Browsing by Author "Al Saai, Salma Mohammed"
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Item Investigation of TDRD7 function in ocular lens development(University of Delaware, 2020) Al Saai, Salma MohammedClear eyesight requires maintenance of a transparent ocular lens tissue throughout the life of an individual. Loss of lens transparency, termed cataract, is the leading cause of blindness worldwide. Depending on the age of their onset, cataracts can be age related (common above the age of 70) or congenital (present at birth or within the first year of life). The current cataract treatment option is surgery, which is costly but is successful in restoring vision, especially in adults. Treatment of congenital cataract in children is clinically challenging due to long-term complications of surgery such as retinal detachment and secondary glaucoma. Additionally, timely restoration of vision is critical because an opaque lens in early childhood may cause amblyopia, sensory deprivation, which can prevent normal eye development and cause permanent blindness. About 25%-50% of congenital cataracts are estimated to be caused by an underlying genetic alteration and over 40 genes have been linked to congenital cataract. ☐ Mutations in the TDRD7 (Tudor domain containing 7) gene are linked to human congenital cataract, and TDRD7 polymorphisms are associated with age related cataract. Additionally, multiple mouse models that are Tdrd7 deficient have been described and they exhibit fully penetrant cataract, with defects similar to that observed in human. Tdrd7 encodes a ribonucleoprotein / RNA granule component protein that is predicted to bind to other proteins through its Tudor domains and OST-HTH/LOTUS domains and closely associate with RNA. The identification of Tdrd7 mutations linked to congenital cataract suggests that regulators of post-transcriptional gene expression control are critical for lens development and maintenance of transparency in vertebrates. However, the pathobiology of the Tdrd7-deficienct cataract defects and mechanistic basis of Tdrd7 function in the lens is not fully understood. My dissertation work sought to address this knowledge gap by studying Tdrd7 targeted knockout mouse (Tdrd7-/-) as a model of this human disease. ☐ Phenotypic characterization of Tdrd7-/- mouse lens identified fully penetrant cataracts by postnatal age 22 (P22). Analysis by scanning electron microscopy revealed that Tdrd7-/- lenses at P18, which are non-cataractous, exhibit fiber cell defects. However, the nature of the fiber cell defect was undefined. By detailed characterization of Tdrd7-/- lenses using molecular and cellular approaches, I have uncovered a novel function for Tdrd7 in regulating fiber cell morphology. My confocal microscopy data on Tdrd7-/- lens cross-sections stained for Phalloidin (to visualize F-actin) shows that severe F-actin cytoskeletal defects specifically in fiber cells in late stages of post-nuclear degradation. Integrated bioinformatics analysis of RNA-seq data coupled with protein two-dimensional fluorescence difference in-gel electrophoresis and mass spectrometry screen on Tdrd7-/- lens 14 days prior to the onset of cataract, identified Tdrd7 to be upstream of the heat shock protein Hspb1. My experiments confirmed the reduction of Hspb1 mRNA and protein in the Tdrd7-/- lens. Further, I also performed confocal microscopy to characterize Hspb1 spatiotemporal expression in normal lens development. I also identified Tdrd7 protein to be associated with the mRNA of Hspb1 in the lens by both biochemical (i.e. RNA-immunoprecipitation coupled with RT-PCR) and fluorescence microscopy (i.e. small RNA imaging coupled with immunostaining) approaches. Together, these findings in my dissertation have led to a model where Tdrd7 sustains optimal high levels of the Hspb1 in maturing lens fiber cells that have undergone organelle degradation. These lens fiber cells in terminal stages of maturation can be considered to resemble a stress-like condition. Moreover, Hspb1 is known to interact with F-actin and function in maintaining the cytoskeleton under conditions of stress. Thus, the model derived from my data further suggests that Tdrd7-based control of Hspb1 levels may be necessary for maintenance of F-actin cytoskeleton and cellular morphology of fiber cells post-nuclear degradation. Together these findings serve to expand the role of Tdrd7-family proteins in cellular differentiation during organogenesis. ☐ Next, to gain further molecular insights into the alterations caused by the deficiency of Tdrd7, I have generated and investigated the transcriptome (by RNA-seq) and the proteome (by TMT-coupled with Mass spectrometry) of Tdrd7-/- mouse lens 7 days prior to the onset of cataract (postnatal day P15). Using a systematic integrated approach to carefully analyze both data sets I was able to identify several new high-priority candidate genes (Bfsp1, Bfsp2, Cap2) and proteins in P15 Tdrd7-/- lens, several of which may contribute to the cataract defects as well as may serve to further explain the cytoskeletal defects. These data suggests a role for Tdrd7 in maintaining several aspects of the lens cytoskeleton. In addition, these new data have identified a long noncoding RNA (Snhg12) to be severely reduced in Tdrd7-/- P15 lens transcriptome. These findings further supports the importance of Tdrd7 as a key regulatory factor important for maintaining lens transparency. ☐ Finally, I have examined a new high-priority target downstream of Tdrd7, namely Cap2 (Cyclase Associated Proteins2). The P15 Tdrd7-/- lens RNA-seq and proteome analysis identified Cap2 among the top upregulated candidates. By iSyTE analysis, I showed that Cap2 is highly expressed and enriched in the mouse lens across several embryonic and postnatal stages. I performed cellular and molecular characterization of Cap2-/- mouse lens. Interestingly, Cap2-/- lenses exhibited lens fiber cell morphological defects, including F-actin aggregates, in the same region of lens that exhibited defects in Tdrd7-/- mice, namely the region containing maturing fiber cells, post-nuclear degradation. Thus, I have identified Cap2 as a new factor critical for lens biology. Together, these data provide new evidence that Tdrd7 functions in the control of the cellular morphology of maturing fiber cells at the beginning of the organelle degradation zone in the lens. ☐ In sum, work in this dissertation has uncovered the cellular and molecular basis of the lens defects in Tdrd7-/- mice, in turn leading to the advancement of our understanding of the pathobiology of Tdrd7-deficiency based cataracts.