Cardiac morphogenesis is a complex process governed by evolutionarily conserved
transcription factors and signaling molecules. The Drosophila cardiac tube is linear,
made of 52 pairs of cardiomyocytes (CMs), which express specific transcription
factor genes that have human homologues implicated in Congenital Heart Diseases
(CHDs) (NKX2-5, GATA4 and TBX5). The Drosophila cardiac tube is linear and
composed of a rostral portion named aorta and a caudal one called heart,
distinguished by morphological and functional differences controlled by Hox genes,
key regulators of axial patterning. Overexpression and inactivation of the Hox gene
abdominal-A (abd-A), which is expressed exclusively in the heart, revealed that abd-A
controls heart identity. The aim of our work is to isolate the heart-specific cisregulatory
sequences of abd-A direct target genes, the realizator genes granting heart
identity. In each segment of the heart, four pairs of cardiomyocytes (CMs) express
tinman (tin), homologous to NKX2-5, and acquire strong contractile and automatic
rhythmic activities. By tyramide amplified FISH, we found that seven genes, encoding
ion channels, pumps or transporters, are specifically expressed in the Tin-CMs of the
heart. We initially used online available tools to identify their heart-specific cisregutatory
modules by looking for Conserved Non-coding Sequences containing
clusters of binding sites for various cardiac transcription factors, including Hox
proteins. Based on these data we generated several reporter gene constructs and
transgenic embryos, but none of them showed reporter gene expression in the heart. In
order to identify additional abd-A target genes, we performed microarray experiments
comparing the transcriptomes of aorta versus heart and identified 144 genes
overexpressed in the heart. In order to find the heart-specific cis-regulatory regions of
these target genes we developed a new bioinformatic approach where prediction is
based on pattern matching and ordered statistics. We first retrieved Conserved Noncoding
Sequences from the alignment between the D.melanogaster and
D.pseudobscura genomes. We scored for combinations of conserved occurrences of
ABD-A, ABD-B, TIN, PNR, dMEF2, MADS box, T-box and E-box sites and we
ranked these results based on two independent strategies. On one hand we ranked the
putative cis-regulatory sequences according to best scored ABD-A biding sites, on the
other hand we scored according to conservation of binding sites. We integrated and
ranked again the two lists obtained independently to produce a final rank. We
generated nGFP reporter construct flies for in vivo validation. We identified three 1kblong
heart-specific enhancers. By in vivo and in vitro experiments we are determining
whether they are direct abd-A targets, demonstrating the role of a Hox gene in the
realization of heart identity. The identified abd-A direct target genes may be targets also
of the NKX2-5, GATA4 and/or TBX5 homologues tin, pannier and Doc genes,
respectively. The identification of sequences coregulated by a Hox protein and the
homologues of transcription factors causing CHDs, will provide a mean to test
whether these factors function as Hox cofactors granting cardiac specificity to Hox
proteins, increasing our knowledge on the molecular mechanisms underlying CHDs.
Finally, it may be investigated whether these Hox targets are involved in CHDs.
| Identifer | oai:union.ndltd.org:unibo.it/oai:amsdottorato.cib.unibo.it:646 |
| Date | 09 June 2008 |
| Creators | Tevy, Maria Florencia <1978> |
| Contributors | Capovilla, Maria |
| Publisher | Alma Mater Studiorum - Università di Bologna |
| Source Sets | Università di Bologna |
| Language | Italian |
| Detected Language | English |
| Type | Doctoral Thesis, PeerReviewed |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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