Abstract
The Drosophila transcription factor Tramtrack (Ttk) is involved in a wide range of developmental decisions, ranging from early embryonic patterning to differentiation processes in organogenesis. Given the wide spectrum of functions and pleiotropic effects that hinder a comprehensive characterisation, many of the tissue specific functions of this transcription factor are only poorly understood. We recently discovered multiple roles of Ttk in the development of the tracheal system on the morphogenetic level. Here, we sought to identify some of the underlying genetic components that are responsible for the tracheal phenotypes of Ttk mutants. We therefore profiled gene expression changes after Ttk loss- and gain-of-function in whole embryos and cell populations enriched for tracheal cells. The analysis of the transcriptomes revealed widespread changes in gene expression. Interestingly, one of the most prominent gene classes that showed significant opposing responses to loss- and gain-of-function was annotated with functions in chitin metabolism, along with additional genes that are linked to cellular responses, which are impaired in ttk mutants. The expression changes of these genes were validated by quantitative real-time PCR and further functional analysis of these candidate genes and other genes also expected to control tracheal tube size revealed at least a partial explanation of Ttk's role in tube size regulation. The computational analysis of our tissue-specific gene expression data highlighted the sensitivity of the approach and revealed an interesting set of novel putatively tracheal genes.
Highlights
Transcription factors play critical roles in all aspects of development
Microarray analysis of ttk mis-expression mutants In order to identify direct or indirect Ttk target genes that could explain the diverse tracheal phenotypes seen in mutant embryos, we pursued RNA profiling experiments on embryos that are homozygous for the mutant ttkD2-50 allele and control embryos at developmental stages 11 to 16
Mutants and controls, expressed a membrane tethered RFP under the control of the btl enhancer from the Red Fluorescent Protein-moesin construct. This enabled a fluorescence-activated cell sorting (FACS) strategy, which enabled us to obtain a cell population that was enriched for tracheal cells that were subject to microarray gene expression analysis
Summary
Transcription factors play critical roles in all aspects of development They control the gene batteries that lead to cellular events such as proliferation, cell fate specification and differentiation, cell migration, cell morphological changes and apoptosis. Given this wide spectrum of functions, very often transcription factors exhibit a high degree of pleiotropy that hinders a comprehensive functional characterisation in a given tissue or developmental stage. We aimed to identify these downstream targets using microarray transcriptome profiling This enabled us to find direct and indirect transcriptional targets, including those which are difficult to identify in traditional mutant screens due to pleiotropy and/or functional redundancy. To identify targets specific to the tracheal system, and to separate the tracheal action of Ttk from action on other target systems, we combined whole-embryo expression profiles with transcription profiles of embryonic cell isolates, which were enriched for tracheal cells by fluorescence-activated cell sorting (FACS)
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