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Dev Biol
2008 Oct 01;3221:74-85. doi: 10.1016/j.ydbio.2008.07.012.
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The lmx1b gene is pivotal in glomus development in Xenopus laevis.
Haldin CE
,
Massé KL
,
Bhamra S
,
Simrick S
,
Kyuno J
,
Jones EA
.
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We have previously shown that lmx1b, a LIM homeodomain protein, is expressed in the pronephric glomus. We now show temporal and spatial expression patterns of lmx1b and its potential binding partners in both dissected pronephric anlagen and in individual dissected components of stage 42 pronephroi. Morpholino oligonucleotide knock-down of lmx1b establishes a role for lmx1b in the development of the pronephric components. Depletion of lmx1b results in the formation of a glomus with reduced size. Pronephric tubules were also shown to be reduced in structure and/or coiling whereas more distal tubule structure was unaffected. Over-expression of lmx1b mRNA resulted in no significant phenotype. Given that lmx1b protein is known to function as a heterodimer, we have over-expressed lmx1b mRNA alone or in combination with potential interacting molecules and analysed the effects on kidney structures. Phenotypes observed by over-expression of lim1 and ldb1 are partially rescued by co-injection with lmx1b mRNA. Animal cap experiments confirm that co-injection of lmx1b with potential binding partners can up-regulate pronephric molecular markers suggesting that lmx1b lies upstream of wt1 in the gene network controlling glomus differentiation. This places lmx1b in a genetic hierarchy involved in pronephros development and suggests that it is the balance in levels of binding partners together with restricted expression domains of lmx1b and lim1 which influences differentiation into glomus or tubule derivatives in vivo.
Fig. 2. lmx1b MO1 and MO2 prevent full size glomus formation. (A) lmx1b MO1 (panels b, e) or MO2 (panels h and k) was injected into the V2 blastomere at the 8-cell stage. All injections were carried out using a lineage tracer, either GFP (panels a–f) or LacZ (panels g–l). The size of the glomus was assessed by wt1 in situ hybridisation at stage 35/36. Injection of cMO does not induce any phenotype on the injected side (compare panels a to d and g to j). lmx1b MO1 injected embryos (5 ng) showed a reduced glomus (compare panel b to e). A similar phenotype was observed following the injection of lmx1b MO2 (10 ng) (compare panels h to k). Injection of lmx1b-mut mRNA (2.5 ng) can partially rescue the phenotype of MO1 (panels c and f) and MO2 (panels i and l). The injected side is marked with an asterisk. (B) lmx1b MO1 (panels b, e) or MO2 (panels h and k) was injected into one cell of 2-cell embryos. All injections were done using a lineage tracer, either GFP (panels a–f) or LacZ (panels g–l). The size of the glomus was assessed by nephrin in situ hybridisation at stage 33/34. Injection of cMO does not induce any phenotype on the injected side (compare panels a to d and g to j). lmx1b MO1 injected embryos (5 ng) showed a reduced nephrin-staining area on the injected side (compare panels b to e). A similar phenotype was observed following the injection of lmx1b MO2 (10 ng) (compare panels h to k). Injection of lmx1b-mut mRNA (2.5 ng) can partially rescue the phenotype of MO1 (panels c and f) and MO2 (panels i and l). The injected side is marked with an asterisk.
Fig. 5. Over-expression of lmx1b and its potential binding partners affects wt1 expression. mRNAs of lmx1b and its potential binding partners were injected either alone (panel A) or in combination (panel B), together with lacZ, into one V2 blastomere at the 8-cell stage. Red Gal staining followed by wt1 in situ hybridisation was carried out at stage 35/36 in order to assess any glomus phenotype. For each embryo, the injected side (indicated by an asterisk, panels a–d) was compared to the uninjected side (panels e–h). Injection of lmx1b mRNA alone did not induce any significant glomus phenotype (A, compare panel a to e), whereas injection of lim1 or ldb1 alone, its potential binding partners, resulted in the formation of an enlarged and reduced glomus respectively (A, b and f; A, c and g). These phenotypes can be partially rescued by the co-injection of lmx1b (B, b, arrowhead and f; B, c and g). Interestingly, co-injection of lim1 and ldb1 rescued both phenotypes, resulting in the formation of a more normal glomus (B, a and e). Injection of E47 alone or in combination with lmx1b did not induce any statistically significant phenotype (A, d and h and B, d and h).
Fig. 6. Over-expression of lmx1b and its potential binding partners affects nephrin expression. mRNAs of lmx1b and its potential binding partners were injected either alone (panel A) or in combination (panel B), together with lacZ, into one cell of 2-cell embryos. Red Gal staining followed by nephrin in situ hybridisation was carried out at stage 33/34 in order to assess any glomus phenotype. For each embryo, the injected side (indicated by an asterisk, panels a–d) was compared to the uninjected side (panels e–h). Injection of lmx1b mRNA alone did not induce any significant change in nephrin staining (A, compare panel a to e), whereas injection of lim1 or ldb1 alone resulted in the formation of an enlarged and reduced nephrin domain respectively (A, b and f; A, c and g). These phenotypes can be partially rescued by the co-injection of lmx1b (B, b and f; B, c and g). Co-injection of lim1 and ldb1 rescued both phenotypes, the embryos displayed on the injected side a nephrin domain, similar in area to the uninjected side (B, a and e). Injection of E47 alone or in combination with lmx1b did not induce any statistically significant phenotype (A, d and h and B, d and h).
Fig. 7. Over-expression of lmx1b and its potential binding partners affects the development of tubules. mRNAs of lmx1b and its potential binding partners were injected either alone (panel A) or in combination (panel B), together with lacZ into one V2 blastomere at the 8-cell stage. The morphology of the tubules was assessed at stage 40 by immunohistochemistry using 3G8 (in purple) and 4A6 (in red) monoclonal antibodies (Vize et al., 1995). The injected side was identified by blue β-galactosidase staining and is indicated by an asterisk (a–d). As comparison, the uninjected side of each embryo was photographed (e–h) lmx1b-injected embryos showed no pronephric phenotype (A–a and e). lim1 injection resulted in the formation of an enlarged proximal tubule mass and wider more distal tubules (A–b and f), whereas ldb1 over-expression caused reduction in size of proximal tubules and in some cases affected formation of the more distal tubules (A–c and g). Co-injection of lmx1b with either lim1 or ldb1 partially rescued these phenotypes (B–b and f and B–c and g). Co-expression of lim1 and ldb1 partially rescued both lim1 and ldb1 phenotypes (B–a and e). Injection of E47 resulted in the formation of slightly enlarged proximal tubule mass without affecting the more distal tubules (A–d and h) whereas co-injection of lmx1b and E47 caused the opposite effect with reduction of pronephric proximal tubules (B–d and h).
