In this paper we describe an analysis of the mRNA expression profile of trypanosomes from two discrete bloodstream form stages of the parasite (slender and stumpy forms), as well as during the transition of the stumpy population to the procyclic life-cycle stage. Although previous analyses have compared either cultured or rodent derived bloodstream form parasites with cultured procyclic forms [37, 38], our analysis represents the first comparison of in vivo derived pleomorphic slender cells with genetically identical stumpy forms, and a first analysis of the dynamic changes in mRNA profile that accompany the transition to procyclic forms. Previous analyses of the differentiation from stumpy to procyclic forms have established that this transition follows a defined and highly reproducible temporal programme, with progression through the major events of differentiation occurring with great synchrony in the population . This provides a considerable power to the analysis of regulated mRNA changes because the observed changes at the population level can be used to direct infer changes at the individual cell level. Moreover, by tracking the progressive changes in mRNA abundance between time points in the differentiation programme, trends in the expression of individual or groups of transcripts can be identified, enabling regulated changes in expression to be distinguished from sample-specific variations.
Consistent with current consensus for the best optimisation of microarray analyses , our study focused on maximising the number of biological replicates, with 5 bio-replicates used for the stumpy and differentiating cell populations and 4 bio-replicates used for the pleomorphic slender populations. This allows both measurement and biological variability to be assessed and statistically tested, providing considerable statistical power over technical replicates alone. However, the consequence of this approach is that the ability to detect significantly regulated transcript groups is likely to be somewhat quenched due to the extent of biological variability, such that transcripts known to be differentially regulated may fall outwith the range of statistical significance. This is particularly the case when considering the analysis of stumpy and differentiating populations, which were derived after independent growth in individual mice over six days, likely generating considerable variability in many regulated mRNAs. Nonetheless, by detailed analysis of several cytological events of differentiation, the overall developmental progression of the distinct populations was found to be remarkably consistent enabling the identification of distinct regulated transcript profiles through the transformation process.
Our analyses focussed particularly on mRNAs up-regulated in the bloodstream stumpy form. These are the transmission stage of the trypanosome in the mammalian blood and have not been subject to extensive molecular analysis. A number of characteristics define stumpy forms cytologically: their characteristic morphology , cell-cycle arrest , resistance to proteolytic  and pH stress  and their capacity for differentiation in response to low concentrations of citrate/cis aconitate (CCA) when exposed to cold shock conditions [51, 68]. Consistent with these characteristics, we observed that cell-cycle related transcripts were down-regulated in the stumpy forms (e.g. histone transcripts) whereas the PAD mRNAs (required for CCA reception)  were up-regulated. In addition to these changes we also observed by mRNA analysis and phenotypic validation that the lipid profile of stumpy forms was modified with respect to slender forms, with the expression of IPC representing either pre-adaptation for their differentiation to procyclic forms or early expression of this pathway. Indeed, individual analysis of the expression of each of the four sphingolipid synthase genes showed that one, TbSLS1 (Tb9.211.1030), exhibited particular developmental regulation, indicating that this gene may encode the T. brucei IPC synthase. Based on this prediction, a biochemical analysis has recently confirmed that TbSLS1 has IPC synthase activity, functionally distinguishing it from the closely related TbSLS4, which exhibits sphingomyelin/ethanolamine phosphorylceramide synthase activity  (Professor Jay Bangs, University of Wisconsin, USA; personal communication). Interestingly, squaline monooxygenase utilised in sterol biosynthesis was also up-regulated in stumpy forms. These changes in the lipid composition of stumpy forms perhaps provide explanation for the more robust characteristics of stumpy forms when compared to bloodstream slender forms [29, 30] or demonstrate a requirement for rapid membrane rearrangement upon entry into the tsetse fly. Although a functional analysis of other stumpy-enriched transcripts is needed to dissect their role in stumpy form biology, several of the identified molecules are consistent with the characteristics of this life cycle stage. For example, the enhanced expression of two chloride channel proteins related to mammalian lysosomal chloride channel proteins may reflect the known elevated lysosomal activity of stumpy forms .
In an attempt to distinguish transcripts regulated as a pre-adaptation for the transformation to procyclic forms, we analysed mRNAs elevated in stumpy forms and 1 h into the differentiation programme. Unsurprisingly in these very transient expression profiles, some discrepancy with the validation assays was observed, possibly reflecting both normalisation bias and some non-systematic errors. Nonetheless, the analysis identified a relatively small subset of significantly elevated mRNAs, which comprised surface protein mRNAs, possible gene expression regulators, the aforementioned membrane lipid components and some metabolic enzymes. In all probability these will be a considerable under-representation of those molecules whose expression is enriched in stumpy cells because no account is taken of the differential translational efficiency of mRNAs whose overall abundance does not change significantly. Nonetheless, by bioinformatic comparison of these stumpy enriched mRNAs with the whole cohort of predicted genes in the trypanosome genome, enriched oligonucleotide sequences (SM1, SM2) could be identified in their 3'UTRs. These related motifs were statistically over-represented among stumpy mRNAs and one, SM1, exhibited a positional bias within the 3'UTR, being enriched 150-200 nt from the stop codon. Although the functional significance of over-represented oligonucleotide motifs requires experimental validation, the presence of a positional bias is not expected for a motif or motifs over-represented by chance alone and instead suggests a possible context-dependent function.
As well as focussing on stumpy-enriched mRNAs, we also analysed the dynamic changes in mRNA profiles during synchronous differentiation of stumpy forms to procyclic forms. Analysis of the resulting profiles particularly highlighted the increased abundance of mRNAs associated with the mechanisms of protein synthesis and translational control. This is not surprising: previous analyses of the polysomal profile of slender, stumpy and procyclic forms have established that stumpy forms are particularly quiescent, with metabolic labelling indicating that stumpy forms show only ~25% protein synthesis of bloodstream slender forms , TKS and KM, unpublished observations). After the rapid induction of mRNAs associated with translational processes, the parasites up-regulated mRNAs associated with cell proliferation, including genes required for DNA replication as well as structural components required for cell division. The kinetics of these molecular changes matched well the previously characterised cytological differentiation events, being induced after 6 h in differentiation conditions. Thereafter mRNAs associated with the metabolic changes that accompany differentiation were up-regulated. These changes indicate that there is rapid adaptation in terms of the mRNA profile as cells initiate differentiation to procyclic forms.
Although the tracking of known transcripts during the developmental events is informative and reassuring, the most interesting new information is likely to emerge from the analysis of genes which, although regulated, have no known function assigned or where distinct function cannot be assigned on the basis of sequence alone. Two examples that emerge from our analysis include the differential profile of sphingolipid synthesis genes described above and the observed transient elevation of histone deacetylase 2 during differentiation. Unlike the three other histone deacetylases in T. brucei, gene deletion of histone deacetylase 2 does not generate phenotype in bloodstream forms or during monomorphic cell differentiation ; however more detailed analysis of pleomorphs undergoing synchronous differentiation may uncover a subtle or unexpected role in development, as observed for these molecules in other systems . Finally, although genes with similar expression profiles were not clustered in the genome, analysis of the regulatory motifs governing the co-expression of unlinked genes may allow cryptic regulatory motifs to be identified and post-transcriptional operons to be defined . Hence, analysing genes with regulated expression profiles or which are co-regulated with other genes of known and unknown function should help to dissect the co-ordination of events necessary to generate a successfully differentiated procyclic form cell.