in vertebrates , dickkopf ( dkk ) family genes encode four secreted proteins ( dkk14 ) and a unique dkk3-related protein , soggy or dkk - like1 ( dkkl1 ) . dkks are potent inhibitors of -catenin stabilization in the wnt signaling pathway and suppress the expression of a variety of canonical wnt/-catenin signaling target genes . dkk1 , dkk2 , and dkk4 inhibit the wnt signaling pathway through both their high - affinity binding to the wnt coreceptors lrp-5/6 [ 35 ] and blockade of the signaling cascade downstream of wnt receptor frizzled . in contrast , dkk3 appears to inhibit the wnt signaling pathway by directly binding to -catenin through a complex with a -transducin repeat - containing protein , trcp . we previously reported identifying the dkk3 gene as a molecule that is predominantly expressed in mouse retinal progenitor cells . dkk3 expression is observed during vertebrate development in various organs [ 1 , 811 ] . in our previous report , we used in situ hybridization to show that mouse dkk3 mrnas were detectable in the entire neuroblastic layer ( nbl ) of the retina at embryonic stages , and then in the inner nuclear layer ( inl ) at postnatal stages . moreover , we generated a bac - dkk3-cre transgenic mouse , which utilizes a bacterial artificial chromosome ( bac ) expressing cre recombinase inserted into the dkk3 gene locus . this transgenic mouse line is a powerful tool for ablating a gene of interest in mitotic retinal progenitor cells ( rpcs ) [ 1214 ] . the expression pattern and the possible function of dkk3 suggest that this molecule exerts a role in proliferation , cell fate determination , and/or maintenance of rpcs during mouse development . however , it is still unclear what types of differentiated cells express dkk3 in the developing retina .   here , we report the generation of a bac - dkk3-egfp mouse line , which expresses the egfp gene under the control of dkk3 regulatory elements . this mouse enables us to investigate the expression pattern of dkk3 in the developing retina in detail . in the current study , we found that egfp is specifically and strongly expressed in rpcs at embryonic stages , and in mller glial cells and a subset of amacrine cells at mature stages . a bac clone ( clone i d : rp23 - 12m6 ) containing the entire dkk3 gene was purchased from children 's hospital oakland research institute . according to the ncbi database ( available at http://www.ncbi.nlm.nih.gov/ ) , this bac clone also contained a fragment of ubiquitin - specific peptidase 47 ( usp47 ) , a microtubule - associated monooxygenase called calponin , and a lim domain - containing gene ( mical2 ) . however , neither the transcription initiation site nor the start codon of usp47 is included in the bac clone , but it does contain the 5 untranslated region of mical2 . two homology arms ( 5 arm , 486  bp ; 3 arm , 453  bp ) from exon 1 of the dkk3 gene were amplified by pcr from the bac clone dna and cloned into a t - easy vector ( promega ) . the pcr primers used were 5-ggcgcgcctatgtcgcctgtctag ggactt-3 and 5-cccggggcaagctggatctggtcacgaccgg-3 for the 5 homology arm and 5-ttaattaattgggtgagcggtggtcatcgtc-3 and 5-gg ccggcccagactcaatccctgctggaaaca-3 for the 3 homology arm . the two homologous arms were inserted into both sides of the enhanced green fluorescent protein - polya ( egfp - pa ) cassette in the modified pld53 sca - cre - b shuttle vector . the egfp - pa cassette was introduced into the 5utr of the first exon of the dkk3 gene by homologous recombination ( figure 1(a ) ) . pcr and sequencing were used to confirm the correct insertion of the egfp gene into the dkk3 locus . the entire dkk3-egfp transgene was purified by dialysis on a filter ( millipore , vswp02500 ) floating in an injection buffer ( 10  mm tris , ph 7.5 ; 0.1  mm edta ; 100  mm nacl ) . the purified construct was injected as circular dna into the pronuclei of fertilized one - cell eggs of b6c3f1 mice then implanted into pseudopregnant foster mothers ( icr , japan slc inc ) . all procedures conformed to the arvo statement for the use of animals in ophthalmic and vision research , and the procedures were approved by the institutional safety committee on recombinant dna experiments and the animal research committee of osaka bioscience institute . pcr genotyping for the dkk3-egfp transgene was performed under the following conditions : 94c for 2  min , then 94c , 1  min 60c , 1  min ; 72c , 1  min for a total of 35 cycles and followed by 72c for 10  min ( primers : 5-cagaccatactagtttggcagtac-3 and 5-gcagcttgccggtggtgcagatgaact-3 ) . we extracted total rnas ( retina : 10  g , other tissues : 20  g ) from adult bac - dkk3-egfp transgenic mice using the trizol reagent ( invitrogen ) . a full - length cdna of egfp was used as a radiolabeled probe for hybridization . mouse eye cups were fixed with 4% paraformaldehyde in phosphate - buffered saline ( pbs ) for immunohistochemistry . the samples were cryoprotected , embedded , frozen , and sectioned into 20  m thick sections . we preincubated them with blocking solution ( 4% normal donkey serum and 0.1% triton x-100 in pbs ) for 30  min . then , we incubated the sections with primary antibodies at 4c overnight , rinsed them with blocking solution , and incubated them with secondary antibodies for 30  min . we used the following primary antibodies : rhodopsin ( lsl , lb-5597 ; rabbit polyclonal ; 1  :  10000 ) , s - opsin ( chemicon , ab5407 ; rabbit polyclonal ; 1  :  1000 ) , m - opsin ( oriental bioservice ; rabbit polyclonal ; 1  :  1000 ) , s100 ( sigma , s-2532 ; mouse monoclonal ; 1  :  1000 ) , sox9 ( chemicon , ab5535 ; rabbit polyclonal ; 1  :  750 ) , pax6 ( dshb , p3u1 ; mouse monoclonal ; 1  :  200 ) , and chx10 ( mbl ; rabbit polyclonal ; 1  :  100 ) . we used cy3-conjugated anti - rabbit igg ( jackson immunoresearch laboratories , no . 711 - 165 - 152 ; 1  :  400 ) and anti - mouse igg ( jackson immunoresearch laboratories , no . we also used rhodamine - labeled peanut agglutinin ( pna , vector laboratories , rl-1072 ; 1  :  200 ) . to generate a dkk3 regulatory elements - driven , egfp - expressing mouse , we took advantage of a bacterial artificial chromosome ( bac ) transgenic mouse , carrying bacs modified using homologous recombination in bacteria [ 15 , 17 , 18 ] . we designed a bac construct with the egfp cdna placed in the 5utr of the dkk3 gene by homologous recombination ( figure 1(a ) ) . to verify the tissue - specific egfp expression , total rna from the bac - dkk3-egfp mouse retina and other tissues at the adult stage was analyzed by northern blotting . a single transcript was detected in the retina and cerebrum , but no significant signal was observed in other tissues examined ( figure 1(b ) ) . dkk3 expression was strongly detected in the retina and cerebrum by northern blot analysis using wild - type adult mouse tissues ( figure 1(c ) ) . since the egfp expression level was extremely high in the retina , we even observed the egfp expression as  green eyes  under the room light at the adult stage after mydrin - m ophthalmic solution 0.4% ( santen , no . 084162243 ) was administrated ( figures 1(d ) and 1(e ) ) .   in the bac - dkk3-egfp mouse embryo , we observed an egfp expression in the eye , heart , and spinal cord at embryonic day 9.5 ( e9.5 ) ( figures 2(a ) and 2(b ) ) . an egfp signal was weakly detected in the palate and heart at e16.5 ( figures 2(c ) and 2(d ) ) . in our previous study , dkk3 mrna expression was detected in the entire nbl of the embryonic retina and in the inl in the postnatal retina using in situ hybridization . to confirm this result , we investigated egfp expression in the developing retina of the bac - dkk3-egfp mouse . from e15.5 to postnatal day 0 ( p0 ) , egfp was detected in most mitotic and undifferentiated rpcs throughout the nbl ( figures 2(e)2(g ) ) . at p3 , egfp expression was limited to rpcs , and differentiated retinal cells were not labeled by egfp ( figure 2(h ) ) . at p6 , egfp was expressed in mller glia - like cells , which exhibit long glial fibers and localize their cell bodies in the inl ( figure 2(i ) ) . at p14 , only mller glial cells in the inl - expressed egfp , and we observed a large number of glial fibers extending into the surrounding retinal layers ( figure 2(j ) ) .   it remains to be clarified which cell types express dkk3 in the adult mouse retina . to examine cell types expressing egfp , we performed immunostaining using antibodies against several photoreceptor cell - specific markers ( figure 3 ) . using antibodies against rhodopsin ( rod photoreceptors ) , pna , s - opsin , and m - opsin ( cone photoreceptors ) , we did not detect an egfp signal significantly overlapping with any photoreceptor cell - specific markers ( figures 3(a)3(l ) ) . then , we immunostained the adult retina with another set of retinal cell - type antibodies , including antibodies against s100 , sox9 ( mller glial cells ) , pax6 ( amacrine cells ) , and chx10 ( bipolar cells ) ( figure 4 ) . we observed an egfp signal overlapping with that of s100 in the mller glial fibers extending into other retinal layers ( figures 4(a)4(c ) ) . moreover , cell bodies of the mller glial cells immunostained with sox9 antibody were positive for egfp ( figures 4(d)4(f ) ) . we also found that a small number of amacrine cells express egfp in the inl ( figures 4(g)4(i ) ) . in contrast , bipolar cells were easily distinguished from egfp - expressing cells ( figures 4(j)4(l ) ) . dkk3 was reported to be expressed in rpcs at embryonic stages and inl cells at postnatal stages , as detected by in situ hybridization [ 7 , 10 ] . however , the precise identification of the cell types expressing dkk3 remains to be elucidated . in the current study , we generated a bac - dkk3-egfp transgenic mouse in which dkk3-expressing cells were labeled by egfp and easily distinguished from dkk3-nonexpressing cells ( figures 3 and 4 ) . we showed that almost all rpcs in embryonic retinas and the mller glial cells in postnatal retinas were labeled by egfp ( figures 2 , 3 , and 4 ) , indicating that the bac - dkk3-egfp mouse is a useful tool to specifically identify the mller glial cells in vivo . recently , growing evidence suggests that the mller glial cells appear to contain a similar potential to rpcs in cases of acute injury to the retina [ 19 , 20 ] . in response to excitotoxic damage elicited by n - methyl - d - aspartate ( nmda ) , a large number of mller glial cells undergo dedifferentiation , reentering the cell cycle , and upregulating transcription factors expressed in rpcs including pax6 , chx10 , six3 , sox2 [ 23 , 24 ] , and sox9 [ 23 , 25 ] . these reports suggest that the mller glial cells are a potential source of neurons in retinal regeneration in the adult retina . thus , further studies of the mller glial cells may lead to development of new therapies for retinal damage . the bac - dkk3-egfp mouse may give us clues to address the molecular and cellular nature of the mller glial cells . abnormal overactivation of wnt signaling is a major feature of various human cancers . a large number of studies have focused on the wnt signaling pathway to develop a novel cancer therapy [ 2628 ] . notably , dkk3 has been identified as a downregulated gene in many human cancer cell lines [ 29 , 30 ] . moreover , dkk3 exhibits an ability to suppress cancer cell growth due to its inhibitory function in the wnt signaling pathway , suggesting that dkk3 can be a potential clinical target in cancer therapies [ 2 , 30 ] . the bac - dkk3-egfp mouse may thus be useful for the development of innovative cancer therapies as well as that in the mller glial cell studies , because dkk3 expression can be easily detected by the intense egfp signals . in the current study , we generated the bac - dkk3-egfp transgenic mouse in which dkk3-expressing cells were strongly labeled by egfp . by analyzing the developing retina of this mouse , we showed that almost all rpcs express egfp at embryonic stages and the mller glial cells at postnatal stages . because of the important function of the mller glial cells in retinal regeneration , the bac - dkk3-egfp mouse is a valuable tool for developing regenerative therapies of the retina . furthermore , since dkk3 is an attractive tool for anticancer drug development , the bac - dkk3-egfp mouse may contribute to the advancement of novel cancer therapies .