opposite effect. These data suggest that the non-autonomous cone death in retinitis pigmentosa could, at least in part, be a result of the starvation of cones. Retinitis pigmentosa is a type of inherited retinal degeneration. It is currently untreatable and usually leads to blindness. With over 40 reintitis pigmentosa genes identified, it is the most common type of retinal degeneration caused by a single disease allele (RetNet, http://www.sph.uth.tmc.edu/Retnet/). The phenotype is characterized by an initial loss of night vision as a result of the malfunction and death of rod photoreceptors. This phase is followed by a progressive loss of cones. Because cones are responsible for color and high-acuity vision, it is their loss that leads to a reduction in the quality of life. In many cases, the disease-causing allele is expressed exclusively in rods; nonetheless, cones die as well. Indeed, to date there is no known form of retinal degeneration in humans or mice where rods die and cones survive. In contrast, mutations in cone-specific genes result only in cone death. Several theories have been proposed to explain this finding. For example, cone death could be a result of the release of a toxin produced by dying rods or the loss of a trophic factor that is produced by healthy rods 1‐6 . Alternatively, cone death could be caused by microglia that are mobilized initially during rod death 7 or by oxidative stress 8,9 .O xidative stress might also directly harm cones. The constant flow of oxygen through the retinal pigmented epithelium (RPE) to photoreceptors and the loss of rods, which are 95% of the photoreceptors in human and mouse, may result in an overload of oxygen to the remaining cones 10 . Evidence for all of these mechanisms exists in mice, yet none are able to fully explain why cones may survive for many years in the absence of rods in humans. Nonetheless, rodents are a very good model for this type of retinal degeneration. Although the rodent retina lacks a macula, which is the cone-rich and rod-free area that is present in humans, the macula is not involved in the initial phase of the disease. In humans, retinitis pigmentosa starts outside of the macula, where the distribution of rods and cones is similar to that in mice. To determine the common underlying mechanism for cone death in retinitis pigmentosa, we compared four mouse models harboring mutations in rod-specific genes (Pde6b ‐/‐ (ref. 11), Pde6g ‐/‐ (ref. 12), Rho ‐/‐ (ref. 13) and P23H 14 , which carries a Rho transgene that has an amino acid subsitution of histidine for proline at amino acid 23, as occurs in some human cases of retinitis pigmentosa, see Methods). Affymetrix arrays were used to identify common changes in gene expression that accompany cone death. Changes in a substantial number of genes involved in cellular metabolism coincided with the onset of cone death. These changes were suggestive of cones suffering from a shortage of nutrients. We then found that cones showed signs of autophagy, a cellular self-digestion process, which is consistent with prolonged starvation. We also found that several aspects of the insulin/ mammalian target of rapamycin (mTOR) pathway, an important pathway that regulates cellular metabolism, were affected during the period of cone degeneration. As a result of this finding, we increased and decreased the insulin level and measured the survival of cones in one of the models. Mice treated systemically with insulin had prolonged cone survival, whereas depletion of endogenous insulin had the opposite effect. Therefore, cone starvation is a likely contributor to the slow demise of cones in humans with retinitis pigmentosa. Treatments aimed at improving nutrition of cones are thus a plausible therapeutic avenue. RESULTS Photoreceptor death kinetics and microarray analysis To establish a framework for comparing gene expression in four different models of retinitis pigmentosa, we established the equivalent stages of disease pathology through examination of the kinetics of rod (Fig. 1 and Supplementary Figs. 1 and 2 online) and cone (Fig. 2 and

Homeodomain-containing transcription factors are important regulators of stem cell behavior. HOXB4 mediates expansion of adult and embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically. To define the underlying molecular mechanisms, we performed gene expression profiling in combination with subsequent functional analysis with enriched adult HSCs and embryonic derivatives expressing inducible HOXB4. Thereby, we identified a set of overlapping genes that likely represent “universal” targets of HOXB4. A substantial number of loci are involved in signaling pathways important for controlling self-renewal, maintenance, and differentiation of stem cells. Functional assays performed on selected pathways confirmed the biological coherence of the array results. HOXB4 activity protected adult HSCs from the detrimental effects mediated by the proinflammatory cytokine TNF-α. This protection likely contributes to the competitive repopulation advantage of HOXB4-expressing HSCs observed in vivo . The concept of TNF-α inhibition may also prove beneficial for patients undergoing bone marrow transplantation. Furthermore, we demonstrate that HOXB4 activity and FGF signaling are intertwined. HOXB4-mediated expansion of adult and ES cell-derived HSCs was enhanced by specific and complete inhibition of FGF receptors. In contrast, the expanding activity of HOXB4 on hematopoietic progenitors in day 4–6 embryoid bodies was blunted in the presence of basic FGF (FGF2), indicating a dominant negative effect of FGF signaling on the earliest hematopoietic cells. In summary, our results strongly suggest that HOXB4 modulates the response of HSCs to multiple extrinsic signals in a concerted manner, thereby shifting the balance toward stem cell self-renewal.

The E2f7 and E2f8 family members are thought to function as transcriptional repressors important for the control of cell proliferation. Here, we have analyzed the consequences of inactivating E2f7 and E2f8 in mice and show that their individual loss had no significant effect on development. Their combined ablation, however, resulted in massive apoptosis and dilation of blood vessels, culminating in lethality by embryonic day E11.5. A deficiency in E2f7 and E2f8 led to an increase in E2f1 and p53, as well as in many stress-related genes. Homo- and heterodimers of E2F7 and E2F8 were found on target promoters, including E2f1. Importantly, loss of either E2f1 or p53 suppressed the massive apoptosis in double-mutant embryos. These results identify E2F7 and E2F8 as a unique repressive arm of the E2F transcriptional network that is critical for embryonic development and control of the E2F1-p53 apoptotic axis.

Urinary tract malformations, obstructive uropathy, and hypoplasia/dysplasia are extremely important in terms of pediatric health care costs, with end-stage renal failure in children estimated to cost >$15 billion annually in the United States alone. Even so, little is known regarding the mechanisms that control these processes. Identified was a unique mutant mouse model that develops in utero megabladder, resulting in variable hydroureteronephrosis and chronic renal failure secondary to obstructive uropathy. These animals, designated mgb for megabladder, possess a primary defect in bladder smooth muscle development that is apparent by embryonic day 15. The mgb mouse represents an excellent model for the study of normal and pathogenic bladder development, including the postnatal progression of chronic renal failure that results from the development of in utero obstructive uropathy.

Background Array-based comparative genomic hybridization (aCGH) is a high-throughput method for measuring genome-wide DNA copy number changes. Current aCGH methods have limited resolution, sensitivity and reproducibility. Microarrays for aCGH are available only for a few organisms and combination of aCGH data with expression data is cumbersome. Results We present a novel method of using commercial oligonucleotide expression microarrays for aCGH, enabling DNA copy number measurements and expression profiles to be combined using the same platform. This method yields aCGH data from genomic DNA without complexity reduction at a median resolution of approximately 17,500 base pairs. Due to the well-defined nature of oligonucleotide probes, DNA amplification and deletion can be defined at the level of individual genes and can easily be combined with gene expression data. Conclusion A novel method of gene resolution analysis of copy number variation (graCNV) yields high-resolution maps of DNA copy number changes and is applicable to a broad range of organisms for which commercial oligonucleotide expression microarrays are available. Due to the standardization of oligonucleotide microarrays, graCNV results can reliably be compared between laboratories and can easily be combined with gene expression data using the same platform.

It is well known that spontaneously hypertensive rats (SHR) develop muscle pathologies with hypertension and heart failure, though the mechanism remains poorly understood. Woon et al. (2007) linked the circadian clock gene Bmal1 to hypertension and metabolic dysfunction in the SHR. Building on these findings, we compared the expression pattern of several core-clock genes in the gastrocnemius muscle of aged SHR (80 weeks; overt heart failure) compared to aged-matched control WKY strain. Heart failure was associated with marked effects on the expression of Bmal1, Clock and Rora in addition to several non-circadian genes important in regulating skeletal muscle phenotype including Mck, Ttn and Mef2c. We next performed circadian time-course collections at a young age (8 weeks; pre-hypertensive) and adult age (22 weeks; hypertensive) to determine if clock gene expression was disrupted in gastrocnemius, heart and liver tissues prior to or after the rats became hypertensive. We found that hypertensive/hypertrophic SHR showed a dampening of peak Bmal1 and Rev-erb expression in the liver, and the clock-controlled gene Pgc1α in the gastrocnemius. In addition, the core-clock gene Clock and the muscle-specific, clock-controlled gene Myod1, no longer maintained a circadian pattern of expression in gastrocnemius from the hypertensive SHR. These findings provide a framework to suggest a mechanism whereby chronic heart failure leads to skeletal muscle pathologies; prolonged dysregulation of the molecular clock in skeletal muscle results in altered Clock, Pgc1α and Myod1 expression which in turn leads to the mis-regulation of target genes important for mechanical and metabolic function of skeletal muscle.

MANY macroscopic quantities have microscopic counterparts. The microscopic counterpart of gas pressure, for example, is the instantaneous mechanical force exerted by gas molecules on a unit area. In the analysis of such physical quantities the role of statistical mechanics is conceptually straightforward; it is the calculation of average (macroscopic) values for selected microscopic mechanical quantities A. Such calculations have contributed a great deal to our understanding of the form taken by the equation of state for various materials placed in different environments1.