Wills A et al. (2006), Twisted gastrulation is required for forebrain ...

XB-ART-1014

Dev Biol 2006 Jan 01;2891:166-78. doi: 10.1016/j.ydbio.2005.10.022.

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Twisted gastrulation is required for forebrain specification and cooperates with Chordin to inhibit BMP signaling during X. tropicalis gastrulation.

Wills A , Harland RM , Khokha MK .

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In the developing vertebrate embryo, proper dorsal-ventral patterning relies on BMP antagonists secreted by the organizer during gastrulation. The BMP antagonist chordin has a complex interaction with BMPs that is governed in part by its interaction with the secreted protein twisted gastrulation (tsg). In different contexts, tsg has activity as either a BMP agonist or as a BMP antagonist. Using morpholino oligonucleotides in Xenopus tropicalis, we show that reducing tsg gene product results in a ventralized embryo, and that tsg morphants specifically lack a forebrain. We provide new evidence that tsg acts as a BMP antagonist during X. tropicalis gastrulation since the tsg depletion phenotype can be rescued in two ways: by chordin overexpression and by BMP depletion. We conclude that tsg acts as a BMP antagonist in the context of the frog gastrula, and that it acts cooperatively with chordin to establish dorsal structures and particularly forebrain tissue during development.

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Species referenced: Xenopus tropicalis
Genes referenced: bambi bmp4 bmp7.1 cer1 chrd egr2 emx1l en2 eomes foxg1 fst gsc hoxb9 myf5 myod1 nkx2-1 nkx2-4 nodal3.1 otx2 sox2 szl twsg1 ventx2.2
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	Fig. 1. Expression of tsg in X. tropicalis embryos. Embryos were collected at the 2-cell stage (A), stage 8 (B), stage 9 (C) stage 10.5 (D) stage 11 (E), and stage 22 (F), and assayed for expression of tsg. Panel A is a whole embryo shown in lateral view with animal to the top, panels B, C are bisected embryos shown with animal pole to the top. Panels D, E are bisected embryos shown with animal to the top and dorsal to the right, and panel F is a whole embryo shown laterally with anterior to the left.
	Fig. 2. Tsg morphants are ventralized. (A) Embryos were injected at the two-cell stage with a high dose of tsg MO (g l) and compared to uninjected sibling control embryos (a) at stage 11 for changes in morphology and molecular marker expression as indicated. Arrows in panels e, k indicate the dorsal limit of sizzled expression, while arrows in panels f, l indicate the animal limit of vent-2 expression. Embryos in panels f, l are shown from a dorsal view, all others are vegetal views with dorsal to the top. (B) Embryos injected with low (b, f, j, n, r), intermediate (c, g, k, o, s) or high (d, h, l, p, t) doses of tsg MO were compared to uninjected control siblings (a, e, i, m, q) for changes in morphology and expression of the ventral molecular marker sizzled (e, m) and the dorsal forebrain marker eomesodermin (q t). All embryos are shown in lateral views with dorsal to the top, anterior to the left. Embryos were analyzed at stage 15 (a h) or stage 25 (i t). The arrowhead in panel l marks the cement gland.
	Fig. 3. Co-injection of tsg mRNA rescues the tsg morphant phenotype. (A)NOT SHOWN: Alignment of the target sequence for the tsg MO used in this study with the 5V region of mRNAs for X. tropicalis, X. laevis and Oncorhynchus mykiss (rainbow trout). The translational start site is shown in green, mismatches between O. mykiss and the tsg MO are shown in pink, and mismatches between X. laevis and the tsg MO are shown in blue. (B) NOT SHOWN: Embryos were injected at the two-cell stage with 20 ng tsg MO or 20 ng tsg MO plus 400 pg of O. mykiss tsg mRNA, then assayed at stage 26 for survival and proper blastopore closure. The experiment was repeated with the same result. (C) Embryos were injected with 400 pg O. mykiss tsg mRNA (b, f, j, n, r, v), 20 ng tsg MO (c, g, k, o, s, w), or both (d, h, l, p, t, x) and compared to uninjected sibling control embryos (a, e, i, m, q, u) at stage 11 (a l), stage 19 (m t), or stage 26 (u x) for expression of the molecular markers indicated at left. The view for each marker is indicated at right. For animal and vegetal views, dorsal is to the top while for ventral and lateral views, anterior is to the left.
	Fig. 4. Forebrain patterning is impaired in tsg morphants. Embryos were injected with intermediate (B, E, H, K) or high (C, F, I, L) doses of tsg MO and compared to uninjected sibling control embryos (A, D, G, J). Regional specific markers of the forebrain were assayed including the dorsal telencephalon markers eomesodermin (A) and emx-1 (D), and the ventral telencephalon markers Nkx2.1 (G I) and Nkx2.4 (J L). All embryos are shown at stage 23 and are viewed laterally with anterior to the left.
	Fig. 5. Depletion of chordin and tsg reduces dorsal tissues and results in a cooperative expansion of ventral tissues. Embryos were injected at the two-cell stage with tsg MO (B, F, J, N, R, V, Z, DD), chordin MO (C, G, K, O, S, W, AA, EE), or both MOs (D, H, L, P, T, X, BB, FF) and compared to uninjected sibling control embryos (A, E, I, M, Q, U, Y, CC). Embryos were assayed for expression of the ventral markers BAMBI (E H) and sizzled (I L) and the somite marker myoD (M P). Embryos were also assayed for expression of several regional specific markers of the neural plate: eomesodermin (Q), otx2 (U), en-2 (YB) and krox-20 (CC FF). All embryos are between stages 22 25. Embryos stained for expression of myoD (M P) and krox -20 (CC FF) are viewed dorsally with anterior to the left, all others are viewed laterally with anterior to the left.
	Fig. 6. BMP depletion rescues tsg morphants morphologically. (A) The incidence of blastopore closure was quantified in embryos injected at the two- cell stage with tsg MO, BMP MOs, or varying combinations of tsg and BMP MOs as indicated. The experiment was repeated with similar results. (B) Co- injection of tsg MO with BMP MOs rescues embryos morphology to varying extents. Embryos were injected at the 2-cell stage with 10 ng of tsg MO and either 40 ng of BMP4 MO, 40 ng of BMP7 MO, or 20 ng of both BMP4 and BMP7 MOs. For each combination, the percentage of embryos observed with each degree of rescue was quantified. Representative embryos for each category of rescue are included at right. The category represented in blue indicates embryos with minimal or no rescue of head morphology or embryo length. Embryos represented by the red category had mild to moderate rescue of head morphology and/or embryo length. Embryos represented by the yellow category showed significant rescue of head morphology, embryo length and overall morphology. For panels A and B, all embryos were injected at the 2-cell stage with the combination of MOs indicated, and were assayed at stage 25 for blastopore closure and morphology.
	Fig. 7. BMP depletion rescues patterning defects in tsg morphants. Expression of region-specific markers were analyzed at stage 25 in uninjected embryos (A, E, I, M, Q, U, Y), and in embryos injected with 10 ng of tsg MO (B, F, J, N, R, V, Z), 20 ng each of BMP4 and BMP7 MOs (C, G, K, O, S, W, AA) or a combination of 10 ng tsg MO and 20 ng of BMP4 and BMP7 MOs (D, H, L, P, T, X, BB). Markers assayed included the midbrain hindbrain boundary marker en-2 (E H), the hindbrain marker krox-20 (I L), the spinal cord marker hoxb 9 (M P), the forebrain markers Xbf-1 (Q T) and eomesodermin (U X) and the ventral marker sizzled (YB). All views are lateral with anterior to the left and dorsal to the top.
	Fig. 8. Chordin overexpression rescues tsg morphants. Expression of the ventral mesodermal marker sizzled was examined in stage 25 uninjected embryos (A), and in sibling embryos injected with intermediate doses of tsg MO (B), or an intermediate dose of tsg MO plus chicken chordin mRNA (C). Expression of the dorsal telencephalon marker eomesodermin was also compared among uninjected embryos (D), sibling embryos injected with an intermediate dose of tsg MO (E), and siblings injected with an intermediate dose of tsg MO and chicken chordin mRNA. All views are lateral with anterior to the left and dorsal to the top.
	Supplementary Fig. S1. Rescue of the chordin phenotype by depletion of BMPs. Embryos were injected with chordin MO (B, F, J), BMP 4 and BMP 7 MOs (C, G, K) or the combination of chordin BMP4, and BMP7 MOs (D, H, L), and compared to uninjected sibling controls (A, E, I). Embryos were assayed by morphology (A), expression of eomesodermin (E), or sizzled (I). Embryos are at st. 258 and viewed laterally, anterior to the left, and dorsal to the top.
	Supplementary Fig. S2. BMP depletion rescues tsg morphants morphologically. Embryos were injected with 40 ng of BMP4 MO (B), 40 ng of BMP7 MO (C), or 20 ng of both BMP4 and BMP7 MOs (D), and compared to uninjected controls (A). All views are lateral with anterior to the left and dorsal to the top.
	Fig. 3. Co-injection of tsg mRNA rescues the tsg morphant phenotype. (A) Alignment of the target sequence for the tsg MO used in this study with the 5′ region of mRNAs for X. tropicalis, X. laevis and Oncorhynchus mykiss (rainbow trout). The translational start site is shown in green, mismatches between O. mykiss and the tsg MO are shown in pink, and mismatches between X. laevis and the tsg MO are shown in blue. (B) Embryos were injected at the two-cell stage with 20 ng tsg MO or 20 ng tsg MO plus 400 pg of O. mykiss tsg mRNA, then assayed at stage 26 for survival and proper blastopore closure. The experiment was repeated with the same result. (C) Embryos were injected with 400 pg O. mykiss tsg mRNA (b, f, j, n, r, v), 20 ng tsg MO (c, g, k, o, s, w), or both (d, h, l, p, t, x) and compared to uninjected sibling control embryos (a, e, i, m, q, u) at stage 11 (a–l), stage 19 (m–t), or stage 26 (u–x) for expression of the molecular markers indicated at left. The view for each marker is indicated at right. For animal and vegetal views, dorsal is to the top while for ventral and lateral views, anterior is to the left.
	twsg1 (twisted gastrulation BMP signaling modulator 1 ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 2 (2-cell), lateral view, anterior left, dorsal up.
	twsg1 (twisted gastrulation BMP signaling modulator 1 ) gene expression in bissected Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 8, animal pole up.
	twsg1 ( twisted gastrulation BMP signaling modulator 1 ) gene expression in Xenopus tropicalis embryo, mid-sagittal section assayed via in situ hybridization, NF stage 9, dorsal right, anterior up.
	twsg1 ( twisted gastrulation BMP signaling modulator 1 ) gene expression in Xenopus tropicalis embryo, mid-sagittal section , assayed via in situ hybridization, NF stage 10.5, dorsal right, anterior up. (curators note: no gene expression at this stage)
	twsg1 (twisted gastrulation BMP signaling modulator 1 ) gene expression in Xenopus tropicalis embryo, mid-sagittal section , assayed via in situ hybridization, NF stage 11, dorsal right, anterior up. (Curators note: no gene expression at this stage)
	twsg1 ( twisted gastrulation BMP signaling modulator 1 ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anterior left, dorsal up.