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doi: 10.1242/10.1242/dev.00349

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The zinc-finger protein CNBP is required for forebrain formation in the mouse

Wei Chen1,2, Yuqiong Liang1, Wenjie Deng1, Ken Shimizu1, Amir M. Ashique1,2, En Li3 and Yi-Ping Li1,2,*

1 Department of Cytokine Biology, The Forsyth Institute, Boston, MA 02115, USA
2 Harvard-Forsyth Department of Oral Biology, Harvard School of Dental Medicine, Boston, MA 02115, USA
3 Cardiovascular Research Center, Massachusetts General Hospital, Department of Medicine, Harvard Medical School, Charlestown, MA 02129, USA



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  		Fig. 1. Retroviral insertional mutation of CNBP resulted in anterior patterning and craniofacial defects. (A) Morphology of newborn wild-type mouse. (B) A heterozygote newborn mouse with a short snout and lacking eyes. (C,D) Heterozygotes with a smaller lower jaw (C) and missing eyes (D). (E) A homozygote lacking rostral head structures, including the entire forebrain. (F,G) As early as E7.5, a homozygous mutant embryo is smaller than its wild-type littermate. A constriction is observed between embryonic and extra-embryonic regions in Cnbp-/- mutants (arrow in G). (H,I) By E8.5, forebrain truncation is evident in mutant embryo (arrow in I). (J,K) At E9.5, Cnbp-/- embryos were smaller with forebrain truncations. (L) The integration site of the provirus in the Cnbp gene locus. The flanking sequence, 5' fA8, was cloned by inverse PCR. The Cnbp gene was cloned and characterized by using 5' fA8 as probe. The positions of the 5' fA8 probe and primers 1, 2 and 3 (P1, P2, and P3) for genotyping are shown. (M) Genotype analysis of Cnbp mutant mice by Southern blot. The presence of a single 4 kb fragment represents wild-type allele, while a larger 8 kb fragment, a result of the proviral insertion, represents a mutant allele. (N) Genotype analysis of E6.5 embryos by PCR demonstrates the recovery of wild-type (lanes 1, 5), heterozygous (lanes 3, 4, 6, 8) and homozygous embryos (lanes 2, 7). Primers P1 and P3 amplify a 500 bp wild-type fragment, whereas primers P2 (a Neo insertion-specific primer) and P3 together amplify a 300 bp mutant fragment. (O) Northern blot analysis of total RNA isolated from E9.5 whole embryos derived from Cnbp-heterozygous mutant parents. A 1.65 kb mRNA was detected in the wild-type and heterozygous embryos but was undetectable in the homozygous embryos using Cnbp cDNA as a probe. (P,Q) Immunostaining in tissue sections was performed, using an anti-CNBP polyclonal antibody, to examine CNBP protein levels in E7.25 wild-type and mutant embryos. CNBP protein was localized to the ANE and ADE of a wild-type embryo (brown staining in P) but was absent in the Cnbp-/--mutant embryo (Q).

 


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  		Fig. 2. Identification of early Cnbp-expression pattern and Cnbp-transgene rescue of forebrain defects in Cnbp mutants. (A-D) Cnbp expression was analyzed at pre-gastrulation, gastrulation and post-gastrulation stages by whole-mount in situ hybridization. (A) Cnbp is expressed at the visceral endoderm, anterior to the distal tip of the early embryo at early gastrulation (E6.0; arrow). (B) During primitive streak formation, at E7.0, Cnbp expression localizes to the AVE in the anterior midline from the proximal to the distal region. (C) Cnbp is expressed in anterior axial mesendoderm, ADE and ANE at late-primitive streak stage (E7.5). (D) Sagittal section of an embryo at an approximately similar stage to that shown in C, showing Cnbp expression in the anterior axial mesendoderm, ADE and ANE. (E-G) Cnbp is expressed in the anterior neural folds at 8-10 somite stages. (F) Sagittal section of an embryo at an approximately a similar stage to that shown in E, showing Cnbp expression in the ANE (forebrain) and head mesenchyme. (G) Cnbp continues to be expressed in the headfolds. (H) Cnbp is expressed in the forebrain at E9.25. Transcripts were also detected in the early facial prominences, including the first branchial arch, primitive maxillary region and early frontonasal area. Regions of expression other than the head include the limb bud and tail. (I) The Cnbp transgene comprising the 10 kb mouse Cnbp promoter, the entire Cnbp gene (11 kb) and a 300 bp vector DNA fragment (shaded region on left side) as a tag for genotyping. (J) Transgenic genotyping by PCR analysis using primers P1 and P2, described in I. Lane 5 shows control DNA from wild-type mice. The 300 bp fragment in lanes 1-4 represents recovery of transgenic embryos. (K) Transgenic genotyping by Southern blot analysis using the 300 bp vector DNA as probe. Wild-type embryos are represented in lanes 1, 3 and 5. Genomic DNA from transgenic embryos hybridizes to probe (lanes 2, 4 and 6). (L-M) Transgenic rescue of forebrain defects in Cnbp mutants. Cnbp expression in Cnbp+/+ wild-type embryo (L), Cnbp-/- mutant (M) and TG/Cnbp-/- (n=7) (N) embryos at E9.5. Embryo in M shows forebrain truncation, whereas the transgenic rescued embryo has a normal phenotype and a nearly identical expression pattern as the wild-type embryo (L,N). Cnbp begins to also be expressed in the midbrain shortly before E9.5 (L).

 


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  		Fig. 3. Loss of forebrain in Cnbp mutants. (A,B,K,L) Bf1 mRNA, a marker for the telencephalon was entirely absent in E8.5 (B) and E9.5 (L) Cnbp-/- embryos (n=5). (C-F) Hesx1 and Six3 markers, used here to label the diencephalon, were entirely absent in E8.0 Cnbp-/- embryos (D,F) when compared with wild-type littermates (C,E). (G,H) En1, a marker for midbrain and anterior hindbrain, is normally expressed in E8.5 wild-type and Cnbp-/- mutant embryos. (I,J) Expression of Krox20, a marker for rhombomeres 3 and 5, is observed in E9.0 wild-type and Cnbp-/- mutant embryos. (M,N) Mox1 expression is normal in paraxial mesoderm cells in E9.5 Cnbp-/- mutant when compared with their wild-type littermates. (O,P) Brachyury (T) expression is detected in the notochord and posterior mesoderm cells of mutant embryos at E9.5. T expression in tail was similar in homozygous mutants and their wild-type littermates.

 


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  		Fig. 4. Molecular analyses of the origins of the developmental defects in Cnbp-/- mutants by whole-mount in situ hybridization analysis of marker genes. Lateral views of embryos are shown with anterior (A) to the left and posterior (P) to the right. (A,B) Whole-mount in situ hybridization with Hex probe at E6.0. Hex was expressed normally in the distal end of the epiblast of Cnbp-/- mutant when compared with wild-type littermates. (C,D) Hex was expressed in the displaced AVE of E6.5 wild-type embryos but was retained near the distal end of the epiblast in the mutants (arrow). (E,F) Lim1 is expressed in the AVE and the primitive streak of the E6.5 wild-type embryo; however, transcripts are more towards the distal end of the AVE in Cnbp-/-. (G,H) Hex is expressed in the anterior definitive endoderm (ADE) and AVE of E7.25 wild-type embryos but is not detectable in E7.25 mutant embryos. (I,J) Cer1 expression was undetectable in the ADE and AVE in E7.25 mutant embryos. (K,L) Hex is expressed in the ADE and ANE of E7.5 wild-type embryos but is not detectable in that of E7.5 mutant embryos. (M,N) Cer1 expression was undetectable in the ADE and ANE in E7.5 mutant embryos. (O,P) Expression of Otx2 in the ANE was not observed in mutant embryos.

 


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  		Fig. 5. Whole-mount RNA in situ hybridization staining of AME markers. (A,B) Lim1 is expressed in the ADE and primitive streak at E7.5, but is undetected in the AME of mutant embryos. (C,D) Hnf3b is expressed in the node and prechordal mesoderm in wild-type embryo at E7.5, but only extends a short distance anteriorly from the node in mutant embryos (arrow). (E,F) T is expressed in the primitive streak of the wild-type and mutant embryos. In the mutants, T is only expressed at a short distance from the node. (G,H). At E8.0, Hnf3b expression in mutants is normal in the node and most of the midline but is absent from the anterior head and foregut pocket region. (I-L) Analysis of the of prechordal plate markers Gsc and Dkk1 indicates that the E7.5 and E8.0 mutant embryos lack prechordal plate.

 


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  		Fig. 6. Morphological and cellular basis of the forebrain defects in Cnbp-/-. (A,B) Disorganization of the axial mesendoderm and ANE region of cells (arrow) in E7.5 Cnbp-/- mutants (A) compared with that (arrow) of wild-type littermates (B). (C,D) Lack of ANE and characteristic headfold structure in E8.5 Cnbp-/- embryos compared with that of wild-type littermates. (E-H) Evidence for decreased proliferation rate in mutant head plate by BrdU incorporation analysis in adjacent sections of wild-type (E,G) and Cnbp-/- mutant embryos (F,H). Arrow in F indicates that BrdU-positive nuclei was rarely seen in the anterior region of the mutant compared with that in wild-type embryo (arrow in E). Note that the head regions of wild-type embryos exhibit a high density of BrdU-positive nuclei throughout the axial mesendoderm and ANE regions at E7.5, and in prechordal mesoderm and headfold regions at E8.5. The mutants showed much fewer BrdU-positive nuclei at the same regions. (I-L) TUNEL apoptosis assays in the histologically normal and mutant E7.5 and 8.5 embryos. TUNEL assays showed there was no significant difference of apoptosis in the anterior region of wild-type (I,K) and mutant (J,L) E7.5 and E8.5 embryos. (M-P) E6.0 Cnbp-/- mutant embryos and wild-type littermates were examined for general morphology (M,N) and cell division using a BrdU incorporation assay (O,P). BrdU-positive nuclei in mutant embryos were absent in the AVE of E6.0 mutant embryos (arrow in P). By contrast, the AVE region in normal E6.0 embryos showed the greatest density of BrdU-positive nuclei (arrow in O). (Q) Quantification of BrdU-positive nuclei in the anterior region of E7.5 embryos. The percent of BrdU-positive nuclei was 84% in wild-type embryos compared with 28% in null-mutant embryos. Error bars represent s.e., counts were made of three wild-type embryos (blue bar) and three null-mutant embryos (red bar). These values were determined to be statistically significant (P<0.001).

 


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  		Fig. 7. CNBP positively regulates the endogenous expression of Myc. (A-D) Myc is normally abundantly expressed in the ANE at E7.25 (A) and in the neural folds (arrow, C) at E8.5, but is nearly undetectable in the neural folds (arrow, D) of Cnbp mutants. Expression in the allantois (arrowhead) is not affected in E8.5 mutant embryos. (E,F) At E9.5, Myc is expressed in the forebrain, as well as in the primitive facial prominences of wild-type embryos, but it was undetected in the anterior region of E9.5 mutant embryos. (G) CNBP upregulates Myc promoter activity in embryonic cells. Cnbp+/+ and Cnbp-/- mouse embryonic fibroblast cells (MEFs) were transfected with Myc promoter-luciferase plasmid (columns 1 and 2) or co-transfected with a mouse Cnbp-expression plasmid into Cnbp-/- cells (column 3). A lower level of Myc expression was observed in Cnbp-/- embryonic fibroblasts compared with Cnbp+/+ cells. Transfection of Cnbp-/- embryonic fibroblasts with the Cnbp-expression plasmid resulted in a Myc expression level higher than that found in Cnbp+/+ cells. Results represent luciferase activity related to galactosidase activity. Values are the mean±s.d. of triplicate experiments.

 

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