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Biology The effect of morpholino knockdown of genes TFEB and PKD2 on expression of gene CDH17 and green fluroscent protein (GFP) within the pronephros of Danio Rerio (zebrafish)

Tahseen Rabbani Session number: 00806-046 Henrico High School Country: United States IB region: Americas IB school code: 000806

Supervisor: Charles Falls Word Count: 3,985 October 29th, 2010

On my honor, I certify that I have neither given nor received inappropriate assistance on this assignment and the work is authentically my own. X_______________________

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Abstract
The experimenter investigated the knockdown (cessation of protein production) of two genes, TFEB and PKD2, and the subsequent effect upon the expression gene CDH17, and green fluorescent protein (GFP) within the pronephros (first stage kidney) of Danio Rerio (zebrafish). Implications of results would be vital in assessing the respective genes importance in the pronephric gene regulatory hierarchy. Both genes were knocked down (cessation of protein products) using morpholinos (mRNA inhibitors), and expression of GFP and CDH17 were measured respectively using two different lines of zebrafish and compared against an independently created qualitative scale. A transgenic line of zebrafish was used to study GFP expression, allowing for fluorescent-green visualization within the pronephros, and the second line were wild type AB zebrafish, used to study CDH17 expression by staining of CDH17 mRNA. All 3 genes studied affect pronephric development and thus it was hypothesized that knockdown of PKD2 and TFEB would result in inhibited GFP expression within the pronephros, due to damage, and lesser expression of CDH17, due to the hypothetical dependency of CDH17 expression upon other genes involved with pronephric development. The results indicated a lack of significant difference in GFP expression and CDH17 expression between the experimental (MO injected zebrafish) groups and control group (uninjected zebrafish). One reason for TFEB MO-injected group not displaying any difference in expression could be the existence of an isoformic TFEB protein with a translational start different from what the morpholino was able to block; meaning TFEB might not have been fully knocked down. As for PKD2, perhaps knockdown of CDH17 affects expression of PKD2 rather than vice versa, while GFP expression was not significantly different because PKD2 knockdown was not enough to alter fluorescence. For all future repetitions, different combinations of knockdown amongst the 3 genes would be recommended to possibly yield significant results.

[Word Count: 300 words]

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Table of Contents Section Pg.

Abstract...2 Introduction.....4-9 Variables, methods of analysis.........................10-13 Methods and Materials.....13-17 Results and Data...18-19 Discussion.19-21 Works Cited..22-23 Appendix..............................................24-28 Acknowledgements...27 Supervising letter...28

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The effect of morpholino knockdown of genes TFEB and PKD2 on expression of gene CDH17 and green fluroscent protein (GFP) within the pronephros of Danio Rerio (zebrafish) Introduction Research question: How does antisense morpholino oligo knockdown, or in other words, cessation of protein production for genes TFEB and gene PKD2 affect cadherin-17 (CDH17) and GFP expression in the developing kidneys (pronephros) of zebrafish? Two separate lines of zebrafish will be used to the study the effect cdh-17 and GFP expression, respectively. Analysis of the effects upon CDH17 expression would be important in drawing conclusion on the hierarchy of gene regulation within early-developing kidneys. Hypothesis: If genes TFEB and PKD2 and their respective proteins are knocked down, then the pronephros of ~48 hour developed zebrafish will display down-regulation (reduced expression) of cadherin17 and GFP in the pronephros. The down-regulation of CDH17 is theorized to occur because although TFEBs effects upon the kidney are not well understood, mutations can result in abnormal development of kidneys in mammalia, and lack of PKD2 will result in reduced differentiation and development of the pronephros, both effects which will ultimately damage the function of cdh-17 because it assumed to be below TFEB and PKD2 within the pronephric gene regulatory hierarchy (James Lister, personal communication, September 01, 2001). Dr. Lister, the supervising scientist, remarked the hypothetical regulatory hierarchy could be thought of as: Gene A Gene B Gene C If CDH17 is thought of as gene C; PKD 2 and TFEB as genes A or B, then manipulation of the latter two will result in altered expression of CDH17. CDH17 is assumed to be at the bottom of

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the hierarchy due to the large scope of its phenotypic expression within the pronephros, meaning it is more likely to be dependent on a large range of less-phenotypically expressed genes, such as PKD2 and TFEB. The decreased expression of GFP is hypothesized to occur since knockdown of either TFEB or PKD2 is presumed to cause damage to the kidneys themselves, resulting in decreased visualization of fluorescence. Null Hypothesis: Knockdown of genes TFEB and PKD2 and their respective proteins will neither reduce nor increase expression of cadherin-17 and GFP in the pronephros of ~48 hour developed zebrafish. The Danio rerio, more commonly known as zebrafish, are teleost fish which fall under the class of fish known as Actinopterygii, which are defined by their ray-patterned fins and ability to protrude jaws (Nusslein-Volhard, and Dahm 3-4). Zebrafish in recent years have become model organisms of genetic study for several reasons. It is relatively easy to obtain large clutches of embryos due to the fact female zebrafish can spawn every 2-3 days, and one female can release over a hundred eggs in one clutch. Fertilization and embryological development occur naturally, at a rapid pace, and synchronously amongst one clutch. Embryos develop into free-swimming larvae within 2.5 days (Drummond 299). The genome, which has already been mapped, is 1.7 gigabases long, which is just more than half that of tetrapods, such as human and mouse and the homologous nature between zebrafish genes and mammalian genes makes it a model organism for application of genetic studies to other species (Nusslein-Volhard, and Dahm 3). Cell count and organ development is easily observable due to the fact the chorion (the sac in which the embryo is located) is transparent, allowing for observance of the pronephros (Nusslein-Volhard, and Dahm 3).

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This experiment concerns the pronephric (first stage of kidney formation) development of zebrafish. The kidney of a larval zebrafish consists of two nephrons (regulators of water and solute concentration in the blood) and a glomerulus (a blood-to-urine filter for the nephrons) fused at the embryo midline (Drummond 299). The glomerulus is easily visible given the fact it spans a long length of the larvae. The glomerulus itself connects to tubules which drain into pronophrenic ducts which subsequently release waste into exterior environment (Drummond 300). The propnephros will eventually develop into the mesonephros (approximately at about 4 weeks of development), which controls waste regulation in place of the pronophrenic ducts (Nusslein-Volhard, and Dahm 91). Only the phenotype of pronephros was studied due to the complex structure of the mesonephros. The lab the experiment was performed in had access to a line of transgenic zebrafish, Tg(atp1a1a.4:EGFP)zf34, which produced embryos with strong GFP (green fluorescent protein) signals in the pronephros, which caused the pronephros to fluoresce green under an appropriate GFP filter1 . This transgenic line of fish allowed for one simple method of observing the phenotype of the pronephros. Naturally the signals are designed to fade after the pronephros develops into the mesonephros. A different, non-transgenic line of zebrafish (wild type AB) was to used to study the second dependent variable; CDH17 expression. This experiment sought to find the importance of TFEB and PKD2 on pronephric gene regulation and development through reverse genetic engineering; altering or shutting down a gene of interest and studying its effect on the phenotype (Nusslein-Volhard, and Dahm 141). A popular approach to shut down or knockdown a gene is through the use of antisense morpholino oligonucleotides (otherwise simply known as morpholino or MO); an almost artificially-designed nucleic acid. They consist of standard DNA

"ZFIN ID: ZDB-ALT-070723-1." ZFIN. University of Oregon, n.d. Web. 25 Aug 2010. <http://zfin.org/action/feature/detail?feature.zdbID=ZDB-ALT-070723-1>.

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nitrogenous bases (A, C, G, or T) and hexamer (6 subunits) morpholine rings connected by phosphodiamidate groups as a backbone (Nusslein-Volhard, and Dahm 142). To understand how they function one must consider the central dogma of biology, which states that DNA makes RNA makes protein; the synthesis of the protein begins with the DNA of a specific gene being transcribed into messenger RNA (the resulting transcript is a set of instructions for the assembly of the protein) (Russell 88). The mRNAs sequence is then used to assemble a protein within the ribosomes (translation)(Russell 11). The mRNA itself is single stranded, so the purpose of a morpholino is to bind to the mRNA near the start of where is it to be translated (~25 bases), to prevent assembly of the protein the gene is coding for (Bill, Petzold, Clark, Schimmenti, and Ekker 69) . This is achieved by designing a morpholino which consists of a sequence complementary to the specific mRNA sequence, which will cause it to selectively bind to only the mRNA of the gene of interest. If the gene cannot code for any of its specific protein (s), then it has been knocked down, and the effects of such can be studied in the phenotype. As opposed to natural nucleic acids, morpholinos are uncharged and non -toxic, which subsequently allows for them to be injected at high concentrations, [which] ma y be the secret of their success (Nusslein-Volhard, and Dahm 142). In this experiment, the genes which were knocked down for the purpose of studying cadherin-17 expression were TFEB and PKD2. The key requisite for selection of genes to knockdown was choosing genes which are expressed to some degree within the kidney. Gene TFEB codes for the protein TFEB, a basic helix loop-helix (referring to the structure) protein which forms complexes with similar

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proteins such as TFE3/TFEC and binds to e-boxes (sections of a gene located just above the promoter; the location where transcription for the gene begins) with the sequence CAYGTG ("Human Gene Compendium"). To what effect this binding has on gene regulation is not fully understood. In mice embryos, mutation of TFEB results in a defect of vessels connectin g to the placenta of the mother. This defect results in the death of mice within 11 days, before organs even develop (Steingrimsson, Tessarollo, Reid, Jenkins, and Neal 4607). In humans, an overproduction of TFEB protein is associated with renal sarcoma (malignant tumors of the kidney) , and in early developmental stages of mammalia, TFEB expression can be seen in the kidney tubules (Kuiper et al., 1661; McClive, Pall, Newton, Lee, and Mullins 268). The effect of TFEB knockdown would be important to study in relation to cdh -17 expression, given the acknowledgement TFEB plays a role within the kidney, but the lack of information as to what role it exactly p lays. The second gene knocked down in this experiment was PKD2, a gene which codes for polycystin-2 protein, which in conjunction with polycystin-1 protein (coded by gene PKD1), promotes differentiation, growth and development of cells within the kidneys, including cilia located on the kidney tubules (Genetics Home Reference). 75 mutations of the PKD2 gene are associated with the polycystic kidney disease; resulting in the growth of cysts on the kidney (Genetics Home Reference). Knocking down PKD2 and studying the implicit effects upon CDH17 expression would be important in establishing its important within the pronephric gene regulatory hierarchy.

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Cadherin-17, or gene CDH17, was chosen as marker of study after its respective mRNA proved to be geographically limited within the ducts of the pronephros of 48 hour old zebrafish. Other gene markers considered for study included tfeb, chordin, and gata3, but staining of these markers showed th at they were expressed in areas other than the kidney, which would make it difficult to exclusively analyze the phenotype of the pronephros (kidney). See figure 1 in appendix. Cadherin-17 itself is responsible for maintaining integrity of pronephric ducts in early embryogenesis (Horsfield et al., 2001). Cdh-17 is continually expressed in the kidney even after the pronephros develops into the mesonephros, and is eventually expressed in the liver and intestine of adult zebrafish (Horsfield et al, 2001). Given the scope of its expression, it was theorized that Cdh-17 would be above genes TFEB and PKD2 in the pronephric genetic hierarchy, and thus knockdown of TFEB and PKD2 would result in easily observable changes in cdh -17 expression within the pronephros.

Table 1 Precision of Error for Instruments and Uncertainty Table Instrument or Variable Measurement Uncertainty P1, P10, P20, P200, P1000 pipettes + 0.001 uL (P1) + 0.01 uL (P10, P20, P200 + 0.1 uL (P1000) Clock + 0.5 second Graduated Cylinder + 0.5 mL Injector and air tank + 0.5 psi

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Table 2 Variables Table

Independent Variables Morpholino injection used IV Levels: (Experimental Group) 1st No injection (control) 2th tfeb 1 mg/ml MO 3rd tfeb 4 mg/ml MO 4th tfeb 5 mg/ml MO 5th pkd2 2 mg/ml MO * Tfeb MO sequence 5'GCGCAGGCCGATGCGT GACGACAT 3' pkd2 MO sequence 5' GATCAACCCGTTACC TGACAATACA 3' Uncertainty: + 0.1 micrograms/ + 0.1 microliters Dependent Variable For transgenic line Tg(atp1a1a.4:EGFP)zf34 zebrafish measuring GFP expression: Ratio of embryos with reduced GFP expression in pronephros, scale provided below table. Control Group Embryos not injected with morpholinos; no genes knocked down. Repeated Trials 2 for each level of the IV; 1 trial studying GFP expression, second trial studying cdh-17 expression.

For wild type AB line of zebrafish measuring cdh-17 expression: Ratio of embryos with reduced cdh-17 expression in pronephros, scale provided below table. No uncertainty No uncertainty

Controlled Variables Type of water zebrafish are kept in: Will be kept constant by only keeping zebrafish/embryos in the system water they are accustomed to, which is used in the aquarium room. Line of zebrafish: For measuring GFP expression, only progeny spawned from the Tg(atp1a1a.4:EGFP)zf34 line of zebrafish will be used. For measuring cdh-17 expression, only progeny spawned from the wild type AB line of zebrafish will be used. If not, staining/expression patterns will vary to a great extent. Microscope and camera used: Kept constant by using the same microscope and camera to observe and document the embryos. If not, staining patterns could appear differently under another microscope. Concentrations and types of chemicals in the various solutions: Kept constant by using the same pipettes to administer the same amounts of chemical compounds in every solution used for the different clutches of embryos. If not, results in staining/expression will be discriminatory. Temperature for in situ storage: In situ refers to 48 hour embryos put to death in such a manner that their stage of development and overall form is preserved. In situ embryos will be stored in the same -4 C refrigerator.

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Dependent Variable Measurement Scale for measuring GFP expression:

Reduced/no expression

Full expression: note the fluoresced lines along the pronephros. Kruskal-Wallis measure: Assign full expression a value of 1, assign reduced/no expression a value of 0 Scale for measuring cadherin-17 expression:

Full expression Reduced expression Note the thick accent of cdh-17 in full expression. Kruskal-Wallis measure: Assign full expression a value of 1, assign reduced/no expression a value of 0

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Table 3 Statistical tests performed Statistical Test Mean Percentage

Method of Calculation Significance of test Mean = (number of embryos with full expression / number of embryos total) x 100%. quick visualization of expression of percentage.
First, give all each sample a value of 1 or 0. Follow standard procedure for assigning rank to 0 and 1. For GFP expression, 0 = rank 38, 1 = rank 95. For CDH 17 expression, 0 = rank 4, 1 = rank 41.4 Reduced/no expression given a value of 0 Full expression given a value of 1 H= [ng(MgMall)2] / (N(N+1)/12) (Lowry)

Kruskal-Wallis*

ng = Number of samples for given group

Mg = Rank mean of group Mall = ( Mg)/N
N= number of groups

Compare with Chi-Square table of critical values. Degrees of freedom = 4 (N-1), P = 0.05 If H > critical value, fair assumption to reject null hypothesis. If H<critical value, fair assumption to not reject null hypothesis. Kruskall-Wallis used because distribution of reduced expression and full expression was not ascertained to be normal; Kruskal-Wallis does not assume normal distribution (Lowry). Furthermore, distribution of expressions were not assumed to be independently and identically distributed; Kruskal-Wallis does not make this assumption (Lowry).

* Kruskall-Wallis calculation performed independently by researcher without online generator. Basic operations performed using Casio Fx9860G calculator. Limitations of this experiment included random error, if any of the morpholino becomes biologically inactive, which would be very complex to detect if it has already been injected, but all morpholinos were ensured to be kept in good conditions prior to and after use. Another random error would include embryos dying of natural causes after injection, which would result in different sample numbers for each group, however most standard statistical tests cover this different in sample number, and the Kruskal-Wallis, which was chosen does not even assume

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normal distribution (Lowry). A more technical limitation would be the TFEB morpholino not fully knocking down all isoforms (versions of the protein), thus different concentrations of TFEB morpholino were injected and measured to ensure this limitation would be reduced. Methods and Materials Note: The researcher was trained by Dr. James Lister, the supervising scientist, in all protocol below, and such protocol was approved to be used within the essay, with the researchers own adaptations. All procedures below are standard in nature, and will function with different brands of materials. 1. Injection of morpholino procedure Note: Use latex gloves for full procedure PM before embryo collection: Set up adult fish in spawning tank(s). Use the appropriate line of fish relevant to whether gfp expression of cdh-17 expression will be measured. See variable table to determine which line is needed. Next AM Remove injection trays from refrigerator to warm up.

In the fish/breeding room Transfer one (or more) spawning cage of fish to a cage of fresh water and remove divider. Prop insert up against wall or another tank to create depth gradient, and cover the outsides of the cage (for privacy). Turn on injector. Make sure it is set for Pulsed delivery, with pulse duration 2.0 ms. Turn on the compressed air tank. It should read approximately 40 psi, as should the injector gauge. Turn on the microscope lamp. After several minutes of spawning, check for any embryos at the bottom of the spawning cage. Experimenter used 20-40 embryos per morpholino injected. If fish are not producing enough embryos, or there is limited spawning, do not force it wait and try again the next day. To collect embryos, fill an empty spawning cage with water and transfer the insert containing the fish to this cage.

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Fill cage containing embryos (without zebrafish) with water and pour into sink, until only embryos are left at the bottom. Pour embryos into a petri dish and fill with water. Begin loading needle: take up 1 ul of morpholino to be injected into a pipette (P2 or P10) and attempt to place drop at the top edge of one of the needles, closest to the filament. Liquid should slowly flow down the filament. Note: 1 microliter should be sufficient for a whole dish of embryos. Experimenter injected 1 nanoliter per embryo; sufficient for 1000 embryos. While needles are loading, load embryos into injection trays with an embryo pipette pump. Injection tray should be liquid-free. See Appendix pg. 27 to injection tray creation. Pipet up 20-40 embryos from the petri dish (p1000 with cut tip should suffice). Check each embryo under microscope beforehand to make sure the embryo is healthy. Place microinjection tray under microscope. Hold the pipet pump containing embryos down at a near horizontal angle and dispense into trough, one embryo at a time. Effort should be made space out embryo. No more than 20 embryos in one trough. Absorb excess liquid from the tray, and gently push down embryos with fingertip down into the trough to make sure theyre secure. After as many troughs as needed are filled, fill the depressed area of the tray with system (RO) water. Place filled microinjection needle in needle holder: make sure that micromanipulator is fully retracted, and that screw is loose by turning counter-clockwise. Slide needle in until end can be seen in space beyond the rubber gasket. Tighten screw. Make sure microinjection tray with embryos are within the field of view under the microscope. Use micromanipulator on injector to lower needle into field of view. Adjust knobs on microscope to ensure maximum clarity of needle. Use forceps to gently break off a small portion of the tip of the needle. Use footcontrolled pump to gain an idea of how much liquid is being dispensed from the needle. The amount should be a very small drop when in comparison to the embryo. Use primary manipulator to lower needle next to the first embryo to be injected. Adjust knobs to ensure maximum clarity of both the needle and embryo. Gently continue lowering needle with micromanipulator until it penetrates the chorion (yolk sac) of embryo.

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Use foot-controlled pump to inject a drop of morpholino (appr. 1 nanoliter) into the chorion. Embryo itself may also be injected. Retract needle out of the embryo, and move the next embryo into field of view. Continue said procedure for injecting morpholino with every embryo. When finished with injection, loosen screw holding needle, and remove needle. Place cover on petri dish containing injected embryos. Store dish containing embryos in 37 degrees C incubator for approximately 48 hours. Label all dishes appropriately. At about 48 hours, dechorionate the embryos. This consists of removing the chorions from the embryos using forceps. This must be done with care to ensure the larvae zebrafish remain alive and unscathed.

2. Measuring the results, NOTE: Different Measurement procedures for GFP and CDH17 -----For measuring GFP expression in embryos of the Tg(atp1a1a.4:EGFP)zf34 line-

Place petri dish with dechorionated embryos under stereo dissecting microscope and turn on GFP filter. Record the number of embryos with reduced expression and embryos with full expression of green fluorescent protein in the pronephros, using scale. After recording is finished, add 300 uL of MESAB (3-amino-benzoic acid ethyl ester) to dish water. MESAB is a fish anesthetic. Cut the tip of a P1000 pipette needle to allow aperture to pipette up the embryos. Once all embryos have been pipetted, store embryos in a 1.5 mL Eppendorf tube. Aspirate any remaining solution in the tube and add 1 mL solution of 4% PFA (paraformaldehyde, a fixing agent) in 1xPBS (phosphate buffer saline) to preserve embryos. Note: Use drawn out Pasteur pipette and rubber bulb for all aspirations.

--------For

measuring cdh-17 expression of the wild type AB line, in-situ hybridization is performedIn situ hybridization uses anti-sense digoxigenin (anti-DIG) probes to detect mRNA for a specific gene within a specific organ or tissues. The following procedure details how to hybridize in situ embryos with cdh-17 probe and stain the probe for visualization under a microscope.

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Day 1 Note: ingredients for certain solutions are provided in Appendix, pg. 26. Dechorionate embryos from each dish and using p1000 with cut tip. Add each clutch in separate Eppendorf tubes. Aspirate any left over solution. Embryos must be put through a rehydration series: 1 wash with 66% MeOH (methanol) / 33% 1xPBT (PBS with Tween-20 added) 1 wash with 33% MeOH/ 66% 1xPBT 4 washes with 100% 1xPBT 1 ml total for every wash within this protocol, 5-10 minutes, on gyratory shaker (2030 rpm). Aspirate solution between every wash. Prehybridize embryos in 500 uL 1xHyb mix + tRNA and heparin (Hyb+), for >1 hour at 65 C. Dilute probe stock at a 1:100 to 1:300 dilution in 1xHyb+, preheat at 65 C, total solution can be 500 uL for 10-50 embryos within one tube. Experimenter used antisense digoxigenin-labeled riboprobes designed to detect cdh-17 mRNA. Aspirate prehybridization solution from embryos, and replaced with the prewarmed 1xHyb+ containing diluted probe. Place tubes back in 65 C environment overnight. Day 2 Warm 1xHyb mix without adding tRNA and heparin (Hyb-) at 65 C. Aspirate diluted probe solution from tubes. Note: probe solution can re-used. Embryos must be put through the following wash series in a 65 C water bath 1 quick wash with prewarmed Hyb 1 wash with 66% Hyb- / 33% 2xSSC (saline-sodium citrate buffer) for 10 minutes 1 wash with 33% Hyb-/ 66% 2xSSC for 10 minutes 1 wash with 2xSSC for 10 minutes 2 washes with 2xSSC for 20 minutes Following wash series done on gyratory shaker (20-30 rpm) 1 wash with 66% 0.2xSSC/ 33% 1xPBT for 5 minutes 1 wash with 0.2xSSC/ 66% 1xPBT for 5 minutes 1 wash with 1xPBT for 5 minutes Add blocking solution consisting of 1xPBT + 2 mg/ml BSA + 2% goat serum. 1 mL should suffice. Blocking solution is designed to bind to any protein sites which do not have probes attached, reducing background staining.

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Dilute a small volume of anti-Dig AP (alkaline phosphatase) in block solution; 1:10. Then add 1 uL of diluted anti-Dig AP to 999 uL of block solution. Replace original block solution in tubes with block solution containing diluted anti-dig AP. This antibody is designed to bind to the probes on the embryos and stain blue in the presence of NBT (Nitro-Blue Tetrazolium Chloride) and BCIP (5-Bromo-4-Chloro3'-Indolyphosphate p-Toluidine Salt), which will be added on day 3. Day 3

Put embryos through following wash series on gyratory shakers 15-25 rpm: 1 wash with 1xPBT (quick wash) 5 washes with 1xPBT for 15 minutes each 3 washes with 1x coloration buffer + 0.1% tween-20 solution for 5 minutes Aspirate coloration solution from tubes. Add solution consisting of 1x coloration buffer + 0.1% Tween-20 + NBT/BCIP (4.5 uL/3.5 uL respectively per mL of coloration buffer). Allow staining to develop in dark (place tubes in a box or wrap in foil). Check for color development every 30 minutes. Color reaction should be finished when staining is clearly visible with the naked eye. Aspirate solution, and replace with 1xPBT. Use P1000 tip to pipette up embryos and place in dish to view under microscope. Record the number of embryos with reduced expression and embryos with full expression using DV scale. Cut the tip of a P1000 pipette needle to allow aperture to pipette up the embryos. Once all embryos have been pipetted, store embryos in a 1.5 mL Eppendorf tube. Aspirate any remaining solution in the tube and add 1 mL solution of 4% PFA (paraformaldehyde, a fixing agent) in 1xPBS (phosphate buffer saline) to preserve embryos.

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Results & Data Table 4 The following table displays the percentage of full expression of GFP and CDH17 mRNA amongst embryos according to which gene was knocked down. Tfeb expression* The effect of morpholino knockdown of genes TFEB and PKD2 on Cadherin-17 and green fluroscent protein (GFP) expression in the developing kidneys of Danio Rerio (zebrafish): Percentage of full expression of GFP and CDH17

Morpholino Injected

GFP expression

CDH17 expression

Tfeb expression *

KruskalWallis Value for GFP

KruskalWallis Value for CDH17

Critical Value (df=4)* 9.49, p=0.05

PKD2 MO (2 mg/ml) TFEB MO (1 mg/ml) TFEB MO (4 mg/ml) TFEB MO (5 mg/ml) Control

14/27 = 68.0% 15/25 = 60.0% 14/26 = 53.8% 19/29 = 65.6% 13/25 = 52%

14/15 = 93.3% 14/15 = 93.3% 16/16 = 100% 12/16 = 75.0% 13/14 = 92.9%

13/13 = 100%

H = 1.17

H=1.61

15/15 = 100%

Two clutches of embryos were also hybridized and stained with tfeb riboprobes for purely observational purposes. Not factored into the Kruskal-Wallis calculation. Df = n -1, where n= number of groups. Critical value corresponding to chi-squared table. P=0.05=95% confidence interval. (Frequency Distributions)

Qualitative Observations The tfeb riboprobes detected mRNA for tfeb along a large portion of the embryo, and furthermore, full expression was found in all the embryos.

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Graph 1 The following graph displays the percentage of expression for GFP and cdh-17 amongst every morpholino (MO) used.
The effect of morpholino knockdown of genes TFEB and PKD2 on Cadherin-17 and green fluroscent protein (GFP) expression in the developing kidneys of Danio rerio (zebrafish) 100.00% 90.00% 80.00% 70.00% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00% GFP expression Cdh-17 expression Type of Expression Measured

Percentage of embryos with full expression

PKD2 MO (2 mg/ml) TFEB MO (1 mg/ml) TFEB MO (4 mg/ml) TFEB MO (5 mg/ml) Control

Discussion Significant differences in expression of GFP and cdh-17 in the pronephros were not detected after knockdown of genes TFEB and PKD2, when compared to each other, and when compared to the control (the Kruskall-Wallis compares all groups simultaneously)(Lowry). Both H values, 1.61 and 1.17 did not exceed the critical value of 9.49 according the degrees of freedom (4) and probability 0.05, allowing the null hypothesis to be fairly not rejected (see table 4) . There are possible explanations for these findings. Concerning PKD2, perhaps the gene does not have as large of an effect on genes localized within the pronephros as previously assumed. As noted in table 1, one clutch of embryos with PKD2 were stained for TFEB expression for purely observational purposes, and it was found that 100% of the embryos displayed expression of TFEB. It was also observed that PKD2 did not have a significant effect on the expression of

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TFEB (100% full expression), so perhaps it is understandable that it would not limit the expression of another gene such as CDH-17. However, to fully understand the effects of PKD2 on TFEB and CDH-17, other methods will need to be used, such as increasing the expression of CDH-17 or TFEB, and seeing what effect it would have on PKD2 expression, or up-regulating expression of PKD2 and documenting whether or not it increases/reduces expression of the two other genes. This method would help verify the pronephric gene regulatory hierarchy, rather than relying on the hypothetical hierarchy which was assumed. TFEBs role in the pronephros of zebrafish is still not fully understood, which is why different concentrations of morpholinos were tested. As explained with PKD2, a variety of other methods, such as up-regulating instead of knockdown, would have to be implemented to ascertain the full relationship between these 3 genes, and discovering why knockdown of TFEB had no effect on CDH17 expression. A plausible explanation is that the protein products of TFEB were not completely knocked down. TFEB, located on chromosome 11, normally codes for a protein whose translation begins at exon 1, exon refers to a coding region; actually used in the translation. The morpholino was designed to block the translational start (ATG) at exon 1, however an isoform, or an alternative sequence for tfeb protein can be translated, using an alternative 1st exon which is located between exons 3 and 4, as shown on the map (see figure 2 in appendix). The translational start sequence for this isoform is not complementary to the morpholino which was used, so naturally, this protein product would not have been blocked. It is unknown where this isoform is expressed and to what concentration, but this lack of full knockdown may explain why knockdown of TFEB had no significant effect upon GFP/cdh-17 expression. For future repetitions of this experiment, a morpholino designed for this isoform would be injected along with the original TFEB MO.

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For future repetitions of this experiment, some changes would ensure a wider range of data. Using more repeated trials, and more concentration levels for each type of morpholino is key to generating a wider range of data. Also, perhaps cdh-17 can be knocked to see what effect it has upon tfeb and pkd2 expression. A morpholino designed to block the isoform for tfeb could be injected along with the original TFEB morpholino to ensure fully knocking down the gene. Also, development past 48 hours may also be studied, in case changes in expression prove to occur later on (yet it is important to note that morpholinos can successfully knockdown a gene for only so long)(Bill et al., 69).

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Works Cited Bill, Brent, Andrew Petzold, Karl Clark, Lisa Schimmenti, and Stephen Ekker. "A Primer For Morpholino Use in Zebrafish." Zebrafish. 6.1 (2009): 69-77. Print. Drummond, Ian. "Kidney Development and Disease in the Zebrafish." American Journal of Nephrology. 16 (2005): 299-304. Print. "Frequency Distributions." Department of Biology . Colby College, n.d. Web. 17 Oct 2010. <http://www.colby.edu/biology/BI17x/freq.htmlhttp://www.colby.edu/biology/BI17x/fre q.html>. Horsfield, J., A. Ramachandran, K. Reuter, E. LaVallie, L. Collins-Racie, K. Crosier, and P. Crosier. "Cadherin-17 is required to maintain pronephric duct integrity during zebrafish development.." National Center for Biotechnology Information. U.S. National Library of Medicine, Jul 2001. Web. 25 Aug 2010. <http://www.ncbi.nlm.nih.gov/pubmed/12049763>. Kuiper, Roland, Marga Schepens, Jose Thijssen, Martien Asseldonk, Eva Berg, Julia Bridge, Ed Schuuring, Eric Schoenmak ers, and Ad Kessel. "Upregulation of the transcription factor TFEB in t(6;11)(p21;q13) -positive renal cell carcinomas due to promoter substitution." Human Molecular Genetics 12.14 (2003): 1661-1669. Print. Lowry, Richard. "The Kruskal-Wallis Test for 3 or More Independent Samples ." VassarStats. Vassar College, n.d. Web. 17 Oct 2010. <http://faculty.vassar.edu/lowry/ch14a.html>. McClive, Peter, Gurman Pall, Kathryn Newton, Muriel Lee, John Mullins , and Lesley Forrester. "Gene Trap Integrations Expressed in the Developing Heart: Insertion Site Affects Splicing of the PT1 -ATG Vector." Developmental Dynamics. 212 (1998): 267-276. Print Nusslein-Volhard, Christiane, and Ralph Dahm. Zebrafish. 1st. New York: Oxford University Press, 2002. Print. "PKD2." Genetics Home Reference. U.S. National Library of Medicine, Jun 2006. Web. 25 Aug 2010. <http://ghr.nlm.nih.gov/gene/PKD2>. Russell, Peter. iGenetics. A Molecular approach.. 2nd ed. San Francisco, CA: Pearson Education, 2006. Print. Steingrimsson, Elrikur, Lino Tessarollo, Susan Reid, Nacy Jenkins, and Copeland Neal. "The bHLH-Zip transcription factor Tfeb is essential for placental vascularization." Development. 125. (1998): 4607-4616. Print.

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"transcription factor EB." The Human Gene Compendium. Weizmann Institute of Science, Jun 2006. Web. 26 Aug 2010. <http://www.genecards.org/cgi-bin/carddisp.pl?gene=TFEB>.

* All images of specimen taken by researcher using Olympus DPZ0 camera. Gene map of TFEB provided by supervising scientist, James Lister.

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Appendix

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Figure 1: Left displays chd-17 mRNA staining, while right displays tfeb mRNA staining. Notice how chd-17 staining patterns are geographically limited to the pronephric ducts, while tfeb expression is much broader.

Solution ingredients as preferred by supervising scientist, Dr. James Lister (p. 16 reference) Hyb solution: Formamide 20xSSC Heparin, 25 mg/ml tRNA, 10 mg/ml tween-20, 20% critic acid, 1 M nuclease-free H20 Coloration buffer: Tris-HCl pH 9.5, 1M MgC12, 1M NaCl, 5M Nuclease-free H20 Hyb+ 25.0 mL 12.5 mL 0.1 mL 2.5 mL 0.25 mL 0.46 mL 9.19 mL 50 mL 5mL 2.5 mL 1 mL 41.5 mL 50 mL Hyb25.0 mL 12.5 mL 0.25 mL 0.46 mL 11.79 mL 50 mL

Before use, add Tween-20 5 ul/mL NBT: 50 mg NBT + Anhydrous dimethyl formamide 700 uL + Nuclease-free H20 300 uL BCIP: 50 mg BCIP + 1 mL dimethyl formamide Store NBT/BCIP at -20 degrees Celsius, in dark.

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Creating the agarose trays for embryo micro injection: creating 1 tray. Protocol as trained in by supervising scientist, James Lister. Weigh out 0.45 g of agarose powder. Add 30 mL of RO (reverse osmosis) water to a 250 mL Erlenmeyer flask. Add weighted agarose to water and swirl to mix. Wash any excess powder from the sides of the flask using RO water or dH20 (deionized water). Do not use more than 1-2 mL. Place flask in microwave for 2 minutes to melt the agarose. Stop every 30 seconds to take out flask and swirl. Use heat resistant glove with handling the hot flask. After agarose is done melting, allow flask to sit and cool down for 10 minutes. Add 30 mL of methylene blue (to prevent growth of algae) to the solution in the flask, and swirl to mix. Pour solution into a 10 cm petri dish. Carefully lay down microinjection mold on the surface of the solution starting from a side edge to prevent bubbles forming in the troughs of the mold. Allow 5-10 minutes for solidification. Use spatula or thin blade to pry out mold. Place dish in a refrigerator for 1-2 minutes to ensure agarose is solid.

Figure 2

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Acknowledgements There were many individuals, who with their limitless patience and guidance, made this essay possible to be written. However, the bulk of my appreciation is especially due to Dr. James Lister of Virginia Commonwealth University for the conception of the original project (the role of tfeb on embryonic development) and allowing me to perform research within his lab. Dr. Lister provided me with the necessary equipment; supervision when dealing with adult zebrafish and hazardous chemicals; and training in higher-level protocol necessary for the experiment. All of my questions were thoroughly answered by Dr. Lister, effectively helping me to gain a full grasp over even the most technical areas of my research. I would also like to thank Mr. Charles Falls, my supervisor and physics teacher, and Mrs. Priscilla Biddle, the IB coordinator and my theory of knowledge teacher, for providing ideas for revision and diligently reading through drafts of the essay.