Siu-wai Michael Sung. / Thesis submitted in: December 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 97-111). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations --- p.v / Table of Contents --- p.vii / Chapter Chapter 1 --- General Introduction / Chapter l. 1 --- Drosophila as a model for studying development --- p.1 / Chapter l .2 --- The formation of the body axis in Drosophila --- p.2 / Chapter l .3 --- Maternal genes are essential for development --- p.9 / Chapter 1.4 --- Maternal gene bicoid is essential for formation of the anterior structures in the embryo --- p.13 / Chapter 1.5 --- Establishment of an anterior to posterior bicoid protein gradient --- p.13 / Chapter 1.6 --- The bicoid protein gradient controls the downstream zygotic target genes in a concentration-dependent manner --- p.17 / Chapter 1.7 --- Bicoid protein acts as transcriptional regulators \9 --- p.19 / Chapter 1.8 --- Bicoid protein acts as transcriptional regulators --- p.21 / Chapter 1.9 --- The anterior localization of bcd mRNA --- p.21 / Chapter 1.10 --- Components required for bcd mRNA localization at anterior pole of oocyte / Chapter 1.10.1 --- Cis-acting elements --- p.22 / Chapter 1.10.1.1 --- BLE1 at 3' UTR directs localization of bcd mRNA --- p.23 / Chapter 1.10.2 --- Trans-acting elements / Chapter 1.10.2.1 --- "Exuperantia, swallow, and staufen are necessary for localization for bcd mKNA" --- p.27 / Chapter 1.10.2.2 --- exu protein is an absolute requirement for localization for bcd mRNA --- p.30 / Chapter 1.10.2.3 --- Microtubules dependence of localization --- p.31 / Chapter 1.11 --- Functions of exu in localization of bcd mRNA --- p.32 / Chapter 1.12 --- Characteristics of Bicoid protein and Bic-D gene --- p.33 / Chapter 1.13 --- Aim of Project --- p.36 / Chapter CHAPTER 2 --- Materials and Methods / Chapter 2.1 --- Fly Food --- p.37 / Chapter 2.2 --- Conditions in maintaining the fly stocks and working stocks --- p.37 / Chapter 2.3 --- Localization of exu protein and other intracellular elements by indirect immunofluorescence detection / Chapter 2.3.1 --- Immunohistrochemical distribution of exu and Bic-D protein --- p.38 / Chapter 2.3.2 --- Immunohistrochemical distribution of β-tubulin --- p.39 / Chapter 2.4 --- Preparation of total protein from the female and male flies --- p.41 / Chapter 2.5 --- Analysis of interactions between exu and trans-acting elements / Chapter 2.5.1 --- 35S-methionine metabolic labelling and immunoprecipitation by RIPA buffer --- p.41 / Chapter 2.5.2 --- 35S-methionine metabolic labelling and immunoprecipitation by Mach and Lehmann buffer system --- p.43 / Chapter 2.6 --- Co-immunoprecipitation of exu and Bic-D protein / Chapter 2.6.1 --- Co-immunoprecipitation of exu and Bic-D protein synthesized by in vitro coupled transcription and translation system with modified Mach and Lechmann buffer system --- p.44 / Chapter 2.7 --- in vivo ovary extract co-immunoprecipitation / Chapter 2.7.1 --- in vivo ovary extraction co-immunoprecipitation of exu and Bic-D protein with modified Mach and Lehmann buffer system supplemented with recombinant exu protein --- p.45 / Chapter CHAPTER 3 --- Results / Chapter 3.1 --- Analysis of co-localization of exu and Bic-D protein by double immuno-fluorescence staining on w1118 flies --- p.47 / Chapter 3.2 --- Analysis of co-localization of exu protein and β-tubulin protein by double immuno-fluorescence staining on w1118 flies --- p.51 / Chapter 3.3 --- Analysis of co-localization of exu and Bic-D protein by double immuno-fluorescence staining on Bic-D mutants --- p.55 / Chapter 3.4 --- Co-immunoprecipitation of exu and Bic-D protein synthesized by in vitro coupled transcription and translation system --- p.61 / Chapter 3.5 --- 35S-Methionine metabolic labelling and co-immunoprecipitation of exu and Bic-D protein with RIP A buffer system --- p.65 / Chapter 3.6 --- 35S-Methionine metabolic labelling and co-immunoprecipitation of exu and Bic-D protein with Mach and Lehmann buffer system --- p.68 / Chapter 3.7 --- in vivo ovary extract co-immunoprecipitation of exu and Bic-D protein with modified Mach and Lehmann buffer system supplemented with recombinant exu protein --- p.71 / Chapter CHAPTER 4 --- Discussion / Chapter 4.1 --- Analysis of co-localization of exu protein and other intracellular elements by indirect double immunofluorescence staining detection --- p.74 / Chapter 4.2 --- Analysis of co-localization of exu and BicD protein by double immuno- fluorescence staining on Bic-D mutants --- p.78 / Chapter 4.3 --- Co-immunoprecipitation of exu and BicD protein synthesized by in vitro coupled transcription and translation system --- p.79 / Chapter 4.4 --- Analysis of interactions between exu and trans-acting elements by 35S- Methionine metabolic labelling and immunoprecipitation --- p.82 / Chapter 4.5 --- "in vivo ovary extract coimmunoprecipitation of exu and Bic-D protein with modified Mech and Lehmann buffer system, supplemented with recombinant exu protein" --- p.84 / Chapter 4.6 --- Recent developments on the concept of ribonucleoprotein --- p.86 / Appendix A Supplementary protocols --- p.91 / Appendix B Reagents --- p.95 / Reference --- p.97

Nós humanos estamos inevitavelmente expostos a uma mistura de poluentes, e evidências nos mostram que estes poluentes aumentam a incidência de desordens reprodutivas. Já se sabe que camundongos cronicamente expostos a níveis de poluentes ambientais em São Paulo apresentam: mudanças no ciclo estral, alteração no número de folículos ovarianos, aumento das perdas embrionárias pós implantacionais e alterações na morfologia placentária. Nós hipotetizamos que as alterações na resposta uterina a implantação possa ser mediado por mastócitos e células NK uterinas levando ao aumento na incidência de perdas pós implntacionais associadas a poluição do ar. Para testar essa hipótese, camundongos foram expostos a níveis ambientais de poluentes até os 60 dias de vidas usando câmaras de exposição [uma câmara recebendo ar filtrado e a outra ar ambiente (poluído) não]. Ao atingir a idade reprodutiva os animais foram colocados para acasalar, os machos utilizados neste estudo não foram expostos aos poluentes, e no 6° e 8° dia pós-coito a gestação foi terminada. O desempenho reprodutivo foi avaliado e as fêmeas foram subdivididas em 4 grupos de acordo com a idade gestacional (6º ou 8º dpc) e exposição (ar filtrado ou ar não filtrado). Métodos estereológicos foram usados para avaliar o desenvolvimento dos sítios de implantação, contagem das células NK uterinas e mastócitos. A concentração de média de PM2.5 nas câmaras com o ar não filtrado foi de 27.5 g.m-3 e 6.5 g.m-3 na câmara com ar filtrado (P<0.001). A exposição a poluição do ar nos primeiros dias de gestação mostrou há diferenças no desenvolvimento dos compartimentos do sítio de implantação no 8° dia pós-coito: redução do volume endométrio e volume total do sítio.Quando nós avaliamos o número total de NK uterina o 8° dia pós-coito, observamos que há uma redução no número destas células nos animais expostos a poluição do ar. Os mastócitos no 6° dia gestacional também se mostram em menor número nos animais expostos a poluição, mas essa diferença desaparece com o avanço da gestação. Na avaliação histopatológica do endométrio, verificamos que há uma redução do volume de células deciduais, de trofoblasto, e um aumento do volume de glândulas nos sítios implantacionais no 8° dia pós-coito de animais expostos ao ar poluído. Nossos resultados confirmam os achados de estudos prévios que correlacionam a exposição a poluição do ar com aumento na incidência de falhas implantacionais e fertilidade diminuída. A avaliação da morfologia uterina e do número de células NK uterinas e mastócitos sugerem que os compostos presentes no ar interferem ou prejudicam o processo de implantação embrionária por alterações na resposta do sistema imunológico materno / Humans are inevitably exposed to mixtures of environmental contaminants, and a vast body of evidence now links exposure to these chemicals with an increased incidence of reproductive disorders. We have shown that mice chronically exposed to ambient levels of air pollution (AP) in São Paulo city have negative reproductive performance: resulting in changes in estrous cyclicity, number of follicles and increased post implantation loss rate as well defective placentation. We hypothesized that alterations in uterine response to implantation could be mediated by mast and uNKcells leading to increased incidence of pos implantation losses associated with exposure to air pollution. To test this female mice were exposed to ambient levels of AP from birth to 60 days of age using exposure chambers (receiving filtered air or non filtered situated near to a high traffic crossroad). On reaching the reproductive age, estrous cyclicity, was evaluated and females were allowed to mate to non exposed males and exposures continued until 6ºdpc or 8ºdpc when pregnancy was terminated. Reproductive performance was assessed and females, subdivided in to 4 groups according to gestational day (6ºdpc or 8ºdpc ) and exposure (filtered or non filtered air). Stereological methods and immunohistochemical techniques were used to evaluate implantation sites (IS) development, do cell identification (NK cells and Mast cells) and counts. Mean concentration of PM2.5 in the non-filtered chamber was 27.5 g.m-3 and 6.5 g.m-3; in the filtered chambers (P<0.001). Females exposed to air pollution previously and during the first days of pregnancy showed borderline differences in the development of the compartments of the IS on day 8 of gestation: volume of the endometrium is reduced as well as the IS total volume. When we evaluated the total number of uNk cells (8ºdpc), we observed that there is a decrease in those animals exposed to air pollution The number of mast cells (6º dpc) are also reduced when compared to females exposed to filtered air, but as pregnancy progress this differences in the number of mast cells disappear. The histopathological evaluation was observe the decrease in the volume occupied by decidual and trophoblast cells, and increased the volume of glands, showing development delay in implantation sites in 8dpc.Our data confirm results from previous studies that link exposure to AP and decreased fertility and increased implantation failure. Evaluation of the uterine morphology and the number of mast call and uNkcell suggests that components present in AP interfere or impair embryonic implantation trought changes in maternal immune system responses

Mouse embryonic stem (ES) cells have played a crucial role in studying developmental processes and gene function in vivo. They are extremely useful in the generation of transgenic animals as they can be genetically manipulated and subsequently microinjected into blastocyst stage embryos, where they combine with the inner cell mass and contribute to the developing embryo. Some of the resulting pups are chimaeric, consisting of a mixture of cells derived from the host blastocyst and the injected ES cells. We have identified several ES cell clones arising from gene targeting experiments with an impaired capacity to generate viable chimaeras. When injected into blastocysts, these clones cause embryonic death during mid to late gestation, suggesting that the cells are able to contribute to the embryo but interfere with normal embryonic development. The aim of this work was to identify the underlying changes in the transcriptome, epigenome or cell surface markers that have occurred in these compromised ES cells and to further define the developmental phenotype of the chimaeric embryos. Different stages during development were analysed and whereas there was little difference in embryonic death at gestational day e13.5, there was a significant decrease in embryos surviving to gestational day e17.5. Additionally, severe haemorrhaging was observed in all the dead embryos and small foci of haemorrhaging could also be seen in a number of embryos that were still alive. This was also observed at e13.5, albeit to a less severe extent. Using RNA sequencing to discover differences in the transcriptome between control ES cells and the compromised ES cells, five genes were identified that were downregulated in the compromised cells. Four of these, Gtl2, Rtl1as, Rian and Mirg are all located in the imprinted Dlk1-Dio3 region on chromosome 12 and are normally expressed from the maternal genome. This pattern was also validated in tissues from e17.5 chimaeric embryos. The expression of this locus is to a large extent regulated by a differentially methylated region located approximately 13kb upstream of the Gtl2 promoter, the IG-DMR. Whereas this is usually only methylated on the paternal copy, in the compromised ES cells both the paternal and the maternal copy were fully methylated, likely causing the silencing of Gtl2, Rtl1as, Rian and Mirg. Using the DNA methyltransferase inhibitor 5-azacytidine, expression of Gtl2 could be rescued. Injection of those 5-azacytidine treated cells into blastocysts did partially rescue the embryonic lethal phenotype. Additionally, cell surface markers were analysed in a phenotypic screen using phage display. NGS analysis of the phage outputs indicates that there may be additional differences in cell surface markers between the control and compromised ES cell clones, but their specific details remain to be identified. Overall, we have identified the maternally expressed genes of the Dlk1-Dio3 region as markers that can distinguish between ES cells with normal or compromised developmental potency and propose to include these genes in the pre-blastocyst injection screening routine for experiments involving the production of chimaeras or genetically modified mouse strains.

Nós humanos estamos inevitavelmente expostos a uma mistura de poluentes, e evidências nos mostram que estes poluentes aumentam a incidência de desordens reprodutivas. Já se sabe que camundongos cronicamente expostos a níveis de poluentes ambientais em São Paulo apresentam: mudanças no ciclo estral, alteração no número de folículos ovarianos, aumento das perdas embrionárias pós implantacionais e alterações na morfologia placentária. Nós hipotetizamos que as alterações na resposta uterina a implantação possa ser mediado por mastócitos e células NK uterinas levando ao aumento na incidência de perdas pós implntacionais associadas a poluição do ar. Para testar essa hipótese, camundongos foram expostos a níveis ambientais de poluentes até os 60 dias de vidas usando câmaras de exposição [uma câmara recebendo ar filtrado e a outra ar ambiente (poluído) não]. Ao atingir a idade reprodutiva os animais foram colocados para acasalar, os machos utilizados neste estudo não foram expostos aos poluentes, e no 6° e 8° dia pós-coito a gestação foi terminada. O desempenho reprodutivo foi avaliado e as fêmeas foram subdivididas em 4 grupos de acordo com a idade gestacional (6º ou 8º dpc) e exposição (ar filtrado ou ar não filtrado). Métodos estereológicos foram usados para avaliar o desenvolvimento dos sítios de implantação, contagem das células NK uterinas e mastócitos. A concentração de média de PM2.5 nas câmaras com o ar não filtrado foi de 27.5 g.m-3 e 6.5 g.m-3 na câmara com ar filtrado (P<0.001). A exposição a poluição do ar nos primeiros dias de gestação mostrou há diferenças no desenvolvimento dos compartimentos do sítio de implantação no 8° dia pós-coito: redução do volume endométrio e volume total do sítio.Quando nós avaliamos o número total de NK uterina o 8° dia pós-coito, observamos que há uma redução no número destas células nos animais expostos a poluição do ar. Os mastócitos no 6° dia gestacional também se mostram em menor número nos animais expostos a poluição, mas essa diferença desaparece com o avanço da gestação. Na avaliação histopatológica do endométrio, verificamos que há uma redução do volume de células deciduais, de trofoblasto, e um aumento do volume de glândulas nos sítios implantacionais no 8° dia pós-coito de animais expostos ao ar poluído. Nossos resultados confirmam os achados de estudos prévios que correlacionam a exposição a poluição do ar com aumento na incidência de falhas implantacionais e fertilidade diminuída. A avaliação da morfologia uterina e do número de células NK uterinas e mastócitos sugerem que os compostos presentes no ar interferem ou prejudicam o processo de implantação embrionária por alterações na resposta do sistema imunológico materno / Humans are inevitably exposed to mixtures of environmental contaminants, and a vast body of evidence now links exposure to these chemicals with an increased incidence of reproductive disorders. We have shown that mice chronically exposed to ambient levels of air pollution (AP) in São Paulo city have negative reproductive performance: resulting in changes in estrous cyclicity, number of follicles and increased post implantation loss rate as well defective placentation. We hypothesized that alterations in uterine response to implantation could be mediated by mast and uNKcells leading to increased incidence of pos implantation losses associated with exposure to air pollution. To test this female mice were exposed to ambient levels of AP from birth to 60 days of age using exposure chambers (receiving filtered air or non filtered situated near to a high traffic crossroad). On reaching the reproductive age, estrous cyclicity, was evaluated and females were allowed to mate to non exposed males and exposures continued until 6ºdpc or 8ºdpc when pregnancy was terminated. Reproductive performance was assessed and females, subdivided in to 4 groups according to gestational day (6ºdpc or 8ºdpc ) and exposure (filtered or non filtered air). Stereological methods and immunohistochemical techniques were used to evaluate implantation sites (IS) development, do cell identification (NK cells and Mast cells) and counts. Mean concentration of PM2.5 in the non-filtered chamber was 27.5 g.m-3 and 6.5 g.m-3; in the filtered chambers (P<0.001). Females exposed to air pollution previously and during the first days of pregnancy showed borderline differences in the development of the compartments of the IS on day 8 of gestation: volume of the endometrium is reduced as well as the IS total volume. When we evaluated the total number of uNk cells (8ºdpc), we observed that there is a decrease in those animals exposed to air pollution The number of mast cells (6º dpc) are also reduced when compared to females exposed to filtered air, but as pregnancy progress this differences in the number of mast cells disappear. The histopathological evaluation was observe the decrease in the volume occupied by decidual and trophoblast cells, and increased the volume of glands, showing development delay in implantation sites in 8dpc.Our data confirm results from previous studies that link exposure to AP and decreased fertility and increased implantation failure. Evaluation of the uterine morphology and the number of mast call and uNkcell suggests that components present in AP interfere or impair embryonic implantation trought changes in maternal immune system responses

Newly established F1 hybrid Embryonic Stem cells allow the production of ES cell-derived animals at a high enough efficiency to directly make ES cell based genetics feasible. An F1 hybrid ES cell line, G4 was used to generate transgenic over-expressing cell lines. The consequence of the expression of a panel of transgenes was assessed directly from ES cell-derived embryos produced by the tetraploid complementation assay. The generation of ES cell-derived embryos/animals was very efficient. A sufficient number of mutants for initial phenotypic analyses was derived only a few weeks after the establishment of the cell lines. The genes used in the study had either angiogenic/vasculogenic, anti-angiogenic or unknown properties. Of these transgenic mouse lines VEGF-A and Flt-Fc were used to further elucidate the effects of altered VEGF signaling on cell fate decisions in embryonic development and ES differentiation in two experimental systems. A. Early but transient Flk-1 activation led to enhanced generation of blood progenitors, whereas continuous activation of Flk-1 abolished this effect and enhanced endothelial cell generation. Ex vivo analysis of cells derived from E7.5 embryos demonstrated that sFlt-1-mediated control of Flk-1 activity also impacted the fate of hematopoietic and endothelial cells. The Flt-1-Fc transgenic mouse model was used to alter Flk-1 activation in vivo and show the relevance of the in vitro observations. These results demonstrate that sFlt-1 regulates Flk-1 activation in an oxygen responsive manner. Inhibition of Flk-1 activation by sFlt-1 increases the specification of hemangioblasts to blood cells consistent with a VEGF-independent default mechanism. B. Ubiquitous over-expression of VEGF164 isoform led to E8.75 embryonic lethality. The primary cause of lethality was the failure to form an organized cardiovascular system, which was manifested in three ways: the absence of yolk sac blood vessels, the lack of embryonic-maternal circulation due to the failure of allantochorionic fusion and improper cardiac function. The described phenotypes suggest that VEGF does not inhibit embryonic or extra-embryonic mesoderm formation at gastrulation but perturbs the balance amongst the mesodermal components.

Newly established F1 hybrid Embryonic Stem cells allow the production of ES cell-derived animals at a high enough efficiency to directly make ES cell based genetics feasible. An F1 hybrid ES cell line, G4 was used to generate transgenic over-expressing cell lines. The consequence of the expression of a panel of transgenes was assessed directly from ES cell-derived embryos produced by the tetraploid complementation assay. The generation of ES cell-derived embryos/animals was very efficient. A sufficient number of mutants for initial phenotypic analyses was derived only a few weeks after the establishment of the cell lines. The genes used in the study had either angiogenic/vasculogenic, anti-angiogenic or unknown properties. Of these transgenic mouse lines VEGF-A and Flt-Fc were used to further elucidate the effects of altered VEGF signaling on cell fate decisions in embryonic development and ES differentiation in two experimental systems. A. Early but transient Flk-1 activation led to enhanced generation of blood progenitors, whereas continuous activation of Flk-1 abolished this effect and enhanced endothelial cell generation. Ex vivo analysis of cells derived from E7.5 embryos demonstrated that sFlt-1-mediated control of Flk-1 activity also impacted the fate of hematopoietic and endothelial cells. The Flt-1-Fc transgenic mouse model was used to alter Flk-1 activation in vivo and show the relevance of the in vitro observations. These results demonstrate that sFlt-1 regulates Flk-1 activation in an oxygen responsive manner. Inhibition of Flk-1 activation by sFlt-1 increases the specification of hemangioblasts to blood cells consistent with a VEGF-independent default mechanism. B. Ubiquitous over-expression of VEGF164 isoform led to E8.75 embryonic lethality. The primary cause of lethality was the failure to form an organized cardiovascular system, which was manifested in three ways: the absence of yolk sac blood vessels, the lack of embryonic-maternal circulation due to the failure of allantochorionic fusion and improper cardiac function. The described phenotypes suggest that VEGF does not inhibit embryonic or extra-embryonic mesoderm formation at gastrulation but perturbs the balance amongst the mesodermal components.

The basis of developmental biology lies in the idea of when and how cells decide to divide or to differentiate. Previous studies have established several signaling pathways that determine cell fate decisions, including Notch, Wingless, Hedgehog, Bone morphogenetic protein, and Fibroblast growth factor. Signaling converges on transcriptional factors that regulate gene expression. In mouse embryonic stem cells, I explored how pluripotency and differentiation are regulated through opposing actions of beta-catenin-mediated canonical Wnt signaling, and the mechanisms underlying Sonic hedgehog signaling in generating progenitor cells in the ventral neural tube.

<p>During embryogenesis, a single zygote gives rise to a multicellular embryo with distinct spatial territories marked by differential gene expression. How is this patterning process organized? How robust is this function to perturbations? Experiments that examine normal and regulative development will provide direct evidence for reasoning out the answers to these fundamental questions. Recent advances in technology have led to experimental determinations of increasingly complex gene regulatory networks (GRNs) underlying embryonic development. These GRNs offer a window into systems level properties of the developmental process, but at the same time present the challenge of characterizing their behavior. A suitable modeling framework for developmental systems is needed to help gain insights into embryonic development. Such models should contain enough detail to capture features of interest to developmental biologists, while staying simple enough to be computationally tractable and amenable to conceptual analysis. Combining experiments with the complementary modeling framework, we can grasp a systems level understanding of the regulatory program not readily visible by focusing on individual genes or pathways. </p><p>This dissertation addresses both modeling and experimental challenges. First, we present the autonomous Boolean network modeling framework and show that it is a suitable approach for developmental regulatory systems. We show that important timing information associated with the regulatory interactions can be faithfully represented in autonomous Boolean models in which binary variables representing expression levels are updated in continuous time, and that such models can provide direct insight into features that are difficult to extract from ordinary differential equation (ODE) models. As an application, we model the experimentally well-studied network controlling fly body segmentation. The Boolean model successfully generates the patterns formed in normal and genetically perturbed fly embryos, permits the derivation of constraints on the time delay parameters, clarifies the logic associated with different ODE parameter sets, and provides a platform for studying connectivity and robustness in parameter space. By elucidating the role of regulatory time delays in pattern formation, the results suggest new types of experimental measurements in early embryonic development. We then use this framework to model the much more complicated sea urchin endomesoderm specification system and describe our recent progress on this long term effort. </p><p>Second, we present experimental results on developmental plasticity of the sea urchin embryo. The sea urchin embryo has the remarkable ability to replace surgically removed tissues by reprogramming the presumptive fate of remaining tissues, a process known as transfating, which in turn is a form of regulative development. We show that regulative development requires cellular competence, and that competence is lost early on but can be regained after further differentiation. We demonstrate that regulative replacement of missing tissues can induce distal germ layers to participate in reprogramming, leading to a complete re-patterning in the remainder of the embryo. To understand the molecular mechanism of cell fate reprogramming, we examined micromere depletion induced non-skeletogenic mesoderm (NSM) transfating. We found that the skeletogenic program was greatly temporally compressed in this case, and that akin to another NSM transfating case, the transfating cells went through a hybrid regulatory state where NSM and skeletogenic marker genes were co-expressed.</p> / Dissertation

XB130 is a relatively unstudied novel signal adaptor protein. In the first phase of this study, an in silico search for proteins related to XB130 was conducted. Two other proteins (AFAP and AFAP1L1) were found to have a significant similarity to XB130 and were compared in detail. After an analysis of these three proteins, it was proposed that they are members of a novel protein family, termed the “AFAP family of signal adaptor proteins”. XB130 has previously been found to regulate cell cycle progression, death, and migration in lung epithelial cells. It was therefore hypothesized that XB130 is protective in acute lung injury (ALI) and important for facilitating repair after injury. XB130 was found to be differentially regulated in ALI depending on the initial insult. Engineering XB130 transgenic mice to further characterize the role of XB130 in lung injury/regeneration revealed that this protein could be essential for early embryo development.

XB130 is a relatively unstudied novel signal adaptor protein. In the first phase of this study, an in silico search for proteins related to XB130 was conducted. Two other proteins (AFAP and AFAP1L1) were found to have a significant similarity to XB130 and were compared in detail. After an analysis of these three proteins, it was proposed that they are members of a novel protein family, termed the “AFAP family of signal adaptor proteins”. XB130 has previously been found to regulate cell cycle progression, death, and migration in lung epithelial cells. It was therefore hypothesized that XB130 is protective in acute lung injury (ALI) and important for facilitating repair after injury. XB130 was found to be differentially regulated in ALI depending on the initial insult. Engineering XB130 transgenic mice to further characterize the role of XB130 in lung injury/regeneration revealed that this protein could be essential for early embryo development.