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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.

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Knock-down of tbx5 genes causes cardiac looping defects. (a–c) Embryos injected with control MO or sub-optimal concentrations of tbx5a or tbx5b MOs. (d–d″) tbx5a morphant phenotypes. (e–e′) tbx5b-morphant phenotypes. (f–h″) Double knock-down of tbx5 genes (0.5 ng each MO (f–f″), 1.5 ng each MO (g–g″) and 3 ng each MO (h–h″)). (i) Quantification of the degree of looping phenotypes: wt, complete; phenotype, incomplete looping s.o., sub-otimal. (j) Quantification of the looping orientation phenotypes. A χ2 statistic has been calculated to assess significant differences between groups (**p < 0.001, *p < 0.05). Images are frontal views of 48 hpf embryos, and myl7 expression is used to highlight the developing heart.
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RSOB140014F1: Knock-down of tbx5 genes causes cardiac looping defects. (a–c) Embryos injected with control MO or sub-optimal concentrations of tbx5a or tbx5b MOs. (d–d″) tbx5a morphant phenotypes. (e–e′) tbx5b-morphant phenotypes. (f–h″) Double knock-down of tbx5 genes (0.5 ng each MO (f–f″), 1.5 ng each MO (g–g″) and 3 ng each MO (h–h″)). (i) Quantification of the degree of looping phenotypes: wt, complete; phenotype, incomplete looping s.o., sub-otimal. (j) Quantification of the looping orientation phenotypes. A χ2 statistic has been calculated to assess significant differences between groups (**p < 0.001, *p < 0.05). Images are frontal views of 48 hpf embryos, and myl7 expression is used to highlight the developing heart.

Mentions: tbx5a and, as described recently, tbx5b have been implicated in cardiac looping morphogenesis [14,17]. Analyses of looping phenotypes assayed at 48 h post-fertilization (hpf) had shown that wild-type embryos had undergone complete looping (i.e. the ventricle located at the right-hand side of the embryo with the atrial and ventricular chambers sitting side by side), whereas homozygous tbx5a mutant (hst) and tbx5b morphants failed to do so, indicating that downregulation of tbx5 genes was associated with reduced looping. As tbx5b knock-down on hst mutant embryos did not increase the severity of the defects, it was argued that these paralogues do not have an overlapping function in cardiac development. In addition, looping of these hearts in the reverse orientation was never observed [17]. Similarly, we observed incomplete looping after tbx5a or tbx5b knock-down (figure 1d,e, respectively) in comparison to the characteristic looping observed in control morphants that results in the positioning of the ventricle (v) and the atrium (a) parallel to each other (figure 1a). However, in striking contrast to previous data, defects were observed not only in the degree of looping but also in the orientation of cardiac looping: tbx5 morphants can be classified into three distinguishable heart looping orientation groups (D-loop (right, normal), L-loop (left, reversed) and no-loop). After injection of a control MO, over 99% (n = 158) of the embryos displayed an S-shaped heart with the ventricle lying to the right-hand side of the embryo (D-loop; figure 1a,i). By contrast, 88% (n = 121) of the tbx5a MO-injected embryos had incomplete cardiac looping, and within these 57% showed D-loop, 24% showed L-loop and 19% showed no looping at all (figure 1d–d″,i,j). Similarly, injection of tbx5b MO also caused heart looping defects (81%, n = 108), and these embryos displayed D-looped (52%), L-looped (16%) and no-looped (34%) cardiac morphologies (figure 1e–e″,i,j).Figure 1.


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)

Knock-down of tbx5 genes causes cardiac looping defects. (a–c) Embryos injected with control MO or sub-optimal concentrations of tbx5a or tbx5b MOs. (d–d″) tbx5a morphant phenotypes. (e–e′) tbx5b-morphant phenotypes. (f–h″) Double knock-down of tbx5 genes (0.5 ng each MO (f–f″), 1.5 ng each MO (g–g″) and 3 ng each MO (h–h″)). (i) Quantification of the degree of looping phenotypes: wt, complete; phenotype, incomplete looping s.o., sub-otimal. (j) Quantification of the looping orientation phenotypes. A χ2 statistic has been calculated to assess significant differences between groups (**p < 0.001, *p < 0.05). Images are frontal views of 48 hpf embryos, and myl7 expression is used to highlight the developing heart.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140014F1: Knock-down of tbx5 genes causes cardiac looping defects. (a–c) Embryos injected with control MO or sub-optimal concentrations of tbx5a or tbx5b MOs. (d–d″) tbx5a morphant phenotypes. (e–e′) tbx5b-morphant phenotypes. (f–h″) Double knock-down of tbx5 genes (0.5 ng each MO (f–f″), 1.5 ng each MO (g–g″) and 3 ng each MO (h–h″)). (i) Quantification of the degree of looping phenotypes: wt, complete; phenotype, incomplete looping s.o., sub-otimal. (j) Quantification of the looping orientation phenotypes. A χ2 statistic has been calculated to assess significant differences between groups (**p < 0.001, *p < 0.05). Images are frontal views of 48 hpf embryos, and myl7 expression is used to highlight the developing heart.
Mentions: tbx5a and, as described recently, tbx5b have been implicated in cardiac looping morphogenesis [14,17]. Analyses of looping phenotypes assayed at 48 h post-fertilization (hpf) had shown that wild-type embryos had undergone complete looping (i.e. the ventricle located at the right-hand side of the embryo with the atrial and ventricular chambers sitting side by side), whereas homozygous tbx5a mutant (hst) and tbx5b morphants failed to do so, indicating that downregulation of tbx5 genes was associated with reduced looping. As tbx5b knock-down on hst mutant embryos did not increase the severity of the defects, it was argued that these paralogues do not have an overlapping function in cardiac development. In addition, looping of these hearts in the reverse orientation was never observed [17]. Similarly, we observed incomplete looping after tbx5a or tbx5b knock-down (figure 1d,e, respectively) in comparison to the characteristic looping observed in control morphants that results in the positioning of the ventricle (v) and the atrium (a) parallel to each other (figure 1a). However, in striking contrast to previous data, defects were observed not only in the degree of looping but also in the orientation of cardiac looping: tbx5 morphants can be classified into three distinguishable heart looping orientation groups (D-loop (right, normal), L-loop (left, reversed) and no-loop). After injection of a control MO, over 99% (n = 158) of the embryos displayed an S-shaped heart with the ventricle lying to the right-hand side of the embryo (D-loop; figure 1a,i). By contrast, 88% (n = 121) of the tbx5a MO-injected embryos had incomplete cardiac looping, and within these 57% showed D-loop, 24% showed L-loop and 19% showed no looping at all (figure 1d–d″,i,j). Similarly, injection of tbx5b MO also caused heart looping defects (81%, n = 108), and these embryos displayed D-looped (52%), L-looped (16%) and no-looped (34%) cardiac morphologies (figure 1e–e″,i,j).Figure 1.

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