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. Author manuscript; available in PMC: 2024 Oct 9.
Published in final edited form as: Curr Biol. 2023 Sep 13;33(19):4174–4186.e7. doi: 10.1016/j.cub.2023.08.046

Figure 4. GRL-18 is a novel aECM protein that transiently localizes to nanoscale rings.

Figure 4.

Endogenously-tagged full-length sfGFP-GRL-18 forms rings at the endings of the IL and CEP sense organs in the male head (refer to Figure 1A, right). Views are maximum intensity projections through half of the head nearest the objective, showing three of the six IL sense organs and two of the four CEP sense organs; dashed lines, outline of cuticle. (B) Endogenously-tagged full-length sfGFP-GRL-18 rings are also found at the ray sense organs of the developing male tail during the L4/adult mid-molt stage; dashed lines, outline of cuticle. Insets (i-iii), magnification of rings from the boxed regions. (C) Localization of endogenously-tagged full-length sfGFP-GRL-18 in males through the L4 to adult (Ad) transition, showing early and late molt stages (see Figure S4 for quantification of each localization pattern). Green, sfGFP-GRL-18; magenta, glial endings; dashed lines, outline of cuticle. Arrowheads, sfGFP-GRL-18 rings. (D) Schematic of localization and subsequent disassembly of GRL-18 during patterning of the new cuticle. Cartoons are meant as a guide to the fluorescence images, but the structure and relative positions of the glial ending and cuticle during molting remain unknown. (E) C. elegans gene families distantly related to Hedgehog include ground (grd) and ground-like (grl) genes. (F) Diagram of GRL-18 full-length proteins and fragments containing conserved C-terminal Grl domain [352–445] or non-conserved N-terminal region [31–351] expressed as low-copy transgenes under control of the grl-18 promoter. sfGFP was inserted at the GRL-18 N-terminus after amino acids 1–30 containing the predicted signal sequence (GRL-18[N], GRL-18[31–351]), as an N-terminal fusion preceded by amino acids 1–30 (GRL-18[352–445]), or at the C-terminus. Dark bars, signal sequence; green arrows, sfGFP. (G-J) Localization of sfGFP-GRL-18 at nose tips of L4/adult mid-molt males expressing (G) sfGFP-GRL-18, (H) GRL-18-sfGFP, (I) sfGFP-Grl domain [352–445], and (J) sfGFP-N-terminal region [31–351] transgenes. Green, sfGFP-GRL-18; magenta, glial endings. Arrowheads, sfGFP-GRL-18 rings. All scale bars, 2 μm.