Lohr JL et al. (1997), Left-right asymmetry of a nodal-related gene is...

XB-ART-16700

Development 1997 Apr 01;1248:1465-72. doi: 10.1242/dev.124.8.1465.

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Left-right asymmetry of a nodal-related gene is regulated by dorsoanterior midline structures during Xenopus development.

Lohr JL , Danos MC , Yost HJ .

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Development of asymmetry along the left-right axis is a critical step in the formation of the vertebrate body plan. Disruptions of normal left-right patterning are associated with abnormalities of multiple organ systems, including significant congenital heart disease. The mouse nodal gene, and its homologues in chick and Xenopus, are among the first genes known to be asymmetrically expressed along the left-right axis before the development of organ asymmetry. Alterations in the expression pattern of mouse nodal and the chick homologue (cNR-1) have been associated with defects in the development of left-right asymmetry and cardiac looping (Levin, M., Johnson, R. L., Stern, C. D., Kuehn, M. and Tabin, C. (1995) Cell 82, 803-814; Collignon, J., Varlet, I. and Robertson, E. J. (1996) Nature 381, 155-158; Lowe, L. A., Supp, D. M., Sampath, K., Yokoyama, T., Wright, C. V. E., Potter, S. S., Overbeek, P. and Kuehn, M. R. (1996) Nature 381, 158-161). Here, we show that the normal expression patterns of the Xenopus nodal-related gene (Xnr-1) are variable in a large population of embryos and that Xnr-1 expression is altered by treatments that perturb normal left-right development. The incidence of abnormal Xnr-1 expression patterns correlates well with cardiac reversal rates in both control and experimentally treated Xenopus embryos. Furthermore, dorsal midline structures, including notochord and/or hypochord and neural floorplate, regulate Xnr-1 expression prior to the specification of cardiac left-right orientation by repression of Xnr-1 expression in the right lateral plate mesoderm during closure of the neural tube. The correlation of Xnr-1 expression and orientation of cardiac looping suggests that Xnr-1 is a component of the left-right signaling pathway required for the specification of cardiac orientation in Xenopus, and that dorsal midline structures normally act to repress the signaling pathway on the right side of the embryo.

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Species referenced: Xenopus laevis
Genes referenced: nodal nodal1 not

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	Fig. 1. Xnr-1 expression in control embryos, assayed by whole-mount RNA in situ hybridization. Purple color indicates expression of Xnr-1 RNA. (A) Expression in the left lateral middle and posterior region of stage 21 embryo (dorsal view). (B) Left lateral view of Xnr-1 expression pattern in the same stage 21 embryo. (C) Dorsal view of a stage 24 embryo, and (D) left lateral view of the same embryo. Xnr-1 is expressed in the left anterior lateral region, sweeping from the somites to the cardiac primordia along the dorsoventral axis. Posteriorly, Xnr-1 is expressed just to the left of somites. (E) Xnr-1 expression is apparent in the left lateral plate mesoderm, in a transverse section through stage 24 embryo. Scale bar, 0.2 mm. (F) Xnr-1 expression becomes limited to the left lateral plate mesoderm, fading posteriorly, in a stage 26 embryo. Arrows indicate Xnr-1 expression. Anterior at top of page except in E, where d, dorsal; v, ventral; l, left; r, right.
	Fig. 2. Xnr-1 expression patterns in UV-treated embryos is dependent on dorsoanterior development as scored by the Dorsoanterior Index (Kao and Elinson, 1988; Danos and Yost, 1995). As dorsoanterior development was diminished, the frequency of normal (left-sided, black bars) Xnr-1 expression patterns decreased. The predominant classes of aberrant Xnr-1 expression were bilateral expression (hatched bars) and no expression (vertical lined bars). For each DAI, DAI 5 (n=21), DAI 4 (n=16), DAI 3 (n=35), DAI 2 (n=25), DAI 0-1 (n=12), embryos were fixed and analyzed for Xnr-1 RNA expression patterns by whole-mount in situ hybridization.
	Fig. 3. Representative Xnr-1 expression patterns in Xenopus embryos ventralized by UV irradiation. (A) Normal left expression of Xnr-1 in a DAI 5 embryo. (B) Bilateral expression in the top embryo and left expression in the lower embryo, both at DAI 4. (C) Bilateral expression in two embryos at DAI 3. (D) Bilateral expression in embryos at DAI 2. Dorsal views, anterior at the top in all panels.
	Fig. 4. Xnr-1 expression pattern in Xenopus embryos after extirpation of midline dorsoanterior structures depends on the stage of extirpation. (A) Bilateral Xnr-1 expression in a stage 24 embryo from which dorsal cells had been extirpated at stage 15. (B) Normal left-sided Xnr-1 expression in a stage 24 embryo from which dorsal cells had been extirpated at stage 20. Dorsal view, anterior at top in both panels. (C) Xnr-1 expression patterns are predominately bilateral (red bars) in embryos from which dorsal midline cells were extirpated at stage 15, either in broad extirpations (75% bilateral, s.d.=8%; n=24) or in narrow extirpations (72% bilateral, s.d.=8%; n=27) focused on notochord and prospective floorplate (see Methods), but normal (black bars) in embryos from which dorsal midline cells were extirpated at stage 20 (95% left only, s.d.=8%; n=26). Untreated control embryos from the same experiments demonstrated Xnr-1 expression on the left only in 86% of cases (s.d.=11%; n=52). Results from broad extirpations were statistically identical to those from narrow extirpations at stage 15 (z=0.375, P>0.16) and were significantly different from those at stage 20 (z=5.75, P<0.001).
	Fig. 5. Xnr-1 asymmetric expression is specified in stage 20 lateral plate mesoderm. (A) Left lateral mesoderm explanted at stage 15 (B) Right lateral mesoderm explanted at stage 15. (C) Left lateral mesoderm explanted at stage 20. (D) Right lateral mesoderm explanted at stage 20. Note no Xnr-1 expression in (D) and strong Xnr-1 expression in (A-C). All explants were cultured until sibling embryos were at stage 24, and then fixed and assayed in parallel by in situ hybridization for Xnr-1 expression. Scale bar, 1 mm.
	Fig. 6. Comparison of predicted cardiac reversal rates (based on the equation as defined in the text) versus observed cardiac reversals in control embryos (diamonds), UV-treated embryos (DAI 5-DAI 3) (circles), and midline extirpations (noto) (triangles). The predicted cardiac reversal rates in the data set correlate positively with the observed cardiac reversal rates as defined by the equation y=0.866x - 0.012, r2=0.839.