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Pdlim7 is required for maintenance of the mesenchymal/epidermal Fgf signaling feedback loop during zebrafish pectoral fin development.

Camarata T, Snyder D, Schwend T, Klosowiak J, Holtrup B, Simon HG - BMC Dev. Biol. (2010)

Bottom Line: Knock-down of Pdlim7 function leads to decreased pectoral fin cell proliferation resulting in a severely stunted fin phenotype.Along with the lack of fgf24 in the AER, other critical factors including fgf8 are reduced, suggesting signaling problems to the underlying mesenchyme.These new regulatory mechanisms may have important implications how we interpret Tbx5 function in congenital hand/heart syndromes in humans.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Northwestern University, The Feinberg School of Medicine, Children's Memorial Research Center, Chicago, IL 60614, USA.

ABSTRACT

Background: Vertebrate limb development involves a reciprocal feedback loop between limb mesenchyme and the overlying apical ectodermal ridge (AER). Several gene pathways participate in this feedback loop, including Fgf signaling. In the forelimb lateral plate mesenchyme, Tbx5 activates Fgf10 expression, which in turn initiates and maintains the mesenchyme/AER Fgf signaling loop. Recent findings have revealed that Tbx5 transcriptional activity is regulated by dynamic nucleocytoplasmic shuttling and interaction with Pdlim7, a PDZ-LIM protein family member, along actin filaments. This Tbx5 regulation is critical in heart formation, but the coexpression of both proteins in other developing tissues suggests a broader functional role.

Results: Knock-down of Pdlim7 function leads to decreased pectoral fin cell proliferation resulting in a severely stunted fin phenotype. While early gene induction and patterning in the presumptive fin field appear normal, the pectoral fin precursor cells display compaction and migration defects between 18 and 24 hours post-fertilization (hpf). During fin growth fgf24 is sequentially expressed in the mesenchyme and then in the apical ectodermal ridge (AER). However, in pdlim7 antisense morpholino-treated embryos this switch of expression is prevented and fgf24 remains ectopically active in the mesenchymal cells. Along with the lack of fgf24 in the AER, other critical factors including fgf8 are reduced, suggesting signaling problems to the underlying mesenchyme. As a consequence of perturbed AER function in the absence of Pdlim7, pathway components in the fin mesenchyme are misregulated or absent, indicating a breakdown of the Fgf signaling feedback loop, which is ultimately responsible for the loss of fin outgrowth.

Conclusion: This work provides the first evidence for the involvement of Pdlim7 in pectoral fin development. Proper fin outgrowth requires fgf24 downregulation in the fin mesenchyme with subsequent activation in the AER, and Pdlim7 appears to regulate this transition, potentially through Tbx5 regulation. By controlling Tbx5 subcellular localization and transcriptional activity and possibly additional yet unknown means, Pdlim7 is required for proper development of the heart and the fins. These new regulatory mechanisms may have important implications how we interpret Tbx5 function in congenital hand/heart syndromes in humans.

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Pdlim7 is required for pectoral fin development. A-C: Whole-mount in situ hybridization using antisense RNA to show expression of pdlim7 in the developing pectoral fin mesenchyme at 33 hpf (A), 48 hpf (B), and 72 hpf (C). D-E: Sectioned embryos at 48 hpf of whole-mount in situ hybridization of pdlim7 show expression in fin mesenchyme. Boxed region in D is magnified in E to distinguish mesenchyme (purple color) and AER (arrow). F-G: Dorsal view of wild-type (F) and MO2 injected (G) embryos at 96 hpf. Arrows in G point to position of pectoral fin. H-J: Alcian blue stained cartilage preparations of dissected pectoral fins at 96 hpf. Wild-type (H), severe MO2 phenotype (I), and mild MO2 phenotype (J). I and J from same embryo. cl, cleithrum; pc, postcoracoid process; ed, endodermal disc; sco, scapulocoracoid.
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Figure 1: Pdlim7 is required for pectoral fin development. A-C: Whole-mount in situ hybridization using antisense RNA to show expression of pdlim7 in the developing pectoral fin mesenchyme at 33 hpf (A), 48 hpf (B), and 72 hpf (C). D-E: Sectioned embryos at 48 hpf of whole-mount in situ hybridization of pdlim7 show expression in fin mesenchyme. Boxed region in D is magnified in E to distinguish mesenchyme (purple color) and AER (arrow). F-G: Dorsal view of wild-type (F) and MO2 injected (G) embryos at 96 hpf. Arrows in G point to position of pectoral fin. H-J: Alcian blue stained cartilage preparations of dissected pectoral fins at 96 hpf. Wild-type (H), severe MO2 phenotype (I), and mild MO2 phenotype (J). I and J from same embryo. cl, cleithrum; pc, postcoracoid process; ed, endodermal disc; sco, scapulocoracoid.

Mentions: The PDZ-LIM protein, Pdlim7, was identified as a novel binding protein and regulator of the transcription factor Tbx5 [30,36,38]. Pdlim7 mRNA has been detected in several tissues of the vertebrate embryo including the limbs, heart, and skeletal muscle [30,36]. In the zebrafish embryo during fin development, using whole mount in situ hybridization, we first detected pdlim7 expression in the mesenchyme of the fin field at 33 hours post-fertilization (hpf) which was maintained in the fin up to 72 hpf (Fig. 1A-E; [38]). Expression of pdlim7 was not detected in the AER (Fig. 1D, E). Injection at the one-cell stage with 2 ng of pdlim7 antisense morpholino oligonucleotides, interfering with either protein translation (MO1) or RNA splicing (MO2), resulted in comparable defects in pectoral fin development (Fig. 1 and data not shown; see Methods). At 48 hpf, embryos injected with MO2 produced phenotypes with stunted fin buds as compared to control siblings, indicating a possible defect in forelimb outgrowth ([38]; data not shown). By four days of development in wild-type larvae the pectoral fins are clearly visible, while the pectoral fins of MO2 injected embryos were significantly smaller or absent (Fig. 1F-G). Both control and morphant larvae were stained with Alcian blue to visualize the extent of cartilage differentiation. In controls, all of the major cartilage elements were present in the pectoral fin (Fig. 1H). However, in pdlim7 MO2 injected larvae, cartilage development was severely impeded. In the majority of cases, only a fragment of the cleithrum bone along with limited unidentifiable cartilage condensation could be detected (Fig. 1I). Some phenotypic variability was observed among MO2 treated embryos and occasionally slight differences in phenotype were visualized between left and right pectoral fins within single embryos. Less severe pectoral fin phenotypes in morphant embryos resulted in larvae with elements of the cleithrum, postcoracoid process, and a greatly reduced endochondral disc (Fig. 1J). Based upon mRNA expression and gene knock-down data, Pdlim7 appears to be required for pectoral fin development.


Pdlim7 is required for maintenance of the mesenchymal/epidermal Fgf signaling feedback loop during zebrafish pectoral fin development.

Camarata T, Snyder D, Schwend T, Klosowiak J, Holtrup B, Simon HG - BMC Dev. Biol. (2010)

Pdlim7 is required for pectoral fin development. A-C: Whole-mount in situ hybridization using antisense RNA to show expression of pdlim7 in the developing pectoral fin mesenchyme at 33 hpf (A), 48 hpf (B), and 72 hpf (C). D-E: Sectioned embryos at 48 hpf of whole-mount in situ hybridization of pdlim7 show expression in fin mesenchyme. Boxed region in D is magnified in E to distinguish mesenchyme (purple color) and AER (arrow). F-G: Dorsal view of wild-type (F) and MO2 injected (G) embryos at 96 hpf. Arrows in G point to position of pectoral fin. H-J: Alcian blue stained cartilage preparations of dissected pectoral fins at 96 hpf. Wild-type (H), severe MO2 phenotype (I), and mild MO2 phenotype (J). I and J from same embryo. cl, cleithrum; pc, postcoracoid process; ed, endodermal disc; sco, scapulocoracoid.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Pdlim7 is required for pectoral fin development. A-C: Whole-mount in situ hybridization using antisense RNA to show expression of pdlim7 in the developing pectoral fin mesenchyme at 33 hpf (A), 48 hpf (B), and 72 hpf (C). D-E: Sectioned embryos at 48 hpf of whole-mount in situ hybridization of pdlim7 show expression in fin mesenchyme. Boxed region in D is magnified in E to distinguish mesenchyme (purple color) and AER (arrow). F-G: Dorsal view of wild-type (F) and MO2 injected (G) embryos at 96 hpf. Arrows in G point to position of pectoral fin. H-J: Alcian blue stained cartilage preparations of dissected pectoral fins at 96 hpf. Wild-type (H), severe MO2 phenotype (I), and mild MO2 phenotype (J). I and J from same embryo. cl, cleithrum; pc, postcoracoid process; ed, endodermal disc; sco, scapulocoracoid.
Mentions: The PDZ-LIM protein, Pdlim7, was identified as a novel binding protein and regulator of the transcription factor Tbx5 [30,36,38]. Pdlim7 mRNA has been detected in several tissues of the vertebrate embryo including the limbs, heart, and skeletal muscle [30,36]. In the zebrafish embryo during fin development, using whole mount in situ hybridization, we first detected pdlim7 expression in the mesenchyme of the fin field at 33 hours post-fertilization (hpf) which was maintained in the fin up to 72 hpf (Fig. 1A-E; [38]). Expression of pdlim7 was not detected in the AER (Fig. 1D, E). Injection at the one-cell stage with 2 ng of pdlim7 antisense morpholino oligonucleotides, interfering with either protein translation (MO1) or RNA splicing (MO2), resulted in comparable defects in pectoral fin development (Fig. 1 and data not shown; see Methods). At 48 hpf, embryos injected with MO2 produced phenotypes with stunted fin buds as compared to control siblings, indicating a possible defect in forelimb outgrowth ([38]; data not shown). By four days of development in wild-type larvae the pectoral fins are clearly visible, while the pectoral fins of MO2 injected embryos were significantly smaller or absent (Fig. 1F-G). Both control and morphant larvae were stained with Alcian blue to visualize the extent of cartilage differentiation. In controls, all of the major cartilage elements were present in the pectoral fin (Fig. 1H). However, in pdlim7 MO2 injected larvae, cartilage development was severely impeded. In the majority of cases, only a fragment of the cleithrum bone along with limited unidentifiable cartilage condensation could be detected (Fig. 1I). Some phenotypic variability was observed among MO2 treated embryos and occasionally slight differences in phenotype were visualized between left and right pectoral fins within single embryos. Less severe pectoral fin phenotypes in morphant embryos resulted in larvae with elements of the cleithrum, postcoracoid process, and a greatly reduced endochondral disc (Fig. 1J). Based upon mRNA expression and gene knock-down data, Pdlim7 appears to be required for pectoral fin development.

Bottom Line: Knock-down of Pdlim7 function leads to decreased pectoral fin cell proliferation resulting in a severely stunted fin phenotype.Along with the lack of fgf24 in the AER, other critical factors including fgf8 are reduced, suggesting signaling problems to the underlying mesenchyme.These new regulatory mechanisms may have important implications how we interpret Tbx5 function in congenital hand/heart syndromes in humans.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Northwestern University, The Feinberg School of Medicine, Children's Memorial Research Center, Chicago, IL 60614, USA.

ABSTRACT

Background: Vertebrate limb development involves a reciprocal feedback loop between limb mesenchyme and the overlying apical ectodermal ridge (AER). Several gene pathways participate in this feedback loop, including Fgf signaling. In the forelimb lateral plate mesenchyme, Tbx5 activates Fgf10 expression, which in turn initiates and maintains the mesenchyme/AER Fgf signaling loop. Recent findings have revealed that Tbx5 transcriptional activity is regulated by dynamic nucleocytoplasmic shuttling and interaction with Pdlim7, a PDZ-LIM protein family member, along actin filaments. This Tbx5 regulation is critical in heart formation, but the coexpression of both proteins in other developing tissues suggests a broader functional role.

Results: Knock-down of Pdlim7 function leads to decreased pectoral fin cell proliferation resulting in a severely stunted fin phenotype. While early gene induction and patterning in the presumptive fin field appear normal, the pectoral fin precursor cells display compaction and migration defects between 18 and 24 hours post-fertilization (hpf). During fin growth fgf24 is sequentially expressed in the mesenchyme and then in the apical ectodermal ridge (AER). However, in pdlim7 antisense morpholino-treated embryos this switch of expression is prevented and fgf24 remains ectopically active in the mesenchymal cells. Along with the lack of fgf24 in the AER, other critical factors including fgf8 are reduced, suggesting signaling problems to the underlying mesenchyme. As a consequence of perturbed AER function in the absence of Pdlim7, pathway components in the fin mesenchyme are misregulated or absent, indicating a breakdown of the Fgf signaling feedback loop, which is ultimately responsible for the loss of fin outgrowth.

Conclusion: This work provides the first evidence for the involvement of Pdlim7 in pectoral fin development. Proper fin outgrowth requires fgf24 downregulation in the fin mesenchyme with subsequent activation in the AER, and Pdlim7 appears to regulate this transition, potentially through Tbx5 regulation. By controlling Tbx5 subcellular localization and transcriptional activity and possibly additional yet unknown means, Pdlim7 is required for proper development of the heart and the fins. These new regulatory mechanisms may have important implications how we interpret Tbx5 function in congenital hand/heart syndromes in humans.

Show MeSH
Related in: MedlinePlus