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First published online 27 August 2003
doi: 10.1242/dev.00711

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Functional tests of enhancer conservation between distantly related species

Department of Molecular Biology, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, Wellman 8, Boston, MA 02114, USA

View larger version (88K): [in a new window]   Fig. 1. Expression patterns of fusion genes containing tissue-specific enhancers of Drosophila in C. elegans. (A) Drosophila Gad1::GFP is expressed glial cells (gl) in the head. (B) Drosophila Cha::GFP is expressed in glial cells of labial neurons (gl) and several pharyngeal muscle cells (pha). (C) Drosophila unc-119::GFP is expressed in glial cells of labial neurons (gl), and several dorsal (dhn) and ventral (vhn) head neurons as well as in tail neurons (tn). (D) Drosophila ey::GFP is expressed in IL1D (L, R) and PVT. (E) Drosophila eya::GFP is expressed in PVT, in the gut and certain muscle cells of the pharynx. (F) Drosophila nompA::GFP is expressed in the head hypoderm (hyp) and in several amphid neurons (amph). (G) Drosophila Mef2::GFP is expressed throughout the pharynx (pha) and in a single interneuron AVG. (H) Drosophila eve::GFP is expressed in up to six glial (gl) cells of labial neurons.

View larger version (38K): [in a new window]   Fig. 3. Functional comparisons of unc-47 enhancers between C. elegans and C. briggsae. (A) Schematic representation of expression patterns. 26 GABAergic neurons - four RMEs, AVL, RIS, six DDs, 13 VDs and DVB - are shown in green. SDQ (L, R) are shown in red. (B-E) Expression patterns of ce unc-47::GFP (B,C) and cb unc-47::GFP (D,E) in both C. elegans (B,D) and C. briggsae (C,E). Note that in all four panels most, if not all, of the 26 GABAergic cells express GFP. Arrows indicate SDQ (L, R) in D and SDQL in E.

View larger version (71K): [in a new window]   Fig. 2. Alignments of upstream regulatory sequences of (A) unc-25and (B) unc-47of C. elegansand C. briggsae. —, alignment gaps; *, identical nucleotides. unc-30-binding sites are gray, as shown previously (Eastman et al., 1999). Translated sequences are underlined.

View larger version (10K): [in a new window]   Fig. 4. Functional dissection of cb unc-47 enhancer to identify element(s) responsible for expression in SDQ (L, R). +, strong and consistent expression in particular cells; -, complete lack of expression. (D, V) indicates weak and inconsistent expression of the proximal enhancer in RME (D, V) cells. Note that SDQ (L, R) are the only cells, expression in which is not activated by either of the two shorter enhancer fusion genes.

View larger version (17K): [in a new window]   Fig. 5. Consequences of enhancer-transcription factor co-evolution. (A,B) Sets of orthologous transcription factors control expression of an orthologous target in species A (blue) and B (red). Note that although the order of individual binding sites is rearranged, in both cases transcription factors are co-adapted, as reflected by their different shapes, to form a complex and result in strong activation of expression. (C) If transcription factor 1 in species A is replaced by its ortholog from species B, it could bind to the target previously occupied by its ortholog. It could also interact although less well (as indicated with a broken line) with other transcription factors bound to the enhancer, resulting in weaker transcriptional activation. (D) If an entire enhancer is placed into a heterospecific context, individual transcription factors may be able to bind to their respective target sequences. Their interactions, however, are likely to be greatly hampered, thus resulting in no transcriptional activation or in activation in a different pattern because of serendipitous occurrence of binding sites recognized in other tissues.