Lake BB and Kao KR (2003), Pygopus is required for embryonic brain pattern...

XB-ART-4788

Dev Biol 2003 Sep 01;2611:132-48. doi: 10.1016/s0012-1606(03)00305-1.

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Pygopus is required for embryonic brain patterning in Xenopus.

Lake BB , Kao KR .

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We have identified two Xenopus mRNAs that encode proteins homologous to a component of the Wnt/beta-catenin transcriptional machinery known as Pygopus. The predicted proteins encoded by both mRNAs share the same structural properties with human Pygo-2, but with Xpygo-2alpha having an additional 21 N-terminal residues. Xpygo-2alpha messages accumulate in the prospective anterior neural plate after gastrulation and then are localized to the nervous system, rostral to and including the hindbrain. Xpygo-2beta mRNA is expressed in oocytes and early embryos but declines in level before and during gastrulation. In late neurula, Xpygo-2beta mRNA is restricted to the retinal field, including eye primordia and prospective forebrain. A C-terminal truncated mutant of Xpygo-2 containing the N-terminal Homology Domain (NHD) caused both axis duplication when injected at the 2-cell stage and inhibition of anterior neural development when injected in the prospective head, mimicking the previously described effects of Wnt-signaling activators. Inhibition of Xpygo-2alpha and Xpygo-2beta by injection of gene-specific antisense morpholino oligonucleotides into prospective anterior neurectoderm caused brain defects that were prevented by coinjection of Xpygo-2 mRNA. Both Xpygo-2alpha and Xpygo-2beta morpholinos reduced the eye and forebrain markers Xrx-1, Xpax-6, and XBF-1, while the Xpygo-2alpha morpholino also eliminated expression of the mid-hindbrain marker En-2. The differential expression and regulatory activities of Xpygo-2alpha/beta in rostral neural tissue indicate that they represent essential components of a novel mechanism for Wnt signaling in regionalization of the brain.

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Species referenced: Xenopus
Genes referenced: en2 lsamp pygo2
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	Fig. 1. Comparison of predicted Xpygo-2/ protein sequences with hPygo-2 (NCBI Accession No. AAL91371). Sequence identity between the three proteins is in bold-face. The conserved putative nuclear localization sequence (KKRRK) is double-underlined, while the C-terminal PHD is single-underlined, and the NHD or N-Box is indicated by broken underline.
	Fig. 2. Xpygo-2 / exhibit distinct expression patterns during Xenopus development. (A) RNA from 16 oocytes or embryos were analyzed by RT-PCR using primers specific to both messages (Xpygo-2 / ) and either Xpygo-2 or Xpygo-2 . RT, negative control, without reverse transcriptase. (B) Northern blot of staged embryo RNA probed for both Xpygo-2 isoforms (top arrow, -2 ; bottom arrow, -2 ). The 28S and 18S ribosomal RNAs as well as an RNA ladder are indicated. Stages of development are indicated at the top of each figure (Nieuwkoop and Faber, 1994). Levels of cDNA used or RNA loaded were standardized by using histone (H4) levels.
	Fig. 3. Xpygo-2 / are differentially expressed within the brain. Whole-mount in situ hybridization analysis of staged embryos using probes complementary to both transcripts (A–H) or specific to Xpygo-2 mRNA (I–O), or sense probes, where indicated (B, D, F, H, J, M, O). Specific hybridization is indicated by purple staining reaction. All embryos are shown with anterior to the left. Xpygo-2 mRNA is found early (St 13) in the anterior neural plate (A, dorsal view). By stage 20 and at least until stage 25, messages localized within the anterior neural tube, including prospective brain and eyes (C and E, lateral views) and in the tadpole, in the mid–hindbrain, forebrain, and eyes (G, lateral view). Xpygo-2 transcripts are present throughout blastulae (I, animal pole view), but are restricted to the retinal field at stage 20 (K, dorsal view) and in derivatives of this region (forebrain and eyes) at stage 25 (L, lateral view) and stage 30 (N, lateral view).
	Fig. 4. Targeted overexpression of various Xpygo-2 constructs. (A) The constructs used for subsequent injection experiments encoded wild-type Xpygo-2 and -2, Xpygo-2 NHD (amino acids 1–194), Xpygo-2 NHD (amino acids 1–216), and Xpygo-2 PHD (amino acids 194–389) fused to a strong nuclear localization sequence (NLS). (B) Injection experiments were performed at the eight-cell stage. For targeted expression within ventral mesendodermal derivatives, synthetic RNA was injected into both ventral vegetal blastomeres (VV). To target neural plate and epidermis, RNA or morpholinos were injected into either dorsal (DA) or ventral (VA) animal blastomeres, respectively.
	Fig. 5. Xpygo-2 NHD acts as a Wnt pathway activator to induce dorsal axial structures and suppress anterior neurectoderm. Embryos were either injected in ventral vegetal blastomeres (VV) or dorsal animal blastomeres (DA) with 10 ng of Xpygo-2 PHD RNA (control) (A, C–F), Xpygo-2 / NHD RNA (B, G–J), dominant Wnt activator R85 (K–N), or GSK-3 (O–R). Unlike PHD, overexpression of the NHD generated partial secondary axes when injected ventrally (B). Dorsal animal injected embryos were processed at stage 35 by using pan-neural (2G9; C, G, K, O, lateral view; D, H, L, P, dorsal view), skeletal muscle (12/101; top, lateral view; bottom, dorsal view), and Engrailed-2 (4D9, dorsal view) antibodies. 2G9 stained the anterior forebrain (f), the boundary between forebrain and midbrain (m), hindbrain (h), and spinal cord. The eye (e) was not stained at this stage. The NHD (G, H) and R85 (K, L), unlike GSK (O, P), disrupted anterior most staining of the forebrain (arrows). GSK disrupted or depleted midbrain staining (P, arrow). 12/101 staining indicated that all embryos developed normal somitic muscle (E, I, M, Q). 4D9 staining showed normal or expanded Engrailed-2 localization in embryos injected with NHD (J) and R85 (N) as compared with PHD (F) controls. GSK injection, however, reduced expression of En-2 (R, arrow). Scale bars: 1 mm (C–E); 0.1 mm (F).
	Fig. 6. Xpygo-2 and Xpygo-2 morpholinos (MO) specifically block translation of their target messages. In vitro translation of Xpygo-2 or -2 full-length cDNA constructs was blocked in a graded fashion (200–600 nM MO) by the corresponding MO sequence, but not by the highest concentration of Control MO (600 nM). In addition, neither MO could block translation from synthetic mRNA used in rescue experiments that lacked corresponding complimentary sequences (*). The additional translation product from Xpygo-2 mRNA of similar molecular weight to Xpygo-2 most likely represents the alternative start of translation site provided by the Xpygo-2 start codon, an in vitro artifact.
	Fig. 7. Xpygo-2 and Xpygo-2 antisense morpholinos (MO) cause severe head abnormalities. (A, D, G, J) Head-defective embryos resulting from injection at the 8-cell stage (DA) with a mixture of 20 ng each of both Xpygo-2 MOs and their corresponding fluorescence views optimized for Lissamine (red fluorescence, B, E, H, K) to reveal the -2 MO, and fluorescein (yellow-green fluorescence, C, F, I, L) to reveal the -2 MO. Note the association of MO fluorescence with the head defects seen in the dorsal view (D–F). The coinjection of 1ng each of Xpygo-2 / RNA not targeted by the MOs (J–L) rescued head structures even in the presence of both morpholinos (K, L). Xpygo-2 MO alone (M, N) caused severe head abnormalities including reduced or absent eyes and displaced or expanded cement glands (M, arrow). Xpygo-2 MO (40 ng per embryo; O, P) generated only eye deficits that ranged from reduction and ventral deficits to almost complete loss. Control MO (green fluorescence; 40 ng per embryo; Q, R, lateral view; S, T, dorsal view) did not affect development. Scale bars: 1 mm.
	Fig. 8. Xpygo-2 / MOs inhibit brain development. Immunostaining of tadpoles (st. 43) coinjected with Xpygo-2 MO and Xpygo-2 MO (20 ng each) using the pan-neural antibody 2G9 (A, dorsal view; B, lateral view) indicated anterior neural truncations with loss of retinal tissue as well as fore-, mid-, and hindbrain defects (arrows). These structures were rescued by coinjection of Xpygo-2 / RNA (1 ng each; D, dorsal view; E, lateral view). Xpygo-2 MO alone (40 ng) generated severe neural deficits rostral to and including the hindbrain (F, dorsal view; G, lateral view; arrows), while Xpygo-2 MO alone (40 ng) primarily disrupted and reduced neural tissue rostral to and including the midbrain (I, dorsal view; J, lateral view; arrows). Control MO (40 ng)-injected embryos showed normal 2G9 immunostaining of the eyes (e), forebrain (f), midbrain (m), hindbrain (h), and spinal cord. Undisrupted 12/101 staining indicated that all embryos developed normal muscle (C, H, K, and N; top, lateral; bottom, dorsal). Scale bars: 1 mm.
	Fig. 9. Xpygo-2 / MOs reversibly reduce RNA expression of a subset of brain markers. RT-PCR analysis of tailbud embryos (st. 22–23) injected with Control MO (40 ng), Xpygo-2 MO (40 ng), Xpygo-2 MO (40 ng), and combined Xpygo-2 MO/Xpygo-2 MO (20 ng each) with or without 2 ng of rescuing RNA (1 ng each Xpygo-2 /Xpygo-2 or 2 ng Xpygo-2 NHD). This experiment was repeated four times to confirm reproducibility of results.
	Fig. 10. Xpygo-2 / MOs reversibly eliminated En-2 protein expression. Embryos injected with Xpygo-2 MO/Xpygo-2 Mo (20 ng each) with or without rescuing RNA (2 ng) were fixed (st. 35) and immunostained for En-2 by using the 4D9 monoclonal antibody (A–C; right, dorsal view; left, lateral view). Normal En-2 staining can be seen at the mid–hindbrain junction of Control MO (40 ng)-injected embryos (D, arrows), but is lost in Xpygo-2 MO/Xpygo-2 MO-injected embryos (A). Coinjection of Xpygo-2 / RNA rescues limited expression (B, arrows), while coinjection of Xpygo-2 NHD RNA rescues full expression (C, arrows) of En-2. Scale bar: 0.1 mm.
	pygo2 (pygopus family PHD finger 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, dorsal view, anterior left.
	pygo2 (pygopus family PHD finger 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, lateral view, anterior left, dorsal up.
	pygo2 (pygopus family PHD finger 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.
	pygo2 (pygopus family PHD finger 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.