PMC

Lgl2 and Rab11a control E-cadherin accumulation at KV cell junctions. (A-D) Optical sections showing E-cadherin immunostaining in KV cells of Tg(dusp6:memGFP) embryos. GFP (green arrows) and E-cadherin (red arrows) accumulate at lateral membranes in control (A), Rab11a MO^low (B) and Lgl2 MO^low (C) embryos. E-cadherin membrane staining was reduced in Rab11a MO^low + Lgl2 MO^low embryos (D). Scale bars: 20 μm. (E) Quantification of E-cadherin staining along lateral KV membranes. Average fluorescence intensities from multiple experiments were normalized to controls. Error bars indicate s.d. n, number of embryos analyzed. ^*P<0.05.

Lgl2 localizes to basolateral membranes in KV cells and E-cadherin is disrupted in Lgl2 knockdown embryos. (A) Fluorescence immunostaining using Lgl2 antibodies showed Lgl2 localized at basolateral membranes of KV cells and was excluded from the apical domain enriched with phalloidin staining of filamentous actin (F-Actin). (B) Lgl2:EYFP fusion protein also localized at basolateral membranes of KV cells and near E-cadherin staining. (C,D) In MO control Tg(dusp6:memGFP) embryos, E-cadherin localized at lateral membranes (C), whereas Lgl2 MO knockdown reduced membrane accumulation of E-cadherin (D). Boxes indicate enlarged regions shown in the individual channels. Arrows point out representative lateral membranes. Scale bars: 20 μm.

Working model for how Lgl2 regulates development of ciliated organs. Depletion of Lgl2 disrupted both lumen formation and cilia formation in organs with motile cilia. Our results indicate that Lgl2 interacts with Rab11a-mediated vesicle trafficking to promote E-cadherin accumulation at junctions between ciliated cells in Kupffer’s vesicle. Previous studies have shown that E-cadherin is necessary for Kupffer’s vesicle formation. Genetic interaction studies between Lgl2 and Rab11a revealed that cilia phenotypes are separable from lumen defects, indicating a second role for Lgl2 regulating cilia formation. Whether Lgl2 interacts with vesicle trafficking during ciliogenesis remains an open question.

Lgl2 depletion disrupts KV lumen and cilia formation. (A,B) Ciliated KV cells were labeled by acetylated tubulin immunostaining (red) and GFP expression (green) in Tg(sox17:GFP) embryos injected with control (A) or Lgl2 (B) MO. Scale bars: 20 μm. (C-E) KV cilia number (C), KV cilia length (D) and KV lumen size (E) were significantly reduced in Lgl2 MO embryos compared with MO control and uninjected controls. lgl2 mRNA partially rescued each of these defects. (F) The number of KV cells was similar among control and Lgl2 MO embryos in Tg(dusp6:memGFP) and Tg(sox17:GFP) transgenic lines. Error bars indicate s.d. n, number of embryos analyzed. ^*P<0.05.

Genetic interaction between Lgl2 and Rab11a regulates KV lumen formation uncoupled from ciliogenesis. (A,B) Images of GFP-labeled KV cells were used to determine KV lumen volumes (pseudocolored yellow) in live Tg(sox17:GFP) embryos (A) and acetylated tubulin antibodies were used to stain KV cilia (B) in MO control embryos and embryos injected with sub-optimal Rab11a MO^low, Lgl2 MO^low or co-injected with Rab11a MO^low + Lgl2 MO^low. The dashed circle outlines the approximate boundary of KV lumen. Scale bars: 20 μm. (C) Average volume of KV lumen at the 8-somite stage. (D,E) Analysis of KV cilia number (D) and length (E) showed that KV cilia were not significantly affected in Rab11a MO^low + Lgl2 MO^low embryos. Error bars indicate s.d. n, number of embryos analyzed. ^*P<0.05.

Lgl2 is required for normal left-right asymmetry. (A) Asymmetric heart looping at 2 dpf visualized in Tg(cmlc2:GFP) transgenic embryos that express GFP in the heart. In control embryos, the heart (black arrow) looped to the right, whereas Lgl2 MO knockdown often resulted in reversed or no heart looping (red arrows). (B) Percentage of embryos with heart-looping defects. Injecting a lower dose of Lgl2 MO-1 revealed that looping defects are dose dependent, and co-injecting lgl2 mRNA partially rescued Lgl2 MO-1 and Lgl2 MO-2 heart defects. Injecting lgl2 mRNA alone also disrupted heart looping. (C) RNA in situ hybridization analysis of spaw expression, which was detected in left lateral plate mesoderm (LPM) (arrowhead) and bilaterally in the tail (arrows) at 14 hpf in controls. Lgl2 MO embryos showed disrupted LPM spaw expression, including right-sided, bilateral and absent expression (red arrowheads). (D) Analysis of LPM spaw expression in control and Lgl2 MO embryos. n, number of embryos analyzed. L, left; R, right.

Lgl2 controls development of ciliated organs. (A-H) Whole-mount RNA in situ hybridizations. At the 2-cell stage, antisense lgl2 probes show that lgl2 mRNA is maternally supplied (A). Control lgl2 sense probes showed little background staining (B). lgl2 expression localized in the enveloping layer and dorsal forerunner cells (DFCs) at the 80% epiboly stage (C) and was prominently expressed in Kupffer’s vesicle (KV) at the 2-3 somite(s) stages (D,E). At 24 hpf, lgl2 is highly expressed in the ciliated nasal placodes (np), otic vesicles (ov) and pronephric ducts (pnd) (arrows in F). G and H show higher magnification images of lgl2 expression in the nasal placode and otic vesicle (G) and pronephric duct (H). (I-L) Embryo morphology at 2 dpf. Control embryos (I) had a straight body, whereas Lgl2 MO-1 (J) and Lgl2 MO-2 (K) embryos often showed a curved body. Lgl2 MO embryos also developed hydrocephalus (arrows in J-L) and in some cases had additional severe axial defects (L). (M) Percentage of embryos with body axis defects. n, number of embryos analyzed. (N-S) Fluorescence immunostaining using aPKC antibodies (red) to mark apical membranes of epithelial cells and acetylated tubulin antibodies (green) to label cilia revealed defects in the development of ciliated epithelia in KV (N,O), otic vesicles (P,Q) and pronephric ducts (R,S) in Lgl2 MO embryos (O,Q,S) relative to controls (N,P,R). White boxes indicate areas enlarged in ‘Merge & Zoom’ panels. Scale bars: 20 μm.