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. Thus, identifying the plethora of target genes regulated by transcription factors is key to disentangling their multiple and complex activities. Nowadays, the combination of genome-wide approaches with cell type- or stage-specific isolation provides a powerful strategy to understand how the function of transcription factors is mediated.
Our previous work indicated that the transcription factor Ttk plays multiple relevant roles in the formation of the Drosophila
tracheal system 
. For a more mechanistic view, in the present work we aimed to map the downstream mediator targets of ttk
during embryogenesis with a particular emphasis in the developing tracheal system. Ttk is a zinc-finger transcription factor widely used during the development of a number of different organ systems 
, with a pivotal role in many different morphogenetic events. In the trachea, we observed that Ttk is not only required for tracheal cell identity specification, but in addition it enables several morphogenetic changes, including the cell rearrangements of tube formation, and the proper setting of tube sizes 
. Thus Ttk may govern a complex hierarchy of downstream mediators that execute cellular changes in tracheal development. 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 specifically 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).
In this work we compared microarray expression profiles of wild-type embryos with different ttk
mutant conditions, as well as expression profiles of tracheal cell isolates from both wild-type and mutant embryos. This analysis provided an in vivo
census of genes whose transcription responds to Ttk loss-of-function or over-expression. Several of these genes have predicted Ttk binding sites in their regulatory regions, which make them candidates for direct regulation. To validate our experimental approach, we further analysed effects on the mechanism of tube size regulation, as we established the role of Ttk in this process before 
. The size control of tracheal tubes is complex and involves several molecular mechanisms. It has been shown earlier that the transient assembly of a chitin filament inside the tracheal tubes, and epithelial septate junctions (SJs) play critical roles in the process (reviewed in 
). Our current analysis of microarray data from whole embryos and isolated cells pointed to changes in the expression of chitin metabolism and SJ-related genes in ttk
mutants, which were confirmed by quantitative real-time PCR (qPCR) profiling. In further tests we found that several of these targets are functionally required in tube size control. Thus we could confirm the involvement of Ttk in tube size regulation, and connect Ttk regulation to several downstream genes, which are involved in the control of tube size.
Our results provide entry points for the investigation of the targets of Ttk regulation in further processes. The results also show that our method of cell enrichment is a powerful tool in the search for transcription factor targets.