Limits...
Distinct tissue-specific requirements for the zebrafish tbx5 genes during heart, retina and pectoral fin development.

Pi-Roig A, Martin-Blanco E, Minguillon C - Open Biol (2014)

Bottom Line: However, zebrafish embryos with compromised tbx5 function show a complete absence of pectoral fins, while heart development is disturbed at significantly later developmental stages and eye development remains to be thoroughly analysed.Our data show that distinct relationships between tbx5 paralogues are required in a tissue-specific manner to ensure the proper morphogenesis of the three organs in which they are expressed.Furthermore, we uncover a novel role for tbx5 genes in the establishment of correct heart asymmetry in zebrafish embryos.

View Article: PubMed Central - PubMed

Affiliation: CSIC-Institut de Biologia Molecular de Barcelona, Department of Developmental Biology, Parc Científic de Barcelona, C/Baldiri Reixac, 10, Barcelona 08028, Spain.

ABSTRACT
The transcription factor Tbx5 is expressed in the developing heart, eyes and anterior appendages. Mutations in human TBX5 cause Holt-Oram syndrome, a condition characterized by heart and upper limb malformations. Tbx5-knockout mouse embryos have severely impaired forelimb and heart morphogenesis from the earliest stages of their development. However, zebrafish embryos with compromised tbx5 function show a complete absence of pectoral fins, while heart development is disturbed at significantly later developmental stages and eye development remains to be thoroughly analysed. We identified a novel tbx5 gene in zebrafish--tbx5b--that is co-expressed with its paralogue, tbx5a, in the developing eye and heart and hypothesized that functional redundancy could be occurring in these organs in embryos with impaired tbx5a function. We have now investigated the consequences of tbx5a and/or tbx5b downregulation in zebrafish to reveal that tbx5 genes have essential roles in the establishment of cardiac laterality, dorsoventral retina axis organization and pectoral fin development. Our data show that distinct relationships between tbx5 paralogues are required in a tissue-specific manner to ensure the proper morphogenesis of the three organs in which they are expressed. Furthermore, we uncover a novel role for tbx5 genes in the establishment of correct heart asymmetry in zebrafish embryos.

Show MeSH

Related in: MedlinePlus

The hst mutation is a hypomorphic allele with regards to cardiac laterality. (a) tbx5a variants generated to perform the rescue experiments: a tbx5a full-length (tbx5a FL) version includes the whole N-terminal (N, black rectangle), T- (T, blue rectangle) and C-terminal (C, grey rectangle) domains; a heartstrings version (tbx5a hst) containing the whole N-terminal domain and T-domain and a truncated C-terminal domain; and a tbx5a severely truncated version (tbx5a trunc) that contains the whole N-terminal domain and a truncated T-domain. (b) Quantification of the rescue experiments (wt, left jog; phenotype, right and middle jog). A χ2 statistic has been calculated to assess significant differences between groups (**p < 0.01, *p < 0.05). (c,d) Dorsal views with anterior towards the left of 26 hpf +/+ or +/hst (c) and hst mutant (d) embryos showing normal leftward jogging of the embryonic heart tube highlighted by myl7 expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4043114&req=5

RSOB140014F3: The hst mutation is a hypomorphic allele with regards to cardiac laterality. (a) tbx5a variants generated to perform the rescue experiments: a tbx5a full-length (tbx5a FL) version includes the whole N-terminal (N, black rectangle), T- (T, blue rectangle) and C-terminal (C, grey rectangle) domains; a heartstrings version (tbx5a hst) containing the whole N-terminal domain and T-domain and a truncated C-terminal domain; and a tbx5a severely truncated version (tbx5a trunc) that contains the whole N-terminal domain and a truncated T-domain. (b) Quantification of the rescue experiments (wt, left jog; phenotype, right and middle jog). A χ2 statistic has been calculated to assess significant differences between groups (**p < 0.01, *p < 0.05). (c,d) Dorsal views with anterior towards the left of 26 hpf +/+ or +/hst (c) and hst mutant (d) embryos showing normal leftward jogging of the embryonic heart tube highlighted by myl7 expression.

Mentions: The reasons for the discrepancies between our results, implicating the tbx5 genes in the asymmetry events the zebrafish heart undergoes (namely cardiac jogging first and looping later), and those of others [17] are unclear. One possibility is that we have used a MO against tbx5a to downregulate its function, whereas Parrie et al. [17] used the tbx5a mutant line hst to analyse the effects of this gene loss-of-function. The hst mutation introduces a premature STOP codon at residue 316 of the tbx5a open reading frame, which leaves the mutated protein with intact N-terminal and T-box (DNA binding) domains and part of the C-terminal domain. It is therefore possible that the hst mutation behaves as an hypomorphic allele with regard to the left–right asymmetric development of the heart. In agreement with this hypothesis, most of the TBX5 mutations causing a clear HOS phenotype lie upstream of the predicted hst mutation. To test whether, indeed, the hst mutation behaves as an hypomorphic allele with regards to cardiac development, we developed an assay to assess whether the laterality phenotype of tbx5a morphants could be rescued by introducing specific MO-insensitive forms of the tbx5a mRNA: (i) a full-length tbx5a mRNA that should be able to rescue the tbx5a MO-mediated phenotype, (ii) a tbx5a mRNA that is identical to that produced in hst mutant embryos and (iii) a severely truncated version of tbx5a that we have engineered by introducing a premature STOP codon within the T-box domain (figure 3a). Notably, both the full-length and the hst-like forms of tbx5a were able to partially rescue the cardiac jogging phenotype when co-injected with the tbx5a MO (figure 3b; n = 65 and n = 168, respectively). Similarly, a full-length tbx5b form was able to rescue the jogging phenotype of tbx5b morphants when co-injected with our tbx5b MO (figure 3b; n = 117). By contrast, the severely truncated form of tbx5a was not able to rescue the laterality phenotype (figure 3b, n = 79). Overall, these data demonstrate not only the specificity of the cardiac phenotypes caused by MO-mediated knock-down of tbx5a and/or tbx5b, but also that the hst mutation behaves as a hypomorphic allele with regard to cardiac laterality. We have ourselves analysed heart tube jogging in hst mutants (n = 38) and all of them displayed a normal left-jog as visualized by myl7 expression in 26 hpf embryos (figure 3c,d). These results underline the need for caution when using the hst mutation as a tbx5a loss-of-function allele.Figure 3.


Distinct tissue-specific requirements for the zebrafish tbx5 genes during heart, retina and pectoral fin development.

Pi-Roig A, Martin-Blanco E, Minguillon C - Open Biol (2014)

The hst mutation is a hypomorphic allele with regards to cardiac laterality. (a) tbx5a variants generated to perform the rescue experiments: a tbx5a full-length (tbx5a FL) version includes the whole N-terminal (N, black rectangle), T- (T, blue rectangle) and C-terminal (C, grey rectangle) domains; a heartstrings version (tbx5a hst) containing the whole N-terminal domain and T-domain and a truncated C-terminal domain; and a tbx5a severely truncated version (tbx5a trunc) that contains the whole N-terminal domain and a truncated T-domain. (b) Quantification of the rescue experiments (wt, left jog; phenotype, right and middle jog). A χ2 statistic has been calculated to assess significant differences between groups (**p < 0.01, *p < 0.05). (c,d) Dorsal views with anterior towards the left of 26 hpf +/+ or +/hst (c) and hst mutant (d) embryos showing normal leftward jogging of the embryonic heart tube highlighted by myl7 expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043114&req=5

RSOB140014F3: The hst mutation is a hypomorphic allele with regards to cardiac laterality. (a) tbx5a variants generated to perform the rescue experiments: a tbx5a full-length (tbx5a FL) version includes the whole N-terminal (N, black rectangle), T- (T, blue rectangle) and C-terminal (C, grey rectangle) domains; a heartstrings version (tbx5a hst) containing the whole N-terminal domain and T-domain and a truncated C-terminal domain; and a tbx5a severely truncated version (tbx5a trunc) that contains the whole N-terminal domain and a truncated T-domain. (b) Quantification of the rescue experiments (wt, left jog; phenotype, right and middle jog). A χ2 statistic has been calculated to assess significant differences between groups (**p < 0.01, *p < 0.05). (c,d) Dorsal views with anterior towards the left of 26 hpf +/+ or +/hst (c) and hst mutant (d) embryos showing normal leftward jogging of the embryonic heart tube highlighted by myl7 expression.
Mentions: The reasons for the discrepancies between our results, implicating the tbx5 genes in the asymmetry events the zebrafish heart undergoes (namely cardiac jogging first and looping later), and those of others [17] are unclear. One possibility is that we have used a MO against tbx5a to downregulate its function, whereas Parrie et al. [17] used the tbx5a mutant line hst to analyse the effects of this gene loss-of-function. The hst mutation introduces a premature STOP codon at residue 316 of the tbx5a open reading frame, which leaves the mutated protein with intact N-terminal and T-box (DNA binding) domains and part of the C-terminal domain. It is therefore possible that the hst mutation behaves as an hypomorphic allele with regard to the left–right asymmetric development of the heart. In agreement with this hypothesis, most of the TBX5 mutations causing a clear HOS phenotype lie upstream of the predicted hst mutation. To test whether, indeed, the hst mutation behaves as an hypomorphic allele with regards to cardiac development, we developed an assay to assess whether the laterality phenotype of tbx5a morphants could be rescued by introducing specific MO-insensitive forms of the tbx5a mRNA: (i) a full-length tbx5a mRNA that should be able to rescue the tbx5a MO-mediated phenotype, (ii) a tbx5a mRNA that is identical to that produced in hst mutant embryos and (iii) a severely truncated version of tbx5a that we have engineered by introducing a premature STOP codon within the T-box domain (figure 3a). Notably, both the full-length and the hst-like forms of tbx5a were able to partially rescue the cardiac jogging phenotype when co-injected with the tbx5a MO (figure 3b; n = 65 and n = 168, respectively). Similarly, a full-length tbx5b form was able to rescue the jogging phenotype of tbx5b morphants when co-injected with our tbx5b MO (figure 3b; n = 117). By contrast, the severely truncated form of tbx5a was not able to rescue the laterality phenotype (figure 3b, n = 79). Overall, these data demonstrate not only the specificity of the cardiac phenotypes caused by MO-mediated knock-down of tbx5a and/or tbx5b, but also that the hst mutation behaves as a hypomorphic allele with regard to cardiac laterality. We have ourselves analysed heart tube jogging in hst mutants (n = 38) and all of them displayed a normal left-jog as visualized by myl7 expression in 26 hpf embryos (figure 3c,d). These results underline the need for caution when using the hst mutation as a tbx5a loss-of-function allele.Figure 3.

Bottom Line: However, zebrafish embryos with compromised tbx5 function show a complete absence of pectoral fins, while heart development is disturbed at significantly later developmental stages and eye development remains to be thoroughly analysed.Our data show that distinct relationships between tbx5 paralogues are required in a tissue-specific manner to ensure the proper morphogenesis of the three organs in which they are expressed.Furthermore, we uncover a novel role for tbx5 genes in the establishment of correct heart asymmetry in zebrafish embryos.

View Article: PubMed Central - PubMed

Affiliation: CSIC-Institut de Biologia Molecular de Barcelona, Department of Developmental Biology, Parc Científic de Barcelona, C/Baldiri Reixac, 10, Barcelona 08028, Spain.

ABSTRACT
The transcription factor Tbx5 is expressed in the developing heart, eyes and anterior appendages. Mutations in human TBX5 cause Holt-Oram syndrome, a condition characterized by heart and upper limb malformations. Tbx5-knockout mouse embryos have severely impaired forelimb and heart morphogenesis from the earliest stages of their development. However, zebrafish embryos with compromised tbx5 function show a complete absence of pectoral fins, while heart development is disturbed at significantly later developmental stages and eye development remains to be thoroughly analysed. We identified a novel tbx5 gene in zebrafish--tbx5b--that is co-expressed with its paralogue, tbx5a, in the developing eye and heart and hypothesized that functional redundancy could be occurring in these organs in embryos with impaired tbx5a function. We have now investigated the consequences of tbx5a and/or tbx5b downregulation in zebrafish to reveal that tbx5 genes have essential roles in the establishment of cardiac laterality, dorsoventral retina axis organization and pectoral fin development. Our data show that distinct relationships between tbx5 paralogues are required in a tissue-specific manner to ensure the proper morphogenesis of the three organs in which they are expressed. Furthermore, we uncover a novel role for tbx5 genes in the establishment of correct heart asymmetry in zebrafish embryos.

Show MeSH
Related in: MedlinePlus