| 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.
|
|---|
