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Fig. 4. Microtubule-localized Hmmr determines neural cell polarity. (A) hmmr gene expression in the stage (st.) 16 neural plate. Whole mount in situ hybridization (anterior view). (A′) Transversal histological section as indicated in (A). Arrowheads point to expression in deep neural cells, delineated in white. (B-E) Hmmr protein co-localizes with microtubules (MT) in st. 21 neural cells. (B) Endogenous Hmmr (red) localizes to spindle apparatus in mitotic cells in a dotted pattern. (C) Over-expressed full length Hmmr (hmmr FL; red) localizes with MTs in a pattern typical for over-expressed MAPs. (D) Endogenous Hmmr (red) localizes along MT fibers (arrowheads) and in basal (b) MT lattice detected by anti-Tuba4a antibody (green) as well as to nuclei. (E) Endogenous Hmmr (red) localizes close to MT plus ends apically (a; E1) and basally (E2) as visualized by co-staining for EB1 (Mapre1, green). (F, G) Reduction of Hmmr in hmmr MO-injected cells. Targeted cells identified by lineage tracer (LT) fluorescence (F); LT-positive field of cells indicated by green line in (F’). Quantification and statistical analysis of Hmmr fluorescence, n= eight embryos (G). (H-K, H’-K’) Loss of neural cell polarity in hmmr morphants is rescued by co-expression of FL mRNA. Comparison of cell shape (indicated with white dashed lines) and MT organization (Tuba4a, red) in wildtype (H, H’), morphant (I, I’), hmmr FL (J, J’) and hmmr δN (K, K’) rescued specimens; frequency of phenotypes indicated in each panel. Insets in (I-K) demonstrate LT fluorescence in respective area. Arrows in (H’-K’) indicate elogation / rounding of nuclei. Scale bars: 5 µm. d, dorsal; l, left; r, right, v, ventral. |
