Qiu D et al. (2005), Localization and loss-of-function implicates ci...

XB-ART-1566

Dev Dyn 2005 Sep 01;2341:176-89. doi: 10.1002/dvdy.20509.

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Localization and loss-of-function implicates ciliary proteins in early, cytoplasmic roles in left-right asymmetry.

Qiu D , Cheng SM , Wozniak L , McSweeney M , Perrone E , Levin M .

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Left-right asymmetry is a crucial feature of the vertebrate body plan. While much molecular detail of this patterning pathway has been uncovered, the embryonic mechanisms of the initiation of asymmetry, and their evolutionary conservation among species, are still not understood. A popular recent model based on data from mouse embryos suggests extracellular movement of determinants by ciliary motion at the gastrulating node as the initial step. An alternative model, driven by findings in the frog and chick embryo, focuses instead on cytoplasmic roles of motor proteins. To begin to test the latter hypothesis, we analyzed the very early embryonic localization of ciliary targets implicated in mouse LR asymmetry. Immunohistochemistry was performed on frog and chick embryos using antibodies that have (KIF3B, Polaris, Polycystin-2, acetylated alpha-tubulin) or have not (LRD, INV, detyrosinated alpha-tubulin) been shown to detect in frog embryos only the target that they detect in mammalian tissue. Immunohistochemistry revealed localization signals for all targets in the cytoplasm of cleavage-stage Xenopus embryos, and in the base of the primitive streak in chick embryos at streak initiation. Importantly, several left-right asymmetries were detected in both species, and the localization signals were dependent on microtubule and actin cytoskeletal organization. Moreover, loss-of-function experiments implicated very early intracellular microtubule-dependent motor protein function as an obligate aspect of oriented LR asymmetry in Xenopus embryos. These data are consistent with cytoplasmic roles for motor proteins in patterning the left-right axis that do not involve ciliary motion.

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Species referenced: Xenopus
Genes referenced: actl6a adm dnah11 dnah9 ift88 invs kif3b pkd2 tnfsf10 tuba4b
???displayArticle.antibodies??? alpha and beta-tubulins Ab1 Dnah9 Ab1 Ift88 Ab1 Invs Ab1 Kif3b Ab1 Kif3b Ab2 Pkd2 Ab2

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	Figure 1. Controls and antibody characterization. Sections processed for immunohistochemistry with no primary antibody, no secondary antibody, or with primary antibody pre-incubated with target peptide show no alkaline phosphatase signal in frog embryos (A, sectioned along the AV axis) or chick embryos (B, sectioned across the primitive streak at st. 3). As predicted, the inversin, LRD, and acetylated and detyrosinated alpha -tubulin antibodies correctly label cilia in older embryos (tail epidermis shown with alkaline phosphatase detection (C) or fluorescent secondary antibody, D). Most primary antibodies, such as Kinesin5 (E) reveal symmetrical stain in early embryos. In Western blots, the Polaris antibody reacts with a single band of the predicted size of 95 kDa (F), while the Polycystin-2 antibody reacts with a single band of the predicted size of 110 kDa (G). Red arrows, signal; white arrows, lack of signal; green arrows, additional signal.
	Figure 2. Localization of targets in unperturbed frog embryos. Polaris: (A) section taken perpendicular to the AV axis in the unfertilized Xenopus egg; (B) section taken parallel to the AV axis in an unfertilized egg; (C) section taken parallel to the AV axis in the fertilized egg; (D) in sections taken perpendicular to the AV axis in the 2-cell embryo, one of the two blastomeres shows strong stain near the cell membrane, although the other blastomere occasionally has some stain as well (E). In the 4-cell embryo, all four blastomeres exhibit cell membrane stain (F). Inversin: (G) section taken along the AV axis in the unfertilized egg; (H) section taken perpendicular to the AV axis in the unfertilized egg; (I) signal is seen in the vegetal cortex in fertilized eggs sectioned parallel to the AV axis, and can be detected in the microtubule organizing center (J; brown stain indicated by the green arrow is pigment brought in by the sperm entry trail); (K) section taken perpendicular to the AV axis at the 4-cell stage; (L) section taken along the AV axis at the 8-cell stage, animal pole is upwards. LRD: (M) section taken along the AV axis of the unfertilized egg; (N) section taken along the AV axis of a fertilized egg; (O) section perpendicular to the AV axis taken close to the animal pole reveals staining throughout one of the two blastomeres. P: sections taken through the equator perpendicular to the AV axis show asymmetric staining near the membrane cortex; (Q) some sections taken perpendicular to the AV axis revealed staining between the blastomeres before the completion of the cleavage furrow; (R) sections parallel to the AV axis reveal a rod of signal from the vegetal pole towards the animal pole, in one of the two blastomeres. KIF3B: (S) section along the AV axis of the unfertilized egg; (T) sectioning perpendicular to the AV axis of the fertilized egg reveals staining in the MTOC at nuclear fusion (brown stain is pigment brought in by the sperm entry trail, green arrowhead). Two-cell embryos sectioned perpendicular to the AV axis exhibit signal in the center (U) or in the membrane cortex (V,W) of one blastomere; (X) sections taken perpendicular to the AV axis at the four-cell stage reveal that staining is often absent from one of the four blastomeres on the right side. Polycystin-2: (Y) sections taken along the AV axis of the unfertilized egg show no signal; (Z) section of fertilized egg taken parallel the AV axis; (Aa) section taken perpendicular to the AV axis at the 2-cell stage; (Ab) section taken parallel to the AV axis at the 2-cell stage; (Ac) section taken across the AV axis at the 4-cell stage. alpha -tubulin: (Ad) antibodies recognizing all tubulin subunits stain throughout the cytoplasm of sections taken across the AV axis of the 2-cell embryo. Acetylated alpha -tubulin: (Ae) sections of fertilized and unfertilized eggs along the AV axis revealed spots of signal at the equator; (Af) sections of 2-cell embryos taken perpendicular to the AV axis revealed signal at the membrane cortex in one of two blastomeres; (Ag) section taken near the animal pole, across the AV axis of the 4-cell embryo. Ah: section taken near the vegetal pole, across the AV axis of the 4-cell embryo. Detyrosinated alpha -tubulin: (Ai) section of an unfertilized egg along the AV axis; (Aj) section taken across the AV axis of embryos at the 4-cell stage; (Ak) sections taken parallel to the AV axis at the 2 cell stage (dashed line indicates embryonic midline); (Al) section taken across the AV axis of a 4-cell embryo. Am: In each panel, yellow ovals with their long axis oriented left to right indicate that the section was taken across the AV axis; yellow ovals with their long axes oriented vertically indicate that the section was taken along the AV axis, with the animal pole being upward. Red arrows, signal; white arrows, lack of signal; green arrows, additional signal.
	Fig. 4. Dependence of localization patterns on actin and tubulin organization. Microtubules were targeted by nocodazole from fertilization, as described in the Experimental Procedures section. Actin filaments were targeted by latrunculin from fertilization, as described in the Experimental Procedures section. KIF3B: sections taken perpendicular to the AV axis at the 2-cell stage in control embryos (A), and those in which microtubules (B) or actin filaments (C) were targeted. Polycystin-2: sections taken perpendicular to the AV axis at the 2-cell stage in control embryos (D), and those in which microtubules (E) or actin filaments (F) were targeted. Acetylated -tubulin: sections taken perpendicular to the AV axis at the 4-cell stage in control embryos (G), and those in which microtubules (H) or actin filaments (I) were targeted. LRD: sections taken parallel to the AV axis at the 2-cell stage in control embryos (J), and those in which microtubules (K) or actin filaments (L) were targeted. Polaris: sections taken perpendicular to the AV axis at the 4-cell stage in control embryos (M), and those in which microtubules (N) or actin filaments (O1â€“O4) were targeted. Inversin: sections taken parallel to the AV axis at the 8-cell stage in control embryos (P) or embryos in which microtubules (Q) or actin filaments (R) were targeted; sections taken perpendicular to the AV axis at the 4-cell stage in control embryos (S) and those in which microtubules (T) were targeted. U: In each panel, yellow ovals with their long axis oriented left to right indicate that the section was taken across the AV axis; yellow ovals with their long axes oriented vertically indicate that the section was taken along the AV axis, with the animal pole being upward. Red arrows, signal; white arrows, lack of signal; green arrows, additional signal.