The evolutionarily conserved leprecan gene: its regulation by Brachyury and its role in the developing Ciona notochord.
In Ciona intestinalis, leprecan was identified as a target of the notochord-specific transcription factor Ciona Brachyury (Ci-Bra) (Takahashi, H., Hotta, K., Erives, A., Di Gregorio, A., Zeller, R.W., Levine, M., Satoh, N., 1999. Brachyury downstream notochord differentiation in the ascidian embryo. Genes Dev. 13, 1519-1523). By screening approximately 14 kb of the Ci-leprecan locus for cis-regulatory activity, we have identified a 581-bp minimal notochord-specific cis-regulatory module (CRM) whose activity depends upon T-box binding sites located at the 3'-end of its sequence. These sites are specifically bound in vitro by a GST-Ci-Bra fusion protein, and mutations that abolish binding in vitro result in loss or decrease of regulatory activity in vivo. Serial deletions of the 581-bp notochord CRM revealed that this sequence is also able to direct expression in muscle cells through the same T-box sites that are utilized by Ci-Bra in the notochord, which are also bound in vitro by the muscle-specific T-box activators Ci-Tbx6b and Ci-Tbx6c. Additionally, we created plasmids aimed to interfere with the function of Ci-leprecan and categorized the resulting phenotypes, which consist of variable dislocations of notochord cells along the anterior-posterior axis. Together, these observations provide mechanistic insights generally applicable to T-box transcription factors and their target sequences, as well as a first set of clues on the function of Leprecan in early chordate development.
Dunn
Matthew P
MP
Department of Cell and Developmental Biology, Weill Medical College of Cornell University, 1300 York Avenue, Box 60, New York, NY 10065, USA.
Di Gregorio
Anna
A
D000818:Animals; D001483:Base Sequence; D019521:Body Patterning / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology; D002938:Ciona intestinalis / Q000196:embryology / Q000235:genetics / Q000502:physiology*; D005326:Fetal Proteins / Q000235:genetics / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental; D008562:Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology / Q000196:embryology / Q000502:physiology; D009154:Mutation; D009672:Notochord / Q000166:cytology / Q000196:embryology / Q000502:physiology*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics / Q000502:physiology; D020825:T-Box Domain Proteins / Q000235:genetics / Q000502:physiology*; D000098908:Brachyury Protein
D005326:Fetal Proteins; D008562:Membrane Glycoproteins; D020825:T-Box Domain Proteins; D000098908:Brachyury Protein