Petri N et al. (2022), Abnormal left-right organizer and laterality de...

XB-ART-59443

PLoS One 2022 Jan 01;1711:e0275164. doi: 10.1371/journal.pone.0275164.

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Abnormal left-right organizer and laterality defects in Xenopus embryos after formin inhibitor SMIFH2 treatment.

Petri N , Nordbrink R , Tsikolia N , Kremnyov S .

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Left-right symmetry breaking in most studied vertebrates makes use of so-called leftward flow, a mechanism which was studied in detail especially in mouse and Xenopus laevis embryos and is based on rotation of monocilia on specialized epithelial surface designated as left-right organizer or laterality coordinator. However, it has been argued that prior to emergence of leftward flow an additional mechanism operates during early cleavage stages in Xenopus embryo which is based on cytoskeletal processes. Evidence in favour of this early mechanism was supported by left-right abnormalities after chemical inhibition of cytoskeletal protein formin. Here we analyzed temporal dimension of this effect in detail and found that reported abnormalities arise only after treatment at gastrula-neurula stages, i.e. just prior to and during the operation of left-right organizer. Moreover, molecular and morphological analysis of the left-right organizer reveals its abnormal development. Our results strongly indicate that left-right abnormalities reported after formin inhibition cannot serve as support of models based on early symmetry breaking event in Xenopus embryo.

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Species referenced: Xenopus laevis
Genes referenced: dand5 fmn1 nodal1 pitx2 sox17a sox17b tekt2
GO keywords: cytoskeleton [+]

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	Fig 1. Molecular left-right patterning after formin inhibition. (A) Scheme of time course of chemical treatment and stages of LR analysis. (B, Bâ) Formin inhibitor SMIFH2 had no effect on nodal1 expression patterns in the embryos treated during cleavage (N = 3) and reduced the proportion of left-sided nodal1 expression in the embryos treated during gastrulation and neurulation (N = 6, p = 0,00000313 for 10Î¼M SMIFH2). (B) Summary of experimental data. (Bâ) Representative images of formin-modulated stage 28 tailbud embryos displaying left-sided and abnormal (right, absent and bilateral) expression of nodal1, ventral view. (C, Câ) Formin inhibitor SMIFH2 had no effect on pitx2 expression patterns in the embryos treated during cleavage (N = 4) and reduced the proportion of left-sided pitx2 expression in the embryos treated during gastrulation and neurulation (N = 7, p = 0,0005445 for 10Î¼M SMIFH2). (C) Summary of experimental data. (Câ) Representative images of formin-modulated stage 28 tailbud embryos displaying left-sided and abnormal (right, absent and bilateral) expression of pitx2, ventral view. A, anterior; L, left; P, posterior; R, right. ***, p-value<0.001 compared with the DMSO control, two-proportions z-test with Bonferroni correction; N, number of experiments. Numbers at the base of columns represent number of analyzed embryos.
	Fig 2. Visceral organ situs after formin inhibition. (A) Representative images of formin-modulated stage 46 tadpoles displaying normal organ situs (ss), situs inversion (si), or heterotaxia (ht), as determined by the direction of heart looping (outlined by dashed red line) and gut coiling (outlined by dashed violet line), ventral view. (B) Formin inhibitor SMIFH2 had no effect on organ situs in the tadpoles treated during cleavage (N = 5) and reduced the proportion of tadpoles with normal organ laterality after treatment with 10 Î¼M SMIFH2 during gastrulation and neurulation (N = 5). a, anterior; ht, heterotaxia; l, left; p, posterior; r, right; si, situs inversus; ss, situs solitus. Cyan arrows indicate heart ventricle, yellow arrows indicate truncus arteriosus. ***, p-value = 0,00006552 compared with the DMSO control, two-proportions z-test with Bonferroni correction; N, number of experiments. Numbers at the base of columns represent number of analyzed embryos.
	Fig 3. Formin inhibition at gastrula and neurula stages results in abnormal molecular patterning of GRP. (A-Bâ) Representative images of molecular patterning in the GRP. Dorsal explants of stage 18 neurula embryos after control treatment (A,B) and SMIFH2 administration (Aâ,Bâ) at stages 10â18. In situ hybridisation was performed with probes specific for sox17 (A,Aâ) and tekt2 (B,Bâ). (C and Câ) Representative images of nodal1 expression pattern in the GRP. Dorsal explants of stage 18 neurula embryos after control or SMIFH2 treatment at stages 10â18. (D and Dâ) Histological sections through GRP demonstrate the position of nodal1-positive cells in the prospective somitic mesoderm. Arrows indicate areas of superficial nodal1-positive cells.
	Fig 4. Formin inhibition at gastrula and neurula stages results in morphology defects of left-right organizer at stage 18. (A) Representative scanning electron microscopy images of GRP after exposure to various concentrations of formin inhibitor. (B) Representative histological sections through the GRP after exposure to various concentrations of formin inhibitor. Blue color indicates ectoderm, redânotochord and hypochord, violetâsomitic mesoderm, yellowâendoderm. (C) Cilia polarization, cilia length and percentage of ciliated cells (ciliation rate) in embryos treated with DMSO and SMIFH2 (5 Î¼M and 10 Î¼M). Measurements were performed in notochordal GRP of 11, 14 and 9 embryos respectively (number of analysed cells = 303, 453 and 234 respectively). The apparent decrease of number of ciliated cells is non-significant (p-value = 0,295 for 10 Î¼M, t-test with Bonferroni correction).

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Abe, The development of CRISPR for a mollusc establishes the formin Lsdia1 as the long-sought gene for snail dextral/sinistral coiling. 2019, Pubmed