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First published online 24 August 2005
doi: 10.1242/dev.02007

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Fibulin-1C and Fibulin-1D splice variants have distinct functions and assemble in a hemicentin-dependent manner

¹ Program in Cell Structure and Development, Medical Biotechnology Center, University of Maryland Biotechnology Institute, 725 West Lombard Street, Baltimore, MD 21201, USA
² Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany

View larger version (14K): [in a new window]   Fig. 1. (A) Splicing pattern of nematode fibulin-1C and fibulin-1D. Exons are numbered as in Barth et al. (Barth et al., 1998) with the exception of exons 5 and X, which are labeled 5C and 5D, respectively. Horizontal line indicates region deleted in fbl-1(hd43). Arrows indicate region where GFP or YFP tags were introduced in the fibulin coding sequence. Arrowheads indicate region where mutations were introduced to inactivate specific splice variants (see materials and methods for details). (B) RT-PCR experiment demonstrating that exon 5C is detected in the fibulin-1C variant only, and is replaced by exon 5D in the fibulin-1D variant. Forward and reverse exon specific primers are listed respectively. Lane 1, exons 5C and 14; lane 2, exons 5C and 16; lane 3, exons 5D and 14; lane 4, exons 5D and 16.

View larger version (135K): [in a new window]   Fig. 2. Phenotypic defects in fibulin-1 (hd43) mutants. All panels except for G and H are oriented with dorsal up and anterior to the left. (A,B) Gonad migration in wild-type (A) and fbl-1(hd43) mutant (B) L4 hermaphrodites. Arrows indicate the position of the migrating distal tip cell; arrowheads indicate the width of the proximal gonad. (C,D) Gonad migration in wild-type (C) and fbl-1(hd43) mutant (D) L4 males. Arrows indicate the position of the stationary distal tip cell; arrowheads indicate the position of the seminal vesicle that is severed from the vas deferens and retracted to a dorsal anterior position in mutant. (E,F) Pharyngeal morphology in wild-type (E) and mutant (F) hermaphrodites. Arrows indicate the region of constriction and asymmetry in metacorpus; arrowheads show the abnormal width of the metacorpus and terminal bulb. (G,H) GFP-integrin ß pat-3 distribution in wild-type (G) and fbl-1(hd43) mutant (H) body-wall muscle. Large arrow shows region of GFP-integrin ß pat-3 accumulation at muscle-muscle junctions in wild-type animals only, and small arrows show the gaps at muscle-muscle junctions in the fbl-1(hd43) mutant. (I,J) Intestinal cells expand to fill posterior body cavity in wild-type (I) but not fbl-1(hd43) mutant (J) animals. Arrows in I and J indicate the basal surface of posterior intestinal cells. Scale bars: 10 µm.

View larger version (136K): [in a new window]   Fig. 3. Electron micrographs of mechanosensory neuron and pharyngeal defects in fibulin-1 (hd43) mutants. ALM mechanosensory neuron attachment in wild-type (A) and mutant (B) hermaphrodites. Arrows indicate position of axon; arrowheads indicate extracellular mantle. Also shown is the dorsal body-wall muscle (mus). (C,D) Electron micrographs of cross-sections through the pharynx of wild-type (C) and fbl-1(hd43) mutant (D) animals. Arrows indicate pharyngeal basement membranes that are distorted in mutant animals. Scale bars: 0.5 µm in A,B; 2 µm in C,D.

View larger version (100K): [in a new window]   Fig. 4. Fibulin-1 is secreted by intestine, muscle and hypodermal cells. (A,B) DIC (A) and fluorescence (B) micrographs with arrows showing expression of GFP under the control of the fibulin promoter in intestine. (C-F) Fluorescence micrographs with arrows showing expression in the head (C) and tail (D) muscle, and the spermatheca (E) and hypodermis (F). Scale bar: 10 µm.

View larger version (118K): [in a new window]   Fig. 5. Fibulin-1C::GFP localization. (A-I) Fibulin-1C with an N-terminal GFP tag localizes (arrows) to anterior and posterior intestine (A) and GLR cell surfaces (B), in addition to mechanosensory neuron (C) and uterine (D) attachments, pharynx (E), muscle (F), spermatheca (G), and hermaphrodite (H) and male (I) gonad basement membrane. Scale bars: 10 µm.

View larger version (81K): [in a new window]   Fig. 6. Fibulin-1D::GFP localization. (A-F) Fibulin-1D with an N-terminal GFP tag localizes (arrows) to flexible tracks in the head (A) and GLR cell (B) surfaces, in addition to mechanosensory neuron (C) and uterine (D) attachments, and dorsal (E) and ventral (F) body-wall muscle in the tail. [Fibulin-1D with an N-terminal GFP tag also localizes to dorsal and ventral body-wall muscle in the head (data not shown).] Scale bars: 10 µm.

View larger version (35K): [in a new window]   Fig. 7. Co-localization of fibulin-1D and hemicentin. (A-I) Hemicentin::GFP (A,D,G) and Fibulin-1D with an N-terminal YFP tag (B,E,H) co-localize (C,F,I) on ALM mechanosensory neurons (A-C), flexible tracks (D-F), and uterine attachments (G-I). Scale bar: 10 µm.

View larger version (62K): [in a new window]   Fig. 8. Fibulin-1C and fibulin-1D assembly at uterine attachments and in flexible tracks is dependent on the presence of hemicentin. (A,B) Fibulin-1C with an N-terminal GFP tag localization (arrows) at uterine attachments in wild-type (A) and hemicentin null [him-4(rh319)] mutant (B) animals. (C-F) Fibulin-1D with an N-terminal GFP tag localization (arrows) at uterine attachments (C,D) and flexible tracks (E,F) in wild-type (C,E) and hemicentin null [him-4(rh319)] mutant (D,F) animals. Fibulin-1D is absent from flexible tracks in hemicentin mutants (arrows, E,F), but fibulin-1D assembly on body-wall muscle in the head is not affected. Scale bars: 10 µm.

View larger version (56K): [in a new window]   Fig. 9. Fibulin-1C and fibulin-1D assembly at mechanosensory neuron attachments is dependent on the presence of hemicentin but not on the presence of MEC-1. (A-C) Fibulin-1C with an N-terminal GFP tag localization (arrows) at mechanosensory neuron attachments in wild-type (A), hemicentin null [him-4(rh319); B], and mec-1(e1066) mutant (C) animals. (D-F) Fibulin-1D with an N-terminal GFP tag localization (arrows) at mechanosensory neuron attachments in wild-type (D), hemicentin null [him-4(rh319); E], and mec-1(e1066) mutant (F) animals. Scale bar: 10 µm.

View larger version (87K): [in a new window]   Fig. 10. Hemicentin assembly in some tissues is dependent upon fibulin-1C and/or fibulin-1D expression. (A-L) Hemicentin::GFP localization (arrows) at mechanosensory neuron attachments (A-D), flexible tracks (E-H) and uterine attachments (I-L) in wild type (A,E,I), fibulin-1 (hd43) mutants (B,F,J), and fibulin-1 (hd43) mutants carrying a wild-type fibulin-1D (C,G,K) or fibulin-1C (D,H,L) transgene are shown. Scale bars: 10 µm.