Peptidoglycan fragment microarray platform for human immune system investigation
Date
2021
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
The eye is a complex multicomponent organ, comprising of different tissues such as the cornea, lens, and the retina, which enables high-resolution vision. Disruption of eye development can cause defects such as congenital cataracts (clouding of the ocular lens at birth or within the first year of life), anophthalmia (absence of eye tissue) and microphthalmia (abnormal reduction of eye tissue). Congenital cataract accounts for 5-20% of developmental eye disorders and is detected in 3-4 among 10,000 live births. On the other hand, anophthalmia and microphthalmia (A/M) are detected in 2-6 among 30,000 live births. Moreover, microphthalmia is often associated with other ocular defects such as cataract and coloboma (missing eye structures). However, identification of the underlying genetic changes that are associated with these defects is challenging. Thus far only ~30 genes are linked to A/M defects, while the genetic basis of many A/M cases is undefined. Similarly, mutations or functional compromise of ~50 genes are described to cause non-syndromic or isolated pediatric cataract. ☐ The majority of the genetic changes linked to pediatric cataract have been identified in genes that exhibit highly enriched expression in lens fiber cells suggesting that regulation of their expression in these cells is essential to maintenance of lens transparency. However, so far only a limited number of regulatory factors (e.g., c-Maf, Hsf4, Pax6, Prox1, Sox1, Tdrd7) that are primarily associated with fiber cell gene expression have been linked to lens defects. Similarly, while alterations of several transcription factors have been linked to A/M, our understanding of RNA-binding proteins involved in post-transcriptional gene expression control in eye development and its associated defects remains limited. My dissertation has addressed these critical knowledge-gaps by (1) characterization of two understudied transcription factors (the fiber cell expressed proteins Mafg and Mafk) in embryonic lens development, the perturbations of which result in severe lens defects, (2) characterization of the key downstream target (the molecular chaperone Hspb1) of the pediatric cataract-linked gene Tdrd7, and (3) characterization of an RNA-binding protein (Rbm24) in early eye development, the perturbation of which results in A/M defects. ☐ Deficiency of the small Maf proteins Mafg and Mafk cause multiple defects, namely, progressive neuronal degeneration, cataract, thrombocytopenia, and mid-gestational/perinatal lethality. Previously it was described that Mafg-/-:Mafk+/- compound knockout mice exhibit cataracts from age 4-month onward and Mafg-/-:Mafk-/- double knockout mice develop lens defects significantly early in life, during embryogenesis. However, the pathobiological basis the lens defects in Mafg-/-:Mafk-/- was unknown, representing a key knowledge-gap, which is addressed here in my dissertation. I find that at embryonic day (E) 16.5, the anterior epithelium of the lens (AEL) in Mafg-/-:Mafk-/- animals appears abnormally multilayered as demonstrated by E-cadherin and nuclear staining and is extended toward the posterior region of the lens. Additionally, Mafg-/-:Mafk-/- double knockout lenses exhibit abnormal abundance of F-actin in the region near the “fulcrum” where AEL cells undergo apical constriction prior to elongation and reorientation as early differentiating fiber cells. To uncover the underlying molecular changes associated with these defects, I performed high-throughput RNA-sequencing (RNA-seq) of E16.5 Mafg-/-:Mafk-/- double knockout lenses. Based on rigorous downstream analyses, RNA-seq identified 239 genes that were differentially expressed genes (DEGs) between control and Mafg-/-:Mafk-/- double knockout lenses (with filters of ± 1.5-fold change and p-value < 0.05). The DEGs were further prioritized for their potential role in the lens based on gene ontology (GO) analysis, iSyTE analysis, and the published literature and validated by RT-qPCR and/or immunostaining. These data showed that the Epha5 (Eph receptor A 5), which encodes an Eph receptor protein, was significantly reduced in Mafg-/-:Mafk-/- knockout lenses. Interestingly, deletion of ephrin-A5 (also known as Efna5), a known ligand of the Epha5 receptor, has been associated with lens epithelial cell adhesion defects in other studies. Thus, reduction of Epha5, in the context of other gene expression changes, may contribute to the pathology of Mafg-/-:Mafk-/- knockout AEL multicellularity defects. Additionally, other key factors associated with the cytoskeleton, cell cycle or extracellular matrix (e.g., Cdk1, Cdkn1c, Camsap1, Col3A1, Map3k12, Sipa1l1) were found to be mis-expressed in Mafg-/-:Mafk-/- double knockout lenses, which may also contribute to the lens pathology in these animals. Because factors involved in the cell cycle were found to be mis-expressed in Mafg-/-:Mafk-/- double knockout lenses, I next sought to examine potential alterations in cell proliferation resulting from Mafg and Mafk deficiency. Immunostaining of the established cell proliferation marker Ki67 showed that the number of AEL cells that were proliferating were significantly elevated in Mafg-/-:Mafk-/- knockout lenses, which can partially explain the expansion of the AEL toward the posterior region of the lens. Together, these findings demonstrate a novel role for Mafg and Mafk early in lens development, and furthermore, uncover new downstream regulatory relationships with key cellular factors that potentially may be of significance in non-lens tissues where these transcription factors are expressed. ☐ To advance our understanding of post-transcriptional gene expression control in lens development and its associated defects, I focused on examining candidate RNA-binding proteins and their downstream targets in eye and lens development. Previously, iSyTE identified an RNA-binding protein, Rbm24, as a potential regulator of eye development. In mouse, Rbm24 is expressed in the presumptive lens ectoderm and the optic vesicle at E9.5, and in later stages in the lens and the retina, suggesting that Rbm24 may function in eye development from embryonic early stages. Initial analysis showed that Rbm24GM/GM germline knockout mice exhibit A/M and lens defects. However, Rbm24GM/GM mouse embryos are smaller in size and exhibit early embryonic lethality, and therefore cannot inform on the autonomous requirement of Rbm24 in either the developing optic vesicle or the lens. Therefore, to further understand the role of Rbm24 in the eye, specifically in the optic vesicle, I generated a new Rbm24 conditional knockout mouse line, Rbm24cKOov, in which Rbm24 is deleted from the optic vesicle of the eye. Rbm24cKOov do not exhibit smaller size embryos but exhibit A/M at E10.5. Further, immunostaining demonstrated that the transcription factors Pax6 and Lhx2 exhibited reduced protein levels in the optic vesicle, and furthermore, Pax6 protein was found to be reduced in the lens pit in Rbm24cKOov mouse embryos at E10.5. Together, these data suggest that Rbm24 has an autonomous role in the developing optic vesicle and functions to regulate expression of key transcription factors in the eye. ☐ Besides Rbm24, I worked on another RBP called Tdrd7, which belongs to Tudor family of proteins and is implicated in post-transcriptional control of gene expression. Mutations or deficiency of TDRD7 is linked to congenital cataract in humans as well as in other animal models. Previous work focused on the characterization of molecular changes in the lens in multiple, independently developed Tdrd7 deficient mouse models, which identified the heat shock protein Hspb1 (also known as Hsp27) to be significantly reduced at both mRNA and protein levels. Hspb1 is among the highly expressed small heat shock proteins in humans that is involved in the regulation of cytoskeleton under stress conditions among other cellular processes and is linked to various diseases. However, its involvement in development of the eye and the lens was not characterized. Therefore, I focused on investigating the role of Hspb1 in eye and lens development. ☐ Toward this goal, in collaboration with Dr. Shuo Wei’s laboratory, I generated a new animal model for HSPB1-knockdown using Xenopus tropicalis. I find that HSPB1-knockdown results in microphthalmia and other eye defects such as loss of pigmentation in the retina. Importantly, I show that these defects can be rescued by ectopic expression of mouse Hspb1 mRNA. Further, I find that HSPB1-knockdown results in a reduced expression of Aqp0, an important water channel expressed in lens fiber cells. These data demonstrate that Hspb1 functions in eye and lens development and provide new insights into the mechanism of cataract pathology in Tdrd7-/- lenses. ☐ In summary, my dissertation research has resulted in new advances in the understanding of transcriptional and post-transcriptional gene expression control in the eye and the lens through the characterization of key regulatory proteins such as Mafg, Mafk, Rbm24, and the Tdrd7-target, Hspb1. These data have uncovered new mechanisms underlying the pathology of eye defects such as cataracts, anophthalmia, or microphthalmia.
Description
Keywords
Anophthalmia, Cataracts, Devenopment, Disease, Lens, Microphthalmia