Tbx5a and Tbx5b paralogues act in combination to control separate vectors of migration in the fin field of zebrafish.

The T-box containing family member, TBX5, has been shown to play important functional roles in the pectoral appendages of a variety of vertebrate species. While a single TBX5 gene exists in all tetrapods studied to date, the zebrafish genome retains two paralogues, designated as tbx5a and tbx5b, resulting from a whole genome duplication in the teleost lineage. Zebrafish deficient in tbx5a lack pectoral fin buds, whereas zebrafish deficient in tbx5b exhibit misshapen pectoral fins, showing that both paralogues function in fin development. The mesenchymal cells of the limb/fin bud are derived from the Lateral Plate Mesoderm (LPM). Previous fate mapping work in zebrafish has shown that wildtype (wt) fin field cells are initially located adjacent to somites (s)1-4. The wt fin field cells migrate in opposing diagonal directions placing the limb bud between s2-3 and lateral to the main body. To better characterize tbx5 paralogue functions in zebrafish, time-lapse analyses of the migrations of fin bud precursors under conditions of tbx5a knock-down, tbx5b knock-down and double-knock-down were performed. Our data suggest that zebrafish tbx5a and tbx5b have functionally separated migration direction vectors, that when combined recapitulate the migration of the wt fin field. We and others have shown that loss of Tbx5a function abolishes an fgf24 signaling cue resulting in fin field cells failing to converge in an Antero-Posterior (AP) direction and migrating only in a mediolateral (ML) direction. We show here that loss of Tbx5b function affects initial ML directed movements so that fin field cells fail to migrate laterally but continue to converge along the AP axis. Furthermore, fin field cells in the double Tbx5a/Tbx5b knock-down zebrafish do not engage in directed migrations along either the ML or AP axis. Therefore, these two paralogues may be acting to instruct separate vectors of fin field migration in order to direct proper fin bud formation.

Anterior lateral plate mesoderm gives rise to multiple tissues and requires tbx5a function in left-right asymmetry, migration dynamics, and cell specification of late-addition cardiac cells.

In this study, we elucidate a single cell resolution fate map in the zebrafish in a sub-section of the anterior Lateral Plate Mesoderm (aLPM) at 18 hpf. Our results show that this tissue is not organized into segregated regions but gives rise to intermingled pericardial sac, peritoneum, pharyngeal arch and cardiac precursors. We further report upon asymmetrical contributions of lateral aLPM-derived heart precursors-specifically that twice as many heart precursors arise from the right side versus the left side of the embryo. Cell tracking analyses and large-scale cell labeling of the lateral aLPM corroborate these differences and show that the observed asymmetries are dependent upon Tbx5a expression. Previously, it was shown that cardiac looping was affected in Tbx5a knock-down and knock-out zebrafish (Garrity et al., 2002; Parrie et al., 2013); our present data also implicate tbx5a function in cell specification, establishment and maintenance of cardiac left-right asymmetry.

A transcriptomics analysis of the Tbx5 paralogues in zebrafish.

TBX5 is essential for limb and heart development. Mutations in TBX5 are associated with Holt-Oram syndrome in humans. Due to the teleost specific genome duplication, zebrafish have two copies of TBX5: tbx5a and tbx5b. Both of these genes are expressed in regions of the lateral plate mesoderm and retina. In this study, we perform comparative RNA sequencing analysis on zebrafish embryos during the stages of lateral plate mesoderm migration. This work shows that knockdown of the Tbx5 paralogues results in altered gene expression in many tissues outside of the lateral plate mesoderm, especially in the somitic mesoderm and the intermediate mesoderm. Specifically, knockdown of tbx5b results in changes in somite size, in the differentiation of vasculature progenitors and in later patterning of trunk blood vessels.

Evolutionarily conserved Tbx5-Wnt2/2b pathway orchestrates cardiopulmonary development.

Codevelopment of the lungs and heart underlies key evolutionary innovations in the transition to terrestrial life. Cardiac specializations that support pulmonary circulation, including the atrial septum, are generated by second heart field (SHF) cardiopulmonary progenitors (CPPs). It has been presumed that transcription factors required in the SHF for cardiac septation, e.g., Tbx5, directly drive a cardiac morphogenesis gene-regulatory network. Here, we report instead that TBX5 directly drives Wnt ligands to initiate a bidirectional signaling loop between cardiopulmonary mesoderm and the foregut endoderm for endodermal pulmonary specification and, subsequently, atrial septation. We show that Tbx5 is required for pulmonary specification in mice and amphibians but not for swim bladder development in zebrafish. TBX5 is non-cell-autonomously required for pulmonary endoderm specification by directly driving Wnt2 and Wnt2b expression in cardiopulmonary mesoderm. TBX5 ChIP-sequencing identified cis-regulatory elements at Wnt2 sufficient for endogenous Wnt2 expression domains in vivo and required for Wnt2 expression in precardiac mesoderm in vitro. Tbx5 cooperated with Shh signaling to drive Wnt2b expression for lung morphogenesis. Tbx5 haploinsufficiency in mice, a model of Holt-Oram syndrome, caused a quantitative decrement of mesodermal-to-endodermal Wnt signaling and subsequent endodermal-to-mesodermal Shh signaling required for cardiac morphogenesis. Thus, Tbx5 initiates a mesoderm-endoderm-mesoderm signaling loop in lunged vertebrates that provides a molecular basis for the coevolution of pulmonary and cardiac structures required for terrestrial life.