Biomedical Engineering Reference
In-Depth Information
to the posterior side of the cells before the posterior positioning of the basal
body. Thus, posterior positioning of the basal body to provide posterior tilt
to node cilia is determined by planar polarization mediated by noncanonical
Wnt/PCP signaling.
In a continuation of these studies (unpublished results from our lab and
the lab of Hiroshi Hamada), we discovered the complete loss of all the three
mouse
Dvl
genes in
Dvl1
,
2
,
3
triple null mutants die soon after implantation,
without evidence of gastrulation. In addition, a slightly less severe pheno-
type was observed in
Dvl1
þ
/
þ
;
Dvl2
/
;
Dvl3
/
and
Dvl1
þ
/
;
Dvl2
/
;
Dvl3
/
embryos, where gastrulation is often initiated.Basedon thediscussion
above, these severe gastrulation defects may be due to the disruption of either
the canonical Wnt pathway and or the noncanonical PCP pathway. Further
studies will be required to define these phenotypes and the pathway(s)
important for these severe defects.
14. DVL CONDITIONAL TAGGED ALLELES
To further investigate redundancy, we made fluorescently tagged
transgenic alleles for each of the Dvl genes by BAC recombineering and
transgenesis. The advantages of using the BAC transgenic approach are that
the BAC transgene will likely recapitulate the endogenous gene expression
pattern, as it contains large flanking genomic region where regulatory ele-
ments reside, and because usually only one to two copies will be integrated
into the genome. In addition, each of the
Dvl
BAC transgenic alleles can be
used to see if additional copies of any
Dvl
gene can substitute for other
Dvls
.
First, we produced two BAC transgenic alleles for
Dvl2
(
Wang, Hamblet,
et al., 2006
). Both alleles had in-frame EGFP tags to allow us to examine in-
tracellular distribution of Dvl2 protein, as well as a loxP site in intron 2 to
differentiate this allele from the wild-type and the
Dvl2
knock-out allele.
The second BAC allele also contains a second loxP site in the adjacent
Acadvl
gene. The latter allele acts as a conditional allele
in vivo
.Both
Dvl2
BAC transgenes were able to fully rescue
Dvl1
/
;
Dvl2
/
and
Dvl2
/
;
Lp/
þ
mutants to fertile adults, demonstrating that the
encoded Dvl2-EGFP is appropriately expressed for full phenotypic rescue.
The
Dvl2
-
EGFP
BAC allele is also expressed at levels comparable to en-
dogenous
Dvl2
allele (
Wang, Hamblet, et al., 2006; Wang et al., 2005
).
Similar to the Dvl2-EGFP BAC transgenic allele, we produced
Dvl
fluo-
rescently tagged BAC alleles of
Dvl1
and
Dvl3
. These two BAC alleles,