Muller JK et al. (2003), Left-right asymmetric morphogenesis in the Xeno...

XB-ART-4322

Dev Dyn 2003 Dec 01;2284:672-82. doi: 10.1002/dvdy.10415.

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Left-right asymmetric morphogenesis in the Xenopus digestive system.

Muller JK , Prather DR , Nascone-Yoder NM .

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The morphogenetic mechanisms by which developing organs become left-right asymmetric entities are unknown. To investigate this issue, we compared the roles of the left and right sides of the Xenopus embryo during the development of anatomic asymmetries in the digestive system. Although both sides contribute equivalently to each of the individual digestive organs, during the initial looping of the primitive gut tube, the left side assumes concave topologies where the right side becomes convex. Of interest, the concave surfaces of the gut tube correlate with expression of the LR gene, Pitx2, and ectopic Pitx2 mRNA induces ectopic concavities in a localized manner. A morphometric comparison of the prospective concave and convex surfaces of the gut tube reveals striking disparities in their rate of elongation but no significant differences in cell proliferation. These results provide insight into the nature of symmetry-breaking morphogenetic events during left-right asymmetric organ development.

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Species referenced: Xenopus laevis
Genes referenced: pitx2 stom tnf

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	Fig. 1. Contributions of the left and right lateral plate mesoderm to the digestive organs. A: Diagram of bilateral labeling experiment. Individual squares are designated with a letter and number to indicate their relative dorsoventral (1� 4) and anteroposterior (A�E) position in the lateral plate mesoderm (LPM). D, dorsal; V, ventral; A, anterior; P, posterior. B: The fate of left and right squares A3. The region of the gut shown in brightfield is boxed in the model of the stage 45 gut (upper left). The arrow in the box indicates the anterior view of the slightly rotated and flat-mounted foregut organs shown in the photomicrographs. The fluorescence of the left (DiI) and right (PKH) dyes are shown individually and merged. The orientation of specific organs is indicated in the diagram in the upper right. Faint signals in the intestine are autofluorescence. R, right; L, left; esoph, esophagus; panc, pancreas; stom, stomach; intest, intestine. C: Comparison of the frequency of contribution of the left and right LPM to the digestive system, presented as the percentage of cases in which individual left (orange) and right (green) labeled squares contributed to the different digestive organs at stage 45�46. The coordinates of each labeled square are indicated above and to the left of the graphs, with individual organs indicated below. dp, dorsal pancreas; vp, ventral pancreas; liv, liver; eso, esophagus; stom, stomach; duod, duodenum; int, intestine.
	Fig. 2. Symmetry breaking events in Xenopus digestive organ morphogenesis. A: Three- dimensional cartoons of the key symmetry-breaking morphogenetic events in isolated gut tubes at stages 38�41 (first looping event), 42�43 (second looping event), and 44�45 (intestinal coiling), presented in both ventral and dorsal views. Large scale morphogenetic movements are indicated by black arrows. Open arrowheads indicate the key convex surfaces and filled arrowheads the key concavities of the forming gastroduodenal (GD, purple) and midgut (MG, blue) loops. Asterisks indicate the apex of the MG loop as ascertained by fate mapping studies (data not shown). Final coil origin and coil direction are indicated at stage 45. For clarity, some additional coiling of the hindgut has been omitted from the dorsal view of the stage 45 model. See text for details. fore, foregut; mid, midgut; hind, hindgut; eso, esophagus; st, stomach; duod, duodenum. B: Models of the topologic distribution of the descendants of the left (orange) and right (green) lateral plate mesoderm within the gut tube at stages 40�45. Key concave and convex surfaces are indicated by filled and open arrowheads, respectively, as in A.
	Fig. 3. The role of Pitx2 expression in gastroduodenal (GD) looping. A: At stage 24, Pitx2 is expressed in a defined region of the left (arrow) but not right lateral plate mesoderm (LPM), as indicated by RNA in situ hybridization (top panels). Dye-labeled cells within the Pitx2- expressing region in the left LPM will populate the concavity of the GD loop by stage 40 � 41, while cells from the right LPM occupy a convex surface. Schematic drawings of the left and right sides of a stage 41 gut are shown in the bottom panels. d, duodenum; dp, dorsal pancreas; e, esophagus; i, intestine; liv, liver; LL, left lung; RL, right lung; s, stomach; vp, ventral pancreas. B: Expression profile by RNA in situ hybridization of Pitx2 (arrows) in left and right LPM at stages 28�40. C: Isolated gut tubes from heptanol-treated embryos were fixed at stage 40 and subjected to RNA in situ hybridization with a probe for Pitx2. The positions of the GD loop concavities are indicated by arrows. norm, normal; rev, reversed; sym, symetrical.
	Fig. 4. Ectopic Pitx2 mRNA induces concavities in the gut tube. Embryos were injected with fluorescein dextran (FLDx) alone or in combination with Pitx2 mRNA. At sibling stage 42� 44, embryos were photographed in ventral view using brightfield (bright) and fluorescence (fluor) microscopy, after which guts were isolated and subjected to RNA in situ hybridiza- tion to confirm the presence of ectopic Pitx2 mRNA (Pitx2 ISH). Schematic diagrams of the guts are shown, with individual organs labeled, and ectopic Pitx2 expression domains and areas of abnormal topology indicated by purple arrows. duod, duodenum; int, intestine; liv, liver; pan, pancreas; stom, stomach. A: Control FLDx-injected embryo with normal gut morphology and endogenous Pitx2 expression. Note that Pitx2 is normally expressed bilaterally in the hindgut but only on the left side of the midgut (arrow), in the prospective concavity of the nascent midgut (MG) loop. B: Embryo expressing ectopic Pitx2 mRNA in anterior right foregut, with a small, ectopic right-side concavity (arrow) in the biconcave gastroduodenal (GD) loop and heterotaxic positioning of foregut organs. C: Embryo expressing ectopic Pitx2 mRNA on the right side of the duodenum, with a completely reversed GD loop and foregut organs. D: Embryo expressing ectopic Pitx2 mRNA on the right side of the midgut. A large midgut concavity consumes the right side of the gut tube, reversing the MG loop, and distorting the GD loop. (The Pitx2 ISH expression pattern is shown in right-side view.)
	Fig. 5. The left (L) and right (R) sides of the gut elongate at different rates, with no dif- ference in proliferation. A: The number of mitotic cells on the left and right sides of isolated stage 41, 42, and 43 gut tubes was detected with an anti-phosphohistone H3 antibody. The number of mitotic cells within a fixed square area on each side was av- eraged over multiple samples for each stage. Relative proliferation was deter- mined by arbitrarily setting the number of mitotic cells on the right side to a value of 1. B: The lengths of the left and right sides of the midgut (indicated by the orange and green outlines; photos are not to scale) of stage 41, 42, and 43 embryos were digitally measured. Relative mean lengths were de- termined by arbitrarily setting the right side mean to a value of 1. For comparison, the relative mean lengths of the entire left and right sides of the full gut tube were also measured.