Gene Expression and Regulation in Early Vertebrate Development Xianhui Li

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2007 Gene Expression and Regulation in Early Vertebrate Development Xianhui Li

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Gene Expression and Regulation in Early Vertebrate Development

By XIANHUI LI A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science

Degree Awarded Fall Semester, 2007

The members of the Committee approve the thesis of Xianhui Li defended on October 18, 2007

Curtis R Altmann Professor Co-Directing Thesis

Hank W Bass Professor Co-Directing Thesis

Yoichi Kato Committee Member

Wu-Min Deng Committee Member

Hengli Tang Committee Member

Approved: Timothy S. Moerland, Chair, Department of Biological Science The Office of Graduate Studies has verified and approved the above named committee members.

ACKNOWLEDGEMENTS

I am very grateful to Dr. Curtis Altmann and Dr. Hank Bass, my major professors, for their guidance, support, patience and encouragement. This thesis would not have been completed without their wide knowledge and insightful foresight.

I also appreciate my committee members, Dr. Yoichi Kato, Dr. Wu-Min Deng, Dr. Hengli Tang, and Dr.Nancy Greenbaum for spending their precious time in discussing my project progress, giving invaluable suggestion, and revising my prospectus and thesis.

I thank Dr. George Bates for his support and encouragement.

I thank my colleagues: Ailing Zheng, Tyrone Ryba, Malcolm Klein, Barbara Danner, and Tomomi Kiyota for their help.

I thank my parents, my siblings, my husband and son for their love, sacrifices, patience, and support.

I am appreciative of College of Medicine and Department of Biological Science for their support.

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TABLE OF CONTENTS List of Tables ...... v List of Figures ...... vi Abstract ...... vii

1. Expression and Functional Analysis of LTBP4 in Early Development ...... 1

Introduction ...... 1 Materials and methods ...... 7 Results ...... 13 Discussion ...... 22

2. Identification and Characterization of Downstream Targets of Pax6 in Early Eye Development ...... 30

Introduction ...... 30 Materials and methods ...... 32 Results ...... 35 Discussion ...... 45

REFERENCES ...... 55 BIOGRAPHICAL SKETCH ...... 61

LIST OF TABLES Table 1: The primers that are used for sequencing xtLTBP4 ...... 8 Table 2: Primers for xLTBP2, xLTBP3 and xLTBP4 ...... 15 Table 3: Percentage of secondary axis induced by microinjection of mRNA for hLTBP4 and xtLTBP4 ...... 24 Table 4: Markers and primer sequences used for the animal cap experiment ....28 Table 5: Primers of transcription factors ...... 36 Table 6: Primers of Wnt signal genes ...... 37 Table 7: Primers of receptor genes ...... 37 Table 8: Confirmed pax6 upregulated transcription factors by real time PCR....41 Table 9: Confirmed pax6 upregulated Wnt signal genes by real time PCR ...... 42 Table 10: Confirmed pax6 upregulated receptor genes by real time PCR ...... 42 Table 11: Direct targets of Pax6 ...... 47 Table 12: Phenotype of overexpression of LRP11 ...... 52

LIST OF FIGURES Figure 1: Secretion, activation and targeting of TGF-β ...... 3 Figure 2: The structure of LTBPs ...... 5 Figure 3: Microinjection of Xenopus embryos ...... 11 Figure 4: Expression of xLTBP2, xLTBP3, and xLTBP4...... 16 Figure 5: Whole amount in situ hybridization of xLTBP2-4...... 16 Figure 6: Sequence alignment of Xenopus tropicalis, human, and mouse LTBP4 ...... 18 Figure 7: xtLTBP4 domain structure...... 20 Figure 8: Multiple Alignment of TB domains...... 20 Figure 9: In situ hybridization of xLTBP4...... 21 Figure 10: Injection of hLTBP4 induces a second axis...... 23 Figure 11: Secondary axis is stained by 12-101 and β-gal...... 24 Figure 12: hLTBP4 acts synergistically with Xnr-1...... 26 Figure 13: xtLTBP4 enhances the expression of Xnr1...... 27 Figure 14: hLTBP4 enhances Xnr1 in the mesoderm induction in the animal caps ...... 28 Figure 15: Pitx2 is confirmed by one experiment of PCR...... 39 Figure 16: Model of direct target and indirect target ...... 43 Figure 17: Pax6-GR fusion protein in active condition and inactive condition ..43 Figure 18: Foxd3, a direct target of pax6...... 46 Figure 19: Expression of Pax6, Foxd3 and Irx3 in the eye of the mouse...... 46 Figure 20: Pax6 MO has inhibited exogeneous pax6 in animal caps ...... 47 Figure 21: Expression of Xiro3 in Xenopus requires Pax6...... 48 Figure 22: Overexpression of Xiro3 causes misplaced eyes ...... 49 Figure 23: Overexpression of Xiro3 causes ectopic photoreceptor and muller cells ...... 49 Figure 24: Overexpression Xiro3 induced Pitx2, Six3, Otx2 and Xag...... 50 Figure 25: Overexpression of LRP11 induced ectopic RPE and proximal eye defect ...... 50 Figure 26: Overexpression of LRP11 can induce ectopic photoreceptor cells....51 Figure 27: Spacious expression of LRP11 by in situ hybridization...... 51 Figure 28: Temporal expression of LRP11 by Real time PCR...... 52

ABSTRACT

Gene expression and regulation in early vertebrate development has been examined in Xenopus laevis, a model organism for embryonic developmental biology. Two related projects were carried out and they are described in separate chapters. The first chapter describes the expression and functional analysis of Latent TGF-β Binding Protein 4 (LTBP4) in early development. LTBP4 is expressed in the organizer which is located in the dorsal mesoderm and induces a secondary axis upon overexpressed on the ventral side. The data indicate that LTBP4 enhances the nodal signaling pathway which induces mesoderm in the early development. The second chapter describes the identification and characterization of downstream targets of Paired Box Gene 6 (Pax6) in early eye development. Three groups of Pax6 upregulated genes, identified from a DNA microarray screen, are confirmed by real time PCR. Among the Pax6-activated genes are transcription factors, the genes that are involved in Wnt signaling pathway which is network of proteins implicated in embryogenesis and cancer, and receptor genes. Importantly, seven different genes have been identified as direct targets of Pax6 based on their mRNA accumulation patterns. In addition, functional analysis of some Pax6 regulated genes such as Xiro3 and LRP11 is presented in the second chapter.

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CHAPTER 1

EXPRESSION AND FUNCTIONAL ANALYSIS OF LTBP4 IN EARLY DEVELOPMENT

Introduction The vertebrate embryonic development starts as a fertilized egg, followed by a rapid cell division. The dorsoventral polarity is established immediately after fertilization in Xenopus (reviewed by De Robertis et al., 2000). Once fertilized, the cortex rotates. This rotation establishes parallel microtubules in the vegetal pole that may provide the tracks on which the cortex moves. Disruption of the microtubules by UV inhibits the cortical rotation and causes embryos without axis (Beal et al., 1975). The dorsal side of the embryo (future Spemann organizer) forms opposite the sperm entry site. There are two dorsal signaling centers at the blastula stage. One is the Nieuwkoop center which is involved in dorsal meso-endoderm development. The Nieuwkoop center is located in the dorsal vegetal region and some mesoderm inducers such as nodal related factors are expressed in this region. High level of the nodal can induce dorsal mesoderm. The other center is Blastula Chordin and Noggin Expression (BCNE) center involved in neural specification (Kuroda et al., 2004). It is located at the dorsal animal pole region and is involved in the formation of anterior neural tissue and expression of Chordin, Noggin and Xnr3. Most genes that are expressed in the BCNE center at blastula stage are also expressed in the organizer at the gastrula stage (Agius et al., 2000; Wessely et al., 2001). In the gastrula stage, the three germ layers are formed. In 1924, Spemann and Mangold identified a region in early amphibian embryos that can induce a second axis during gastrulation. This region is called the organizer, located in the dorsal mesoderm. The organizer acts as a signaling center of anterior-posterior and dorsal-ventral patterning. The Spemann organizer secretes growth factor inhibitors such as Chordin, Noggin, Follistatin, ADMP, Frzb-1, sFRP2, Dkk-1, Cerberus, Activin, lefty, Xnrs, Shh, IGFBP5 (reviewed by De Robertis et al., 2004). Among of these molecules, Chordin, Noggin, Xnr3, and Follistatin are bone morphogenetic protein (BMP) signaling pathway inhibitors

(Sasai et al., 1994; Piccolo et al., 1996; Smith and Harland, 1992; McKendry, et al., 1997; Lau, 1995), while Frzb-1, sFRP2, Dkk-1, and Cerberus are Wnt signaingl inhibitors (Leyns et al., 1997; Glinka et al., 1998; Piccolo et al., 1999). ADMP is a BMP family member that is paradoxically expressed in the dorsal center (Reversade et al., 2005), Shh is sonic hedgehog and IGFB5 is an insulin-like growth factor-binding protein that enhances IGF activity (reviewed by De Robertis et al., 2004). 1) The TGF-β subfamily The transforming growth factor beta (TGF-β) signaling pathway can regulate cell fate including the regulation of cell growth, differentiation, apoptosis and morphogenesis (Piek E et al., 1999). The TGF-β superfamily of growth factors includes several subfamilies. Among these are the bone morphogenetic protein (BMP)/growth differentiation factor (GDF) subfamily, the nodal/activin family and the TGF-β subfamily. Members of the TGF-β family are synthesized as larger pro-protein dimers which are proteolytically cleaved to generate a C-terminal mature TGF-β and an N-terminal pro- domain (Sinha et al., 1998). TGF-β subfamily isoforms 1-3 are secreted as latent complexes where the pro-domain remains associated with the mature ligand by a noncovalent bond. The pro-domain, also called the latency associated peptide (LAP), prevents mature ligand binding to the receptor (Sinha et al., 1998). The LAP can associate with Latent TGF-β Binding Proteins (LTBPs) by covalent bonding (Oklu R et al., 2000) as described in Figure 1. 2) The TGF-β signaling pathway Only the mature TGF-β ligand can bind to the receptor and initiate the signaling pathway. Mature TGF-β ligands bind to serine-threonine kinase receptors containing two subunits (Type I and Type II) to activate the TGF-β signaling pathway. The mature ligand binds to the Type II subunit leading to the phosphorylation of the Type I receptor (Massague J, 1998). The Type I receptor in turn phosphorylates Smad regulatory proteins which dimerize with a common Smad (Smad4) and translocate to the nucleus where, together with associated transcription factors, they activate downstream target genes (Massague J, 1998). 3) Regulatory mechanisms of TGF-β signaling The TGF-β signaling pathway is tightly regulated at several levels. Ligand activity is

Figure 1: Secretion, activation and targeting of TGF-β. TGF-β is synthesized as N- terminal LAP and C-terminal TGF-β. Protease will digest it and LAP and mature TGF-β will interact each other by noncovalent bond, which is called small latent TGF-β, this small latent TGF-β will bind LTBP. TGF-β can be secreted as small latent form or large latent form. Large latent TGF-β is not active. Follown proteolytic cleavage, the mature TGF-β is active and can bind to the receptor and active the signaling pathway.

controlled by many secreted proteins, some of which prevent ligand binding, while others affect the activation of the receptors. These secreted proteins include noggin, chordin, follistatin, inhibin, lefty, Cerberus, latent TGF-β binding proteins (LTBPs) and others (Massague J et al., 2002). Receptor activity is also modulated by receptor binding proteins, examples of which include Smurf, Smad 7, and Dapper2 (Massague J et al., 2002). Downstream transcription of various target genes is regulated by FoxG1, DRAP1, and Ski (Massague J et al., 2002). 4) LTBPs The TGF-β isoforms (1-3) are synthesized as larger precursors in which both the pro- region and the mature ligand are secreted as part of a larger complex called the latent TGF-β complex (LTGF-β) as summarized in Figure 2. The LTGF-β complex is covalently linked to LTBP to form the large latent TGF-β (LLTGF-β) complex, which can not bind to the receptor. To date four members of LTBP have been identified (Oklu R et al., 2000). They are secreted glycoproteins with molecular weights ranging from 125KD to 210KD. LTBPs are closely related to fibrillin which is a component of the extracellular microfibrillar network (Oklu R et al., 2000). Both LTBPs and fibrillins have repeated eight-cysteine domains and contain long tandem arrays of epidermal growth factor (EGF)-like domains often found in extracellular matrix proteins. LTBPs play roles in secretion, activation, and targeting of TGF-βs to the extracellular matrix (Oklu et al., 2000) as shown in Figure 1. Much of this work has focused on TGF-β1, β2, and β3 (Saharinen J et al., 1998; Olofsson A et al., 1992) and little is known about any potential role of LTBPs for the remaining members of the large TGF-β superfamily. Other members of the superfamily are predicted to encode LAP related pro-regions including members of BMP, activin, and nodal subfamilies. Anomalous expression of LTBPs has been implicated in a variety of pathological processes including cancer and atherosclerosis (Oklu R et al., 2000). Four members of LTBP family have been identified (LTBP1-4) and have been shown to play important roles in the secretion and activation of TGF-β complexes and the targeting TGF-β ligands to the extracelluar matrix. Loss of function studies in the mouse reveal that disruption of LTBP4 can cause cardiomyopathy, lung emphysema, or colorectal cancer (Sterner-Kock et al., 2002). The research described here is designed to learn more about LTBP4 because it is the least

Figure 2: The structure of LTBPs. Mature TGF-β interacts with LAP by noncovalent bonds to form the small latent TGF-β complex. The LAP is covalently linked to LTBP to form the large latent TGF-β complex.

well chareacterized members of the known LTBPs in early development. 5) Signal pathways involved in secondary axis formation A secondary axis is composed of neural tissue, dorsal mesoderm such as notochord, and the somite. Several signal pathways are involved in secondary axis formation. β-catenin is the earliest known factor that is asymmetrically localized on the future dorsal-ventral axis (Schneider et al., 1996). β-catenin is localized on the dorsal side from the vegetal pole to the animal pole at the blastula (Schneider et al., 1996). It functions as a dorsal determinant in nuclei (reviewed by Schier, 2001). Overexpression of wnt genes on the ventral side of the embryo can stabilize β-catenin and facilitate its translocation into nucleus, inducing a secondary axis. Inhibition of BMP activity can form neural tissue in animal caps (Smith et al., 1992). Low BMP activity levels at the dorsal side induce neuroectoderm and mesoderm such as notochord and muscle, while high BMP activity induces epidermal and kidney. Dorsal-ventral pattering requires repression of BMP activities on the dorsal side by antagonists secreted from the organizer, such as Noggin or Chordin (reviewed by Yamamoto et al., 2004). Nodal Activin signaling induces mesoderm and endoderm. The gradients of nodal activin are important for axis formation (Hainski et al., 1992; Joseph et al., 1997; Ito et al., 2001; Takahashi et al., 2000). High concentration of nodal/activin may forms dorsal mesoderm such as notochord and low concentration will form ventral mesoderm such as blood. BMP inhibitors (Smith et al., 1992; Piccolo et al., 1996; Sasai et al., 1994), wnt inhibitors, wnt signaling, β-catenin, and Activin/nodal signaling play important roles in the formation of the dorsal cell fate (Hainski et al., 1992; Joseph et al., 1997; Ito et al., 2001; Takahashi et al., 2000). If these molecules are overexpressed in the marginal zones of the ventral side, they can induce secondary axis. 6). Xenopus laevis as a model system The experiments employ the frog, Xenopus laevis, as the model system which offers a number of experimental advantages from embryo research. First, Xenopus embryos are very large and readily obtained in large quantities such as thousands of eggs from a female frog one day. These large external embryos can be microinjected and micromanipulated (Vize et al., 1991). Second, fertilization and development occur externally and development proceeds rapidly, which provides visual access to key early

6 stages of tissue differentiation over a relatively short period of time. Third, studies in Xenopus have established a good foundation of knowledge on TGF-β signaling and we can build on this to further examine signal transduction pathway as well as their regulators (Lohr et al., 2000).

Materials and Methods Molecular cloning of a full length Xenopus tropicalis LTBP4 (xtLTBP4) cDNA The forward primer that was used for the cloning was 5’- CTGGTCGACACCATGGCAGGGAAACTGGTGCTTATACTTTTGTC-3’, while the reverse primer was 5’-GCGCGGCCGCGAATGGGTCGTGTCTGTAGTGCTCACTG- 3’. These primers were designed using gene predictions based on the Xenopus tropicalis genomic sequence. The PCR reaction consisted of 30 seconds at 98 °C, followed by 35 cycles of 10 sec at 98 °C, 30 sec at 68 °C, and 2 min at 72 °C, and ended by 10 min of extension of 72 °C. PCR was performed by Phusion High-Fidelity DNA Polymerase (New England Biolabs Inc). The template DNA is RT (see the following method) from Xenopus tropicalis polyA RNA (a gift from Dr. Aaron Zorn) by using oligo-dT. The PCR product was cloned by TOPO subcloning Kit (Invitrogen). The plasmid was purified using cationic detergent cetyl-trimethylammonium bromide (CTAB) method, and sequencing using the primers described in Table 1. Constructions hLTBP4pcDNA3 plasmid was a gift from Dr. Keski-Oja (Saharinen J et al., 1998). hLTBP4pcDNA3 plasmid was digested by Hind III and Sca I and was treated by Klenow fragment, and then digested by the Xbra. Cs2++ vector was digested by Stu I and Xbra. hLTBP4/Hind III blunt/Xbra fragment was inserted into Cs2++/Stu I/Xbra linear vector. The ligation of hLTBP4Cs2++ was transformed into DH5α competent cells and purified by CTAB protocol. hLTBP4Cs2++ construction was digested by Asc I and in vitro transcription was performed by using hLTBP4Cs2++ as a template. Full length xtLTBP4TOPO was digested by Not I and Spe I, Cs2++ vector was digested by Not I and Xbra. Because Spe I and Xbra are compatible, the ligation of xtLTBP4TOPO/Not I/Spe I and Cs2++/Not I/Xbra can be formed. The ligation was transformed into DH5α competent cells. xtLTBP4Cs2++ plasmid was purified by the CTAB protocol. hLTBP4

Table 1: The primers that are used for sequencing xtLTBP4

Name Oligo Sequence 5’ to 3’ xtLTBP4 R525 GCGCAGTTTCCGACATACTT xt LTBP4 F557 CGCAATGTCAGCATCAGTCT xtLTBP4 F3 GACTATTTTCCTGTGCGTGA xtLTBP4 F1049 CAGGGGATATTGGGAGTGTG xtLTBP4 R1470 GCCATGTTCCCCATTGTTAC xtLTBP4 R1072 CCTCCACACTCCCAATATCC xtLTBP4 R2779 GCCTGGAGAGCAGAAACATC xLTBP4 F2309 CCATCCTCCGGAATATCACC xtLTBP4 F3525 CCTAGATATTGATGAGTGTC xtLTBP4 R4005 TTGCTCCCTCATATCCTTGG xtLTBP4 F4183 TCTACTATCCGCCCTGTGCT xtLTBP4 R4738 GGTTTCCAGTGTCTGTAGGTC xLTBP4 F2945 AAGGCACAGACTGCCAAGAT xLTBP4 R3360 GAGGGTGCCTTGTTGACATT xtLTBP4 F1474 AGAGCATTTCTTTGTCCACTG xtLTBP4 R1945 AGGCACACTGACCATTCTCC xtLTBP4 F1924 TTGGAGAATGGTCAGTGTGC xtLTBP4 R2464 GGTTTGGTGACGAGATTCTAC xtLTBP4 F4665 CCTGTGTCCTGGAAGGGATA xtLTBP4 R5210 GCCTCTCTTGGGTCTCCTCT xtLTBP4 F4937 GCCTCCCTCCTTATGAATCC xtLTBP4 R5441 CCATCTGTGTTACGGCATTG xLTBP4 F3422 CTGGCTTGGCATTGCTATG

VSVG Cs2++ was constructed by the following procedure. The forward primer that was used in PCR reaction was: 5’-GCCTCCCATATGGGCCTGA-3’. The two reverse primers were: R1: 5’- CGGTTCATTTCGATATCAGTGTAGGCCCGTGGTCGTGCGGGC-3’. R2: 5’- TACGCGGCCGCCTACTTACCCAGGCGGTTCATTTCGATATCA-3’. The PCR reaction consisted of 30 seconds at 98 °C, followed by 24 cycles of 10 sec at 98 °C, 30 sec at 68 °C, and 2 min at 72 °C, and ended by 10 min of extension of 72 °C. PCR was performed by Phusion High-Fidelity DNA Polymerase (New England Biolabs Inc). Primers that were used in this reaction were the mixture of forward primer, R1, and R2 primer. The ratio of R1 and R2 was 1:64. The template DNA was 1ng of hLTBP4Cs2++. Then PCR product was digested with Nde I and Not I. hLTBP4Cs2++ was partially digested with Nde I and filled in with klenow fragment DNA polymerase to kill the Nde I site in the vector. Then hLTBP4 Cs2++Nde I filled in was digested with Nde I and Not I. PCR fragment/Nde I/Not I and hLTBP4 Cs2++ Nde I filled in/Nde I/Not I were ligated together. The ligation was transformed into DH5α competent cells. hLTBP4VSVGCs2++ plasmid was purified by the CTAB protocol. Purification of DNA plasmid by using CTAB Bacterial cells were grown in 50ml TB overnight and the cells were centrifuged by 6000×G for 10 minutes. The bacterial pellet was resuspended with 10mL P1 (50mM Tris-HCl pH 8, 10mM EDTA, 100µg/mL RNAse A) for 5 minutes and the bacterial solution was lysed with 10mL P2 (200mM NaOH, 1% SDS) for 5 minutes, and then the lysis was neutralized with 10mL P3 (3.0M KoAc pH 5.5) for 10 minutes on ice. The neutralization solution was centrifuged by 3200×G for 15 minutes at 40C. The supernatant was transferred into a fresh tube and 3.5mL 2% CTAB was added. The DNA pellet can be obtained by centrifuging for 15 minutes and be resuspended with 2.5mL 1.2M NaCl. The pellet was reprecipitated with 6.25mL 100% ethanol. The pellet was washed with 70% ethanol and dried at 37 degree. The pellet was resuspended in 500µL TE (10mM Tris-HCl, pH 7.5, 1mM EDTA). Generation of embryos Ovulation was induced by injection of 500 IU of human chorionic gonadotropin (hCG). About 16-18 hours later, the induced female frog starts to lay eggs. The eggs were

collected manually and fertilized in vitro with male testis. Embryos were staged according to Nieuwkoop and Faber (1967). Microinjection and microsurgery of embryos RNA for injection was prepared for in vitro transcription using Ambion mMESSAGE mMACHINETM High Yield Capped RNA Transcription Kit. The template DNAs were from hLTBP4Cs2++, xtLTBP4Cs2++ and hLTBP4VSVGCs2++ and were linearized by Asc I and transcribed by SP6 polymerase. Xnr1 template DNA was from HAXnr1- PCs2++ and linearized by Not I. Xnr1 RNA was also transcribed by SP6 RNA polymerase. For cap samples, Xnr1 capped RNA with or without LTBP4 capped RNA was injected into two cells of the animal pole at the two cell stage. Animal caps were isolated at stage 8.5. Caps were cultured until sibling embryos reached stage 10 as described in Figure 3 A. Total RNA was extracted at this stage and the cDNA template was produced with reverse transcriptase. Real time PCR was performed with the cDNA template at this stage. The caps were grown until sibling embryos reached stage 24. For detecting phenotype, 2ng of hLTBP4 or xtLTBP4 capped RNA were co-injected with β- galactosidase into ventral marginal zone of 4-cell stage (Figure 3 B). The embryos were grown until stage 36 and then fixed in MEMFA (0.1M MOPS pH 7.4, 2mM EGTA,

1mM MgSO4, 3.7% formaldehyde). The embryos were stained by X-gal, a chemical that will form a permanent blue precipitation in the presence of β-galactosidase. Total RNA isolation and in vitro reverse transcription Total RNA isolation was performed by the RNA-Bee protocol. The whole embryos or caps samples were broken in solution D (4M guanidinium thiocyanate, 25 mM Sodium Citrate, pH 7.0, 0.5% sarcosyl, 0.1M 2-mercaptoethanol) and then RNA-Bee (Chomczynski et al., 1987) was added. RNA was extracted with chloroform and precipitated with 1 volume of isopropanol. The pellets were resuspended and reprecipitated by 7.5M LiCl. The pellets were washed with 70% ethanol and resuspended in DEPC-treated water. Once total RNA was extracted, 500 pg total RNA was reverse transcribed in 20µL using random hexamer primers and 100 Units of Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase (NEW ENGLAND Biolabs) at 420C for 30 minutes. Real time PCR

Figure 3: Microinjection of Xenopus embryos. A: Microinjection for cap assay. B: microinjection for LTBP4 phenotype assay.

The Real time PCR reaction consisted of 40 cycles of 20 sec at 95 °C, 10 sec at 60 °C, and 10 sec at 72 °C, 32 sec at 80°C by using 0.1µL of the RT reaction. Each 25µL PCR reaction contained 1× Biorad Real Time PCR mix with SYBR green, 1× Rox, 0.1µL RT mix, and 40µM gene-specific primers (upstream and downstream). The real-time PCR reaction was run on an ABI lightCycler and normalized by Ornithine decarboxylase PCR product. In situ hybridization xLTBP2 antisense RNA probe was prepared by linearizing xLTBP2PBSSKII with Eco RI and transcribing with T7 RNA polymerase according to manufacturer’s instructions with digoxygenin (DIG Quantification Teststrips, Roche). xLTBP3 antisense RNA probe was prepared by linearizing xLTBP3PCMVSPOR6 with Sal I and transcribing with T7 RNA polymerase. xLTBP4 antisense RNA probe was prepared by linearizing xLTBP4PBSSKII with Eco RI and transcribing with T7 RNA polymerase according to manufacturer’s instructions with digoxygenin (DIG Quantification Teststrips, Roche). Whole mount in situ hybridization was performed as described by Harland (1991). Immunocytochemistry The embryos were fixed in MEMFA for 1-2 hours and then embryos were transferred into 100% methanol. Embryos were rehydrated in 1×PBS. Next, the embryos were washed in PBT (2mg /mL bovine serum albumin, 0.1% triton X-100 in 1×PBS) and blocked in PBT+ 20% Heat-Inactived (HI) Goat Serum. Then the embryos were incubated with primary antibody (12-101) at 1:500 at 40C for overnight and washed 10 minutes several times with 5mL of PBT. Embryos were then incubated with secondary antibody (mouse fluorescein IgG) at 1:10,000 for 4-6 hours at room temperature and washed 10 minutes several times with 5mL of PBT. Co-IP experiment and western bloting 2ng hLTBP4-VSVG and 1ng Xnr1-flag capped RNAs were injected into two cells at the two-cell stage at the animal poles. The embryos were cultured until stage 8.5 and caps were isolated at this stage. The protein was extracted from stage 10 caps with extraction buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% Triton X-100, 4ng/mL Pepstatin A, 1µg/mL Leupeptin, 1µg/mL Aprotinin, 1mM PMSF). The samples were precleared at 40C for half an hour by using 20µL protein G-adsorbed beads

(Eliasson, M., et al, 1988). To prepare the protein G-conjugated beads for antibody adsorption, 20µL of beads per immunoprecipitation reaction were incubated with 300µL extraction buffer including 1µL of 1µg/µL anti-VSVG or anti-flag antibodies for 1 hour with rotation at room temperature. Next, the supernatant was removed and precleared extraction sample was added. The beads, antibody and the sample mixture were incubated at 40C for 3-6 hours with rotation. The sample was washed several times with extraction buffer. Next 4× reducing sample buffer was added to each tube to a final 1×concentration. The samples were boiled at 95°C for 3 minutes. Tubes were centrifuged for 30 seconds at 12,000×G and samples were subjected to western blotting. For western blotting, proteins were separated by 8% SDS-PAGE and transferred onto a PVDF membrane (Sigma). The membranes were blocked in 5% non-fat dry milk in PBS-T (1× PBS with 0.05% Tween 20) for 1 hour at room temperature. Primary antibody concentration is 1:1000 in blocking solution and secondary antibody (peroxidase conjugated) is 1:10,000. Primary antibody and secondary antibody were incubated at room temperature separately for 1 hour. Membranes were washed several times in PBS-T and reacted with a chemiluminescent substrate for 1-2 minutes. Exposures of membrane to X-ray film lasted between 1-5 minutes typically.

Results 1. The expression of LTBP 2, 3, 4 is regulated temporally Studies on LTBP1 have been previously published (Altmann, et al.,2002). Using a bioinformatic approach, Xenopus laveis cDNA clones which matched LTBP2, 3, and 4 were obtained and confirmed by sequence analysis. The DNA sequences for LTBP2 (partial cDNA) and LTBP-3 (complete cDNA) had moderate homology to human LTBP genes. The xLTBP4 clone had a lower homology and matched only the C-terminal region. While, the complete cDNA of Xenopus tropicalis LTBP4 has been cloned and sequenced. The analysis confirmed the conclusion that we had identified the Xenopus homology of three uncharacterized LTBPs. Oligonucleotide primers were designed and reverse transcription polymerase chain reaction assays (RT-PCR) were performed using total RNA from various stages of development. As shown in Figure 4, xLTBP2 and xLTBP3 were initially expressed at low levels followed by increased expression

beginning at late gastrula stages (stage 12) and eventually high levels of expression at neural stage (stages 18-21). Expression continued through tadpole stages (stage 25). In contrast, lower levels of xLTBP4 mRNA were detected at all tested developmental stages. The mRNA level of xLTBP4 was relatively high at stage 10 (early gastrula). The expression of LTBPs shows a temporal pattern of regulation during early development. The primers that were used in this experiment are shown in Table 2. 2. Whole mount in situ hybridization of xLTBP2, 3, 4 To examine the spatial pattern of expression for each gene, whole mount in situ hybridization was performed. Expression was observed for all three xLTBP family members at gastrula and neural stages (Figure 5). Importantly, at gastrula stages, LTBP3 and LTBP4 were expressed in the organizer region of the developing embryo (dorsal mesoderm) similar to the pattern of expression of LTBP1 (Figure 5 panel B and C, blue). LTBP2 was expressed in whole mesoderm, not limited dorsal mesoderm (Figure 5 panel A blue). Examination of embryos at neural stage shows a temporally regulated pattern of expression (Figure 5 D-F). These results demonstrate that LTBP transcripts exhibit spatial and temporal regulation and that the specific patterns of different family members are distinct. 3. Identification of Xenopus tropicalis LTBP-4 We isolated a full length LTBP4 clone from Xenopus tropicalis (xtLTBP4) with a conserved domain structure characteristic of LTBPs. The full length xtLTBP4 shows 53% conservation at DNA level with human LTBP4, and even higher conservation within the conserved domains, especially the TB domains (Figure 7). Surprisingly, the 3rd TB domain, known to be critical for the interaction of TGF-β1LAP with LTBP shows the greatest divergence while the 2nd TB is the most conserved (Figure 8). This suggests that LTBP4 may interact weakly, if at all, with TGFβ1 ligand. Moreover, the Xenopus 3rd TB domain lacks the conserved two amino acid insertion (Figure 8) proposed to play a critical role in the formation of the covalent cysteine bond between LTBPs and TGF-β1 LAP (Chen Y et al., 2005) and which are present in all other known 3rd TB domains (Chen Y et al., 2005). These results are consistent with the hypothesis that the other TB domains than 3rd TB may interact with other TGF-β ligands or that xtTB3 interacts with

Table 2: Primers for xLTBP2, xLTBP3 and xLTBP4. Primers are designed using the primer 3 software and tested by netprimer software. U indicates upstream primers. D indicates downstream primers.

Figure 4: Expression of xLTBP2, xLTBP3, and xLTBP4 by reverse transcription polymerase chain reaction assays (RT-PCR). Each lane shows different embryo stages. Each row shows different gens. ODC stands for Ornithine decarboxylase and is a loading control.

Figure 5: Whole amount in situ hybridization of xLTBP2-4. Gastrula stage embryos show staining in the prospective mesoderm for each gene (xLTBP2 A,D; xLTBP3 B,E; xLTBP4, C,F). Gastrula A-C, Neurula D-F, Staining was not observed in sense strand negative controls controls (data not shown).

TGF-β ligands primarily through non-covalent interactions. This is significant in view the observation that some of these alternative spliced forms of LTBP4 are unable to interact with TGF-β1 (Koli K et al., 2001). 4. xLTBP4 is expressed in the organizer, neural plate and head Since xLTBP4 was expressed strongly in the organizer, it was chosen for further analysis. Different stages of embryos were used for whole mount in situ hybridization. Figure 9 shows that LTBP4 is expressed in the organizer at the gastrula stage and in the head at the early tadpole stage. This suggests LTBP4 may be involved in the dorsal and anterior extension of the body axis. RT-PCR data shows that LTBP4 is expressed at the gastrula, neural, and early tadpole stages, though expression of LTBP4 at the neural stage is low (Figure 4). 5. LTBP4 induces a partial secondary axis Since LTBP4 is strongly expressed in the organizer, we hypothesized that LTBP4 may induce axis formation. This process of body axis formation in the embryo arises from a series of inductive cell-cell interactions. In TGF-β superfamily, misexpression of activin and nodal (Thomsen et al., 1990), or inhibition of BMP can also induce a body axis (Xanthos et al., 2002). When 2ng hLTBP4 capped mRNA was co-injected with β- galactosidase mRNA in the ventral marginal zone, a partial secondary axis was observed to form (Figure 10). Uninjected or only β-galactosidase injected embryos showed no such secondary axis induction. We also stained the injected embryos with X-gal and the secondary axis stained blue. This indicated that injection of LTBP4 mRNA could induce secondary axis. To confirm that this secondary axis exhibited features of a normal secondary axis, we also did immunocytochemstry with the 12-101 antibody which detects somites. The somite is typical dorsal mesoderm tissue that is present in nomal axis. The results show that both primary axis and secondary axis are stained by 12-101 (Figure 11 C and D). This suggests that the secondary axis includes somite tissue and therefore resembles a real axis. In 397 hLTBP4 injected embryos, about 27% embryos have secondary axis, while in 132 xtLTBP4 injected embryos, about 12% embryos show the secondary axis (Table 3). 6. LTBP4 enhances Xnr1 Since Activin nodal signaling can induce secondary axis, we hypothesized that LTBP4

(1) 1 10 20 30 40 50 60 70 80 98 mLTBP4 (AAN04661) (1) MRRPGLGGPCPLLLLLLLPAATSASGSSPSPSPSPIEKAVVPSHQAGVAACHCCLDQTPKSSRCTRASCRVRNCPPAKCTGLEGCLTPTPSVPSPSRS hLTBP4(NP_003564) (1) ------MGDVKALLFVVAARARRLGG------AAAS--ESLAVS------EAFCRVRSCQPKKCAGPQRCLNPVPAVPSPSPS xtLTBP4 (1) ------MAGKLVLILLSLNCLWWTPASG Consensus (1) G LL LL A G AA A CRVR C P KC GL CLSP PAVPSPS S

(99) 99 110 120 130 140 150 160 170 180 196 mLTBP4 (AAN04661) (99) PVEKSQVSLNWQPLTLQEARALLRQRRPRGPWARALLKRRPPHRAPAGQARVLCPLICHNGGVCVKPDRCLCPPDFAGKFCQLHSSGARPPAPAMPG- hLTBP4(NP_003564) (64) -VRKRQVSLNWQPLTLQEARALLKRRRPRGPGGRGLLRRRPPQRAPAGKAPVLCPLICHNGGVCVKPDRCFCPPDFAGKFCQLHSSGARPPAPAVPG- xtLTBP4 (23) QVEKVKVMFTPMVCRLRCLGDRCTNQCERGNMTTVYSDELSGNNGEHGFRAFLCPLLCQNGGVCLKKDKCLCPPNFTGKFCQIPISTDGGNIKQDHKN Consensus (99) VEK QVSLNWQPLTLQEARALLKNRRPRGP ARALLKRRPPNRAPAG A VLCPLICHNGGVCVKPDRCLCPPDFAGKFCQLHSSGARPPAPAMPG

(196) 196 210 220 230 240 250 260 270 280 293 mLTBP4 (AAN04661) (196) ------LTRSVYTMPLANHRDDEHGVASMVSVHVEHPQEASVVVHQVERVSGPWEEANPEALARAEAAARAEAAAPYTVLAQSAPREDGYSDASGF hLTBP4(NP_003564) (160) ------LTRSVYTMPLANHRDDEHGVASMVSVHVEHPQEASVVVHQVERVSGPWEEADAEAVARAEAAARAEAAAPYTVLAQSAPREDGYSDASGF xtLTBP4 (120) NSSVEQNTMTKSVHTLLLSNYHPEKGGAASVVKVHVEHPPEASVNIHQVERIDG--QSGDRARNGIQNHEQLGARIPLYGVQAQSSPRINGYTENSGF Consensus (196) LTRSVYTMPLANHRDDEHGVASMVSVHVEHPQEASVVVHQVERVSGPWEEAD EALARAEAAARAEAAAPYTVLAQSAPREDGYSDASGF

(294) 294 300 310 320 330 340 350 360 370 380 391 mLTBP4 (AAN04661) (286) GYCFRELRGSECASPLPGLRTQEVCCRGEGLAWGVHDCHPCAEHLRNSNQVSGPNGPCPPGFERVNGSCVDVDECATGGRCQHGECANTRGGYTCVCP hLTBP4(NP_003564) (250) GYCFRELRGGECASPLPGLRTQEVCCRGAGLAWGVHDCQLCSERLGNSERVSAPDGPCPTGFERVNGSCEDVDECATGGRCQHGECANTRGGYTCVCP xtLTBP4 (216) GYCFRRLENGQCASPIPGLRTQEVCRRSSGVAWGVHNCTPCNGHQGDVLSASVIDTPCPKGFERINGTCIDIDECTDSTLCVNGDCTNTRGSYTCMCR Consensus (294) GYCFRELRGGECASPLPGLRTQEVCCRGAGLAWGVHDC PCAEHLGNS VSAPDGPCP GFERVNGSCIDVDECATGGRCQHGECANTRGGYTCVCP

(391) 391 400 410 420 430 440 450 460 470 488 mLTBP4 (AAN04661) (383) PDGFLLDSSRSSCISQHVISEAKGPCYRVLHDGGCSLPILRNITKQICCCSRVGKAWGRGCQLCPPYGSEGFREICPAGPGYHYSASDLRYNTRPLNQ hLTBP4(NP_003564) (347) PDGFLLDSSRSSCISQHVISEAKGPCFRVLRDGGCSLPILRNITKQICCCSRVGKAWGRGCQLCPPFGSEGFREICPAGPGYHYSASDLRYNTRPLGQ xtLTBP4 (313) REGYLLDSSRSSCISHHVISEVKGPCFRILRDGKCSLPILRNITKQICCCSRVGKAWGKGCEQCPPFGTEGFKETCPAGPGYHYSASDLRINTRYVGQ Consensus (391) PDGFLLDSSRSSCISQHVISEAKGPCFRVLRDGGCSLPILRNITKQICCCSRVGKAWGRGCQLCPPFGSEGFREICPAGPGYHYSASDLRYNTRPLGQ

(489) 489 500 510 520 530 540 550 560 570 586 mLTBP4 (AAN04661) (481) DPPRVTFNQP---RVPPATPRPPTGFLPTRRPEPRPDPGPQPEPRPRPEPRPRPESRPRPEPRPRPEPRPQPESQPRPESRPRPESQPWPEFPLPSIP hLTBP4(NP_003564) (445) EPPRVSLSQP---RTLPATSRPSAGFLPTHRLEPRPEP------R----P------DP------RPGPELPLPSIP xtLTBP4 (411) DQARVPLLRQPASRGFPFTTASPVYHAVTGHITVERQQ------DI Consensus (489) DPPRVSL QP R PATSRPP GFLPT RIEPRPDP R P DP P PE PLPSIP

(587) 587 600 610 620 630 640 650 660 670 684 mLTBP4 (AAN04661) (576) AWTGPEIPESGPSSSMCQRNPQVCGPGRCVPRPSGYTCACDPGFRLGPQDTRCIDIDECRRVPTPCAPGRCENTPGSFRCVCGTGFQAGPRATECLDV hLTBP4(NP_003564) (496) AWTGPEIPESGPSSGMCQRNPQVCGPGRCISRPSGYTCACDSGFRLSPQGTRCIDVDECRRVPPPCAPGRCENSPGSFRCVCGPGFRAGPRAAECLDV xtLTBP4 (451) DQSRISSPNQRVPS----REP--VSANRSISGPDRVSVLPAPVPTETQKEAPSQDIDECSTDQTPCDNGRCENTPGSYRCFCSPGFTLNPQGKSCIDI Consensus (587) AWTGPEIPESGPSS MCQRNPQVCGPGRCISRPSGYTCACDPGFRLSPQDTRCIDIDECRRVPTPCAPGRCENTPGSFRCVCGPGF AGPRA ECLDV

(685) 685 690 700 710 720 730 740 750 760 770 782 mLTBP4 (AAN04661) (674) DECRRVPP-PCDRGRCENTPGSFLCVCPAGYQAAPHGASCQDVDECTQSPGLCGRGVCENLPGSFRCVCPAGFRGSACEEDVDECAQQPPPCGPGRCD hLTBP4(NP_003564) (594) DECHRVPP-PCDLGRCENTPGSFLCVCPAGYQAAPHGASCQDVDECTQSPGLCGRGGCKNLPGSFRCVCPAGFRGSACEEDVDECAQEPPPCGPGRCD xtLTBP4 (543) DECRISPRRFCLSGRCENTPGSFLCVCPVGYTANPQGTDCRDTDECRQTPRICGSG------Consensus (685) DECRRVPP PCD GRCENTPGSFLCVCPAGYQAAPHGASCQDVDECTQSPGLCGRG C NLPGSFRCVCPAGFRGSACEEDVDECAQ PPPCGPGRCD

(783) 783 790 800 810 820 830 840 850 860 870 880 mLTBP4 (AAN04661) (771) NTAGSFHCACPAGFRSRGPGAPCQDVDECSRSPSPCAYGRCENTEGSFKCVCPTGFQPNAAGSECEDVDECENRLACPGQECVNSPGSFQCRACPVGH hLTBP4(NP_003564) (691) NTAGSFHCACPAGFRSRGPGAPCQDVDECARSPPPCTYGRCENTEGSFQCVCPMGFQPNTAGSECEDVDECENHLACPGQECVNSPGSFQCRTCPSGH xtLTBP4 (599) ------RCQNTPGSFRCSCPVGYRLTPQGNECIDINECENPGACAGQECLNTPGSFQCRQCHSGY Consensus (783) NTAGSFHCACPAGFRSRGPGAPCQDVDECARSP PC YGRCENTEGSFKCVCPMGFQPN AGSECEDVDECEN LACPGQECVNSPGSFQCR CPSGH

(881) 881 890 900 910 920 930 940 950 960 978 mLTBP4 (AAN04661) (869) HLHRGRCTDVDECSSGTP-CGLHGQCTNTKGSFHCSCSTGYRAPSGQPGPCADINECLEGDFCFPHGECLNTDGSFTCTCAPGYRPGPRGASCLDVDE hLTBP4(NP_003564) (789) HLHRGRCTDVDECSSGAPPCGPHGHCTNTEGSFRCSCAPGYRAPSGRPGPCADVNECLEGDFCFPHGECLNTDGSFACTCAPGYRPGPRGASCLDVDE xtLTBP4 (658) RLQNRRCVDINECQTDSA-CGRHEKCINTEGSFECECLPGYHLNDRN--RCTDINECLEGDFCFPRGECQNTEGSYICVCAEGYVTTPDGASCVDKDE Consensus (881) HLHRGRCTDVDECSSGSP CG HG CTNTEGSF CSCAPGYRAPSGNPGPCADINECLEGDFCFPHGECLNTDGSF CTCAPGYRPGPRGASCLDVDE

(979) 979 990 1000 1010 1020 1030 1040 1050 1060 1076 mLTBP4 (AAN04661) (966) CSEEDLCQSGICTNTDGSFECICPPGHRAGPDLASCLDIDECRERGPALCGSQRCENSPGSYRCVRDCDPGYHPGPEGTCDDIDECREYGSAICGAQR hLTBP4(NP_003564) (887) CSEEDLCQSGICTNTDGSFECICPPGHRAGPDLASCLDVDECRERGPALCGSQRCENSPGSYRCVRDCDPGYHAGPEGTCDDVDECQEYGPEICGAQR xtLTBP4 (753) CQQGTLCQGGRCINTQGSFQCQCPTGFRVTFNKAACTDVDECLEYGRSICGTKRCENTPGSYRCISDCEPGYQLS------Consensus (979) CSEEDLCQSGICTNTDGSFECICPPGHRAGPDLASCLDVDECRERGPALCGSQRCENSPGSYRCVRDCDPGYH GPEGTCDDIDEC EYG ICGAQR (1077) 1077 1090 1100 1110 1120 1130 1140 1150 1160 1174 mLTBP4 (AAN04661) (1064) CENTPGSYRCTPACDPGYQPTPGGGCQDVDECRNRSFCGAHAMCQNLPGSFQCVCDQGYEGARDGRHCVDVNECETLQGVCGSALCENVEGSFLCVCP hLTBP4(NP_003564) (985) CENTPGSYRCTPACDPGYQPTPGGGCQDVDECRNRSFCGAHAVCQNLPGSFQCLCDQGYEGARDGRHCVDVNECETLQGVCGAALCENVEGSFLCVCP xtLTBP4 (828) SS------GDCTDINECLNKTVCGDHAMCHNLAGTYQCLCDQGYEGARGERHCVDVNECLTLQSVCGTALCENVEGSFLCTCP Consensus (1077) CENTPGSYRCTPACDPGYQPTPGGGCQDVDECRNRSFCGAHAMCQNLPGSFQCLCDQGYEGARDGRHCVDVNECETLQGVCGSALCENVEGSFLCVCP

(1174) 1174 1180 1190 1200 1210 1220 1230 1240 1250 1260 1271 mLTBP4 (AAN04661) (1161) PNSPEEFDPMTGRCVPPRAPAGTFPGSQPQAPASPSLPARPPAPPPPRRPSPPRQGPVSSGRRECYFDTAAPDACDNILARNVTWQECCCTVGEGWGS hLTBP4(NP_003564) (1082) PNSPEEFDPMTGRCVPPRTSVGMSPGSQPQAPVSPVLPARPPPPPLSRRPRKPRKGPVGSGCRECYFDTAAPDACDNILARNVTWQECCCTVGEGWGS xtLTBP4 (904) PNSKEEFDPMSGKCIR-----S------NSTIRPVLPSLP------SPSQVDTLTSGHKECYYNLEESEVCGNVLARNVSRDECCRSIGEGWGQ Consensus (1174) PNSPEEFDPMTGRCVPPR G PGSQPQAPISPVLPARPP PP RRPSPPR GPVSSG RECYFDTAAPDACDNILARNVTWQECCCTVGEGWGS

Figure 6: Continued

Figure 6: Continued (1370) 1370 1380 1390 1400 1410 1420 1430 1440 1450 1467 mLTBP4 (AAN04661) (1357) TVGSYHCTCEPPLVLDGSRRRCVSNESQSLDDNLGVCWQEVGPDLVCSRPRLDRQATYTECCCLYGEAWGMDCALCPAQDSDDFEALCNVLRPPAYGP hLTBP4(NP_003564) (1278) TVGSYHCTCEPPLVLDGSQRRCVSNESQSLDDNLGVCWQEVGADLVCSHPRLDRQATYTECCCLYGEAWGMDCALCPAQDSDDFEALCNVLRPPAYSP xtLTBP4 (1073) TVGSYYCTCNPPLVLDSTQRRCVANTSQTIDENLSYCWQEIGADMLCKRPLLERQTTYTECCCHYGEAWGLHCALCPGRDTDDFESLCIDWRPTDTGN Consensus (1370) TVGSYHCTCEPPLVLDGSQRRCVSNESQSLDDNLGVCWQEVGADLVCSRPRLDRQATYTECCCLYGEAWGMDCALCPAQDSDDFEALCNVLRPPAYGP (1272) 1272 1280 1290 1300 1310 1320 1330 1340 1350 1369 mLTBP4 (AAN04661) (1259) GCRIQQCPGTETAEYQSLCPHGRGYLVPSGDLSARRDVDECQLFQDQVCKSGVCVNTAPGYSCYCSNGFYYHAHRLECVDNDECADEEPACEGGRCVN hLTBP4(NP_003564) (1180) GCRIQQCPGTETAEYQSLCPHGRGYLAPSGDLSLRRDVDECQLFRDQVCKSGVCVNTAPGYSCYCSNGYYYHTQRLECIDNDECADEEPACEGGRCVN xtLTBP4 (981) NC--ERCPAIDTAEYQALCPRGGGYTISQ--Q-GLKDINECQIFGNQLCKGGFCLNKVPSFSCYCSNGYYYDVQRLECVDNDECHEEELCE-GGNCIN Consensus (1272) GCRIQQCPGTETAEYQSLCPHGRGYLIPSGDLSARRDVDECQLF DQVCKSGVCVNTAPGYSCYCSNGYYYH QRLECVDNDECADEEPACEGGRCVN

(1468) 1468 1480 1490 1500 1510 1520 1530 1540 1550 1565 mLTBP4 (AAN04661) (1455) PRP------GGFGIPYEYGPDIGPPYQSLPYGPDLYPPPVLPYDPYPPPPGPFARREAPYGAPPFDMPDFEDDGGPYGESE------hLTBP4(NP_003564) (1376) PRP------GGFGLPYEYGPDLGPPYQGLPYGPELYPPPALPYDPYPPPPGPFARREAPYGAPRFDMPDFEDDGGPYGESE------xtLTBP4 (1171) PGPPQPSLYDYGTEYGGYGVPYGPDAFANPPPRIVRPGYESYPAAPGGMSFGSRPSLPYGPRDSLYSLPPYESPDFDSDIYYPDPSVEEPRNPFRRTD Consensus (1468) PRP GGFGIPYEYGPDIGPPYQ LPYGPELYPPP LPYDPYPPPPGPFARREAPYGAPPFDMPDFEDDGGPYGESE

(1565) 1565 1570 1580 1590 1600 1610 1620 1630 1640 1650 1662 mLTBP4 (AAN04661) (1530) ----TPDPPSRGTGWPYRSRDTRGSFPEPE-----ESSERGS------YTGALSEPYEGLEAEECGILDGCPHGRCVRVPEGFTCD hLTBP4(NP_003564) (1451) ----APAPPGPGTRWPYRSRDTRRSFPEPE-----EPPEGGS------YAGSLAEPYEELEAEECGILDGCTNDRCVRVPEGFTCR xtLTBP4 (1268) DSSRDPNPYPSAPRSRSRSTDISSRFEVDAGGRRVQPRRPGSRDPGSVSSISSWRGDPREAFDDRYEQFEGLQAEECGILNGCENGRCIRVPEGYTCD Consensus (1565) P PP GTRWPYRSRDTR SFPEPE EP E GS Y GALAEPYEGLEAEECGILDGC NGRCVRVPEGFTCD

(1631) 1631 1640 1650 1660 1670 1680 1690 1700 1710 1728 mLTBP4 (AAN04661) (1569) EPYEGLEAEECGILDGCPHGRCVRVPEGFTCDCFDGYRLDITRMSCVDVNECDEAEATSPLCINARCVNTDGSFRCICRPGFAPTHQPHHCAPARPRA hLTBP4(NP_003564) (1490) EPYEELEAEECGILDGCTNDRCVRVPEGFTCRCFDGYRLDMTRMACVDINECDEAEAASPLCVNARCLNTDGSFRCICRPGFAPTHQPHHCAPARPRA xtLTBP4 (1334) EQFEGLQAEECGILNGCENGRCIRVPEGYTCDCYDGYRLDITLMACVDINECDEAEGLSLLCLNGQCRNTDGSYECVCPRGYVLSRHHNYCLPIQQ-- Consensus (1631) EPYEGLEAEECGILDGC NGRCVRVPEGFTCDCFDGYRLDITRMACVDINECDEAEA SPLCINARCLNTDGSFRCICRPGFAPTHQPHHCAPARPRA Figure 6: Sequence alignment of Xenopus tropicalis, human, and mouse LTBP4. Yellow color indicates the conserved sequences in all three species, blue color indicates the conserved sequences in two species and green color indicates the similarity. Top numbers in each line indicates the amino acid residue of mouse LTBP4. mLTBP4 stands for mouse LTBP4, hLTBP4 stands for human LTBP4, and xtLTBP4 stands for Xenopus tropicalis LTBP4.

Figure 7: xtLTBP4 domain structure. xtLTBP4 domain structure exhibits a structure similar to that of human. It includes a signal sequence, 4 eight cysteine TB domains and multiple EGF like repeats.

Figure 8: Multiple Alignment of TB domains from LTBP4 in mouse, human and Xenopus tropicalis. TB domains of LTBP4 in mouse, human and Xenopus are conserved.

Figure 9: In situ hybridization of xLTBP4. In gastrula stage, LTBP4 was expressed in dorsal mesoderm, which is the organizer. In the neural stage, xLTBP4 was expressed in the neural plate. In the early tadpole stage, xLTBP4 was expressed in the head, eye, and neural tube. A to D are gastrula embryos. E to H are neural stage embryos. I to L are early tadpole stage embryos.

21 may enhance this signaling pathway. We injected 5pg Xnr1 mRNA, 2ng hLTBP4 mRNA (or xtLTBP4) separately and co-injected Xnr1 and hLTBP4 into the animal pole of the two-cell stage. Then we isolated the caps at stage 8.5 and grew them until stage 10. We isolated RNA at stage 10 and use it to prepare cDNA. We performed Real time PCR to detect the expression of target genes of Xnr1, for example, Chordin, Goosecoid, Xbra, Sox17. The primers used are listed in Table 4 and the results are shown in the Figure 12 and Figure 13. We found that both hLTBP4 and xtLTBP4 seem to increase Xnr activity in the expression of Chordin, Xbra and Sox17. hLTBP4 also enhances Xnr1 activity in the expression of Goosecoid, but xtLTBP4 does not (data are not shown). Nodal ligand genes such as Xnr1 can induce mesoderm in animal caps which makes animal caps enlongate (Joseph et al., 1997; Ito et al., 2001; Takahashi et al., 2000). We injected lower amount of Xnr1 mRNA with and without hLTBP4 mRNA to see if hLTBP4 can enhance this phenotype. The results are shown in Figure 14. 25pg of Xnr1 mRNA is not sufficient to make animal caps enlongate, but coinjection of Xnr and hLTBP4t did induce animal caps to enlongate (Figure 14). These results suggest that LTBP4 enhances Xnr1 activity. Co-expression of Activin and LTBP4 did not enhance Activin in mesoderm induction. 7. LTBP4 does not inhibit BMP LTBP4 could induce a secondary axis by inhibiting the BMP signaling pathway. To test this idea, we injected LTBP4 mRNA in the animal pole and isolated the caps at stage 8.5. We grew the caps until stage 20 and isolated RNA. Real time PCR by using different neural markers did not reveal hLTPB4 induction on neural tissue markers in animal caps. This suggests that LTBP4 is not an inhibitor of BMP signal. 8. LTBP4 physical interaction with Xnr1 is not detected hLTBP4 was tagged by VSVG by using PCR as described in methods. Xnr1-flag was a gift from Dr Chenbei Chang (unpublished data). We coinjected 2ng hLTBP4-VSVG mRNA and 1ng Xnr1-flag mRNA into the animal pole at the two-cell stage and isolated caps at stage 8.5. We grew the caps until stage 10, extracted the protein at this stage, and found via Co-IP assay no evidence for an interaction between hLTBP4 and Xnr1.

Discussion

Figure 10: Injection of hLTBP4 induces a second axis. A: uninjected control embryos, B: embryos co-injected with 2ng hLTBP4 mRNA and 100pg β-gal mRNA into the ventral marginal zone at the four-cell stage. The arrows indicate the positions of secondary axis formation.

Figure 11: Secondary axis is stained by 12-101 and β-gal. A: uninjected control embryo, B: injected embryo with 2ng hLTBP4 mRNA and 100pg β-gal mRNA into the ventral marginal zone at the four-cell stage, C: injected embryo immunostained with 12- 101 antibody staining and visualized by fluorescent microscope, D: uninjected control embryo, F: section of hLTBP4 injected embryo showing β-gal stained secondary axis. Embryos were fixed at stage 32 and stained with X-gal.

Table 3: Percentage of secondary axis induced by microinjection of mRNA for hLTBP4 and xtLTBP4

1. The role of LTBP4 during TGF-β signaling LTBPs have previously been shown to play roles in TGFβ1, TGFβ2 and TGFβ3 signaling pathways. From our in situ results, xLTBP4 was expressed in the organizer. Most of BMP inhibitors were also expressed in the organizer. Previously, we thought that LTBP4 may be a BMP4 inhibitor. However, from our cap experiment, LTBP4 did not induce neural tissue in the animal cap. This result suggests that LTBP4 is not an inhibitor of the BMP signaling pathway. Our data indicates that LTBP4 enhanced Xnr1 activity and this may be how LTBP4 can induce a secondary axis. We previously thought that LTBP4 may physically interact with Xnr1, but we did not observe an interaction by co-IP. This negative result does not necessarily mean that LTBP4 does not interact with Xnr1. If LTBP4 interacts LAP, we would not able to detect this interaction since our Xnr1 tagged at C-terminal region. Another possibility is that LTBP4 may act as a morphogen and concentrate Xnr1 in the organizer region (discuss in the following paragraph). We also co-injected LTBP4 with Activin and we found that LTBP4 did not enhance Activin (Activin data not shown), while LTBP1 was expressed in the organizer and enhanced both Xnr1 and activin (Altmann et al., 2002). However, LTBP1 did not induce secondary axis. It suggests that LTBP1 can enhance both the Activin and the Xnr signaling pathway, but not enough to induce a secondary axis. Taken together, these findings suggest that LTBP4 is sufficient to induce a secondary axis. 2. Role of LTBP4 in the organizer LTBP1, LTBP3 and LTBP4 are expressed in the organizer. LTBP1 and LTBP4 act as agonists in nodal signaling pathway. LTBP1 also enhanced Activin. Since LTBP1, LTBP3 and LTBP4 have similar expression patterns and LTBP1 and LTBP4 have similar functions, LTBPs therefore may function redundantly and be required for the formation and functioning of the organizer region. This could be tested by loss of function of LTBPs. We predict that a loss of function of any single member of LTBPs may not affect the formation of organizer. Interestingly, some antagonists of Activin and nodal signaling are also expressed in the organizer at the same time such as follistatin (Lau, 1995). The consequences of the expression of these competing factors are not known, but they may regulate the concentration of activin and nodal ligand during normal embryonic development.

Figure 12: hLTBP4 acts synergistically with Xnr-1. TGF-β ligand Xnr-1 was injected at 5 pg with or without hLTBP4 at the two cell stage. Tissue explants were prepared at the blastula stages. Explants were harvested when sibling embryos were at the gastrula stages and real time quantitative PCR was performed and normalized to ODC expression.

Figure 13: xtLTBP4 enhances the expression of Xnr1 by real time PCR. TGF-β ligand Xnr-1 was injected at 20 pg or 40 pg with or without xtLTBP4 at the two cell stage. Tissue explants were prepared at the blastula stages. Explants were harvested when sibling embryos were at the gastrula stages and real time quantitative PCR was performed and normalized to ODC expression.

Figure 14: hLTBP4 enhances Xnr in the mesoderm induction in the animal caps by elongating the animal caps. TGF-β ligand Xnr-1 was injected at 25 pg with or without hLTBP4 at the two cell stage. Tissue explants were prepared at the blastula stages. Explants were grown until sibling embryos were at the stage 24. A: Xnr1 25pg, B: xnr1 25pg+1ng hLTBP4, C: uninjection. D: 1ng hLTBP4.

Table 4: Markers and primer sequences used for the animal cap experiment

3. Morphogen thresholds Concentration gradients of morphogens are thought to establish early embryo pattern by specifying the cell fates. For example, BMP4 is though to be morphogen that causes ectoderm cells to differentiate into epidermis and neural tissue (Wilson et al., 1997). Secondary axis formation resulted from LTBP4 expression in ventral marginal zone. We did not see the phenotype when we injected LTBP4 in the dorsal marginal zone, animal pole, or vegetal pole. In addition, we found that when we injected closer to ventral marginal zone, the number of phenotype increased, when the injection site was closer to dorsal marginal zone, the number of phenotype decreased (data not shown). Therefore LTBP4 may play a role in morphogen thresholds. This suggests that LTBP4 may have a highest concentration in dorsal marginal zone and lower concentrate gradients from dorsal to ventral side in vivo, while when we overexpressed in ventral marginal zone, LTBP4 has highest concentration in ventral marginal zone and lower concentrate gradients from ventral marginal zone to dorsal side. This may be the reason that we see the decreasing number of phenotype when the injection sites are closer to the dorsal marginal zone.

CHAPTER 2

IDENTIFICATION AND CHARACTERIZATION OF DOWNSTREAM TARGETS OF PAX6 IN EARLY EYE DEVELOPMENT

Introduction Pax6 is a highly conserved transcription factor in the family of paired box genes that contain both a paired box domain and a homeobox domain (Cvekl A et al., 2004). Pax6 is involved in central nervous system, pancreas and eye development (Manuel et al., 2005; St-Onge et al., 1997). Pax6 is necessary and sufficient for eye formation in Xenopus (Chow et al., 1999). Mutations in Pax6 result in aniridia in human and small eye phenotypes in rodents (Hill et al., 1991; Glaser et al., 1992). The role of Pax6 in eye development is well established, but the associated molecular mechanisms are poorly understood. In particular, few early downstream targets of Pax6 have been identified, nor have many direct targets been characterized. A major aim of the research described in this chapter is to identify and characterize the downstream targets of Pax6 in early eye development. Medical significance of eye and pancreas development Millions of Americans suffer from problems and diseases of the eye and approximately 150 million Americans are using some sort of corrective eyewear (Shoemaker, 2002). More severe vision problems include blindness, cataracts, glaucoma, diabetic retinopathy, hypertensive retinopathy, and macular degeneration affect more than 30 million people (Shoemaker, 2002). However, the mechanisms of most of these severe eye diseases remain to be elucidated and an effective therapy is not known. The developing eye is an excellent model to understand many eye diseases and biological problems including the genesis of some eye diseases (Cowell et al., 1999). Studies on eye development and gene regulation show that Pax6 plays essential roles in the developing eye. Pax6 is also involved in pancreas development (St-Onge et al., 1997). One of the most serious pancreas diseases is diabetes. Pax6 knockout mice showed less glucagon-positive cells

30 and less insulin-positive cells (reviewed by Dohrmann et al., 2000). Our research is focused on identification and characterization of Pax6 regulated genes in early eye development. Early eye development The tissues of the eye are derived from neuroectoderm, surface ectoderm, and mesoderm. The first sign of the eye development is the appearance of the optic pit in mammals or optic primordium in amphibians in the neural stage, which forms the optic vesicles (Chow and Lang, 2001). With the growing of optic vesicles, the overlying surface ectoderm becomes thick and forms lens placodes, and the forebrain becomes constricted into an optic stalk. The invagination of the optic vesicles leads to formation two layers of optic cup. The inner layer becomes neural retina and the outer layer becomes the retina pigment epithelia (RPE). The invigination of the central region of lens placode results in the formation of lens vesicles (Chow and Lang, 2001). Multiple molecular signals regulate the progression of early eye development. Hedgehog is important for ventral neural specification. In human, loss of function in sonic hedgehog (SHH) can cause holoprosencephaly, which includes cyclopia in extreme cases (Belloni et al., 1996, Roessler et al., 1996). Gain-of-function experiments show that SHH may play a role in ventralization of the eye fields (Ekker et al., 1995). SHH also promotes rod photoreceptor cell differentiation in mammalian retinal cells in vitro (Levine et al., 1997). The fibroblast growth factor (FGF) signaling can specify retina development and is required for lens induction and FGF is necessary for neural retina differentiation in chick embryos (Pittack C et al., 1997). Gain and loss of function show that FGF signaling is required for lens fiber cell differentiation (McAvoy et al., 1991). TGF-β signaling is also important for lens development. Loss of function of BMP7 causes a failure to form lens placode along with a loss of expression of Pax6 (Wawersik et al., 1999). Wnt signaling plays important roles in lens development promoting fiber cell differentiation (Lyu and Joo, 2004) and both Wnts and Dkks are co-expressed early in the lens primordia (Ang et al., 2004). Pax6 gene Pax6 is a transcription factor that belongs to the family of paired box genes that contain both a paired domain (PD) and a homeobox domain (HD). The paired domains and

homeobox domains of all Pax6 proteins are highly conserved (Callaerts et al., 1997). Pax6 is expressed widely in vertebrates and invertebrates. In vertebrates, the first expression of Pax6 is seen on embryonic day 8 in mouse, stage 10 in humans, and in stage 7 in chicken when the first somites are formed and the neural folds begin to close (Grindley et al., 1995; Walther & Gruss, 1991; Gerard et al., 1995; Li et al., 1994). Expression of Pax6 is seen in the forebrain, hind brain, spinal cord, and the neural tube. The expression is also seen in the neural parts of the eye and nose and in the pancreas (Li et al., 1994; Walther & Gruss, 1991; Turque et al., 1994). Pax6 is initially expressed throughout optic cup, but it is expressed only in ganglion and amacrine in the differentiated retina (Grindley et al., 1995; Belecky-Adams et al., 1997). Misexpression of Pax6 genes (eyeless in Drosophila) in Drosophila, mice, Xenopus and squid leads to ectopic eye formation (Halder et al., 1995; Tomarev et al., 1997; Chow et al.,1999). The misexpression of Pax6 can also induce ectopic expression of early eye development genes such as Otx2, Rx, and Six3 in Xenopus (Chow et al., 1999). Mutation of the mouse Pax6 gene and mutations of Sey in heterozygous mice cause small eye phenotype and homozygous mouse are anophthalmic and die at birth (Hill et al., 1991; Hogan et al., 1986; Grindley et al., 1995; Matsuo et al., 1993). Similar mutations in humans lead to aniridia (Glaser et al., 1992; Hanson et al., 1993; Jordan et al., 1992). Mutation of eyeless gene in Drosophila causes loss of eyes (Halder, 1995). Pax6 is also essential for lens induction. Pax6 is involved in the activation of crystallin (Altmann et al., 1997). Pax6 is expressed in the endocrine cells and can be detected throughout pancreas development. Though Pax6 plays important roles in early development, early downstream targets of Pax6 are not known well. Few direct transcriptional targets of Pax6 are known.

Materials and Methods Constructions Pax6-flag GR-Cs2++ construction is made from PCR. PCR was performed by using forward primer 5’-CGAGTGTCTACCAGCCAA-3’ and reverse primer with Xho I site 5’-CCGCTCGAGCGGCTTGTCATCGTCGT-3’. The template was Pax6-flagCs2++ plasmid. PCR condition was described as above. The PCR fragment was digested by

Acc I and Xho I and resubcloned into Pax6 flag Cs2++/Acc I/Xho I to get new Pax6flagCS2++ which was digested by Xba and Xho I. GRCs2++ vector was digested by Xho I and Xba. The fragment of Pax6 flag/Xho I/Xba was cloned into GRCs2++/Xba/Xho I to get Pax6 flag GR Cs2++. Since Pax6 flag GR Cs2++ has a frame shift, Pax6 flagGRCs2++ was digested by Xho I and filled in by klenow fragment of DNA polymerase I and religated to get Pax6flagGRCs2++ Xho I filled in version. Pax6falgGRCs2++ Xho I filled in version are in frame. Transformation and purification of DNA plasmid by using CTAB Plasmid was transformed into DH5α competent cells. The next day, one colony was picked up from the plate and the cells were grown in 50mL TB overnight. Then next day, the cells were centrifuged by 6000×G for 10minutes. The bacterial pellet was resuspended with 10mL P1 (50mM Tris-HCl pH 8, 10mM EDTA, 100µg/mL RNAse A) for 5 minutes and the bacterial solution was lysed with 10ml P2 (200mM NaOH, 1% SDS) for 5 minutes, and then the lysis was neutralized with 10mL P3 (3.0M KoAc pH 5.5) for 10 minutes on ice. The neutralization solution was centrifuged by 3200×G for 15 minutes at 40C. The supernatant was transferred into a fresh tube and 3.5mL 2% CTAB was added. The DNA pellet can be obtained by centrifuging for 15 minutes and be resuspended with 2.5mL 1.2M NaCl. The pellet was reprecipitated with 6.25mL 100% ethanol. The pellet was washed with 70% ethanol and dried at 37 degree. The dried pellet was resuspended in 500µL TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). Generation of embryos Ovulation was induced by injection of 500 IU of human chorionic gonadotropin (hCG). About 16-18 hours later, the induced female frog starts to lay eggs. The eggs were collected manually and fertilized in vitro with male testis. Embryos were staged according to Nieuwkoop and Faber (1967). Embryos for injection were incubated at 0.1×

MMR (0.01M NaCl, 0.2mM KCl, 0.1mM MgSO4, 0.2mM CaCl2 0.5mM HEPES pH7.8, 0.01mM EDTA) or 0.5× MMR. Embryos were prepared for in situ hybridization by removal of the vitelline membrane manually, fixation with MEMFA (0.1M MOPS pH

7.4, 2mM EGTA, 1mM MgSO4 3.7% formaldehyde) and dehydration with 100% ethanol. Microinjection and microsurgery of embryos

RNA for injection was prepared for in vitro transcription using Ambion mMESSAGE mMACHINETM High Yield Capped RNA Transcription Kit. The template DNAs were from Pax6-flag-GR-Cs2++ Xho I filled in version and Xiro3Cs2++, vectors were linearized by Asc I and in vitro transcribed by SP6 RNA polymerase. LRP11Pcs2+ was linearized by Not I and in vitro transcribed by SP6 RNA polymerase. For detecting phenotype, RNAs were microinjected into one cell in dorsal animal pole at 4-cell stage. The injected embryos were grown until stage 45 and were fixed by MAMFA. For Real Time PCR samples, RNAs were injected in 2 cells at the animal pole at 2-cell stage. Caps were isolated at stage 8.5 and cultured until sibling embryos reached stage 14 or stage 16. Total RNAs were extracted at these stages and Real Time PCR was performed. Total RNA isolation and in vitro reverse transcription Total RNA isolation was performed by RNA-Bee protocol. The whole embryos or caps samples were broken in solution D (4M guanidinium thiocyanate, 25mM Sodium Citrate, Ph 7.0, 0.5% sarcosyl, 0.1M 2-mercaptoethanol) and then RNA-Bee (Chomczynski et al., 1987) was added. RNA was extracted with chloroform and precipitated with 1 volume of isopropanol. The pellets were resuspended and reprecipitated by 7.5M Licl. The pellets were washed by 70% ethanol and resuspended in DEPC treated water. Once total RNA was extracted, 500pg total RNA was reverse transcribed in a final volume (20µL) of reaction sample by using random hexamer primers and 100 Units of Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase (NEW ENGLAND Biolabs). The RT reaction was incubated at 420C for 30 minutes. Real time PCR The Real time PCR reaction consisted of 40 cycles of 20 sec at 95 °C, 10 sec at 60 °C, and 10 sec at 72 °C, 32 sec at 80°C by using 0.1 µL of the RT reaction. Each 25 µL PCR reaction contained 1× Biorad Real Time PCR mix with SYBR green, 1× Rox, 0.1 µL RT mix, and 40 µM gene-specific primers (upstream and downstream). The real-time method was run on an ABI lightCycler. The samples were normalized by histone 4, histone 2A or EF1α. The gene sequences were downloaded from NCBI and primers were designed using primer 3 software. The primers are listed in Table 5, Table 6, and Table 7. In situ hybridization

LRP11 antisense RNA probe was prepared by linearizing xLRP11- PBSSKII with Eco RI and transcribing with T7 RNA polymerase according to manufacturer’s instructions with digoxygenin (DIG Quantification Teststrips, Roche). Xiro3 antisense RNA probe was prepared by linearizing Xiro3Pcs2+ with Hind III and transcribing with T7 RNA polymerase according to manufacturer’s instructions with digoxygenin (DIG Quantification Teststrips, Roche). Whole mount in situ hybridization was performed as described by Harland (1991). Fluorescence Immunocytochemstry The injected embryos were fixed at stage 46 with 4% paraformaldehyde in Phosphate- Buffered Saline (PBS) for one to two hours. The embryos were washed in PBS 10 minutes three times and incubated in 30% sucrose in PBS overnight at 40C. The embryos were frozen in tissue-Tek freezing medium, and sectioned with a cryostat (10µM-14 µM) at -200C. The sections were refixed with 4% paraformaldehyde for 15 minutes and washed with PBS. The sections were blocked in 5% heat inactive (HI) goat serum, 0.3% Triton X-100/PBS at least one hour and then incubated with primary antibody 1:1000 in 5% goat serum, 0.1% Triton X-100/PBS at 40C overnight. The primary antibody was washed with 0.3% Triton X-100/PBS for 15 minutes three times. The sections were incubated with Fluorescein or Rhodopsin labeled secondary fluorescent antibody 1:10,000 in 5% HI goat serum/PBS one hour at room temperature. The slides were then washed with PBS 10 minutes three times, and mounted in mounting medium.

Results 1. Confirmed Pax6 regulated genes by Real Time PCR More than 500 Xenopus genes are upregulated by Pax6 according to a recent DNA microarray experiment (Altmann, unpublished data). Among these genes, 50 are transcription factors, 25 are involved in the Wnt signaling pathway and 22 are receptor genes. Twenty-five transcription factors, seven Wnt signaling genes, and five receptor genes have been confirmed as follows by real time PCR (Table 8, 9, and 10). One nanogram of Pax6 capped RNA was injected in two cell stage and caps were isolated at stage 8.5 (blastula stage). The explants were allowed to develop until sibling embryos reached stage 20 (neural stage) and total RNA was isolated at this stage. The cDNA was

Table 5: Primers of transcription factors Human gene Xenopus gene name name Upstream primer sequence (5’ to 3’) Downstream primer sequence (5’ to 3’) Six6 Six6 CAGCAGGTCTTGTCCCAGG GCCAGTGTGATGATGTCCC Otx2 Otx2 GGATGGATTTGTTGCACCAGTC CACTCTCCGAGCTCACTTCTC Otx2 Otx5b TGCCACTGACTGCCTTGATT TGTTTGTTCCATTATTAGATTTCCA Six3 Six3 ATAGGAGCCCTGATCTGCCT CAGCACCAGTCTATCGGACA Sox2 Sox2 ACCGCTATGATGTCAGTG GCCAGTGTGATGATGTCCC Zic2 Zic2 CAGCCCACCACCATCATCATC ATGGTTGCTCTGCTCTGGCC Otx1 Otx1-A AGCAAGCCACCGCCTCCTC TAGCCCACAAAGCCCATTA Hes1 Esr-6e GTCCAACAGCATCTCCATCA TTCCATAGCATTCAAGGTG Sox9 Sox9 GATTATGCCGTTTCTCCTTTC ATTGCTCGCCGTATTGTCTA Pitx2 Pitx2 CCCACTGTCCTCCCAGAGTA AGGGAAGGGTTGCTGAGATT Nkx3-1 Nkx3-1 AGCAGATGGCAGGGAGAGT TGGATAAAGCAGGCAAGGG Hbox10-A AGAGGGTTGTGCGAGTGAAG AGAGCGACGAGGAAGAGGAG Gli2 Gli2 AGGCCCATGTGACCAAGAAG ATGGGGAAGGCTCACTGCTG Irx3 Xiro3 CTCGGAAGTCTCGGATGGGT GGGTTTATGGGCTACGGGCA Tgif Tgif CGACGCAAACAACTTCACAG TTTGAGAGCAACATCCACCA Mixl1 Bix4 CAAGGAACCAATCAGAGCAAG GATTCAGGGCAAGACAGGAG Etv1 Xer81 AGCTCTCTGAACCGTGCAAT GTGGTTGAGGGTAGGCTTGA Hoxd3 Hoxd3 CCAACATAAACAGGGCTGCC ACATGTATGGGCTGGCTGCT Xhox3 ATACCGGACAGCCTTCACC CTGTCGCTTGTCCTTCATCC Foxd3 Foxd3 GTCTGCTCCCTCAACGAGTC GATCTGCGAGTTCATCAGCA Nkx2-3 Zax CCTCACCTCCTTCTCCATTC GAGTCAGTGGCGGGTTCTC Spr-1 Xspr-2 AAACACTCCTAAACACAACACGG AACTCTTCCCACATAACATCCAG Tcfl1 Xtcf-3b GCATCACAGCCTTCATACCTC GCCACTACCTTAGCCCTCATC Cdx1 Xcad2 CGCCTTCTATAAATTCCTCTGTTC GCTGCTAGTTCTGCCTTCC Insm1 Insm1 GCACAGACACAGATTCCGAG CCGACCACAGCAGCCATAGG

Table 6: Primers of Wnt signaling genes Human gene Xenopus gene Upstream primer sequence (5’ to Downstream primer sequence (5’ name name 3’) to 3’) Frizzled 7 Frizzled 7 GCTGTTCTGCTGCCTATTCC GTGGAGCCAACACAAAACCT Dab 2 Dab2 AGATCCCGTTACGTGTCTGC CAAACCCAAAAACTCCTGCT Dkk 1 Dkk1 CCTACCCGCTCTACAGTTGC TTGGAAGCGCTCTTGATCTT Prickle Prickle CGCACAAATGACCTACGATG ACTCTTGCCCATCCGTACAC APC APC TGTATTGCTATGCGCCAGTC CTGGTCCATTCCTTGTTCGT LRP 6 LRP6 AACAGTCCTTCCACCCACAG ATCGCTGATGTATCCCTTCG Wif-1 Wif-1 TGCTGCTCACAATGCCTCTA TGGTGTGGATGTTGACAGGT

Table 7: Primers of receptor genes Human gene Upstream primer sequence (5’ to downstream primer sequence (5’ name Xenopus gene name 3’) to 3’) LRP11 LRP11 CTTTCACGGCCATCAAGAAT TGGTGGAACAATCAGGACAA GPR56 GPR56 CTCAGCCCCATGTAAAGCAC GCACTCGCACAACACAGAAG RET RET ACCACAGCTACCCATTCAGG CACAGGTGTGGAGTCAGCAT NPRC NPR3 TGACTCTGACTGTGGCAACC AACATGGCCAGGAACATCTC P2RX4 P2RX4 ATGTCTAGGGACGGCTGCT GACACCCAGTTCCGTTGTG

made by reverse-transcriptase and the real time PCR was performed by using the cDNA as a template, and each of these experiments have been replicated in triplicate. Genes regulated by two fold or more were considered to be confirmed. Figure 15 is an example of real time PCR. The figure shows that Pitx2 is upregulated by Pax6 at least two fold. For each time point, ODC, H4, H2a or EF1α are the loading controls. Among these 25 transcription factors confirmed genes, 10 genes have been shown to be involved in eye development. Specifically, it has been known that Pax6 induced ectopic Rx, Six3, Otx2 and endogenous Pax6 by in situ hybridization, but was not confirmed by PCR (Chow et al., 1999). The other 15 genes have not been characterized in the eye development. Among the Wnt signaling confirmed genes, Frizzle, Prickle and Wif1 have been shown to be involved eye development, while Dab2, Dkk, APC and LRP6 have not been shown to be involved in eye development. Most of confirmed receptor genes have not been shown to be involved in eye development and therefore represent interesting new discoveries. 2. Direct targets Pax6 plays an important role in the eye development in both vertebrates and invertebrates (Chow and Lang, 2001). Understanding the molecular pathway of Pax6 regulation is critical for deciphering the genetic programs of visual systems. However, the regulation of Pax6 at the molecular level is not well characterized. The known direct targets include Sox2, Eya, Six3 and crystallin genes (Ashery-Padan et al., 2000; Xu et al., 1997; Goudreau et al., 2002; Duncan et al., 1998). Identification of Pax6 direct targets is critical for understanding eye development. Seven Pax6 direct targets have been identified by using Pax6 inducible protein in the presence or absence of the translation inhibitor cycloheximide (CHX). CHX can be used to distinguish whether a gene is direct or indirect target as summarized in Figure 16. A. Pax6 inducible protein Most of Pax6 upregulated genes are expressed after the gastrula stage and the embryos can not survive in cycloheximide solution longer than 2 hours. So in this experiment, we used Pax6-GR construct to control Pax6 expression. We have made Pax6-flag with a glucocorticoid receptor hormone DNA binding domain fusion protein (Pax6-GR). When Pax6-GR mRNA was injected into the cell, mRNA would be translated into protein. An

Figure 15: Pitx2 is confirmed by one experiment of PCR. Green and purple lines are H4 loading control. Red line shows the sample of Pax6 injected sample and blue line shows the sample of uninjection.

inhibitor, heat shock protein 90 (HSP90), will bind to the GR-DNA binding domain and prevent Pax6-GR translocation into the nucleus. In the presence of dexamathasone (DEX), DEX will displace HSP90 and Pax6-GR will enter the nucleus and become active allowing us to check for Pax6-regulated genes as summarized in Figure 17. B. Identification of direct targets and indirect targets One nanogram of Pax6-GR mRNA was microinjected into two cells in the animal pole at the 2-cell stage. Embryos were grown until stage 8.5 and the animal caps were isolated at this stage. We kept growing animal caps and at stage 14 at which point Dexamethasone (1µg/mL) and CHX (5µg/mL) were added. The caps were grown until the sibling embryos reached stage16 at which point total RNA was isolated. Real time PCR was performed on cDNA as follows. Eight cDNA samples including negative control were generated, which were Pax6 U (Pax6 injected and untreated), Pax6 DEX (Pax6 injected and DEX treated), Pax6 CHX (Pax6 injected and CHX treated), Pax6 DEX CHX (Pax6 injected DEX and CHX treated), UU (uninjected untreated), U DEX ( uninjected DEX treated), U CHX (uninjected CHX treated), U DEX CHX (uninjected DEX and CHX treated). The increasing level of induction of targets in the samples of Pax6 DEX and Pax6 DEX CHX were identified as the direct targets. The increasing level of induction of the targets in the sample Pax6 DEX rather than Pax6 DEX CHX are the indirect targets. By this experiment, we detected 25 confirmed transcription factors and identified seven direct targets (Table 11). Among these targets, Foxd3, Xiro3 and Krox20 are not previously known to be involved in eye development. Foxd3 induction in Pax6 injected sample is about 70 folds compared with uninjected sample (Figure 18) and it was involved in formation of neural crest (Sasai et al., 2001). Both Xiro3 and Foxd3 are expressed in retina in mouse (Figure 19), but not known to play a role in early eye development. C. Expression of Xiro3 in Xenopus requires Pax6 Xiro3 is expressed in the prospective hindbrain and spinal cord areas during neural stage, while in the early tadpole stage, the expression is detected in the brain, extending from anterior to the midbrain-hindbrain junction and decreasing in the spinal cord (Bellefroid et al., 1998). Xiro3 is also expressed weakly in the eye (Bellefroid et al., 1998). To test whether Pax6 was required for the expression of Xiro3, we used a Pax6 morpholino oligo

Table 8: Confirmed pax6 upregulated transcription factors by real time PCR human gene Xenopus gene name name Gene description Six 6 Six 6 sine oculis homeobox homolog 6 Otx2 Otx2 orthodenticle homolog 2 Otx2 Otx5b orthodenticle homolog 2 Six-3 Six-3 sine oculis homeobox homolog 3 Sox2 Sox2 SRY (sex determining region Y)-box 2 Zic2 Zic2 Zic family member 2 Otx1 Otx1-A orthodenticle homolog 1 Hes1 ESR-6e hairy and enhancer of split 1 Sox9 Sox9 SRY (sex determining region Y)-box 9 Nkx-3-1 Nkx3-1 homeobox protein NKX-3.1 Hbox10-A orthopedia homolog Gli2 Gli2 GLI-Kruppel family member Irx3 Xiro3 iroquois homeobox protein 3 TGIF TGIF TGFB-induced factor MIXL1 Bix4 Mix1 homeobox-like 1 paired-like homeodomain transcription Pitx2 Pitx2 factor 2 Etv1 Xer81 ets variant gene 1 HoxD3 HoxD3 homeobox D3 Foxd3 Foxd3 forkhead box D3 Nkx2-3 Zax NK2 transcription factor related, locus 3 SPR-1 XSPR-2 SPR-1 protein XTCF-3b TCF7L1 transcription factor 7-like 1 caudal type homeobox transcription factor CDX1 xCAD2 1 INSM1 INSM1 insulinoma-associated 1

Table 9: Confirmed pax6 upregulated Wnt signal genes by real time PCR Human gene name Xenopus gene name Gene discription Frizzled 7 Frizzled 7 Frizzled homolog 7 (Drosophila) Dab2 Dab2 disabled homolog 2 Xdkk1 Dkk1 Dickkopf homolog 1 Prickle Prickle LIM protein prickle APC APC APC (adenomatous plyposis coli) LRP6 (low density lipoprotein receptor relative protein LRP6 LRP6 6) Wif-1 Wif-1 Wif-1 (Wnt inhibitory factor-1)

Table 10: Confirmed pax6 upregulated receptor genes by real time PCR Human gene name Xenopus gene name Gene discription LRP11 (low density lipoprotein receptor relative LRP11 LRP11 protein11) GPR56 GPR56 G protein-coupled receptor 56 RET RET Ret proto-oncogene NPRC NPR3 Natriuretic peptide receptor type C NPR-C P2RX4 P2RX4 ATP-gated ion channel subunit P2X4

Figure 16: Model of direct target and indirect target in the presence of CHX or in the absence of CHX. CHX stands for cycloheximide. B can be a factor or a protein complex.

Figure 17: Pax6-GR fusion protein in active condition and inactive condition. DEX stands for dexamathasone. HSP90 stands for heat shock protein 90.

(Pax6 MO) to specifically reduce Pax6 protein level. Pax6 MO or Pax6-HA with β-gal was coinjected intone cell of four-cell stage. The negative controls in this experiment were uninjected embryos and standard MO (that should have no target and no significant biological activity) injected embryos. The injection site was targeted in left side and the dorsal animal pole. We grew embryos until stage 20~22 and stained with red gal to indicate the injection site. Subsequent in situ hybridization was performed with Xiro3 RNA as a probe. We predicted that Pax6-HA would expand the expression of Xiro3, while Pax6 MO would inhibit the expression of Xiro3. After injections the caps were isolated at stage 8.5 and grown until stage 10. The protein was extracted from the caps and western blotting was performed as shown in Figure 20. The Pax6 MO treatment blocked translation of exogenous Pax6-HA, while the standard MO treatment did not block the translation of exogenous Pax6-HA. Figure 21 shows that Pax6-HA increased the expression of Xiro3, while Pax6 MO inhibited the expression of Xiro3. The control standard MO did not change the expression of Xiro3. These results provide evidence that the accumulation of Xiro3 mRNA requires Pax6. D. Overexpression of Xiro3 causes misplaced eyes and induces ectopic photoreceptor cells It is known that Xiro3 induces anteria neural tissue (Bellefroid et al., 1998), but it is not known whether Xiro3 induces eye tissues. We overexpressed Xiro3 to learn more about its role in eye development and retina cellular phenotypes. Xiro3 capped RNA was injected into one cell of the four-cell stage embryos. Embryos were grown until stage 45 to detect the resulting eye phenotypes. 250pg or 500pg of Xiro3 capped RNA and β-gal were co-injected. Embryos were fixed at stage 45 and stained by X-gal. The embryos show misplaced eyes and a small eye phenotype as shown in Figure 22. Figure 23 illustrates that these treatments disrupted the orderly arrangement of cells in the retina. Xiro3 appeared to induce ectopic photoreceptor and müller cells as described in Figure 23. E. Overexpression of Xiro3 in animal caps induces cement gland marker and some eye gene markers 500pg Xiro3 capped RNA was injected into two cells at the animal pole at the 2-cell stage of the embryo. Caps were cut at stage 8.5 and RNA was isolated at stage 20. Real

time PCR was performed to assay for changes in the levels of mRNA for several genes of interest, including Six3, Six6, Pitx2 and Xag. The results showed that overexpression of Xiro3 induced an increase in the expression of Pitx2, Six3, Otx2, and Xag as described in Figure 24. 3. LRP11 induced ectopic eyes Overexpression of LRP11 was found to induce ectopic retinal pigment epithelia (RPE) and proximal defects of eyes. LRP11 was overexpressed by microinjecting with 10ng, 5ng, 2.5ng into 4- and 16-cell stage embryos. β-gal coinjection was used as a cell lineage tracer for these experiments. Embryos were fixed at stage 45. LRP11 induced ectopic retina pigment epithelium (RPE) (Figure 25 A-D). Some embryos showed proximal eye defect in the left eye displaying an extension of RPE (Figure 25 E and F). Some embryos showed misplaced eyes (data not shown). More than one third of the injections resulted in a defectable phenotype as summarized in Table 12. The injected eyes were sectioned and immunostained for the presence of XAP1, a photoreceptor marker. Figure 26 shows that overexpression of LRP11 induced ectopic photoreceptor cells. In addition, we detected spatial and temporal expression of LRP11 by in situ hybridization and real time PCR (Figure 27 and Figure 28). Our results showed that LRP11 was expressed maternally, decreased during the early neural stage, and increased slightly during the late neural stage. LRP11 was expressed in the mesoderm at the gastrula stage (Figure 27 A) and neural plate in neural stage (Figure 27 D, F, and G). In the early tadpole stage, LRP11 was expressed in the neural tube, head, and eyes (Figure 27 C, E, H, I, J, and K).

Discussion 1. Identified Pax6 regulated genes from Microarray and Real time PCR From Pax6 overexpression microarray analyses, 500 genes were found to exhibit Pax6- induced over expression. These genes include transcriptional factors, Wnt signal genes, and receptor genes. We verified the induction by real time PCR, and not that several of these represent novel findings. Their role in eye development will be important to establish. Wnt signaling has been known to be important for eye development in both invertebrates and vertebrates. In particular, Wnt signaling plays important roles in lens development promoting fiber cell differentiation and both Wnt and Dkks are co-

Figure 18: Foxd3, a direct target of pax6. Introduction of Foxd3 in Pax6-GR Dex sample and in Pax6 Dex Chx sample is high, while introduction of Foxd3 in Pax6-GR U, Pax6-GR Chx and all uninjected sample is low.

Figure 19: Expression of Pax6, Foxd3 and Irx3 in the eye of the mouse. From UCSC VisiGen, in mouse Pax6 and Foxd3 are both expressed in retina and lens epithelium lay. Irx3 (Xiro3 in Xenopus) is expressed in retina ganglion cells which are also overlapped by Pax6.

Table 11: Direct targets of Pax6 pax6 U pax6 dex pax6 chx pax6 dex U U U dex U chx U dex chx chx Six3 1.02 8.70 4.44 15.27 1.00 1.14 1.80 2.10 Foxd3 0.37 84.76 0.94 78.95 1.00 2.33 1.08 2.87 Xiro3 0.31 5.06 0.57 12.49 1.00 1.08 0.62 1.89 Otx2 0.57 303.19 0.44 392.73 1.00 3.94 9.60 11.70 Krox20 4.16 17.78 6.63 23.41 1.00 2.12 4.42 5.26 Six6 1.98 7.35 2.04 14.70 1.00 1.66 0.84 0.99 Sox9 1.77 6.61 0.86 7.44 1.00 0.59 1.58 1.18

Figure 20: Pax6 MO has inhibited exogeneous Pax6 in animal caps. while standard MO and only Pax6 injected sample have not inhibited Pax6 in the western blotting.

Figure 21: Expression of Xiro3 in Xenopus requires Pax6. Embryos were injected Pax6HA 500pg (A and B), Pax6MO 40ng (C and D), or standard MO (E), fixed at stage 22, stained by red-gal and performed in situ hybridization by using Xiro3 RNA probe. F is uninjected embryo. A and B show that overexpression of Pax6HA causes expansion of Xiro3 expression. C and D show that Pax6 MO inhibits Xiro3 expression compared with uninjected side. E and F are standard MO injection and uninjection, which did not affect Xiro3 expression.

Figure 22: Overexpression of Xiro3 causes misplaced eyes which locate closer to the center. The misplaced eyes look like smaller than normal eye. The arrow indicates the injected sides.

Figure 23: Overexpression of Xiro3 causes ectopic photoreceptor cells and muller cells. Green is anti-rhodopsin and red is anti-glutamine synthetase which detects muller cells (A-D Xiro3 injected embryos, E and F uninjected embryos).

Figure 24: Overexpression Xiro3 induced Pitx2, Six3, Otx2 and Xag. Overexpression of Xiro3 in animal caps induced Pitx2, Six3, Otx2 and Xag compared with uninjected caps

Figure 25: Overexpression of LRP11 induced ectopic RPE and proximal eye defect. A-D Ecotopic retina pigment epithelium (arrow). E and F Proximal eye defect (arrow).

Figure 26: Overexpression of LRP11 can induce ectopic photoreceptor cells. A and B are the sections of eyes without cross phenotype labeled by anti-XAP2 which is an antibody of photoreceptor cells.

Figure 27: Spacious expression of LRP11 by in situ hybridization. xLRP11 is expressed in pan mesoderm and animal pole in gastrula stage (A). In neural stage, it is expressed in neural tube (D F G). In early tadpole stage, xLRP11 is expressed in neural tube, eye, ear and heart region (B, C, E, H, I, J, K). F, G, H, I, J, K embryos are in Benzyl Benzoate.

Table 12: Phenotype of overexpression of LRP11 No. embryo Percentage

Total injected 133 100%

Ectopic RPE 15 11%

Proximal eye defect 22 17%

Eye misplaced 12 9%

No eye phenotype 27 20%

Miss injected 49 37%

Dead 86%

Total phenotype 49 37%

Figure 28: Temporal expression of LRP11 by Real time PCR. xLRP11 is expressed highest before MBT (the Midblastula Transition). It is expressed lower at gastrula and early neural stage. It is expressed higher at late neural stage.

expressed early in the lens primordial (Lyu and Joo, 2004). So far, there is no evidence that the Wnt pathway is regulated by Pax6. In this thesis, we confirmed seven Wnt pathway genes that are upregulated by Pax6. They are receptors (Frizzled 7, LRP6, and LRP11), soluble inhibitors (Dkk1, Wif1,) and a variety of intracellular components (Dab2, Prickle, and APC). Interestingly, a new potential co-receptor (LRP11) was identified (He et al., 2004; Wehrli et al., 2000; Pandur and Kuhl, 2001) as well as the LDL receptor adaptor protein ARH (Zhou et al., 2003; He et al., 2002; Garcia et al., 2001). Among the up regulated genes involved in Wnt signaling are Prickle, a gene involved in the non- canonical Wnt pathway and in cellular migration (Carreira-Barbosa et al., 2003; Veeman et al., 2003; Takeuchi et al., 2003). In addition to transcription factors and Wnt pathway genes, we also identified some receptor genes that are upregulated by Pax6. They are involved in signal transduction and may reveal the presence of newly discovered signaling pathways in eye development. 2. Identification and functional analysis of Pax6 direct targets genes The Eya, Six3 and crystallin genes are the only known Pax6 direct targets (Xu et al., 1997; Goudreau et al., 2002; Duncan et al., 1998). In this thesis, we have identified 7 seven direct targets. Previously unknown targets include Foxd3, Irx3 (Xiro3 in Xenopus), Otx2, Krox20, Six3 and Sox9. From our results, we did not detect Eya upregulation by Pax6. Foxd3 and Irx3 (Xiro3 in Xenopus) have expression patterns that overlap with that of Pax6 mouse (Figure 19). This is consistent with our findings in Xenopus that Foxd3 and Xiro3 are direct targets of Pax6. 3. LRP11 plays a role in eye development Interestingly, a new potential co-receptor (LRP11) was identified (He et al., 2004; Wehrli et al., 2000; Pandur and Kuhl, 2001) as well as the LDL receptor adaptor protein ARH (Zhou et al., 2003; He et al., 2002; Garcia et al., 2001). LRP11 is upregulated by Pax6, but the function of LRP11 is not known. When LRP11 was overexpressed in the dorsal animal pole, it induced ectopic eye tissues. This was confirmed by immunocytochemistry. Therefore we conclude that LRP11 may play an important and primary role in eye development. A genetic test of this idea, such as the analysis of a LRP11 loss of function mutation, would provide further insight. 4. Xiro3 is involved eye development

We found that Xiro3 is a direct target of Pax6. In addition, Xiro3 was involved in anterior neural induction (Bellefroid et al., 1998). In mouse, Irx3 was expressed with distinct spatio-temporal patterns during neurogenesis (Cohen et al., 2000). There is no published evidence showing that Xiro3 plays a role in eye development. We showed that overexpression of Pax6 increased the expression of Xiro3. Loss of Pax6 decreased the expression of Xiro3. Xiro3 overexpression affected eye formation and caused a small eye phenotype along with induction of ectopic eye tissue. Xiro3 induced some eye markers such as Pitx2, Six3 and Otx3 in molecular level. It is not known why overexpression of Xiro3 caused small eyes and induced ectopic eyes, but future genetic analyses should be informative.

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BIOGRAPHICAL SKETCH

Education 2004-2007 Florida State University. M.S. Dept. of Biological Science, degree expected 12/07 1990-1995 Beijing University of Chinese Medicine, Beijing, China, M.D., major: clinical medicine

Research Experience 2004-present Florida State University, Research assistant, Dept. of Biological Science 1999-2002 Guang-an-men Hospital of Beijing, Research Assistant, Dept. of Cardiovascular Diseases Teaching Experience 2006-2007 Teaching assistant for PCB3063 general genetics in FSU (twice) 2006 Teaching assistant for Biological Science II BSC2011.01 in FSU 2006 Teaching assistant for ZOO 4753C Histology in FSU 1998-2002 Teacher for training medical school students and new resident doctors in Beijing Guan-an-men Hospital

Work Experience 2001-2002 Guang-an-men Hospital of Beijing, Dept. of Cardiovascular Diseases Attending Physician. 1995-2001 Guang-an-men Hospital of Beijing, Resident Doctor,

Honors and Awards 2001 Patients Choice Award for outstanding doctors (Top 5 in 200 doctors), Guang-an-men Hospital in Beijing. 1997 Awarded Excellent Resident Doctor during rotation (Top 1 in 20 rotated resident doctors), Guang-an-men Hospital in Beijing. 1995 Graduated as TOP 10% students in the class, Beijing University of Chinese Medicine. 1990-1994 “High Honors” Scholarship for four consecutive years, Beijing University of Chinese Medicine.

Publications 1. Wang X., Wang J., Chen J., Zhou S., Ni S., Li X., Li J., Li X., Li Y., Zhang Y., Wu F., Hu Q., Zhu X., Zhu J., Yang S., and Qi J. (2002) The comprehensive study on the effect of Xiaochaihu Soup on the complicated syndromes. P 134-180. China Press of Chinese Medicine, Beijing, P.R.China 2. Fu Y., Gao G., and Li X. (2000) Coronary Disease. P300-400. Scientific and Technical Documents Publishing House, Beijing, P.R.China 3. Li X. (2000) The new usage of Guizhilongmu Soup. Forum on Traditional Chinese Medicine 15, 10

4. Li X., and Wang F. (1996) The recent progress in treating Sjogren Syndrome by Chinese Medicine (review). Journal of Chinese Medicine 37, 117-118 5. Chai R., and Li X. (1995) The methods of Doctor Chai to treat hypertension unresponsive to the general antihypertension drugs. China Journal of Chinese Medicine 10, 58