Manojlovic Z et al. (2017), La-related protein 6 controls ciliated cell dif...

XB-ART-54515

Cilia 2017 Jan 01;6:4. doi: 10.1186/s13630-017-0047-7.

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La-related protein 6 controls ciliated cell differentiation.

Manojlovic Z , Earwood R , Kato A , Perez D , Cabrera OA , Didier R , Megraw TL , Stefanovic B , Kato Y .

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BACKGROUND: La-related protein 6 (LARP6) is an evolutionally conserved RNA-binding protein. Vertebrate LARP6 binds the 5' stem-loop found in mRNAs encoding type I collagen to regulate their translation, but other target mRNAs and additional functions for LARP6 are unknown. The aim of this study was to elucidate an additional function of LARP6 and to evaluate the importance of its function during development. METHODS: To uncover the role of LARP6 in development, we utilized Morpholino Oligos to deplete LARP6 protein in Xenopus embryos. Then, embryonic phenotypes and ciliary structures of LAPR6 morphants were examined. To identify the molecular mechanism underlying ciliogenesis regulated by LARP6, we tested the expression level of cilia-related genes, which play important roles in ciliogenesis, by RT-PCR or whole mount in situ hybridization (WISH). RESULTS: We knocked down LARP6 in Xenopus embryos and found neural tube closure defects. LARP6 mutant, which compromises the collagen synthesis, could rescue these defects. Neural tube closure defects are coincident with lack of cilia, antenna-like cellular organelles with motility- or sensory-related functions, in the neural tube. The absence of cilia at the epidermis was also observed in LARP6 morphants, and this defect was due to the absence of basal bodies which are formed from centrioles and required for ciliary assembly. In the process of multi-ciliated cell (MCC) differentiation, mcidas, which activates the transcription of genes required for centriole formation during ciliogenesis, could partially restore MCCs in LARP6 morphants. In addition, LARP6 likely controls the expression of mcidas in a Notch-independent manner. CONCLUSIONS: La-related protein 6 is involved in ciliated cell differentiation during development by controlling the expression of cilia-related genes including mcidas. This LARP6 function involves a mechanism that is distinct from its established role in binding to collagen mRNAs and regulating their translation.

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Species referenced: Xenopus
Genes referenced: arl13b b9d1 cetn2 col1a1 fkbp3 foxj1 gmnc hes5 larp6 mab21l3 maml1 mcc mcidas notch1 odad4 odc1 rfx2 rfx4 rfx7 sox2 strap tsc1 tuba4b tubg1
GO keywords: cilium organization
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	Fig. 1 LARP6 morphants have neural tube closure defects. a Temporal expression profiles of LARP6 gene. b Spatial expression profiles of LARP6 gene. c LARP6 MO (L6MO) but not control MO (conMO) depleted X. laevis LARP6 protein (xlL6-2HA) in late neurula stage embryos. conMO or L6MO was injected with xlL6-2HA mRNA, and LARP6 was detected by immunoblotting with anti-HA antibody. d Human LARP6 (HA-hL6) was not knocked down by L6MO in stage 10 or 20 embryos. e Depletion of LARP6 resulted in neural tube closure defects. conMO, L6MO (15 ng/blastomere) and/or HA-hL6 mRNA (250 pg/blastomere) were injected into two dorsal blastomeres of 4-cell stage embryos (between animal pole and marginal zone). Images are dorsal views of late neurula stage embryos. f The summary of rescue experiments: the C-terminus of LARP6 is important for neural tube closure. LAM La motif, RRM RNA recognition motif, SUZC SUZ-C motif. n indicates number of embryos examined. 5′SL binding shows the interaction between LARP6 and the 5′ stem-loop region of collagen RNA. F phenylalanine, A alanine, E glutamic acid, a anterior, p posterior, b brain, e eye. ODC and β-actin were used as a loading control
	Fig. 2 LARP6 is required for ciliogenesis in the neural tube. No cilia are detected in the neural tube of LARP6 morphants. conMO, L6MO and/or HA-hL6 mRNA were injected into two dorsal blastomeres of 4-cell stage embryos and fixed at stage 20. Cilia were stained by anti-acetylated α-tubulin antibody. Following staining, 100-μm transverse sections of specimens were prepared and images were taken by confocal microscopy. memRFP mRNA was injected as a lineage tracer. White dots indicate the lumen of the neural tube. The scale bar represents 10 μm. n indicates number of embryos examined
	Fig. 3 LARP6 is necessary for MCC differentiation. a Absence of cilia on epidermal MCC of LARP6 morphants. L6MO (15 ng/blastomere) with memRFP mRNA or conMO with memGFP mRNA were injected into the right side or the left side, respectively. Embryos were fixed at stage 25. Cilia were stained with an anti-acetylated tubulin antibody. Following staining, lateral explants were prepared from embryos and images from the right (L6MO) or left (conMO) lateral side were taken by confocal microscopy. b Rescue experiments with hL6 or δ1-478 mRNA in LARP6 morphant MCCs. c–e B9D1, γ-tubulin and centrin proteins were not detected at the LARP6 morphant epidermis. Axonemes and ciliary transition zones or basal bodies were stained by an anti-acetylated tubulin and anti-B9D1, anti-γ-tubulin or anti-centrin antibody, respectively. Following staining, lateral explants were prepared from embryos and images were taken by confocal microscopy. Arrowheads indicate examples of centrioles. f The depletion of LARP6 did not change total amounts of γ-tubulin and centrin proteins in embryos. g The localization of LARP6 in MCCs. N nucleus. An Arrowhead indicates an example of a cilium. Scale bars 20 µm in (a), 10 µm in (b–e, g). n indicates number of embryos examined
	Fig. 4 LARP6 regulates the expression of mcidas in ciliogenesis. a The depletion of LARP6 blocked ciliogenesis in a Notch-independent manner. dn-mam1 RNA and/or L6MO with memRFP mRNA or conMO with memGFP mRNA were injected into the right side or the left side, respectively. Embryos were fixed at stage 25. Cilia were stained with an anti-acetylated tubulin antibody. Following staining, lateral explants were prepared from embryos and images were taken by confocal microscopy. The scale bar represents 20 μm. b Quantification of number of MCCs from a. Three embryos were randomly chosen from each of three independent experiments. ≤0.05, ***≤0.0001. c mcidas expression in LARP6 morphants at stage 14 by WISH. Images from the left (conMO) or right (L6MO) lateral side were taken from the same embryo. Red-gal signals indicate L6MO injected side. a anterior, p posterior, d dorsal, v ventral. d Rescue experiments with mcidas mRNA (500 pg/blastomere) in LARP6 morphant MCCs. e LARP6 does not control the expression of mab21-l3 and gmnc genes. L6MO was injected into two blastomeres of 2-cell stage embryos and RNAs were isolated at stage 13 for semi-quantitative RT-PCR. f The model of LARP6 function in ciliogenesis. LARP6 controls the expression of mcidas in a Notch-independent manner. n indicates number of embryos examined
	Figure S1. LARP6 controls the expression of cilia-related genes in a Notch-independent manner.
	larp6 (La ribonucleoprotein domain family member 6 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 9, animal view
	larp6 (La ribonucleoprotein domain family member 6 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior up.
	larp6 (La ribonucleoprotein domain family member 6 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.

References [+] :

Bayfield, Conserved and divergent features of the structure and function of La and La-related proteins (LARPs). 2010, Pubmed