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A pathway for unicellular tube extension depending on the lymphatic vessel determinant Prox1 and on osmoregulation

Abstract

The mechanisms regulating the extension of small unicellular tubes remain poorly defined. Here we identify several steps in Caenorhabditis elegans excretory canal growth, and propose a model for lumen extension. Our results suggest that the basal and apical excretory membranes grow sequentially: the former extends first like an axon growth cone; the latter extends next as a result of an osmoregulatory activity triggering peri-apical vesicles (a membrane reservoir) to fuse with the lumen. An apical cytoskeletal web including intermediate filaments and actin crosslinking proteins ensures straight regular lumen growth. Expression of several genes encoding proteins mediating excretory lumen extension, such as the osmoregulatory STE20-like kinase GCK-3 and the intermediate filament IFB-1, is regulated by ceh-26 (here referred to as pros-1), which we found essential for excretory canal formation. Interestingly, PROS-1 is homologous to vertebrate Prox1, a transcription factor controlling lymphatic vessel growth. Our findings have potential evolutionary implications for the origin of fluid-collecting organs, and provide a reference for lymphangiogenesis.

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Figure 1: Extension of the excretory canal involves its basal, but not luminal, surface.
Figure 2: Ultrastructure of a control excretory canal in normal conditions.
Figure 3: Variations in osmotic conditions promote canal extension.
Figure 4: Intermediate filaments are required to maintain excretory canal structure.
Figure 5: A null mutation in the Prospero/PROX1 homologue impairs excretory canal formation.
Figure 6: Excretory canal elongation and lumen formation are severely compromised in pros-1 mutants.
Figure 7: Identification of potential PROS-1 targets.
Figure 8: Proposed model of excretory canal formation and extension.

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Acknowledgements

We thank M. Buechner (University of Kansas, USA), J. Culotti (Mt. Sinai Hospital, Toronto, Canada), I. Hope (University of Leeds, UK), V. Göbel (Massachusetts General Hospital, Boston, USA), K. Strange (Mount Desert Island Biological Laboratory, Salisbury Cove, USA), C. Eckmann (MPI-CBG, Dresden, Germany) and the CGC for strains, the Sanger Center for cosmids; P. Schultz for access to his EMPACT2 high-pressure system and Technai microscope for electron microscope tomography; L. Bianchetti, F. Klein and G. Garber for help with bioinformatics; M. Koch, P. Kessler and D. Hentsch for imaging advice; C. Spiegelhalter for assistance with electron microscopy; E. Troesch and D. Busso for directed mutagenesis; and M. Diem for technical assistance. We thank L. Broday, O. Pourquié, C. Gally and S. Quintin for critical reading of the manuscript; and L. Broday, C. Gally, I. Biryukova and G. Volohonsky for useful suggestions during the course of this work. This work was supported by grants from the Institut National du Cancer to M.L., and by a fellowship from the Fondation pour la Recherche Médicale to I.K. We wish to dedicate this work to R. Kolotueva.

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Authors and Affiliations

Authors

Contributions

I.K. and M.L. designed the experiments. I.K. developed markers to monitor canal outgrowth, discovered the canal pearling response, identified the molecular nature of mc41, and characterized the role of IFB-1 in the canal. Y.S. and V.H. carried out most electron microscopy experiments with help from I.K. V.H. and D.R. examined the influence of osmotic shocks on growth and characterized PROS-1 potential targets with input from I.K. and M.L. M.L. wrote the manuscript on the basis of a draft by I.K.

Corresponding author

Correspondence to Michel Labouesse.

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Supplementary information

Supplementary Information

Supplementary Information (PDF 1358 kb)

Supplementary Tables 1–3

Supplementary Information (XLS 204 kb)

Kinetics of excretory canal growth in a wild-type L1 larva.

Video of a young transgenic vha-1p::gfp L1 larva during excretory canal extension (see Fig. 1e for timing). (AVI 28 kb)

3D reconstruction of the left and right excretory canal extensions in a wild-type L1 larva.

3D models were constructed from 50 (V2) or 40 (V3) 60 nm-thick serial sections. Individual electron micrographs were aligned with Adobe Photoshop, exported as an image stack, then processed with the 3Dmod module of the Imod suite for manual segmentation of identifiable structures and 3D rendering (see also Fig. 1f–f’). (MPG 3982 kb)

3D reconstruction of the left and right excretory canal extensions in a wild-type L1 larva.

3D models were constructed from 50 (V2) or 40 (V3) 60 nm-thick serial sections. Individual electron micrographs were aligned with Adobe Photoshop, exported as an image stack, then processed with the 3Dmod module of the Imod suite for manual segmentation of identifiable structures and 3D rendering (see also Fig. 1f–f’). (MPG 2642 kb)

3D-tomography reconstruction of the excretory canal in a wild-type adult.

Electron tomograms were computed from tilt series using the etomo module of the Imod suite. The contours of the structures of interest were then manually traced and rendered with 3Dmod (see also Fig. 2d). (AVI 40113 kb)

Movement of vesicles within puffs after recovery from hypertonic conditions in a wild-type adult.

Video of a transgenic vha-1p::gfp adult recovering from a 500 mM NaCl osmotic shock on control plates with 50 mM NaCl. Images captures on a Leica-SP5 confocal microscope with 11 focal planes every 30 s (only one focal plane shown). Some vesicles are highly mobile within pearls (see also Supplementary Fig. S2b). (AVI 10756 kb)

3D reconstruction of the excretory canal in standard salt conditions.

Electron tomograms were processed as indicated in the legend to Supplementary Videos S4 (canaliculi vesicles connected to the lumen are shaded pale blue as the lumen; see also Fig. 3e–e’). (MPG 9541 kb)

3D reconstruction of the excretory canal on standard medium after recovery from high salt conditions.

(canaliculi vesicles connected to the lumen are shaded pale blue as the lumen; see also Fig. 3f–f’). (MPG 11421 kb)

Kinetics of excretory canal retraction after depletion of IFB-1 by RNAi.

Video of a double transgenic vha-5::mrfp vha-1p::gfp larva after ifb-1(RNAi) treatment during larval development; the movie shows only the green channel in false colours (see Fig. 4d for timing). (AVI 130 kb)

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Kolotuev, I., Hyenne, V., Schwab, Y. et al. A pathway for unicellular tube extension depending on the lymphatic vessel determinant Prox1 and on osmoregulation. Nat Cell Biol 15, 157–168 (2013). https://doi.org/10.1038/ncb2662

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