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First published online 1 September 2004
doi: 10.1242/dev.01374

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Met and Hgf signaling controls hypaxial muscle and lateral line development in the zebrafish

¹ MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
² The Victor Chang Cardiac Research Institute, 384 Victoria Street, Darlinghurst 2010, NSW, Australia

View larger version (74K): [in a new window]   Fig. 1. Transplantation of zebrafish somites reveals a restriction to anterior somites for appendicular and PHM muscle formation. (A) Transplantation of anterior somites from a 13-17 somite stage donor embryo carrying a transgene that drives GFP expression (green) from a skeletal muscle-specific promoter into the non-transgenic host at the level of somite 4. Successful transplant immediately after transplantation. (B) The same transplanted embryo as in A, but 30 hours later, revealing the contribution of migratory myoblasts to the fin (f) musculature. (C) Similar transplant to that in A here shown 4 hours after transplantation, but this time at the level of somite 5, revealing a contribution of this somite to PHM formation at 48 hpf (arrows in D). (E) Similar transplant as in A but now encompassing both somites 4 and 5, which, 24 hours later, contribute to both the fin musculature (f) and the PHM (see F). (F) Unbroken lines indicate the level of the sections in the G and H. (G) Section of 48 hpf embryo transplanted in F, at the level of somite 4 (SOM) showing the successful nature of the transplant into the ventral region. The dermis has completely healed around the transplanted tissue. (H) Section taken at the level of the fin showing the contribution of the transplanted tissue to the fin (F) and muscle mass (mm), and also to the PHM. NC, notochord.

View larger version (115K): [in a new window]   Fig. 2. Posterior hypaxial muscle morphogenesis. (A,C,E,G,I,K,M) Various fluorescent images of the PHM within the actin GFP (green) transgenic embryos and larvae at different stages of development. (B,D,F,H,J,L,N) corresponding DIC bright field images of the developing embryo and larvae. (A,B) Oblique lateral view, anterior towards the left, of a 40 hpf embryo, with the first detectable expression of GFP in the migrating PHM (arrow) evident adjacent to somite 5. (C,D) Dorsal view anterior to the top of a 45 hpf embryo, where the PHM (arrows) continues to migrate over the yolk. fm, fin muscle. (E,F) Views as in C,D but of a 52 hpf embryo, where the PHM has reached the level of the fin. (G,H) Oblique lateral view, anterior towards the top of the page, of the same 52 hpf embryo, showing the oblique ventral migration trajectory of the PHM and its anterior turn towards its attachment site at the cleithrum (arrow). (I,J) Oblique lateral view, anterior towards the left, of a 96 hpf embryo where the PHM has reached its attachment point at the cleithrum (arrow). (K,L) High-magnification view of the same embryo as in I and J showing attachment to the cleithrum anteriorly by the sternohyal muscle and posteriorly by the PHM. (M,N) Ventral view, anterior towards the top of the same embryo in I to L showing the relationship of the attachment of the PHM at the cleithrum (arrows) to the rest of the muscles of the head.

View larger version (60K): [in a new window]   Fig. 3. The posterior hypaxial muscle undergoes an unusual set of morphogenetic movements. (A-I) Selected images from the accompanying timelapse movie (Movie 1 in the supplementary material), shown as near simultaneous bright field and fluorescent image capture on the acting GFP transgenic embryos with the tip of the migrating PHM muscle indicated. t is the time in minutes from the initiation of the timelapse. (J-M) High-resolution images of the most anterior of the migrating cells of the PHM revealing movement of filopodial protrusions at the leading edge. Images are taken 20 minutes apart in continuous timelapse.

View larger version (81K): [in a new window]   Fig. 4. The zebrafish receptor tyrosine kinase, met and its ligand hgf are expressed within tissues crucial for controlling hypaxial and appendicular muscle formation and PLLP deposition. (A) Phylogenetic analysis revealing the relationship of the zebrafish met gene to known met homologs (zebrafish met Accession Number, AY687384). (B) Phylogenetic analysis revealing the relationship of the encoded zebrafish Hgf proteins to known Hgf proteins and the more distantly related and distinct Hgf-Like (Hgfl) proteins in other species [zebrafish hgf sequences, Accession Numbers AY690480 (hgf1) and AY690481 (hgf2)]. Phylogenetic trees were constructed by the neighbor-joining method based on the proportion of amino acid sites at which sequences compared were different using MEGA (version 2.1; http://www.megasoftware.net/). The reliability of each interior branch of a given topology was assessed using the bootstrap interior branch test with 1000 bootstrap replications. (C-L) met is expressed within fin and PHM muscle precursors, as well as the PLLP. (C,D) Lateral view, anterior towards the left, dorsal towards the top, of a 26-somite stage embryo hybridized with an antisense RNA probe to the met gene. Arrowheads indicate expression within the migrating lateral line primordia and arrows within the ventral lateral regions of somites 4-6. (E) Cross-section at the level of somite 4, dorsal towards the top, of a 24 hpf embryo similarly stained for met expression. The arrowhead indicates expression within the PLLP and the arrow, expression within fin muscle precursors. (F,G) At 28 hpf, the PLLP still expresses a high level of met and has traversed a number of somites to be positioned at the level of yolk extension. The trailing edge of the PLLP (bracket) has downregulated met expression prior to deposition. (F) Lateral view, anterior towards the left, dorsal towards the top. (G) Dorsal view, anterior towards the left. (H,I) At 28 hpf, expression of met is evident in fin muscle precursors (fmp) migrating towards the fin and the posterior hypaxial muscle (phm). (I) Magnification of the area boxed in H. (J,K) Expression of met within a 36 hpf embryo. At this stage, expression is evident within cells of the PHM, but only as they first exit the somite, as well as expression within the fin myoblast. (J) Dorsal view, anterior towards the top. (K) Lateral view anterior towards the right, the fin (f) is outlined by a broken line. (L) Expression of met within a 48 hpf embryo reveals its restriction at this stage to the dorsoventral muscle masses of the fin (arrows). (M-Q) Expression of hgf during hypaxial myoblast and PLLP migration. At 22 hpf, expression of hgf is expressed at somite boundaries and at lower levels through the somite (M) and at the caudal tip of the tail, and is localized exclusively within the notochord (N); lateral views, anterior towards the left. (O) By 30 hpf, hgf transcripts can be detected through out the fin bud mesenchyme with expression in the fin increasing at 36 (P) and 48 hpf (Q). In situ hybridization with mRNA to the sense strand of the hgf gene did not detect any of these regions of expression (data not shown).

View larger version (48K): [in a new window]   Fig. 5. Injection of Met morpholino perturbs formation of the hypaxial muscles in which met is expressed. (A,B) Wild-type uninjected actin GFP transgenic embryo at 44 hpf, revealing normal fin formation (arrow) in a bright-field DIC image (A) and the corresponding fluorescent image showing GFP expression (B, green) in the dorsal and ventral muscle masses of the fin as well as the PHM (arrows). (C,D) Similar views as in A and B, revealing that injection of a morpholino against the met ATG sequence does not perturb fin formation (C, arrow) but results in a lack of muscle within the fin, and PHM (D). The arrowhead in D indicates a small amount of fin muscle present in a single muscle mass, with the arrow on the contralateral side showing a complete lack of fin muscle. (A-D) Dorsal views anterior towards the left. (E-G) GFP expression in an uninjected actin GFP transgenic embryo at 48 hpf (E) and 96 hpf (F,G) with fin muscle and the PHM arrowed. (H-J) A similar stage of met morpholino injected embryos showing a complete lack of fin and PHM musculature at 48 hpf (H) and a lack of PHM attachment at the clethirum at 96 hpf (I,J). The lack of attachment at the cleithrum at 96 hpf also appears to affect the sternohyal muscle (arrows), which, although still present, is retracted from its attachment to the rostral point of the cleithrum (arrows). (E,H) Dorsal views, anterior towards the left. (F,I) Ventral views, anterior towards the top: (I) a more dorsal view than that in F. (G,J) Lateral views anterior towards the left. (K,L) Expression of lbx1 in 22 hpf uninjected (K) and met morpholino-injected embryos (L) with somites 4 and 5 arrowed. No difference can be seen at this stage between injected and uninjected embryos, suggesting fin myoblasts are initially specified normally in met morphant embryos. (M,N) Expression of lbx1 in 36 hpf uninjected (M) and met morpholino injected embryos (N), revealing that expression of lbx1 within migrating fin and PHM myoblasts is absent in morpholino-injected embryos (arrows), but migration from anterior somites where met is not expressed (denoted *) is unaffected. (O,P) Expression of myod in 48 hpf uninjected (O) and met morpholino injected embryo (P) reveals a lack of fin myoblast differentiation in morpholino injected embryos. (K-P) Dorsal views, anterior towards the top.

View larger version (51K): [in a new window]   Fig. 6. Met is required to control deposition of neuromasts from the PLLP. (A-C) Embryos (48 hpf) stained with the vital dye DASPEI (Whitfield et al., 1996), which marks hair cells within deposited neuromasts. (A) Wild-type embryos possess six to eight deposited hair cell clusters per side (arrowheads) at this stage of development. (B,C) Embryos injected with morpholinos against met possess a deficit in neuromast formation either blocking deposition almost entirely (B) or severely altering timing and spacing of neuromast deposition (C). (D,E) met morpholino injection (E) results in a reduced number of hair cells within neuromast clusters, when deposition does occur, when compared with uninjected embryos (D). (F-I) Staining with an antisense probe to the follistatin gene reveals a concomitant deficit in support cells within met morpholino-injected embryos. (F,G) Lateral views of a wild-type 48 hpf embryo, revealing follistatin expression in the deposited neuromasts (F), including the terminal neuromasts of the tail tip (G). (H,I) Injection of a met morpholino results in a lack of follistatin staining in a similar stage embryo, both along the body axis (H) and at the tip of the tail (I), indicating that support cells are absent in met morpholino-injected embryos. (J,K) prox1 expression specifically marks the migrating primordia and at 36 hpf reveals that the PLLP has traversed the majority of the length of the axis within a wild-type embryo. Expression within the PLLP is downregulated within trailing edge cells (bracket, K) prior to deposition. (L,M) Within met-deficient embryos, PLLP migration is not perturbed but the primordium appears enlarged at the end of the migratory process. Furthermore, prox1 is not downregulated at the trailing edge of the PLLP. Compare the wild-type primordium (K) with the met-deficient PLLP (M). All panels are lateral views anterior towards the left.

View larger version (91K): [in a new window]   Fig. 7. Gain- or loss-of-function of the Met ligand Hgf perturbs hypaxial muscle formation. (A) Implantation of a Hgf-soaked bead (b) adjacent to the fin at 20 hpf results in the formation of an ectopic spur (s) of muscle adjacent to somite 4 and the presence of ectopic muscle fibers (ef, arrow) on the yolk at the level of somite 4, as revealed by an anti MyHC antibody (brown). (B) High-magnification view of the region boxed in A. (C) Similar view to that in B, but of the unaffected contralateral side where no delaminating fibers can be detected at this stage. (A-C) Dorsal views, anterior towards the top. (D) Implantation of a Hgf bead into an actin GFP-expressing embryo also reveals an ectopic spur of differentiating muscle (arrow, S) from somite 4 and ectopic fibers (ef) adjacent to somite 4. (E) Paired bright-field DIC image revealing the position of the implanted bead (b). (D,E) Lateral views, anterior towards the right. (F) Uninjected actin GFP embryo at 48 hpf. (H) Similar stage injected sibling to the embryo in F into which anti-Hgf antibody has been injected, on the right-hand side of the axis, at 20 hpf and allowed to develop to 48 hpf. (G,I) The corresponding bright-field DIC images for F and H, revealing normal development of the fin within injected and uninjected embryos. (J) actin GFP transgenic embryo into which the anti-Hgf antibody has been injected adjacent to the left side of the axis at 28 hpf and allowed to develop to 48 hpf. A gap is evident in the migrating PHM (bracket).

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