Worm Breeder's Gazette 16(1): 58 (October 1, 1999)
Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Ave. Bronx NY 10461
Members of the caudal-like proteins are required for proper patterning of the posterior body region. In mouse and Xenopus, caudal-like proteins are also required for setting up the correct expression domains of Hox genes. For example, the expression boundaries of Hox genes are shifted posteriorly in cdx-1 and cdx-2 knockout mice.
The C. elegans caudal homolog, pal-1, is required for specifying the identity of embryonic posterior blastomere P2. The null allele, pal-1(ct224), which deletes most coding sequence, causes embryonic lethality and severely disrupts posterior development (Nob phenotype)1,2. pal-1 is also involved in specifying cell fate in a postembryonic lineage descended from the anterior embryonic AB blastomere. This role was revealed by a viable allele, e2091, which results in missing male rays. In pal-1(e2091), PAL-1 is absent from seam cell V6. As a consenquence, Hox gene mab-5, a putative transcriptional target of PAL-1, is not expressed in V6 descendants, leading to loss of rays3.
Several previous attempts to identify the mutation in e2091 were unsuccessful, and revealed that the protein sequence is intact. We have now shown that e2091 is a T to C transition, which is located in the middle of the last intron. We amplified the 10Kb pal-1 genomic DNA from wild type, which consists of about 4Kb upstream sequence and about 1Kb downstream sequence, and found it can rescue the V6 ray loss phenotype of pal-1(e2091). The genomic counterpart from pal-1(e2091), as expected, gives no rescue. In the whole rescuing fragment, only two point mutations in the last intron were found: T to C(24264, the number corresponding to the cosmid C38D4) and C to T(23370). We sequenced the intron of a presumptive intragenic revertant, pal-1( e2091 bx89), and found the muation at 24264 has been reverted to wild type: C to T. To further prove that intron sequence was required for pal-1 function in V6, we used a genomic-cDNA hybrid construct pSC16, which lacks the last intron of pal-1. pSC16 is capable of rescuing pal-1 null embryonic lethality, but we found that it cannot rescue the V6 ray loss phenotype of pal-1(e2091). These results suggest that an enhancer element essential for transcription of pal-1 in V6 resides within the last intron.
Thanks Dr. Lois Edgar for sending us pSC16 and pal-1(ct224)
1. Hunter and Kenyon (1996) Cell 87:217-226
2. Edgar et al. 1993 Meeting Abstract, p.10.
3. Hunter et al (1999) Development 126:805-814