Abstracts - Society for Developmental Biology

157
rax mutant was recently found in Xenopus tropicalis via a target-selected TILLING screen, and is the first mutation of its
kind in Xenopus. Similar to other vertebrates (human, mouse, and zebrafish), homozygous mutant animals from this line
fail to form eyes, most fail to complete metamorphosis and all die before reaching sexual maturity. We have developed a
fast, efficient method of transgenesis for use in Xenopus using Bacterial Artificial Chromosomes (BACs) to transiently
drive specific and accurate transgene expression, and use this method to drive a rax-GFP fusion transgene capable of
rescuing the mutant phenotype. One goal of this work is to identify the transcriptional activators leading to the initial gene
expression of master eye genes like rax. We, along with others, have identified a highly conserved region upstream of the
rax proximal promoter that appears to be necessary for initial rax activation. This region contains a number of conserved
putative transcription factor binding sites, but it remains unclear which sites are necessary for rax activation, as this has not
been tested systematically using an in vivo, functional assay. In these experiments we use our BAC transgenesis rax rescue
assay to identify key transcription factor binding sites necessary for early rax activation by individually mutating highly
conserved sites found in the proximal upstream enhancer and testing each contruct’s ability to rescue the mutant
phenotype.
Program/Abstract # 474
Imprinting centre acts simultaneously as promoter for lncRNA-mediated epigenetic silencing and insulator
function in vivo
Lefebvre, Louis; Gu, T.; Bogutz, A.B.; Jones, MJ, University of British Columbia, Vancouver, Canada
Several imprinted genes are silenced on the paternal allele by the large non-coding RNA (lncRNA) Kcnq1ot1 on mouse
Chr7. We described a GFP insertional allele in this domain, termed Tel7KI, behaving as a maternally expressed genes. The
silencing of Tel7KI occurs in post-implantation embryos. Imprinting of the paternal is recapitulated in differentiating
mESC where a role for the lncRNA Kcnq1ot1 was suggested via the introduction of a targeted deletion of the lncRNA
promoter (IC2) in cis of the GFP allele in +/Tel7KI mESC. The +/Tel7KI-IC2KO mESC undergo an epigenetic switch and
fail to silence the GFP reported in embryoid bodies. We have now obtained a meiotic recombinant between the Tel7KI and
IC2KO alleles, bringing both alleles in a cis configuration. Paternal transmission of the Tel7KI-IC2KO haplotype provided
in vivo confirmation that IC2 is required for the silencing of the paternal Tel7KI since we observed an epigenotype switch
on the paternal Tel7KI-IC2KO haplotype. Consequently embryos of two different genotypes can express the GFP:
Tel7KI/+ and +/Tel7KI-IC2KO. Surprisingly, these two genotypes exhibit different tissue-specific expression patterns in
post-implantation embryos. We propose a model in which IC2 simultaneously plays a dual role: (i) as the imprinted
Kcnq1ot1 promoter, and (ii) as a paternal allele-specific insulator element, regulating the expression patterns of linked loci.
Whether the IC2 boundary element is also implicated in the epigenetic silencing of Kcnq1ot1 targets remains to be tested.
Our work has important implications for the structure and evolution of imprinted centres and established Tel7KI as a
sensitive reporter for postulated chromosomal looping during murine development.
Program/Abstract # 475
Proteolytic carving of the mammalian head by the Taspase1-TFIIA-CDNKN2A Axe
Takeda, Shugaku, Memorial Sloan-Kettering Cancer Center, New York, United States; Sasagawa, Satoru (Osaka Medical
Center for Cancer and Cardiovascular Diseases, Osaka, Japan); Hsieh, James (Memorial Sloan-Kettering Cancer
Center, New York, NY, United States)
The build of a mammalian head represents one of the most elegant creations by the Mother Nature and demands
unimaginably complex yet precise signaling events, explaining our current limited understanding of this developmental
process. Here our genetic and biochemical data indicate that Taspase1 a protease functions as a key orchestrator which
cleaves TFIIA a so-called general transcription factor to assure the needed rapid expansion of forebrain during
embryogenesis. Specifically, we discovered that both p16/Ink4a and p19/Arf are aberrantly upregulated in mice that are
either knocked out of Taspase1 or knocked in of non-cleavable TFIIA. Most remarkably, the exhibited severe craniofacial
defects can be largely rescued by genetic deficiency of the CDKN2A allele that encodes both p16/Ink4a and p19/Arf.
Interestingly, the genetic non-cleavage of both MLL1 and MLL2, two known Taspase1 substrates, did not incur
conspicuous craniofacial defects. Altogether, our study demonstrates a critical craniofacial program, which emanates from
Taspase1 through TFIIA to CDKN2A that involves a highly conserved protease, a general transcription regulator, and a
key cell cycle regulatory genetic locus. In conclusion, this sophisticated proteolysis-transcription-division circuit
apparently constitutes a previously unknown critical genetic framework buried in the contemporary genetic construction
blueprint for a mammalian head.
Program/Abstract # 476
Ectoderm-mesoderm separation is controlled through selective repulsion generated by specific pairs of ephrins and