Branford WW et al. (2000), Regulation of gut and heart left-right asymmetr...

XB-ART-10731

Dev Biol 2000 Jul 15;2232:291-306. doi: 10.1006/dbio.2000.9739.

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Regulation of gut and heart left-right asymmetry by context-dependent interactions between xenopus lefty and BMP4 signaling.

Branford WW , Essner JJ , Yost HJ .

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The Lefty subfamily of TGFbeta signaling molecules has been implicated in early development in mouse, zebrafish, and chick. Here, we show that Xenopus lefty (Xlefty) is expressed both bilaterally in symmetric midline domains and unilaterally in left lateral plate mesoderm and anterior dorsal endoderm. To examine the roles of Xlefty in left-right development, we created a system for scoring gut asymmetry and examined the effects of unilateral Xlefty misexpression on gut development, heart development, and Xnr-1 and XPitx2 expression. In contrast to the unilateral effects of Vg1, Activin, Nodal, or BMPs, targeted expression of Xlefty in either the left or the right side of Xenopus embryos randomized the direction of heart looping, gut coiling, and left-right positioning of the gut and downregulated the asymmetric expression of Xnr-1 and XPitx2. It is currently thought that Lefty proteins act as feedback inhibitors of Nodal signaling. However, this would not explain the effects of right-sided Xlefty misexpression. Here, we show that Xlefty interacts with the signaling pathways of other members of the TGFbeta family during left-right development. Results from coexpression of Xlefty and Vg1 indicate that Xlefty can nullify the effects of Vg1 ectopic expression and that Xlefty is downstream of left-sided Vg1 signaling. Results from coexpression of Xlefty and XBMP4 indicate that XLefty and XBMP4 interact both synergistically and antagonistically in a context-dependent manner. We propose a model in which interactions of Xlefty with multiple members of the TGFbeta family enhance the differences between the right-sided BMP/ALK2/Smad pathway and the left-sided Vg1/anti-BMP/Nodal pathway, leading to left-right morphogenesis of the gut and heart.

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Species referenced: Xenopus
Genes referenced: acvr1 bmp4 gdf1 lefty nodal nodal1 pitx2 tgfb1

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	FIG. 2. Xlefty expression during Xenopus development. (A) Whole-mount in situ localization of Xlefty transcripts at gastrula through tailbud stages. (A) Vegetal pole view, dorsal at top. (B, C, E, J, L, and N) Dorsal view, anterior at left. (D, F, G, K, M, O, P) Left lateral view, anterior at left. (C, D), (E, F), (I, J), and (M, N) show a dorsal and a left lateral view of the same embryo. Stage of embryo(s) depicted is shown at the bottom right. (A) During early gastrulation, Xlefty expression predominantly localized to the dorsal blastopore margin. Note the punctate expression in other regions around the blastopore (arrowheads). (B) As gastrulation proceeded, Xlefty expression became localized to the prospective floor plate and prechordal plate, but was still present around the closing blastopore (arrowheads). (C, D) During early neurulation, expression continued to be localized to the prospective floor plate and prechordal plate (arrows), in addition to expression around the ventrolateral yolk plug (arrowhead). (E, F) As neurulation proceeded, the dorsal midline expression of Xlefty was downregulated centrally (arrows), resulting in an anterior and posterior domain of expression (arrowheads). (G) Following neurulation, Xlefty expression appeared in the posterior midline in the floor plate (arrowhead), notochord (asterisk), and hypochord (arrow). (H) The expression in the floor plate and hypochord remained through tailbud stages (H), while the notochord expression was transient and variable in intensity (G). The floor plate and hypochord expression progressed in a posterior-to-anterior direction (G) and receded in the opposite direction (P; data not shown). At st. 23, unilateral Xlefty expression became apparent in the left lateral plate mesoderm (arrowhead in H), then migrated anteriorly (I), and eventually became localized to the left cardiac field (arrowhead in O). Between st. 24 and st. 25, another unilateral domain of Xlefty expression emerged in the left anterior dorsal endoderm (arrowheads in K, L) and remained into early tailbud stages (K). Variable levels of bilateral posterior endoderm expression (arrow in K) were also present from approximately st. 245 through tailbud stages (K; data not shown).
	FIG. 1. The amino acid sequence predicted from Xlefty is conserved in vertebrate evolution. The amino acid sequences of Xenopus (X) Lefty-a and Lefty-b are compared to zebrafish (z) Lefty1 and 2, chick (c) Lefty, mouse (m) Lefty1 and 2, and human (h) LEFTY A and B. Light gray shading represents conservative amino acid substitutions, and medium gray indicates amino acid identity. Putative cleavage sites, with RXXR consensus sequence, and conserved cysteines, predicted to form the characteristic cysteine knot of the TGFb superfamily, are shown in black. The position of the missing cysteine in the Lefty subfamily is indicated with an arrow.
	FIG. 2. Xlefty expression during Xenopus development. (A–P) Whole-mount in situ localization of Xlefty transcripts at gastrula through tailbud stages. (A) Vegetal pole view, dorsal at top. (B, C, E, J, L, and N) Dorsal view, anterior at left. (D, F, G–I, K, M, O, P) Left lateral view, anterior at left. (C, D), (E, F), (I, J), and (M, N) show a dorsal and a left lateral view of the same embryo. Stage of embryo(s) depicted is shown at the bottom right. (A) During early gastrulation, Xlefty expression predominantly localized to the dorsal blastopore margin. Note the punctate expression in other regions around the blastopore (arrowheads). (B) As gastrulation proceeded, Xlefty expression became localized to the prospective floor plate and prechordal plate, but was still present around the closing blastopore (arrowheads). (C, D) During early neurulation, expression continued to be localized to the prospective floor plate and prechordal plate (arrows), in addition to expression around the ventrolateral yolk plug (arrowhead). (E, F) As neurulation proceeded, the dorsal midline expression of Xlefty was downregulated centrally (arrows), resulting in an anterior and posterior domain of expression (arrowheads). (G) Following neurulation, Xlefty expression appeared in the posterior midline in the floor plate (arrowhead), notochord (asterisk), and hypochord (arrow). (H–P) The expression in the floor plate and hypochord remained through tailbud stages (H–P), while the notochord expression was transient and variable in intensity (G–I). The floor plate and hypochord expression progressed in a posterior-to-anterior direction (G–I) and receded in the opposite direction (P; data not shown). At st. 23, unilateral Xlefty expression became apparent in the left lateral plate mesoderm (arrowhead in H), then migrated anteriorly (I–N), and eventually became localized to the left cardiac field (arrowhead in O). Between st. 24 and st. 25, another unilateral domain of Xlefty expression emerged in the left anterior dorsal endoderm (arrowheads in K, L) and remained into early tailbud stages (K–O). Variable levels of bilateral posterior endoderm expression (arrow in K) were also present from approximately st. 24–25 through tailbud stages (K–P; data not shown).
	FIG. 3. Normal and left–right reversed cardiac orientation in st. 46 Xenopus embryos. (A, B) Ventral views of immunofluorescent hearts, with anterior at the top, stained with a monoclonal antibody against bovine cardiac troponin T. (A) Normal left–right orientation of the Xenopus heart, with the outflow tract (conotruncus; ct) arising from the right anterior aspect of the ventricle (v) and looping to the left. (B) A left–right reversed Xenopus heart, with the outflow tract arising from the left anterior aspect of the ventricle and looping to the right.
	FIG. 4. Normal and left–right reversed gut classes and subclasses in st. 46 Xenopus embryos. (A–L) Ventral views of Xenopus guts, with anterior at the top, as viewed by light microscopy. (A, B, and I), (C, D, and J), (E, F, and K), and (G, H, and L) depict different views of the same gut. The pancreas is marked by an asterisk in A, C, E, and G. (A, C) Normal, right gut coil origins (RO class). (E, G) Reversed, left gut coil origins (LO class). (B, F) Normal, counterclockwise gut coil direction (CCW class). (D, H) Reversed, clockwise gut coil direction. (I) Normal left–right orientation of the Xenopus gut (RO/CCW subclass). (J) Abnormal Xenopus gut with a normal, right coil origin and a reversed, clockwise coil direction (RO/CW subclass). (K) Abnormal Xenopus gut with a reversed, left coil origin and a normal, counterclockwise coil direction (LO/CCW subclass). (L) “Mirror-image,” left–right reversed Xenopus gut (LO/CW subclass).
	FIG. 5. Loss of XPitx2 lateral plate mesoderm expression in Xlefty-injected embryos. (A–D) Whole-mount in situ localization of XPitx2 transcripts at st. 26 in embryos unilaterally injected on the left or right with 25 pg of Xlefty DNA. (A, C, D) Left lateral view, anterior to left. (B) Dorsal view, anterior to left. (A, B) Normal XPitx2 expression in the left lateral plate mesoderm (arrows), as well as in the head and dorsal midline (arrowheads). (C, D) Loss of XPitx2 expression in the left lateral plate mesoderm of embryos injected on the left (C) or right (D) with Xlefty DNA. Note the presence of the other endogenous domains of XPitx2 expression in these embryos (arrowheads).
	FIG. 6. Summary of results from Xlefty DNA injections (25 pg) and Xlefty/BVg1 and Xlefty/XBMP4 DNA co-injections. The percentage of left–right reversals observed for each experimental group is given in parentheses. The arrows indicate an increase or decrease in reversal percentage from the percentage given in the preceding line.