Hoppler S et al. (1996), Expression of a dominant-negative Wnt blocks in...

XB-ART-17480

Genes Dev 1996 Nov 01;1021:2805-17. doi: 10.1101/gad.10.21.2805.

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Expression of a dominant-negative Wnt blocks induction of MyoD in Xenopus embryos.

Hoppler S , Brown JD , Moon RT .

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During gastrulation of Xenopus embryos the prospective mesoderm is induced initially with domains of dorsal and ventral fate, then further patterned to generate somitic mesoderm by signals from the gastrula organizer. Although Xwnt-8 is expressed in future ventrolateral mesoderm and induces prospective epidermis to differentiate in vitro as ventral mesoderm, no loss-of-function studies have demonstrated a requirement for Wnt signaling for the normal expression of mesodermal genes in the gastrula. We report development of a dominant-negative Wnt (dnXwnt-8) that inhibits embryonic responses to Wnt signaling in a cell-nonautonomous fashion. By expressing dnXwnt-8 in Xenopus embryos, we uncover a requirement of Wnt signaling for localized expression in prospective mesoderm of XMyoDa and Xenopus-posterior (Xpo). Because ectopic expression of functional Xwnt-8 in the dorsal marginal zone of the gastrula induces ectopic XMyoDa and Xpo, both gain-of-function and loss-of-function experiments support a model in which endogenous Xwnt-8 functions to induce expression of genes involved in specification of ventral and somitic mesoderm.

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Species referenced: Xenopus
Genes referenced: gsc h4c4 myc myod1 nodal3.1 nodal3.2 post wnt1 wnt8a

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	Figure 1. Truncated Wnt protein sequences. Alignment of the amino acid sequences for Dwnt-1 (wingless), mouse Wnt-1, and Xwnt-8. Periods represent gaps in the sequence alignment; the position of conserved cysteine residues are indicated by the letter C below the aligned sequences. The underlined letters represent the sequence of the truncated protein encoded by the Dwnt-1 mutant alleles $84 and CE7 and the amino acid sequence of dnWnt-1 and dnXwnt-8, truncated Wnt-1 and Xwnt-8 proteins, respectively, with dominant-negative activity. A truncation of Xwnt-8 at position 230, which results in a 30-amino-acid-shorter protein than dnXwnt-8, had only weak inhibitory activity.
	Figure 2. dnXwnt-8 inhibits gsc induction and axis duplication by Wnts. Development of tadpoles (A, C,E, G, anterior to the left) and gsc in situ hybridization of gastrula-stage embryos (B,D,F,H). Embryos were injected at the four-cell stage with -300 pg of control RNA (A-D) or dnXwnt-8 RNA (E-H) into each ventral blastomere and again at the 8- to 16-cell stage into the ventral marginal zone with -25 pg of RNA encoding Xwnt-8 (C-F) or -60 pg of A[3-catenin RNA (G,H). The RNA amounts were based on first establishing the lowest amount of each required to elicit a duplication of the axis in most embryos. Embryos injected with control RNA develop with a single head {A) and a single domain of gsc expression (B, indicated by arrowhead). When injection of control RNA is followed by a second injection of RNA encoding wild-type Xwnt-8, embryos develop with a duplication of the embryonic axis (C, two heads indicated by arrowheads) and with an ectopic domain of gsc expression (D, two gsc expression domains indicated by arrowheads). When dnXwnt-8 RNA injection precedes that of RNA encoding wild-type Xwnt-8, axis duplication is repressed; embryos develop with one head (E) and with a single domain of gsc expression (F, indicated by arrowhead). Injection of dnXwnt-8 RNA does not affect the axis duplicating activity of subsequently injected A[3-catenin RNA, and tadpoles develop with two heads (G, indicated by arrowheads) and with an ectopic domain of gsc expression (H, two gsc expression domains indicated by arrowhead). Note that dnXwnt-8 RNA injections specifically inhibit Wnt-induced axis duplication (see also Table 2).
	Figure 3. dnXwnt-8 blocks embryonic responses to Xwnt-8 in a cell nonautonomous manner. (A,B) Embryos were first injected at the four-cell stage with either -300 pg of control RNA (A) or dnXwnt-8 RNA (B) into the marginal zone of both ventral blastomeres and again at the 16-cell stage (A,B) into a single ventral marginal blastomere with -50 pg of Xwnt- 8myc RNA. At gastrula stage 10, embryos were fixed and stained for expression of the c-myc epitope (dark brown staining), which revealed that dnXwnt-8 does not affect the expression of Xwnt-8myc [cf. B with A, control), although it blocks its axis-duplicating activity (not shown, but identical to Fig. 2E, F). (C) Drawing (Nieuwkoop and Faber 1967) explaining the design of the nonautonomy experiment in D-F, with five cells (red hatched) being injected at the early 16-cell stage (indicated by arrows) with lineage tracer (phenol red and 13-galactosidase RNA) and ~250 pg of RNAs [either dnXwnt-8 RNA or a control RNA). Then, at the late 16-cell stage the central cell [white cell within the red hatched area indicated by an asterisk ()] was injected with -25 pg of Xwnt-8myc RNA. (D) Blastulastage embryos injected as in C were processed for [3-galactosidase activity (blue) and c-myc epitope localization (light brown). Note that the blue [3-galactosidase RNA-injected cells [indicated by arrowhead) do not overlap with the brown Xwnt-8myc RNA-injected cells [indicated by an asterisk ()]. (E) An endogenous and an ectopic domain of gsc expression [solid arrowheads) are evident when control RNAs are injected into the five red cells as in C, followed by Xwnt-8myc in the central cell. (F) In contrast, injection of dnXwnt-8 into the five red cells as in C, followed by Xwnt-8myc into the central cell, had greatly reduced or no ectopic gsc expression (the shaded arrowhead indicates an ectopic domain of reduced gsc expression in the embryo at right). Note that dnXwnt-8 is able to block the induction of gsc (cf. F with E, control), even if the proteins are expressed in exclusive populations of cells, as shown with the complementary staining in D.
	Figure 4. dnXwnt-8 inhibits Xwnt-8 expressed after the blastula stage. (A) RT-PCR analysis of gene expression in control stage 12 animal explants for a control marker (histone H4), a dorsal marginal zone marker (gsc), and a ventral marginal zone marker (Xpo). Embryos were injected at the two-cell stage into the animal pole of both blastomeres with -800 pg of control RNA or dnXwnt-8 RNA (as indicated at top) and -80 pg of CSKA Xwnt-8 plasmid DNA (where indicated at top), and animal caps were explanted and treated with activin (where indicated by + ) and subjected to analysis with RT-PCR. (B-D) Histological sections through animal pole explants. Embryos were injected at the two-cell stage into the animal pole of both blastomeres with -200 pg of control RNA (B), with -200 pg of control RNA, and -30 pg of CSKA Xwnt-8 plasmid (C), or -200 pg of dnXwnt-8 RNA and -30 pg of CSKA Xwnt-8 plasmid (D). Animal pole explants were prepared at stage 8, then cultured until sibling embryos reached stages 38-40. Note that dnXwnt-8 RNA represses the formation of vesicular structures induced by CSKA Xwnt-8 (see Table 3A). (E-G) Stage 40 embryos (anterior to the left and dorsal on the top) that developed from embryos injected at the four-cell stage into the marginal zone of both dorsal blastomeres with -200 pg of control RNA (E), -200 pg of control RNA mixed with -30 pg of CSKA Xwnt-8 plasmid (F), or -200 pg of dnXwnt-8 RNA mixed with -30 pg of CSKA Xwnt-8 plasmid (G). Note that dnXwnt-8 interferes with the Xwnt-8-mediated repression of dorsoanterior structures such as cement gland (indicated by small arrowheads} and eyes (see also Table 3B).
	Figure 5. dnXwnt-8-injected embryos develop dorsal mesoderm but form defective somites. (A,C) Injection of -3 ng of control RNA into cleavage-stage embryos leads to development of normal stage 36--38 embryos, both in terms of external morphology {A, anterior to left, dorsal on top) and internal anatomy, as examined in histological cross section through the trunk region (C, dorsal on topJ. (B,D) When 1.5-3 ng of dnXwnt-8 RNA is injected, development is highly aberrant, embryos develop with a shortened axis as well as a relatively enlarged head region (B), and histological cross section through such embryos (D) reveals that the notochord is enlarged and that somitic muscle tissue is reduced and disorganized. (E) Injection into the marginal zone of all four blastomeres of four-cell embryos with 1.5-3 ng of RNAs encoding either control protein (bottom) or dnXwnt-8 {top) results in normal expression of gsc. (F) Similarly, 1.5-3 ng of dnXwnt-8 (top) does not significantly reduce the level of expression of Xnr3 compared with control embryos (bottom). (en)Endoderm; (no)notochord; (nt) neural tube; {sin) somitic muscle.
	Figure 6. dnXwnt-8 blocks induction of XMyoDa and Xpo in the ventral and lateral marginal zone, whereas ectopic Xwnt-8 induces ectopic expression of these markers in the dorsal marginal zone. In situ hybridization (purple staining) of stage 101/4-101/2 embryos with probes for XMyoDa (A,C,E,G,I) and Xpo (B,D,F,H,J). (A,B) Control RNA (-3 ng) injections into the marginal zone of all four blastomeres of four-cell embryos lead to normal expression of both the lateral mesodermal marker XMyoDa (A) and the ventrolateral mesodermal marker Xpo (B). (C,D) When 0.8-3 pg of dnXwnt-8 RNA is injected, expression of XMyoDa is abolished (C) and localized Xpo RNA staining is greatly reduced (D) (see Table 4A). (E,F) Overexpression of wild-type Xwnt-8 (CSKA Xwnt-8, -100 pg) in one side of the ventrolateral mesoderm of dnXwnt-8 RNA (-800 pg)-injected embryos rescues the expression of the mesodermal markers XMyoDa (E, indicated by arrowheads) and Xpo (F, indicated by arrowheads) predominantly on the side of injection (see also Table 4B). (G-J) In a gain-of-function analysis, CSKA Xwnt-8 DNA (-100 pg) was injected into the marginal zone of both dorsal blastomeres at the four-cell stage (L J) to ectopically express Xwnt-8 in the dorsal prospective mesoderm. Whereas ventral injected embryos have the normal dorsal gap of XMyoDa expression [G, indicated by an open arrowhead; vegetal view (left) and dorsal view (right) of the same embryo; see Table 4C], ectopic Xwnt-8 expression results in the ectopic expression of XMyoDa in most dorsal prospective mesoderm [L indicated by a solid arrowhead; vegetal view (left) and dorsal view (right) of the same embryo]. Similarly, whereas uninjected embryos show only weak Xpo staining on the dorsal side [H, indicated by an open arrowhead; vegetal view (left) and dorsal view (right) of the same embryo; see also Table 4C], ectopic Xwnt-8 expression also causes strong expression of Xpo in the dorsal prospective mesoderm [J, indicated by a solid arrowhead, vegetal view (left) and dorsal view (right) of the same embryo].