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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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tbx5 genes are required for dorsoventral retina organization. (a) Schematics of our quantification method. Expression of efn2a and ephB2 in control (ctrl) embryos (b–b′), tbx5a morphants (c–c′) and tbx5b morphants (d–d′). (e–g′) Expression of efn2a and ephB2 in embryos co-injected with different concentrations of both tbx5a and tbx5b MOs. (h–i) Quantification of the results obtained for the expression of efn2a (h) and ephB2 (i). (j–m) Retinal projections of 48 hpf ath5:GFP embryos injected with control, tbx5a, tbx5b or tbx5a and tbx5b MO. (n) Optic nerve diameter quantifications. Data are represented as the mean ± s.e. A Kruskal–Wallis test was used to determine statistical differences among experimental groups (*p < 0.05, **p < 0.001). D, dorsal; N, nasal; T, temporal; V, ventral.
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RSOB140014F4: tbx5 genes are required for dorsoventral retina organization. (a) Schematics of our quantification method. Expression of efn2a and ephB2 in control (ctrl) embryos (b–b′), tbx5a morphants (c–c′) and tbx5b morphants (d–d′). (e–g′) Expression of efn2a and ephB2 in embryos co-injected with different concentrations of both tbx5a and tbx5b MOs. (h–i) Quantification of the results obtained for the expression of efn2a (h) and ephB2 (i). (j–m) Retinal projections of 48 hpf ath5:GFP embryos injected with control, tbx5a, tbx5b or tbx5a and tbx5b MO. (n) Optic nerve diameter quantifications. Data are represented as the mean ± s.e. A Kruskal–Wallis test was used to determine statistical differences among experimental groups (*p < 0.05, **p < 0.001). D, dorsal; N, nasal; T, temporal; V, ventral.

Mentions: We analysed the expression of the dorsally expressed ephrin, efnb2a, and the ventrally expressed ephrin receptor, ephB2, because restricted ephrinB/ephB expression along the dorsoventral axis has been shown to play a key role in retinotectal topographic map formation [24,25]. To quantify the extent of efnb2a expression, we measured the angle of expression of this gene by setting a ‘hinge’ in the centre of the lens (figure 4a). In control embryos, the efnb2a expression domain was measured to form an average angle of 63° (figure 4b,h). Knock-down of either tbx5a or tbx5b caused a reduction of the efnb2a angle of expression leading to an average angle of 54°, although this decrease was not found to be statistically significant (figure 4c,d,h). To investigate whether both paralogues function in conjunction to determine the extent of dorsal efnb2a expression, we co-injected sub-optimal doses of tbx5a and tbx5b MOs. Remarkably, the efnb2a expression domain was greatly reduced to an average angle of 37°, a statistically significant 41% reduction compared with control embryos (figure 4e,h). Injection of increasing concentrations of both MOs caused slightly more severe effects than those observed after co-injection of sub-optimal doses of tbx5a and tbx5b MOs (figure 4f,g,h). Notably, further statistical analyses showed that significant differences are found between the injection of single tbx5a and tbx5b MOs with respect to their co-injection at these higher doses (1.5 and 3 ng each; figure 4h), suggesting that tbx5 paralogues act redundantly in the dorsal retina to ensure efnb2a expression in this territory. To better map the decrease of efnb2a expression observed in tbx5a- and/or tbx5b-morphant retinas, we divided the angles obtained into two, a dorso-nasal angle and a dorso-temporal angle, by setting the dorsal-most point as the point lying dorsal to the ventrally located choroid fissure (D in figure 4a). This showed that both dorso-nasal and dorso-temporal borders of expression similarly decreased accordingly to the total angle of efnb2a expression measured (electronic supplementary material, figure S2a–c).Figure 4.


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)

tbx5 genes are required for dorsoventral retina organization. (a) Schematics of our quantification method. Expression of efn2a and ephB2 in control (ctrl) embryos (b–b′), tbx5a morphants (c–c′) and tbx5b morphants (d–d′). (e–g′) Expression of efn2a and ephB2 in embryos co-injected with different concentrations of both tbx5a and tbx5b MOs. (h–i) Quantification of the results obtained for the expression of efn2a (h) and ephB2 (i). (j–m) Retinal projections of 48 hpf ath5:GFP embryos injected with control, tbx5a, tbx5b or tbx5a and tbx5b MO. (n) Optic nerve diameter quantifications. Data are represented as the mean ± s.e. A Kruskal–Wallis test was used to determine statistical differences among experimental groups (*p < 0.05, **p < 0.001). D, dorsal; N, nasal; T, temporal; V, ventral.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140014F4: tbx5 genes are required for dorsoventral retina organization. (a) Schematics of our quantification method. Expression of efn2a and ephB2 in control (ctrl) embryos (b–b′), tbx5a morphants (c–c′) and tbx5b morphants (d–d′). (e–g′) Expression of efn2a and ephB2 in embryos co-injected with different concentrations of both tbx5a and tbx5b MOs. (h–i) Quantification of the results obtained for the expression of efn2a (h) and ephB2 (i). (j–m) Retinal projections of 48 hpf ath5:GFP embryos injected with control, tbx5a, tbx5b or tbx5a and tbx5b MO. (n) Optic nerve diameter quantifications. Data are represented as the mean ± s.e. A Kruskal–Wallis test was used to determine statistical differences among experimental groups (*p < 0.05, **p < 0.001). D, dorsal; N, nasal; T, temporal; V, ventral.
Mentions: We analysed the expression of the dorsally expressed ephrin, efnb2a, and the ventrally expressed ephrin receptor, ephB2, because restricted ephrinB/ephB expression along the dorsoventral axis has been shown to play a key role in retinotectal topographic map formation [24,25]. To quantify the extent of efnb2a expression, we measured the angle of expression of this gene by setting a ‘hinge’ in the centre of the lens (figure 4a). In control embryos, the efnb2a expression domain was measured to form an average angle of 63° (figure 4b,h). Knock-down of either tbx5a or tbx5b caused a reduction of the efnb2a angle of expression leading to an average angle of 54°, although this decrease was not found to be statistically significant (figure 4c,d,h). To investigate whether both paralogues function in conjunction to determine the extent of dorsal efnb2a expression, we co-injected sub-optimal doses of tbx5a and tbx5b MOs. Remarkably, the efnb2a expression domain was greatly reduced to an average angle of 37°, a statistically significant 41% reduction compared with control embryos (figure 4e,h). Injection of increasing concentrations of both MOs caused slightly more severe effects than those observed after co-injection of sub-optimal doses of tbx5a and tbx5b MOs (figure 4f,g,h). Notably, further statistical analyses showed that significant differences are found between the injection of single tbx5a and tbx5b MOs with respect to their co-injection at these higher doses (1.5 and 3 ng each; figure 4h), suggesting that tbx5 paralogues act redundantly in the dorsal retina to ensure efnb2a expression in this territory. To better map the decrease of efnb2a expression observed in tbx5a- and/or tbx5b-morphant retinas, we divided the angles obtained into two, a dorso-nasal angle and a dorso-temporal angle, by setting the dorsal-most point as the point lying dorsal to the ventrally located choroid fissure (D in figure 4a). This showed that both dorso-nasal and dorso-temporal borders of expression similarly decreased accordingly to the total angle of efnb2a expression measured (electronic supplementary material, figure S2a–c).Figure 4.

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