Molecular and genetic analyses of Fgf signaling during cardiac outflow tract development

Epithelial-mesenchymal tissue interactions regulate the development of derivatives of the caudal pharyngeal arches (PAs) to govern the ultimate morphogenesis of the aortic arch and outflow tract (OFT) of the heart. Disruption of these signaling pathways is thought to contribute to the pathology of a significant proportion of congenital cardiovascular defects in humans. In this study, I tested whether Fibroblast Growth Factor 15 (Fgf15), a secreted signaling molecule expressed within the PAs, is an extracellular mediator of tissue interactions during PA and OFT development. Analyses of Fgf15−/− mouse embryonic hearts revealed abnormalities primarily localized to the OFT, correlating with aberrant cardiac neural crest cell behavior. The T-box-containing transcription factor Tbx1 has been implicated in the cardiovascular defects associated with the human 22q11 Deletion Syndromes, and regulates the expression of other Fgf family members within the mouse PAs. However, expression and genetic interaction studies incorporating mice deficient for Tbx1, its upstream regulator, Sonic Hedgehog (Shh), or its putative downstream effector, Fgf8, indicated that Fgf15 functions during OFT development in a manner independent of these factors. Rather, analyses of compound mutant mice indicated that Fgf15 and Fgf9, an additional Fgf family member expressed within the PAs, genetically interact, providing insight into the factors acting in conjunction with Fgf15 during OFT development. Finally, in an effort to further characterize this Fgf15-mediated developmental pathway, promoter deletion analyses were employed to isolate a 415bp sequence 7.1Kb 5′ to the Fgf15 transcription start site both necessary and sufficient to drive reporter gene expression within the epithelium of the PAs. Sequence comparisons among multiple mammalian species facilitated the identification of evolutionarily conserved potential trans-acting factor binding sites within this fragment. Subsequent studies will investigate the molecular pathway(s) through which Fgf15 functions via identification of factors that bind to this element to govern Fgf15 gene expression. Furthermore, targeted deletion of this element will establish the developmental requirement for pharyngeal epithelium-derived Fgf15 signaling function. Taken as a whole, these data demonstrate that Fgf15 is a component of a novel, Tbx1-independent molecular pathway, functioning within the PAs in a manner cooperative with Fgf9, required for proper development of the cardiac OFT. ^

Molecular genetic analyses of extracellular signaling pathways during murine retinal development

Extracellular signaling pathways initiated by secreted proteins are important in the co-ordination of tissue interactions in multi-cellular organisms, particularly during embryonic development. These signaling cascades direct diverse cellular events, including proliferation, differentiation and migration, in both autocrine and paracrine modes. In adult animals, abnormal function of these proteins often results in degenerative and tumourigenic syndromes. In this study, I have focused on elucidating the role of Bone Morphogenetic Protein (Bmp) signal transduction during neuronal specification and differentiation in the vertebrate embryo, using the mouse retina as a model. Using tissue-specific conditional knock-out approaches, the consequences of genetic loss-of-function of this signaling pathway on retinal physiology were examined. Mutant mice lacking Bmp type I receptor function displayed a range of retinal phenotypes, each of which appeared to be regulated at a different threshold of Bmp receptor activity. Novel essential functions for Bmp signaling were uncovered for retinal neurogenesis, cell survival, and axonal pathfinding at the optic disc. Further, BmprIa and BmprIa exhibited genetic interactions suggestive of functional redundancy. To further characterize the underlying molecular bases for the pleiotropic effects of Bmp receptors, retina-specific loss-of-function mutants of the obligate Bmp-activated transcriptional mediator Smad4 were generated. A comparison of the retina-specific Smad4 mutant phenotypes with those of the Bmp receptor mutant retina revealed that only a subset of retinal phenotypes, namely optic disc axon pathfinding and axial patterning were common for both classes of mutant animals. Thus, these results suggest that, contrary to the classic scheme of Bmp signal transduction, Smad4-independent pathways may be operative downstream of the type I receptors. Indeed, such alternative intracellular signaling cascades may constitute a molecular basis for the multiple cellular responses elicited by Bmp signaling. Finally, I tested whether the potential Bmp pathway targets, the extracellular ligands Fgf9 and Fgf15, mediate essential cellular processes in the retina. The analyses of Fgf9 −/−; Fgf15−/− mutant mice posit a novel shared role for these genes in intra-retinal axon pathfinding. Collectively, these studies have elucidated part of the molecular machinery directing mammalian neuro-retinal development, and provided useful in vivo models to study visual function. ^

Structure-function analyses of {\it PAX6\/} and the molecular bases of aniridia

PAX6 is a transcription activator that regulates eye development in animals ranging from Drosophila to human. The C-terminal region of PAX6 is proline/serine/threonine-rich (PST) and functions as a potent transactivation domain when attached to a heterologous DNA-binding domain of the yeast transcription factor, GAL4. The PST region comprises 152 amino acids encoded by four exons. The transactivation function of the PST region has not been defined and characterized in detail by in vitro mutagenesis. I dissected the PST domain in two independent systems, a heterologous system using a GAL4 DNA-binding site and the native system of PAX6. In both systems, the results show consistently that all four constituent exons of the PST domain are responsible for the transactivation function. The four exon fragments act cooperatively to stimulate transcription, although none of them can function individually as an independent transactivation domain. Combinations of two or more exon fragments can reconstitute substantial transactivation activity when fused to the DNA-binding domain of GAL4, but they surprisingly do not produce much activity in the context of native PAX6 even though the mutant PAX6 proteins are stable and their DNA-binding function remains unaffected. I conclude that the PAX6 protein contains an unusually large transactivation domain that is evolutionarily conserved to a high degree, and that its full transactivation activity relies on the cooperative action of the four exon fragments.^ Most PAX6 mutations detected in patients with aniridia result in truncations of the protein. Some of the truncation mutations occur in the PST region of PAX6, resulting in mutant proteins that retain their DNA-binding ability but have no significant transactivation activity. It is not clear whether such mutants are true loss-of-function or dominant-negative mutants. I show that these mutants are dominant-negative if they are coexpressed with wild-type PAX6 in cultured cells and that the dominant-negative effects result from enhanced DNA-binding ability of these mutants due to removal of the PST domain. These mutants are able to repress the wild-type PAX6 activity not only at target genes with paired domain binding sites but also at target genes with homeodomain binding sites.^ Mutations in the human PAX6 gene produce various phenotypes, including aniridia, Peters' anomaly, autosomal dominant keratitis, and familial foveal dysplasia. The various phenotypes may arise from different mutations in the same gene. To test this theory, I performed a functional analysis of two missense mutations in the paired domain: the R26G mutation reported in a case of Peters' anomaly, and the I87R mutation identified in a patient with aniridia. While both the R26 and the I87 positions are conserved in the paired boxes of all known PAX genes, X-ray crystallography has shown that only R26 makes contact with DNA. I found that the R26G mutant failed to bind a subset of paired domain binding sites but, surprisingly, bound other sites and successfully transactivated promoters containing those sites. In contrast, the I87R mutant had lost the ability to bind DNA at all tested sites and failed to transactivate promoters. My data support the haploinsufficiency hypothesis of aniridia, and the hypothesis that R26G is a hypomorphic allele. ^

An ecologic comparison study of the impact of economic disadvantage on Texas Assessment of Knowledge and Skills performance, graduation rates, and readiness for higher education for students attending public and charter schools in Texas from 2004 to 2006

Purpose. No Child Left Behind aimed to "improve the academic achievement of the disadvantaged." The primary research question considered how academic achievement of those from economic disadvantage compared to those not from disadvantage? ^ Economically disadvantaged students can potentially have added academic disadvantage. Research shows low academic achievement can potentially result in drug abuse, youth violence, and teen pregnancy. ^ Methods. To compare the student populations, measures included TAKS results and academic indicator data collected by the Texas Education Agency. ^ Results. T-test analyses showed a significant difference between the economically and non-economically disadvantaged student populations in meeting the TAKS passing standard, graduation, and preparation for higher education.^ Conclusions. The achievement gap between students remained as indicated by the Texas testing program. More research and time are needed to observe if the desired impact on those from economic disadvantage will be reflected by academic achievement data.^

A review of economic analysis in the vaccination program; decision making and governance: Adivisory Committee on Immunization Practices (ACIP)

The Advisory Committee on Immunization Practices (ACIP) develops written recommendations for the routine administration of vaccines to children and adults in the U.S. civilian population. The ACIP is the only entity in the federal government that makes such recommendations. ACIP elaborates on selection of its members and rules out concerns regarding its integrity, but fails to provide information about the importance of economic analysis in vaccine selection. ACIP recommendations can have large health and economic consequences. Emphasis on economic evaluation in health is a likely response to severe pressures of the federal and state health budget. This study describes the economic aspects considered by the ACIP while sanctioning a vaccine, and reviews the economic evaluations (our economic data) provided for vaccine deliberations. A five year study period from 2004 to 2009 is adopted. Publicly available data from ACIP web database is used. Drummond et al. (2005) checklist serves as a guide to assess the quality of economic evaluations presented. Drummond et al.'s checklist is a comprehensive hence it is unrealistic to expect every ACIP deliberation to meet all of their criteria. For practical purposes we have selected seven criteria that we judge to be significant criteria provided by Drummond et al. Twenty-four data points were obtained in a five year period. Our results show that out of the total twenty-four data point‘s (economic evaluations) only five data points received a score of six; that is six items on the list of seven were met. None of the data points received a perfect score of seven. Seven of the twenty-four data points received a score of five. A minimum of a two score was received by only one of the economic analyses. The type of economic evaluation along with the model criteria and ICER/QALY criteria met at 0.875 (87.5%). These three criteria were met at the highest rate among the seven criteria studied. Our study findings demonstrate that the perspective criteria met at 0.583 (58.3%) followed by source and sensitivity analysis criteria both tied at 0.541 (54.1%). The discount factor was met at 0.250 (25.0%).^ Economic analysis is not a novel concept to the ACIP. It has been practiced and presented at these meetings on a regular basis for more than five years. ACIP‘s stated goal is to utilize good quality epidemiologic, clinical and economic analyses to help policy makers choose among alternatives presented and thus achieve a better informed decision. As seen in our study the economic analyses over the years are inconsistent. The large variability coupled with lack of a standardized format may compromise the utility of the economic information for decision-making. While making recommendations, the ACIP takes into account all available information about a vaccine. Thus it is vital that standardized high quality economic information is provided at the ACIP meetings. Our study may provide a call for the ACIP to further investigate deficiencies within the system and thereby to improve economic evaluation data presented. ^