A GENOMIC SCREEN FOR ZIC1 TARGET GENES IN NEURAL DEVELOPMENT by Shuzhao Li A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biological Sciences MONTANA STATE UNIVERSITY Bozeman, Montana July 2006 ©COPYRIGHT by Shuzhao Li 2006 All Rights Reserved ii APPROVAL of a thesis submitted by Shuzhao Li This thesis has been read by each member of the thesis committee and has been found to be satisfactory regarding content, English usage, format, citations, bibliographic style, and consistency, and is ready for submission to the Division of Graduate Education. Dr. Christa Merzdorf Approved for the Department of Cell Biology and Neuroscience Dr. Gwen Jacobs Approved for the Division of Graduate Education Dr. Joseph Fedock iii STATEMENT OF PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a master’s degree at Montana State University, I agree that the Library shall make it available to borrowers under rules of the Library. If I have indicated my intention to copyright this thesis by including a copyright notice page, copying is allowable only for scholarly purposes, consistent with “fair use” as prescribed in the U.S. Copyright Law. Requests for permission for extended quotation from or reproduction of this thesis in whole or in parts may be granted only by the copyright holder. Shuzhao Li July 2006 iv TABLE OF CONTENTS 1. INTRODUCTION.......................................................................................................1 2. GENOMIC SCREEN WITH DNA MICROARRAYS...............................................4 Experimental Design..................................................................................................4 Results from Spotted cDNA Arrays with CHX..........................................................6 Affymetrix genechips: Early Response without CHX...............................................9 Affymetrix Genechips: Early Response with CHX..................................................12 Data Processing and Cross Comparisons.................................................................15 Materials and Methods.............................................................................................16 3. SCREEN RESULTS AND ANALYSIS....................................................................19 Sequence Analysis....................................................................................................19 Cloning and in situ Hybridizations...........................................................................19 Confirmation with PCR: Technical Development....................................................20 Confirmation with PCR: Results..............................................................................22 Discussion.................................................................................................................26 Materials and methods..............................................................................................30 4. COMPUTATIONAL ANALYSIS OF GENE EXPRESSION..................................32 Studying promoters of Confirmed Genes.................................................................32 Evolutionary View of Zic1 Functions......................................................................33 Regulators of Zic1....................................................................................................35 5. XFEB AS A NOVEL TARGET OF ZIC1.................................................................36 Xfeb is a Direct Target of Zic1.................................................................................36 Xfeb Contains Five SCP Domains and May be a Serine Protease...........................38 Xfeb is Expressed in the Developing Hindbrain Region..........................................42 Xfeb Represses otx2 and hoxB1...............................................................................45 Discussion.................................................................................................................47 Materials and Methods.............................................................................................48 REFERENCES CITED.................................................................................................51 v LIST OF TABLES Table Page 2.2.1 Top candidates from spotted cDNA array experiment.......................................7 2.3.1 Top candidates from Affymetrix experiment without CHX, 20 genes.............10 2.4.1. Top candidates from Affy experiment with CHX, 28 genes...........................12 3.2.1 Genes tested by in situ hybridization. .............................................................20 3.4.1. 49 genes tested with PCR, 5 confirmed..........................................................22 3.4.2. With data from more genechips,16 genes were tested, 6 confirmed...............25 3.4.3. Normalized QPCR data for six confirmed genes............................................25 vi LIST OF FIGURES Figure Page 2.1.1. Schematic of cDNA microarray experiment.....................................................6 3.4.1 Several direct targets of Zic1............................................................................24 3.5.1 Emerging regulatory relationships in neural crest development......................28 3.6.1. Schematic of RT-PCR assays..........................................................................31 4.1.1. An example output of MEME.........................................................................33 4.1.2. Identification of conserved non-coding regions through comparative genomics....................................................................................................................34 4.3.1. An example of identifying TF binding site through phylogenetic footprinting ...................................................................................................................................35 5.1.1. Xfeb is activated by Zic1. ...............................................................................37 5.1.2. Zic1 regulates Xfeb in whole embryos............................................................38 5.2.1. Xfeb contains five tandem SCP-like domains that may have serine protease activity.......................................................................................................................41 5.3.1. A developmental profile of Xfeb expression...................................................43 5.3.2. Spatial expression of Xfeb..............................................................................44 5.3.3. The gap between Xfeb and wnt1 closes up as embryos age............................45 5.4.1. Xfeb represses expression of the anterior neural gene otx2 and hindbrain gene hoxB1........................................................................................................................46 5.4.2. Xfeb affects snail expression...........................................................................47 vii ABSTRACT The transcription factor Zic1 plays important roles in patterning the neural plate in early vertebrate development. A crucial step toward understanding the mechanisms of Zic1 function is to identify the downstream target genes. We misexpressed Zic1 in animal caps from Xenopus embryos, and screened for differential gene expression with DNA microarrays. Candidate genes from these microarray data were further validated by quantitative PCR. Through this study, eleven genes have been shown to be directly upregulated by Zic1. One of the direct targets of Zic1 is a novel gene that we have named Xfeb. Xfeb is expressed in the presumptive hindbrain region during neurula stages and in somite tissues later in development. Xfeb represses the hindbrain gene hoxB1 and the anterior neural gene otx2, suggesting that Xfeb is involved in regionalizing the neural plate, possibly by ensuring a posterior expression limit for otx2. 1 INTRODUCTION Early neural development has been proposed as a two-step model, with neural induction as the first step, followed by initiative patterning of the resulting neural plate (Sasai and De Robertis, 1997). For neural induction, besides inhibition of BMP (bone morphogenetic protein) signaling, roles of positive signals, such as FGFs (fibroblast growth factors), Wnts and IGFs (insulin-like growth factors, Pera et al. 2001), have been suggested in recent studies (reviewed in Harland, 2000; Bainter et al, 2001; Munoz- Sanjuan and Brivanlou, 2002. Stern, 2002; Bally-Cuif and Hammerschmidt, 2003). Many effector genes, supposingly linkers between neural induction and initial patterning of nervous system, have entered the picture in the last few years. These include zinc-finger transcription factors Gli, Zic, XSIP1/ZEB2, Kheper; winged helix family XBF; homeodomain proteins Iroquois; HMG-domain proteins Sox; several basic helix-loop- helix (Atonal, Achaete-scute and Olig) factors; POU2; Geminin, etc. (Sasai, 1998; Bainter et al, 2001; Bally-Cuif and Hammerschmidt, 2003) However, the patterning step might well be interconnected with neural induction by sharing genetic circuits, since distinct regions of the nervous system to become are already discernable at gastrulation stage, and many of these effector genes are already expressed by then and show neural inducing activities. Zic genes encode C2H2 zinc finger transcription factors, homologous to odd-paired in fruit fly. At least five zic genes have been are found in human, mouse and frog. A few 2 zic orthologs