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After the primary anterior-posterior patterning of the neural plate, a subset of wnt signaling molecules including Xwnt-1, Xwnt-2b, Xwnt-3A, Xwnt-8b are still expressed in the developing brain in a region spanning from the posterior part of the diencephalon to the mesencephalon/metencephalon boundary. In this expression field, they are colocalized with the HMG-box transcription factor XTcf-4. Using antisense morpholino loss-of-function strategies, we demonstrate that the expression of this transcription factor depends on Xwnt-2b, which itself is under the control of XTcf-4. Marker gene analyses reveal that this autoregulatory loop is important for proper development of the midbrain and the isthmus. Staining for NCAM reveals a lack of dorsal neural tissue in this area. This reduction is caused by a reduced proliferation rate as shown by staining for PhosphoH3 positive nuclei. In rescue experiments, we demonstrate that individual isoforms of XTcf-4 control the development of different parts of the brain. XTcf-4A restored the expression of the mesencephalon marker genes pax-6 and wnt-2b but not the isthmus marker gene en-2. XTcf-4C, in contrast, restored en-2, but had only weak effects on pax-6 and wnt-2b. Thus, autoregulation of canonical Wnt signaling and alternative expression of different isoforms of XTcf-4 is essential for specifying the developing CNS.
Fig. 1. Knockdown of Xwnt-2b results in reduced expression of tcf-4 and en-2. (A) Alignment of Xwnt-2b with the Xwnt-2b EGFP construct containing the 5′-UTR binding site for the Xwnt-2b morpholino (Wmo) but not for the control morpholino (Como). (B) Wnt-2b morpholino (Wmo), but not control morpholino (Como), represses the translation of exogenously expressed Xwnt-2b (Xwnt-2bEGFP). NOP lysates of a single embryonic equivalent of stage 13 (according to Nieuwkoop and Faber, 1967) stained with α-EGFP antibody. Counterstaining of the blot with α-Gemin was used as loading control. (C) Wnt-2b morpholino blocks the expression of XTcf-4 protein in the midbrain. Ten picomoles of the morpholino were injected together with 100 pg of myc-tagged EGFP mRNA into one blastomere of two-cell stage embryos. Transverse sections through the midbrain region of stage 30 embryos were stained with α-Tcf3/4 primary antibody and visualized with cy3-labeled secondary antibody. The EGFP signal indicates the injected side. (D) In situ hybridization of stage 30 embryos reveals that the Wnt-2b morpholino blocks the expression of tcf-4 and en-2, but not otx-2 and pax-6. Ten picomoles of the morpholino were injected together with 100 pg of myc-tagged EGFP mRNA into one blastomere of two-cell stage embryos. At late neurula stages, embryos were sorted by the fluorescence signal of EGFP. At stage 30, embryos were fixed and analyzed by whole-mount in situ hybridization. f: forebrain, m: midbrain, h: hindbrain.
Fig. 2. XTcf-4 expression is regulated by Wnt/β-Catenin signaling. (A) Xwnt-2b and Xwnt-8, but not Xwnt-5A or Xwnt-11, restore the expression of tcf-4 in Wnt-2b-depleted embryos. Ten picomoles of Wnt-2b morpholino were coinjected with 100 pg of myc-tagged EGFP mRNA and 100 pg cDNA encoding for the indicated Xwnts. At late neurula stages, embryos were sorted by the fluorescence signal of the coinjected EGFP for the injected side. At stage 30, embryos were fixed and stained by in situ hybridization for the expression of tcf-4. Arrowheads mark the reduced tcf-4 expression at the injected side. (B) In situ hybridizations as shown in A were evaluated for tcf-4 expression at the injected side. Given is the percentage of not reduced (wild-type) tcf-4 expression compared to the control side. n indicates the number of analyzed embryos.
Fig. 4. Knockdown of XTcf-4 results in reduced proliferation of dorsal braintissue. (A) XTcf-4 morpholino, but not control morpholino, blocks the expression of NCAM protein in the dorsal midbrain. Ten picomoles of the morpholino were injected together with 100 pg of myc-tagged EGFP mRNA into one blastomere of two-cell stage embryos. Transverse sections of stage 30 embryos were incubated with α-NCAM and visualized with a Cy3-coupled goat anti-mouse antibody. The injected side is traced by EGFP. Note: At the XTcf-4 morpholino-injected side, the NCAM signal is missing in the dorsal half of the midbrain (arrow). Consistently, the dorsal part appears to consist of less tissue (arrow, brightfield). (B) The pan-neural marker gene nrp-1 is still expressed in the Tcf-4-depleted, size-reduced dorsal midbrain. Fifty-micrometer transversal sections through the midbrain region of stage 30 embryos stained for nrp-1 reveal that the dorsal neural tissue (arrow) is less thick at the injected side (asterisk). (C) XTcf-4 morpholino blocks cell proliferation in the dorsal brain. Transversal sections of stage 25 embryos were stained with the M-phase marker phosphoH3 and visualized with Cy-3-coupled secondary antibody. The injected side is marked by α-myc staining for the coinjected GFPmyc. The white line illustrates the subdivision of the midbrain into ventral (v) and dorsal (d) part for the quantification in C. (D) Quantification of the phosphoH3-positive nuclei in the brain reveals that on the injected side (filled bars), the number of proliferating cells is decreased, while at the ventralmidbrain cell proliferation was not altered. The phosphoH3-positive nuclei at the uninjected side (open bars) were set as 100%.
Fig. 5. XTcf-4 regulates the expression of midbrain and isthmus marker genes. Ten picomoles of XTcf-4 morpholino were coinjected with 100 pg EGFP-RNA. At late neurula stages, embryos were sorted by the fluorescence signal of the coinjected EGFP. At stage 30, embryos were fixed and stained by in situ hybridization for the expression of the indicated genes. The midbrain marker genes wnt-2b, otx-2, and pax-6 and the isthmus marker genes en-2 and fgf-8were markedly reduced at the XTcf-4 morpholino (Tmo)-injected side (marked with an asterisk), while the forebrain marker bf-1 and the rhombomere 3 and 5 marker krox-20 were not reduced. The control morpholino (Como) did not reduce the expression of any of the marker genes analyzed. In early neurula (stage 13), the XTcf-4 morpholino did not alter the expression of pax-6. f: forebrain, m: midbrain, h: hindbrain.
Fig. 7. XTcf-4A and XTcf-4C differ in restoring midbrain and isthmus defects. (A) In situ hybridizations of stage 30 embryos for marker gene expression specific for midbrain (wnt-2b, pax-6) or isthmus (en-2, wnt-1) demonstrate that the individual isoforms of XTcf-4 restore different regions of the brain. Dorsal view of wnt-2b, pax-6, and en-2 stained embryos reveal that the isoform XTcf-4A restored the expression of midbrain markers (wnt-2b and pax-6), whereas XTcf-4C restored the expression of the isthmus marker en-2. Transversal sections through the isthmus (row 4) demonstrate that en-2 expression was restored by XTcf-4C but not by XTcf-4A in XTCF-4-depleted embryos. The curved expression domain of wnt 1 posteriorly demarcating the isthmus (arrowhead) is best seen in the lateral view (see uninjected side, row 5). At the injected side (row 6), this curved wnt-1 expression was restored by XTcf 4C, but not by XTcf 4A. One blastomere of two-cell stage embryos was coinjected with EGFP and the indicated construct. At late neurula stages, embryos were sorted by the fluorescence of EGFP. The asterisks mark the injected side. m: midbrain, h: hindbrain. (B) Quantification of the ability of the XTcf-4 isoforms to restore the expression of wnt-2b, pax-6, en-2, fgf-8, and wnt-1 in XTcf-4-depleted embryos. The number of analyzed embryos is given.
