ANALISIS FODA

Unlike with protein coding regions, which are distinguished by 3bp codons, Methionine start sites and STOP codons, and splice site identifiers, gene regulatory regions have a much looser set of rules. Still, variations in such gene regulatory regions can result in drastic changes in gene

expression, and the effect of mutations within promoter regions with regards to the correct

expression of genes is now acknowledged to be as potentially harmful as those within coding regions

Figure 1.5. The embryology and development of Ciona intesitinalis

(A) Schematic view of gastrulation of the Ciona embryo. The specification of all cell types of the tadpole larva occurs prior to gastrulation. (i-iv) show vegetal/posterior views of

gastrulation stage embryps, whilst (I’-iv’) display lateral views. (B) (i-iv) Schematic showing cell and tissue fates through developing gastrula to mid tailbud embryos. Schematics are made from traces of the embryos in (figure 5.9 Q E, F, M, O), though numbers of cells displayed may not be absolutely accurate due to cell membranes not being visible in different focal planes. (v) Represents a transverse section through the plane shown by the dotted line in (iv), showing the location of the three rows of lateral muscle cells, dorsal nerve cord, notochord and endodermal strand. (C) (i) Image of a swimming Ciona larva, and schematic (ii) showing the fates of all cell types depicted in (A). (D) Image of a mature Ciona adult with visible filled sperm duct. (E)

schematic showing ascidian metamorphosis. The tadpole attaches to the substrate via the palps, and reabsorption of the tail occurs, whilst the body axis rotates during metamorphosis to the juvenile stage. Legend: Presumptive notochord cells are shown in red, endoderm yellow, muscle orange, epidermis grey, nerve cord dark blue, sensory vesicle light blue and the palps in green. Lower case lettering refers to the stage of development; g, gastrula; en, early neurula; itb, initial tailbud; mtb, mid tailbud. Figures taken and adapted from (Jeffery and Swalla, 1997; Munro et al., 2006; Sato et al., 2012)

40 (reviewed in Wray et al. (2003)). Though gene promoters contain a basal promoter region, specified by a 100bp region containing a TATA box as well as various transcription factor binding sites (TFBS) (Lee and Young, 2000; Wray, 2003; Wray et al., 2003), there are no other defining features and nucleotide sequence has proven to be a poor indicator of Promoter function or efficiency beyond these basic features.

Perhaps the most famous example of the importance of cis-regulatory regions to the correct expression of genes is in the case of the Drosophila Bithorax complex (BX-C) locus and the even- skipped (eve) gene. Within the BX-C locus of Drosophila, several non-coding regions, both within introns and intergenic, were found to direct expression of the Ultrabithorax (Ubx) and abdominal-a (abd-A) genes (Bender et al., 1983; Karch et al., 1985; Simon et al., 1990; White and Wilcox, 1985). The authors identified the abx/bx region (Ubx intronic), the bxd/pbx region (upstream of Ubx), the iab-2 region (abd-A intronic) and the iab-3 region (abd-A upstream). Each of these regulatory regions was able to drive expression of a LacZ construct within distinctive parasegments of the Drosophila embryo, within parasegments 5 (abx/bx), 6 (bxd/pbx), 7 (iab-2) and 8 (iab-3) respectively (Simon et al., 1990). A similar, but much more in depth study, has been carried out specifically for the eve gene, where the regulatory region has been thoroughly characterised into discrete ‘modules’ directing the expression of specific eve expression bands. Though eve is activated in a ubiquitous manner, these modules act as repressors within specific regions to give eve its characteristic stripped pattern. One module, directed by Knirps defines the third and seventh stripes, with Knirps repressing the posterior of stripe three and the anterior of stripe seven. Hunchback then sets the anterior limit of stripe three and the posterior limit of stripe seven by causing repression at the anterior and posterior ends of the embryo. Similar, but separate, modules define the expression for all of the stripes of eve, resulting in a complex array of repression and activation from relatively simple individual modules, giving the characteristic seven stripes pattern of Drosophila eve (Arnosti et al., 1996; Small et al., 1996).

Even small changes and mutations in the regulatory landscape can lead to novel domains of expression and phenotypic consequences, as seen in the pigmented wing patterns of the Yellow gene in drosophilids, where subtle changes within cis-regulatory elements, and the co-option of new transcription factors have led to novel and repeated changes in wing pigment patterns (Gompel et al., 2005). It is these regulatory inputs, in the form of transcription factor binding sites, that seem to be the only common factor between promoters, enhancers and repressors, and the suite of binding sites within a cis-regulatory region are key to directing both the temporal and spatial expression of the target gene. This regulatory input results in the transcriptional activation of the target gene if a

41 certain threshold is reached, whether that is an amount of a single factor, or the combination of specific factors binding the cis-regulatory region (Davidson, 2001).

The cascade of signalling molecules and transcription factors that result in gene activation or repression results in a complex web of gene regulatory networks (GRNs) that interact and lead to the repression and activation of genes in a variety of cell types and developmental contexts (Davidson, 2010; Davidson and Erwin, 2006). For example, the identification of conserved factors within the endomesoderm GRNs of both sea urchin and Xenopus (Hinman et al., 2003; Loose and Patient, 2004) suggests that signalling molecules and their downstream transcription factors could be used to deduce ancestral regulatory interactions. Such GRNs have also been described for other tissues such as the neural crest (Sauka-Spengler et al., 2007) and have even been described for specific

developmental events, such as the specification of the midgut-hindgut boundary in the sea urchin (Annunziata and Arnone, 2014). This last study holds particular relevance due to the involvement of the ParaHox genes Xlox and Cdx, and the presence of the Xlox/Cdx midgut-hindgut boundary throughout the deuterostomes as discussed earlier. It is therefore possible to envisage how such gene regulatory networks might be used to identify the cis-regulatory elements involved in the network, through identification of relevant TFBS, as well as inform the identification of such regulatory interactions in other species. This approach could prove more fruitful than traditional cross-species sequence comparisons, as such approaches have proved to be unreliable for the identification of cis-regulatory elements. Even within the vertebrates there are relatively few cis- regulatory elements that display conserved sequence (Woolfe et al., 2005), and this becomes much harder, though possible in a few cases, as comparisons between larger evolutionary distances are made (Makunin et al., 2013; Pascual-Anaya et al., 2008; Vavouri et al., 2007; Woolfe et al., 2005).