Tbx20 Is an Essential Regulator of Embryonic Heart Growth in Zebrafish

Abstract

The molecular mechanisms that regulate cardiomyocyte proliferation during embryonic heart growth are not completely deciphered yet. In a forward genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we identified the recessive embryonic-lethal zebrafish mutant line weiches herz (whz). Homozygous mutant whz embryos display impaired heart growth due to diminished embryonic cardiomyocyte proliferation resulting in cardiac hypoplasia and weak cardiac contraction. By positional cloning, we found in whz mutant zebrafish a missense mutation within the T-box 20 (Tbx20) transcription factor gene leading to destabilization of Tbx20 protein. Morpholino-mediated knock-down of Tbx20 in wild-type zebrafish embryos phenocopies whz, indicating that the whz phenotype is due to loss of Tbx20 function, thereby leading to significantly reduced cardiomyocyte numbers by impaired proliferation of heart muscle cells. Ectopic overexpression of wild-type Tbx20 in whz mutant embryos restored cardiomyocyte proliferation and heart growth. Interestingly, ectopic overexpression of Tbx20 in wild-type zebrafish embryos resulted, similar to the situation in the embryonic mouse heart, in significantly reduced proliferation rates of ventricular cardiomyocytes, suggesting that Tbx20 activity needs to be tightly fine-tuned to guarantee regular cardiomyocyte proliferation and embryonic heart growth in vivo.

Loss of the novel Vcp (valosin containing protein) interactor Washc4 interferes with autophagy-mediated proteostasis in striated muscle and leads to myopathy in vivo

“…For histology, fixed embryos were embedded in JB-4 (Polysciences, Inc., 00226–1) and 5-µm sections were cut, dried and stained with hematoxylin & eosin [47]. For transmission electron microscopy analyses, zebrafish embryos were fixed in 2.5% glutaraldehyde and 1% paraformaldehyde (Sigma-Aldrich, P6148) in 0.1 M phosphate buffer (pH 7.3).…”

Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish

“…All uninjected embryos were phenotypically normal (n = 52). This observation, together with the fact that tbx20 deficiency leads to severe cardiac defects [22, 25, 26], enabled us to conclude that the integration of two loxP sites does not significantly impair tbx20 expression. Among embryos injected with Cre mRNA, approximately 25% displayed severe cardiac defects at 3 dpf in two independent experiments.…”

“…In zebrafish, tbx20 is expressed in bilateral cardiac precursors prior to their migration and coalescence. Expression appears to persist through larval development, and is upregulated in response to injury in adult hearts [22, 25–27, 40]. In mouse, cardiomyocyte-specific deletion of Tbx20 in adults leads to lethal cardiomyopathy and arrhythmia, indicating that TBX20 is required not only for cardiac development, but also for homeostasis [24].…”

Gene profiling of head mesoderm in early zebrafish development: insights into the evolution of cranial mesoderm

“…tbx20 encodes a T-box transcription factor reported to be expressed in the ALPM [58] and encodes an essential regulator of embryonic heart growth in zebrafish [59]. In chick embryos, Tbx20 is expressed in the lateral plate mesoderm at early developmental stages and subsequently in primitive heart tube [60, 61].…”