UNIVERSITY OF CINCINNATI _____________ , 20 _____ I,______________________________________________, hereby submit this as part of the requirements for the degree of: ________________________________________________ in: ________________________________________________ It is entitled: ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ Approved by: ________________________ ________________________ ________________________ ________________________ ________________________ DOWNSTREAM EFFECTORS OF THE HOMEOBOX TRANSCRIPTION FACTOR HOXA 11 A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (Ph.D.) in the Department of Developmental Biology of the College of Medicine 2000 by M. Todd Valerius B.S. University of Cincinnati, 1993 Committee Chair: S. Steven Potter Abstract Homeobox transcription factors play critical roles in the genetic cascade of development. Critical to understanding their function is describing the expression and identifying the downstream targets of homeobox genes. The initial characterization of the dispersed homeobox gene Gsh-1 is reported in chapter 2. In situ hybridization showed developmental expression was limited to the CNS. In the hindbrain and neural tube stripes of Gsh-1 were seen in neuroepithelial tissue. The diencephalon and mesencephalon also showed Gsh-1 expression including the future thalamus and hypothalamus. Using a fusion protein approach, the in vitro consensus DNA binding site of Gsh-1 was determined. Chapter 3 reports the characterization of kidney cell lines derived from Hoxa 11- SV40 T antigen transgenic mice. Molecular marker analysis determined the mK3 and mK4 cell lines represent early metanephric mesenchyme and differentiated epithelial-like cells respectively. Co-culture experiments with isolated ureteric bud demonstrated mK3 cells retained early metanephric mesenchyme function by supporting ureteric bud growth. Expression profile comparisons of the mK3 and mK4 cell lines identified 121 differentially expressed genes. Several of these were previously described in the differentiation of metanephric mesenchyme, validating the approach. The remaining genes, consisting of both known and unknown, are now implicated in this process. Chapter 4 describes the identification of candidate downstream targets of Hoxa 11. The Hoxa 11-SV40 T antigen transgene described in chapter 3 was bred into the Hoxa 11/Hoxd 11 double mutant mouse line. A kidney cell line, mK10, was isolated from mutant transgenic E18.5 embryos (Hoxa 11-/- Hoxd 11 -/- Hoxa 11/SV40 Tag +). The mK10 cells, as well as HEK293 cells, were transfected with expression constructs containing the Hoxa 11 cDNA to create cell populations with and without Hoxa 11 for each. Differential Display, GDA arrays, and GeneChip microarrays were used for expression profile comparisons. These screens identified nine genes that were reproducibly altered in expression with the addition of Hoxa 11 expression. Integrin alpha 8 (ItgA8) was altered in both cell lines. In situ hybridization of E13.5 Hoxa 11/Hoxd 11 mutant kidneys showed ItgA8 expression was altered, consistent with ItgA8 being a downstream target of Hoxa 11. Acknowledgements I would first like to thank my primary advisor Dr. S. Steven Potter. Without his mentoring and endless patience I would not have accomplished this lofty goal. I would also like to thank my committee, Dr. Robert Arceci, Dr. Jun Ma, Dr. Anil Menon, and Dr. Jeff Robbins for their guidance and support. Many thanks to the other faculty and staff of the Molecular and Developmental Biology Program. My future accomplishments in science will in no small way be a result of the excellent training I have received here. To past and present members of the Potter laboratory I am very appreciative of the friendship and help I have always received. Finally, a most special thanks goes to my wife Wendy and the rest of my family. I can not imagine having the will to finish this program without the love and support of these wonderful people. I am blessed to have such a family. Table of Contents Chapter 1 Introduction 2 References 9 Chapter 2 Gsh-1 : A novel murine homeobox gene expressed in the central nervous system 14 Abstract 15 Introduction 16 Methods 20 Results 23 Discussion 39 Acknowledgements 44 References 45 Chapter 3 Defining the genetic basis of metanephric mesenchyme differentiation 55 Abstract 56 Introduction 57 Methods 61 Results 66 Discussion 80 Acknowledgements 83 References 84 Chapter 4 Hoxa 11 downstream target candidates identified by expression profile analysis 94 Abstract 95 Introduction 96 Methods 98 Results 101 Discussion 114 Acknowledgements 117 References 118 Chapter 5 Additional Discussion 127 References 132 - 1 - Chapter 1 Introduction - 2 - Homeobox genes encode transcription factors that contain a 60 amino acid helix- turn-helix DNA-binding motif called the homeodomain. These genes function by regulating batteries of downstream targets responsible for organogenesis of specific structures (Lewis, 1978; McGinnis and Krumlauf, 1992). Some homeobox genes are found in clusters and in Drosophila this is known as the homeotic complex (HomC). Mammalian homologs of the Drosophila homeotic genes, the Hox genes, are well conserved in homeodomain sequence and their clustered chromosomal organization (Sharkey et al., 1997). While there is a single homeotic complex of 8 genes in Drosophila, mammals have four Hox clusters and a total of 39 Hox genes located on different chromosomes with each cluster containing 9-11 genes. These four complexes are believed to have formed through evolution via duplication of a single ancestral homeobox cluster. The position of the Hox genes within a cluster is conserved allowing them to be aligned into 13 paralogous groups by homeodomain sequence homology. In addition, the Abdominal-B (Abd-B) type Hox genes are expanded in mammals with groups 9-13 sharing homology with the single Drosophila Abd-B gene. In Drosophila the homeotic genes act as master regulators specifying body segment identity along the A/P axis of the embryo. This is illustrated by the classic antennapedia mutation in flies in which an extra pair of wings is formed in a body segment that normally would not have wings (Lewis, 1978). This phenomenon of homeotic tranformation is also seen in Hox gene mutations in mouse, notably in skeletal elements where A/P axis phenotypes are easily studied. Both loss of function (Post et al., 1993; Kanzler et al., 1998; Small and Potter, 1993) and gain of function (Charite et al., 1994; Zhang et al., 1994) studies support this general role for A/P axis identity in vertebrates as well. The conserved - 3 - chromosomal organization and role in segment identity in such distantly related species as Drosophila and mouse point to the important role Hox genes play in development. In vitro DNA-binding studies have determined the consensus binding sequence for Hox genes is rather small, consisting of a TAAT core (Benson et al., 1995; Maconochie et al., 1996; Pellerin et al., 1994; Valerius et al., 1995) (See Chapter 2). In addition, due to the high similarity between Hox homeodomains there is very little variation in the consensus element. In some cases specificity is conferred by Hox cofactors that may modify DNA-binding via protein-protein interactions or by cooperative DNA-binding, effectively expanding the consensus site or favoring one Hox protein over another (Castelli-Gair, 1998; Li et al., 1999; Mann and Chan, 1996; Shen et al., 1997; Shen et al., 1999). The small amount of binding site information from these in vitro DNA-binding studies has made the search for the downstream targets by sequence searches difficult. These studies on the whole demonstrate that the complete complex of cofactors must be understood to identify the proper DNA sequence target, and this may vary tissue to tissue with the complement of cofactors and Hox proteins. Furthermore, it would require knowledge of all Hox cofactors and their expression to use an in vitro approach to determine target sequence sites for a given cell or tissue, and this is not known today. In Drosophila only about a dozen Hox targets are known and these can be broken into three categories including signaling molecules (e.g. dpp, wingless), transcription factors like empty spiracles (e.g. distaless), and structural or cell adhesion genes (Graba et al., 1997). Regulation of other Hox genes as well as autoregulation has also been described previously (Gould et al., 1997; Popperl et al., 1995). In mammals the number - 4 - of known Hox targets has recently caught up to the fly. Early reports identified thyroid transcription factor 1 (TTF1) as a target of Hoxb 3 (Guazzi et al., 1994) and LCAM as a target of Hoxd 9 (Goomer et al., 1994). More recently studies have shown Hoxa 5 to directly regulate p53 and the progesterone receptor (Raman et al., 2000a; Raman et al., 2000b). Promoter studies have suggested targets for several Hox genes. Hoxa 10 has been shown to bind the p21 promoter with MEIS1 and PBX1 and activate transcription (Bromleigh and Freedman, 2000). It has also been shown that Hoxc 8 can bind the osteopontin promoter and repress transcription unless displaced by Smad1 signaling (Shi et al., 1999). Hoxc 6 was shown to regulate a NCAM promoter/reporter construct in osteoclast