Recommended
More Related Content
What's hot
What's hot (18)
Similar to SigmaXiShowcase_S_Patel_2020
Similar to SigmaXiShowcase_S_Patel_2020 (20)
Recently uploaded
Recently uploaded (20)
SigmaXiShowcase_S_Patel_2020
- 1. Rbm24 Function in Early Eye and Lens Development Shaili Patel Ph.D./MBA Candidate Laboratory of Dr. Salil A. Lachke Department of Biological Sciences University of Delaware
- 2. Eye is a complex multi-component organ Cornea Retina Lens Optic nerve • The eye consists of the cornea and the lens in the anterior segment and the retina in the posterior segment • The lens and the cornea are transparent tissues that enable light to be focused on the retina • The focused light, is then converted to electric impulses that are relayed to the brain via the optic nerve and that’s how we can see
- 3. Eye development in mammals • Eye development starts during gastrulation from the neural ectoderm tissue • Interactions and signaling between each developing tissues makes it possible for the eye to develop normally • Any disruption of these morphogenetic events by genetic or environmental influences can affect the eye at a cellular level (both transcriptional and/or post- transcriptional level) and potentially cause growth and structural defects resulting in congenital eye disorders Dash et al. (2016), WIREs RNA
- 4. Anophthalmia and microphthalmia are congenital eye disorders • Anophthalmia- Absence of eye tissue (a) • Microphthalmia- Decreased size of eye (b) • They account for ~3-12% visual defects in children- 2-6 in 30,000 live human-births • They can both be unilateral or bilateral- various ocular anomalies (c) • These phenotypes arise because of defects in signaling and transcription factor function • Signaling and transcription in general has been well studied in ocular development, however, post-transcriptional control is not well visited Shah et al. (2011b) Philippa Harding and Mariya Moosajee, Aug.2019 * Verma and Fitzpatrick (2007) Gonzalez et al. (2011) False eye
- 5. Transcriptional regulation in eye development • (B) shows a transcriptional and signaling regulatory gene network during eye development between embryonic days E9.5 to E10.5 in two specific parts of the eye called the optic vesicle (OV) and Preplacodal Lens Ectoderm (PLE) (A) • Gene map in (B) shows that transcriptional regulation and signaling is well studied in the eye during development • However, post- transcriptional regulation is not well studied OV PLE S. A. Lachke and R. L. Maas, (2010), WIREs Systems Biology and Medicine B A
- 6. Post-transcriptional control of gene expression by RNA-binding proteins (RBPs) GENOME PROTEOME TRANSCRIPTOME NUCLEUS CYTOSOL 3’ END CLEAVAGE, POLYADENYLATION EXPORT STABILITY DECAY TRANSLATION TRANSCRIPTION CAPPING +1 (A)n LOCALIZATION 5’ 3’ RBP RBP RBP GENEPROTEIN Pre-mRNA 5’ UTR 3’ UTR SPLICING RBP RBPRBP RBP RBP RBP (A)n RBPRBP RBP RBP (A)n RBP (A)n (A)n RBPRBP (A)n RBP RBP Dash et al. (2016), WIREs RNA • Post-transcriptional regulation is facilitated by two major classes of molecules (1) miRNA and (2) RBPs (in yellow ovals in the figure ) • The focus of our lab is to study RBPs • RBPs play an important role throughout the life cycle of an mRNA starting from its birth to death, thus regulating the proteome of a cell which eventually drives development
- 7. RNA-binding proteins Tdrd7 and Celf1 are essential for vertebrate lens development Siddam et al. (2018), PLOS Gen.Lachke et al. (2011), Science • Our lab has characterized post-transcriptional regulators such as Tdrd7 and Celf1 are important for normal eye and lens development • (A) shows that deficiency or mutations in Tdrd7 results in Cataracts in Human, Mouse and Chicken; whereas (B) shows that deficiency of Celf1 results in cataracts or microphthalmia in Mouse, fish, and frog A B Barnum*, Al Saai*, Patel*, et al. (2020), in press, Hum. Mol. Gen.
- 8. How does our lab find these genes to study eye development and disease? Kakrana et al. (2018), Nucleic Acid Res.Lachke et al. (2012), IOVS • Our lab designed a bioinformatics tool called iSyTE (integrated systems tool for eye gene discovery). iSyTE predicts genes that are important (present) in the lens of the eye compared to the whole body • Since they are eye specific genes, it means that when deleted, they can specifically cause eye diseases • Using this tool we find a new RBP called Rbm24, which is my gene of interest • Rbm24 is highly enriched and expressed in the lens of the eye during and after development Aryal et al. (2020), Hum. Genet. The redder or pink the higher the lens enrichment in the lens; the bluer, lower the lens enrichment.
- 9. What is Rbm24? • Rbm24 is RNA-binding motif protein 24. It is highly conserved in vertebrates (Fish, Frog, Mouse, and Humans (Top to bottom as shown in the figure) • It consists of two main domains, the RRM domain which is known to bind to RNA and could be important for stabilizing, degrading, and/or for the translational control; whereas the function of Alanine rich regions is not well studied
- 10. Function of Rbm24 in vertebrate development • Zebrafish Knockdown (reducing protein production) results in 1) Eye defects (based on my current data) 2) Cardiac defects 3) Defects in somite 4) Abnormal craniofacial morphology • Xenopus Knockdown results in 1) Eye defects (based on my current data) 2) Cardiac defects • Mouse Knockout (deleting it completely) results in 1) Eye defects (based on my current data) 2) Cardiac defects 3) Skeletal defects
- 11. Rbm24 post-transcriptionally controls key downstream regulatory factors in distinct tissues in the developing eye, disruption of which leads to ocular defects Global Objective
- 12. Rbm24 protein is expressed during mouse eye development Rbm24 protein is expressed in different tissues of the eye such as the lens (lpt, lv, e, f), surface ectoderm (se),and optic vesicle (ov), and the retina (oc, r) E10.5 oc lpt oc lpt B B’ E11.5 r lv r lv C C’ Rbm24 DAPI A A’ ov se se ov E9.5 E14.5 e f e f D’ D Dash, Brastrom, Patel et al. (2019) Hum. Mol. Gen.
- 13. Rbm24 deletion in mouse results in anophthalmia and microphthalmia Dash, Brastrom, Patel et al. (2019) Hum. Mol. Gen. • Deleting Rbm24 from the whole body, results in a 100% penetrance of anophthalmia (B’) (no eye) and/or microphthalmia (abnormally small eye (D’)) • Because Rbm24 plays an important role in heart development, deleting it from the whole body also results in embryonic lethality starting embryonic (E) day E7.5 and the mice are completely lethal by E14.5
- 14. rbm24a is also expressed in zebrafish eye rbm24arbm24arbm24a 2dpf BA 20 som. stage l r 2dpf C rbm24a Dash, Brastrom, Patel et al. (2019) Hum. Mol. Gen. • Because there is 80% conservation between zebrafish and mouse Rbm24 , we decided to test whether the expression of Rbm24 is also conserved in fish • In zebrafish, one of the Rbm24 orthologs, rbm24a, is expressed in early eye development beginning at 20-somites stage and, analogous to mouse expression, rbm24 is later enriched in the lens at 2-day post fertilization (dpf)
- 15. rbm24a knockdown and knockout zebrafish exhibit ocular defects rbm24aKDrbm24aKO • Because there is 80% conservation between zebrafish and mouse Rbm24, we decided to knockdown and knockout Rbm24 expression in fish • We observe a similar small eye phenotype (B’ D, D’) Dash, Brastrom, Patel et al. (2019) Hum. Mol. Gen.
- 16. Rbm24 deletion results in downregulation of eye development markers A B C B’ C’A’ • Deleting Rbm24 from the whole-body results in eye defects • Eye development proteins such as Paired box 6 (Pax6), a master regulator of the eye, E-cadherin (E-cad), a protein important for normal lens development, and γ-Crystallin (γ-Cry), the most abundant proteins in the eye that help maintain the eye transparency, are all significantly reduced in expression in absence of Rbm24 lv- lens vesicle- tissue from which the lens develops e- epithelial fiber cells of the lens f- fiber cells of the lens oc- optic cup- tissue from which the retina develops r- retina Asterisk * shows eye defects Dash, Brastrom, Patel et al. (2019) Hum. Mol. Gen.
- 17. Rbm24 deletion results in severely reduced expression of Sox2 in mouse C DControl Rbm24-/- A B E9.5 Control Rbm24-/- Sox2 DAPI se ov se ov E14.5 e f r e f r C D • Sox2 protein is significantly downregulated in Rbm24 deletion mice during early eye development at E9.5 • Mutations in SOX2 gene cause anophthalmia and microphthalmia in humans • 20% of human anophthalmic cases are linked to mutations in the SOX2 gene alone • 73% reported SOX2 mutations lead to unilateral anophthalmia and the remaining 27% are affected with unilateral/bilateral microphthalmia Dash, Brastrom, Patel et al. (2019) Hum. Mol. Gen.
- 18. Deletion of Rbm24 results in downregulation of Sox2 mRNA in mouse and fish B * * * Sox2mRNA(f.c.) Fish E14.5 Sox2mRNA(f.c.) * A Mouse Dash, Brastrom, Patel et al. (2019) Hum. Mol. Gen.
- 19. Rbm24 regulates Sox2 expression by post- transcriptional mechanism Dash, Brastrom, Patel et al. (2019) Hum. Mol. Gen. In press 1 410 1369 2453 5’ UTR 3’ UTRExon 1 Sox2 mRNA 1418 1526 1732 ARE sites A1 A2 A3 A • (A) Sox2 mRNA contains AU Rich Elements (AREs) in its 3’UTR (Rbm24 is known to bind to AREs in Colorectal Carcinoma cells • Therefore, we wanted to test if Rbm24 binds to Sox2 mRNA to control its stability (because that’s what RNA-binding proteins do! • They help in post-transcriptional regulation (remember slide 6? ) Dash, Brastrom, Patel et al. (2019) Hum. Mol. Gen.
- 20. Rbm24 regulates Sox2 expression stabilizing its mRNASox2mRNAIP/Input IgG Rbm24 RIP B * RNA Immunoprecipitation (RIP RNA EMSAC Unbiotinyl. WT ARE (AUUUUA) Biotinyl. WT ARE (AUUUUA) - + - - + + + + + Unbiotinyl. MT ARE (AGGGGA) - + + - - - + RNA-Protein complex Free RNA Rbm24 protein (B) Sox2 mRNA binds to Rbm24 protein, showing there is interaction between them C) Rbm24 binds to Sox2 3’UTR (band on the right, left band shows wildtype competitive). For more data see paper cited below Dash, Brastrom, Patel et al. (2019) Hum. Mol. Gen.
- 21. Rbm24 deletion results in reduced expression of Lhx2 E9.5 ov se ov se Lhx2 DAPI B C E9.5 Control Rbm24-/- Lhx2mRNA(f.c.) A E14.5 * Mouse • In addition to lens proteins, deletion of Rbm24 results in reduced expression of LIM Homeobox 2 (Lhx2), a retina protein important for retina development • Lhx2 expression is also significantly reduced at a mRNA level Dash, Brastrom, Patel et al. (2019) Hum. Mol. Gen.
- 22. Rbm24 role in other vertebrates Control MO Rbm24 MO InjectedUninjected Microphthalmia Patel et al. in prep. • Reducing Rbm24 protein expression in frogs results in eye defects • Frog embryos injected with a Rbm24 morpholino (MO) exhibit smaller eyes (microphthalmia) • MO is a type of oligomer molecule used in molecular biology to modify gene expression
- 23. Conclusions • Rbm24 is expressed in the eye during development • Deleting Rbm24 in mouse, fish and frogs results in eye defects • Sox2 mutations results in eye defects in humans, which accounts for 20% anophthalmia/microphthalmia cases • Rbm24 binds to Sox2 and is important for stabilizing Sox2 mRNA, showing that Rbm24 is important for eye development • Rbm24 also controls other eye developmental proteins such as Pax6, E-Cad and γ-Cry, which are all important for normal development of different eye tissues
- 24. Current working model of Rbm24’s role in early eye and lens development Jag1Sox2 Lhx2 Pax6 Sox2 Cell SurvivalOptic vesicle GRN Lens placode GRN Rbm24 Optic vesicle/Optic cup Lens placode/Lens vesicle Birc2 Cell Surviv al Birc2 Rbm24 • Working model for Rbm24 function in Eye Development is that, Rbm24 regulates Sox2 and Lhx2 in the retinal lineage which regulates the Retinal gene regulatory network • It also regulates Pax6 an Sox2 in the lens lineage which regulate each other and the lens GRN. Further Rbm24 regulates Jag1 (data not shown here) which along with Sox2 regulates cell survival • Thus our work has enabled an overlay of transcriptional regulation network with that of a post-transcriptional regulator. This shows that like transcription factors, RBPs form a distinct regulatory network to control the proteome of a cell and development of a tissue type AnteriorPosterior
- 25. Acknowledgements Advisor: Dr. Salil Lachke, Associate Professor and Chair Collaborators: Dr. Shuo Wei Lab, University of Delaware Dr. Diane Slusarski Lab, University of Iowa Thank you for taking the time out to look at my application!
Editor's Notes
- Fig 2. Development of ocular tissue in mammals from anterior neural plate (mouse stage E8.5) to lens, cornea and retina (Graw, 2009). Eye development in vertebrates begins late during gastrulation from the ectodermal tissue when the anterior neural plate bifurcates to two optic sulci in the eye field at E8.5 in mouse. This is followed by evagination of lateral walls of diencephalon leading to the formation of the optic vesicle (OV) on both sides. The OV signals to the overlying surface ectoderm inducing it to thicken to form a lens placode. Reciprocally, the lens placode signals to OV resulting in the formation of a double-layered optic cup (OC). The inner cell layer of the OC will form the neural retina and the outer cell layer will differentiate into the retinal pigment epithelium later in development. At the same time, lens placode invaginates the developing OC to form the lens pit. Subsequently, it separates from the surface ectoderm to form the lens vesicle and the overlying ectoderm contributes to the formation of cornea. While anterior cells of the lens vesicle form the anterior epithelium of the lens (AEL), the posterior cells of the lens vesicle terminally differentiate into primary fiber cells. These fiber cells elongate to fill the lumen of the lens vesicle. Cells of AEL that are near the equator of the lens exit cell cycle and initiate differentiation into secondary fiber cells. This process is continuous through out the lifetime of an individual
- These phenotypes arise because of defects in signaling and TF function. Signaling and transcription in general has been well studied in ocular development, however, post-transcriptional control is not well visited. Let me take a step back and explain what I mean by post transcriptional control Because it is clinically prevelant impacting 3-12% of the population it is important to study the mechanism of how this develops. In order to do so our lab studies post-transcriptional… (title of next slide) They can be unilateral or bilateral, and when unilateral, the contralateral eye may be normal or have various ocular anomalies, including cataract or coloboma Microphthamlia- clinically confirmed if the axial length of the eye is more than two standard deviations (SD) below the age adjusted population mean; < 21 mm in adults and < 14 mm in newborns Microphthalmia is classed as severe if the corneal diameter is < 4 mm and is associated with a total axial length < 10 mm at birth or < 12 mm after 1 year of age Figure: Clinical images of anophthalmia and microphthalmia. (a) Bilateral anophthalmia. (b) Bilateralmicrophthalmia. (c) Unilateral anophthalmia with shell. SO because A/M are prevalent, it is important to study what leads to these eye disorders during development. Therefore to study diseases like these, our lab focuses on understanding the post-transcriptional regulation of eye development.
- SO because A/M are prevalent, it is important to study what leads to these eye disorders during development. Therefore to study diseases like these, our lab focuses on understanding the post-transcriptional regulation of eye development. Post transcriptional regulation is facilitated by two major classes of molecules (1) miRNA and (2) RBPs. Our lab focuses on RBPs. So why are RBPs important? RBPs control different decisions in the life of an mRNA. For example they can influence 5’ capping, 3’ polyadenylation, splicing, transport and storage or degradation. RBPs assist in one or more of these processes that control the expression of the protein from the mRNA, thus regulating the proteome of a cell which eventually drives development. Our lab focusses on identifying and characterizing RNA-binding proteins in eye development. We have found two such RBPS to be essential for eye development.
- in recent years, our lab has shown that in addition to transcriptional regulation, post transcriptional regulators are also important. We have characterized post-transcriptional regulators such as tdrd7 and celf1 and we have shown that they are imp for normal lens development. Tdrd7 rna-associated protein
- Because we saw an isyte prediction of high enrichment of rbm24 in the lens, we investigated the Rbm24 protein expression in the ocular tissue in the E10.5, E11.5 and E14.5 developing lens. Also noticed that Rbm24 is expressed in the optic cup and the retina. But when we analyzed earlier developmental stages, we found the expression of Rbm24 in the surface ectoderm and the optic vesicle, suggesting that it may be involved in the function of both these during early eye development.
- RATIO of eye size? Here, we demonstrate that in addition to these phenotypes Rbm24-/- mice exhibit severe ocular defects at 100% penetrance. At E11.5, approximately half of the Rbm24-/- mice exhibit anophthalmia (absent/rudimentary ocular tissue due to arrested eye development) on one side and microphthalmia (small eye) on the other, while the remainder of the mutants exhibit bilateral microphthalmia. On phenotypic analysis we observed Rbm24-/- mice die at variable frequency after E7.5 and do not survive after E14.5, and further, after stage E9.5 they are smaller compared to control.
- Because there is 80% conservation between Zebrafish and mouse Rbm24 , we decided to test whether the expression of Rbm24 is also conserved in fish. All zebrafish experiments were performed in collaboration with Dr. Diane Slusarski’s lab at University of Iowa. First off, because of genome duplication in zebrafish, there are two orthologs in rbm24 in zebrafish, rbm24a and rbm24b. In zebrafish, one of the Rbm24 orthologs, rbm24a, is expressed in early eye development beginning at 20-somites stage and, analogous to mouse, is later enriched in the lens at 2-day post fertilization (dpf). The other zebrafish Rbm24 ortholog, rbm24b, exhibits high expression in somites but low expression in the developing eye tissue. This data was suggestive of rbm24a as the ortholog that likely functions in the eye,
- This data identifies Rbm24 as a new post-transcriptional regulator necessary for eye development in vertebrates and suggests rbm24a is the ortholog likely functions in the eye In a dose-dependent manner, rbm24a morphants exhibit progressively severe microphthalmia at high frequency (1 ng: 76%; 2 ng: 92%) or anophthalmia (rudimentary or diffused ocular tissue) at 1.5% penetrance at 3dpf stage Therefore in zebrafish, we used morpholinos and CRISPR/Cas9 to generate rbm24a-knockdown and deletion, respectively. In a dose-dependent manner, rbm24a morphants exhibit progressively severe microphthalmia at 3dpf. To confirm the zebrafish rbm24a-knockdown phenotypes, CRISPR/Cas9 was used to generate deletions in rbm24a, which resulted in microphthalmia in F0 embryos at 3dpf stage. Rbm24a- In addition a subset of rbm24 KD morphants exhibited features of aphakia which is the absence of the lens tissue. Together, these data identify Rbm24 as a new post-transcriptional regulator necessary for eye development in vertebrates. Rbm24 a – starts with eye defect and then heart defect No crispr with b- even though we observed the microopthal, microopth disorder is coming after the heart phenotype- and that’s lethal and its not an eye specific phenotype compared to the other one, in rbm1- you see eye first then growth retardation and then heart
- To answer if Sox2 is important for eye development in absence of Rbm24, I performed immunostaining on Rbm24 knockout mice with Sox2. Interestingly, Sox2 protein is significantly downregulated in Rbm24-/- mice during early eye development at E9.5 in the presumptive lens ectoderm and optic vesicle (Fig. 4A-B’), and is undetected in knockout ocular tissue at later developmental stages
- At E14.5 when there is some eye structure formed, there is a milder kind of defect, because this is the surviving tissue. But even in the milder kind of defect, when you do the whole eye lystate qRT-PCR, Sox2 is downregulated. While Sox2 mRNA is expectedly found to be severely down-regulated in Rbm24-/- eye tissue, it is also reduced in whole embryonic tissue (Fig. 4F). Moreover, sox2 mRNA is also found to be downregulated in rbm24a morphants zebrafish
- Interestingly, Sox2 mRNA 3’UTR has three AU-rich elements (AREs) and Rbm24 can bind AREs in other mRNAs (namely p21 and p63) in human colorectal carcinoma cells . first tested the possibility that Rbm24 directly binds to Sox2 ARE by performing electrophoretic mobility shift assay (EMSA).
- We find that the full-length Rbm24 protein binds, in a sequence-specific manner, to a 20-bp biotin-labeled RNA-oligomer that contains the Sox2 3’UTR ARE site A1 The Rbm24 protein-Sox2 3’UTR RNA complex can be competed out with a 1000-fold excess of the non-biotinylated wild-type ARE oligomer, but not with the non-biotinylated mutant ARE oligomer Furthermore, we performed RNA-immunoprecipitation (RIP) assay using Rbm24 antibody on wild-type whole eye lysates that shows enrichment of Sox2 mRNA in the Rbm24 antibody pulldown (Fig. 4I). n=6 eyes per biological replicate; total three biological replicates Together, these findings demonstrate that Rbm24 directly binds to Sox2 mRNA through interaction with its 3’UTR ARE. 8% of mRNAs encoded by the human genome contain AREs in their 3’UTRs
- Next, to understand the key regulators that maybe regulated by Rbm24, I looked at Lhx2, which is also linked to anophthalmia in mouse. Lhx2 is severely downregulated in both the protein and the mRNA level.
- Rbm24 microinjections result a loss of pigmentation phenotype in Rbm24 MO vs. control Xenopus embryos. We observe a loss of pigmentation phenotype in morpholino injected side of the embryo compared to uninjected side and control morpholino.