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PROGRAM | Biological Sciences

Investigating Transcriptional and Post-transcriptional Control in Eye and Lens Development and their Associated Defects

By: Shaili Patel Chair: Salil Lachke


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 cataracts (clouding of the ocular lens), anophthalmia (absence of eye tissue) and microphthalmia (abnormal reduction of the eye). Depending on its onset, cataract is termed as congenital/pediatric or age-related. 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 ~52 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 transcription factors (e.g. Pax6, c-Maf, Prox1, Sox1 and Hsf4) 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 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.

It is known that the 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 (KO) mice exhibit cataracts from age 4-month onward and Mafg-/-:Mafk-/- double KO 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-/- KO 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 next sought to uncover the underlying molecular changes associated with these defects, I performed high-throughput RNA-sequencing (RNA-seq) of E16.5 Mafg-/-:Mafk-/- KO lenses.  Based on downstream analysis, RNA-seq identified 239 genes that were differentially expressed genes (DEGs) between control and Mafg-/-:Mafk-/- KO 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 Eph receptor ligand Epha5 was significantly reduced in Mafg-/-:Mafk-/- KO lenses.  Deletion of Epha5 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-/- KO 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-/- KO lenses, which may also contribute to lens pathology.  Because factors involved in the cell cycle were found to be misexpressed in Mafg-/-:Mafk-/- KO lenses, I 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-/- KO 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.

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, the transcription factors Pax6 and Lhx2 were both reduced in the optic vesicle and Pax6 was reduced in the lens pit in Rbm24cKOov mouse embryos at E10.5. Together, these data suggest that Rbm24 has an autonomous role in the developmental optic vesicle and functions to regulate expression of key transcription factors in the eye.

In my research dissertation, 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 independently developed Tdrd7 null 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 lens and eye 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. Importantly, I show that these defects can be rescued by ectopic expression of the mouse Hspb1 mRNA. Further, I find that Hspb1-knockdown results in a reduced expression of Aqp0, a water channel in the lens. 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/or lens through the characterization of key regulatory proteins such as Mafg, Mafk, Rbm24, and the Tdrd7 target, Hspb1. These data have uncovered new mechanisms of eye defects such as cataracts, anophthalmia, or microphthalmia.

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