Martin BL and Harland RM (2006), A novel role for lbx1 in Xenopus hypaxial myoge...

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Development 2006 Jan 01;1332:195-208. doi: 10.1242/dev.02183.

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A novel role for lbx1 in Xenopus hypaxial myogenesis.

Martin BL , Harland RM .

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We have examined lbx1 expression in early X. laevis tadpoles. In contrast to amniotes, lbx1 is expressed in all of the myoblasts that contribute to the body wall musculature, as well as in a group of cells that migrate into the head. Despite this different expression, the function of lbx1 appears to be conserved. Morpholino (MO) knockdown of lbx1 causes a specific reduction of body wall muscles and hypoglossal muscles originating from the somites. Although myoblast migratory defects are observed in antisense MO injected tadpoles targeting lbx1, this results at least in part from a lack of myoblast proliferation in the hypaxial muscle domain. Conversely, overexpression of lbx1 mRNA results in enlarged somites, an increase in cell proliferation, but a lack of differentiated muscle. The control of cell proliferation is linked to a strong downregulation of myoD expression in gain-of-function experiments. Co-injection of myoD mRNA with lbx1 mRNA eliminates the overproliferation phenotype observed when lbx1 is injected alone. The results indicate that a primary function of lbx1 in hypaxial muscle development is to repress myoD, allowing myoblasts to proliferate before the eventual onset of terminal differentiation.

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Species referenced: Xenopus laevis
Genes referenced: lbx1 myf5 myod1 ncam1 pax3 pax8 tbx3 znrd2
???displayArticle.antibodies??? H3f3a Ab9 Somite Ab1
???displayArticle.morpholinos??? lbx1 MO1 lbx1 MO2

Phenotypes: Xla Wt + Dre.lbx1 (Fig.11 C, D) [+]

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	Fig. 1. Xenopus hypaxial muscles and lbx1 expression. Stage 37 (A) and 45 (B) tadpoles were stained with the 12/101 antibody. A is a lateral view, while B is a ventral view with anterior to the left. The hypaxial muscles consist of the rectus abdominus, rectus cervicus and geniohyoideus. (C-H) Expression of lbx1 from stage 17 to 42. (C) Neural expression precedes myoblast expression (stage 17, dorsal view). (D) Early myoblast expression can be seen at stage 26 (arrowhead). (E,F) Myoblast expression expands posteriorly and ventrally as development procedes. (G) At stage 37, lbx1-positive cells can be seen migrating around the hyoid (arrow). (H) By stage 42, very few lbx1-positive myoblasts are seen in the ventral body wall region (arrowheads).
	Fig. 3. Ventral body wall myoblasts appear to migrate as mesenchymal cells. (A) lbx1-expressing myoblasts of a stage 37/38 tadpole emerge at the somite cleft (arrows, most posterior somite with emerging myoblasts shown). (B) Another hypaxial myoblast-specific marker, tbx3, can be seen expressed in cells migrating out of the somite clefts in a stage 31 tadpole (arrows). Myotomes in A and B are labeled with 12/101 (brown). (C,D) lbx1 expression is found in long-range type hypaxial myoblasts. lbx1 expression in the geniohyoideus can be seen in a ventral view of a stage 42 tadpole (C, arrowheads), which co-localizes with 12/101 (D). (E,F) lbx1 is also found in the migrating muscles of the hindlimb of a stage 53 tadpole (E, arrows), similar to pax3 expression at the same stage (F, arrows) (limbs are shown from a lateral view, distal end towards the left).
	Fig. 4. lbx1 is required for hypaxial muscle formation. RT-PCR was performed on tadpoles injected with lbx1-splice MO into one out of two cells or both cells at the two-cell stage. (A) A specific loss of the properly spliced lbx1 transcript is seen. Embryos were injected into one cell at the two-cell stage with a control MO (B-E), the lbx1-splice blocking MO (F-I) or the lbx1-translation blocking MO (J-M) along withβ -galactosidase mRNA as a lineage tracer (red). Tadpoles were stained with the 12/101 antibody (brown). The uninjected (B,D,F,H,J,L) and injected (C,E,G,I,K,M) sides of each tadpole are shown, where the uninjected sides are pictured with anterior towards the right. The control MO-injected tadpoles do not show a difference in the presence of rectus abdominus muscles on the injected side compared with the uninjected side at stage 37 (compare B with C) or stage 40 (D compared to E). Tadpoles injected with the lbx1-splice MO exhibit a loss of rectus abdominus and rectus cervicus muscles on the injected side at stage 37 (compare F with G). At stage 40, these muscles appear but are greatly reduced in the lbx1-splice MO-injected tadpoles (compare H with I). Similar results were observed for the lbx1-trans MO-injected tadpoles (J-M). (N,O) Stage 40 control injected (N) and lbx1-splice injected (O) embryos are shown from a ventral view, with the MO injected side downwards (embryos were injected into one cell at the two-cell stage). The geniohyoideus muscle is lost on the lbx1-splice injected side of the tadpole (O, arrow), whereas both are present in the control injected MO (N, arrow). Myoblast migration defects are observed in lbx1-splice injected tadpoles. (P-S) Tadpoles in P and R are stained for pax3 mRNA (purple) and then merged with the 12/101 antibody (green, Q,S). A control tadpole is pictured in P and Q, which shows the movement of pax3-positive myoblasts away from the differentiated epaxial myotome. In lbx1-splice-injected tadpoles (R,S), a large number of pax3-positive myoblasts remain adjacent to the differentiated myotome.
	Fig. 5. myf5 expression is affected by the gain or loss of lbx1. (A,B) A transverse section through an lbx1-splice-injected tadpole (left side uninjected, right injected) reveals not only a loss of ventral body wall muscles (B, arrow), but also a loss of a more dorsal domain of epaxial muscle (B, arrowhead). A corresponding loss of myf5 expression is seen in stage 28 lbx1-splice injected tadpoles. (C,D) An lbx1-splice injected tadpole where C is the uninjected side and D is the injected side. (E,F) Control MO-injected tadpole where E is the uninjected side and F is the injected side. All of these panels are stained for myf5 expression. A loss of myf5 expression in the region that lbx1 is normally expressed is seen in the lbx1-splice-injected tadpole on the injected side (D, arrow). The similar expression domains for lbx1 and myf5 in the hypaxial myoblasts can be seen in G-J. (G,H) A transverse section of a stage 28 tadpole at approximately the level of trunk somite 3. The section is stained for lbx1 (G) and merged with 12/101 staining (H, green). (I,J) Similarly, a transverse section from a stage 28 tadpole at approximately the level of trunk somite 3. This section is stained for myf5 (I) and merged with 12/101 staining (J, green). Both transcripts are found in the ventral hypaxial myoblast region of the somite (H,J, arrows). Overexpression of zebrafish lbx1 causes an increase in myf5 expression and larger somites, but a decrease in differentiated muscle. (K,L) A transverse section of a stage 28 tadpole, showing myf5 staining (K) merged with 12/101 staining (L, green). Embryos were injected into one cell at the two-cell stage. The injected side is to the right. (M,N) Stage 29 tadpole co-injected with lbx1-splice MO and lbx1 mRNA. The uninjected side is shown in M and injected side in N. The loss of myf5 expression is rescued when lbx1 mRNA is added back to lbx1-splice injected cells.
	Fig. 7. Lbx1 downregulates myoD. Embryos were injected with lbx1 mRNA in one cell at the two-cell stage and stained for myoD (A-E), pax3 (F-J) or lbx1 (K-M). Red staining is the β-galactosidase lineage tracer. At stage 22, myoD is downregulated on the injected side (A), while pax3 expression is higher (F). These embryos are shown from a dorsal view, with the injected side on top. Transverse sections of stage 24 embryos show a complete loss of myoD expression on the injected side (right side), despite there being an enlarged somite (B), while pax3 expression is higher on the injected side (right side) (G). At stage 33, expression of myoD is still downregulated in regions of theβ -galactosidase lineage tracer (C-E). The uninjected side is shown in C, injected side in D, and a transverse section in E (injected side to the right). pax3 expression is also still upregulated at stage 33 in regions of the lineage tracer (H,I). The uninjected side is shown in H, injected side in I, and a transverse section in J (injected side to the right). At stage 24, endogenous lbx1 expression is lost on the injected side of the embryo (L), when compared with the uninjected side (K). At stage 33, ectopic lbx1 expression in the posterior somites (red staining) fails to upregulate endogenous lbx1 expression (M). (N) A stage 26, tadpole injected with lbx1-splice MO (right side) shows a mild upregulation of myoD in the ventrolateral domain of the somite (arrow). (O-T) Transverse sections of stage 31 (O-Q) and 41 (R-T) tadpoles injected with lbx1 into one cell at the two-cell stage were stained for injected lbx1 mRNA (O,R), myoD (P,S) and p27 (Q,T, arrow in Q indicates region of strong expression on the uninjected side). When injected lbx1 mRNA is still present (O), myoD and p27 are repressed on the injected side (P,Q). When the injected mRNA is no longer detectable (R), myoD and p27 are upregulated on the injected side (S,T).
	Fig. 8. The engrailed repressor domain of lbx1 is necessary for its function. (A-F) mRNA from a mutant lbx1 construct that lacks the engrailed homology domain was injected into one cell at the two-cell stage. The injected sides are on the right. Phenotypes opposite of wild-type lbx1 overexpression were observed. At stage 24, a transverse section reveals an increase in myoD staining on the injected side (A, arrow), while a transverse section through a stage 31 tadpole exhibits a decrease in Pax3 staining (B, arrow). Transverse sections through stage 29 tadpoles (C-F) were stained with the pH3 antibody alone (C,E) or with 12/101 (D,F). No increase in the number of mitotic nuclei is observed on the injected sides (C,E), and 12/101 staining (D,F) indicates a slightly shorter, yet wider, differentiated myotome (arrows).
	Fig. 11. Enlarged myotomes of lbx1-injected tadpoles is not the result of recruitment of non-myogenic lineages. (A,B) Injection of myoD (A) or myoD + lbx1 (B) mRNA does not cause an increase in cell proliferation on the injected side (right side), as judged by pH3 staining on transverse sections of stage 29 tadpoles. Instead, non-myogenic lineages such as the pronephros are recruited to the myotome. (C,D) Transverse sections through stage 31 tadpoles indicate that both NCAM staining in the neural tube (C, arrow) and Pax8 staining in the pronephric tubules (D, arrow) are present on the injected side. (E,F) Lateral views of a tadpole injected with lbx1 show the presence of pronephric tubules on both the uninjected (E) and injected (F) sides (arrows). (G-J) Stage 31 tadpoles injected with myoD alone (G,H) or myoD + lbx1 (I,J), and stained with pax8, show normal pronephric tubules on the uninjected sides (G,I, arrows), but are lacking on the injected sides (H,J). However, tadpoles injected with lbx1 alone do not show a lack of tissues surrounding the myotome, despite there being a larger somite.
	lbx1 (ladybird homeobox 1) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 17, dorsal view, anterior left.
	lbx1 (ladybird homeobox 1) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 26, lateral view, anterior left.
	lbx1 (ladybird homeobox 1) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 28, lateral view, anterior left.

	lbx1 (ladybird homeobox 1) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 42, lateral view, anterior left.

	Fig. 2. The geniohyoideus is a hypaxial muscle and originates from the anterior trunk somites. DiI labeling (red) and 12/101 immunofluorescence (green) was used to track the migration of cells from the anterior trunk somites. (A) A schematic of the stage 28 injections [modified, with permission, from Niewkoop and Faber (Niewkoop and Faber, 1967)]. A live tadpole is visualized at stage 37 (B) and 45 (C). The original site of the injection is marked by an arrowhead, while labeled cells that have migrated into the head are marked with arrows. (C-G) Ventral views of the head. (D-G) The migratory population of cells coming from the anterior trunk somites co-localize with the geniohyoideus muscle at stage 45. A light-field image is shown in D, DiI labeling in E, 12/101 in F and the merge of the DiI and 12/101 images in G.
	Fig. 6. Myoblast cell proliferation is controlled by lbx1. An anti-phospho histone H3 antibody (brown) in combination with 12/101 (green) shows cells proliferating in the hypaxial muscle region (A-D, arrows). A transverse section through a stage 24 tadpole, before lbx1 expression is on in myoblasts, shows very little cell proliferation in the ventrolateral region of the somite (A, arrow). At stages 28 (B), 33 (C) and 37 (D), an increasing number of proliferating cells can be seen in the ventrolateral region of the somite (arrows). The remaining panels show tadpoles injected in one cell at the two-cell stage with either lbx1- splice MO (E,F), lbx1 mRNA (G,H,K-N) or a control MO (I,J). All of these panels are transverse sections with the injected side on the right. Stage 33 tadpoles that have been injected with lbx1-splice MO show a decreased number of proliferating cells in the ventrolateral region of the somite (E). Consistent with the loss of proliferating cells in E, a slightly smaller differentiated muscle mass can be seen in the same section in F (12/101, green). (G) Stage 28 tadpoles injected with lbx1 mRNA exhibit a dramatic increase in the number of proliferating cells in the region of the lineage tracer (red). (H) Active cell proliferation seen in G inhibits muscle differentiation (12/101, green). (I,J) Control injected tadpoles at stage 33 show no difference in cell proliferation or size of differentiated muscle. (K,L) By stage 37, tadpoles injected with lbx1 mRNA have the same amount of cell proliferation, but the differentiated muscle mass on the injected side is much larger. (M,N) A stage 41 tadpole injected with lbx1 mRNA has a larger differentiated muscle mass on the injected side in both the trunk (M) and tail (N).
	Fig. 9. Co-injection of myoD mRNA with lbx1 mRNA eliminates overproliferation and pax3 upregulation. (A- O) Embryos were injected into one cell at the two-cell stage with, lbx1 (A-C), myoD (D-F), myf5 (G-I), lbx1 + myoD (J-L) and lbx1 + myf5 (M-O). Injected embryos were grown until stage 29, sectioned in the transverse plane, and analyzed with anti-phospho-histone H3 antibody (A,D,G,J,M), 12/101 (B,E,H,K,N) or pax3 expression (C,F,I,L,O). The injected side of each tadpole is on the right, marked by Beta- galactosidase (red). Anti-phospho-histone H3 and 12/101 staining were performed on the same sections, while pax3 staining was carried out on different tadpoles. Injection of lbx1 causes an increase in proliferation (A), decrease in differentiated muscle (B) and increase in pax3 (C). Injection of myoD causes no change in proliferation (D), an increase in differentiated muscle (E) and a decrease in pax3 expression (F). Injection of myf5 causes no significant change in cell proliferation (G), a slight increase in differentiated muscle (H) and no change in pax3 expression (I). Injection of lbx1 + myoD (J-L) produces a phenotype consistent with injection of myoD alone, while injection of lbx1 + myf5 (M-O) causes a phenotype like that of lbx1 alone.