PgmNr Z6051: Zebrafish ambra1a and ambra1b silencing affects heart development.

Authors:
G. Meneghetti 1 ; T. Skobo 2 ; N. Facchinello 1 ; P. Bonaldo 2 ; F. Cecconi 3 ; L. Dalla Valle 1


Institutes
1) Department of Biology, University of Padua, Italy; 2) Department of Molecular medicine, University of Padua, Italy; 3) Department of Biology, University of Rome Tor Vergata, Italy.


Abstract:

In zebrafish two paralogous genes, ambra1a and ambra1b, both required for the autophagic process and during development, encode the protein Ambra1, a positive regulator of early steps of autophagosome formation. As evidenced by whole mount in situ hybridization (WMISH), both transcripts are expressed in the heart-forming region. In this work, we analyse the ambra1a and ambra1b knockdown effects on heart development by means of morpholino oligonucleotides (MOs). Silencing of the two proteins by ATG-MOs affects heart morphogenesis resulting in a small, string-like heart with pericardial edema, whereas treatment with SPLIC-MOs does not result on clear cardiac phenotypes, indicating the importance of maternally supplied ambra1 transcripts. Co-injection of both ATG-MOs determines a more severe phenotype with a prominent pericardial edema. Cardiac defects are effectively rescued by co-injection of MOs with human AMBRA1 mRNA showing the conservation of Ambra1 functions during evolution. WMISH of myosin light chain 7 (myl7) transcripts as well as confocal analysis of ambra1a and ambra1b morphants of the transgenic line Tg-myl7:egfp reveal, at 24 hpf, defects in the heart jogging process followed by imperfect cardiac looping with a high percentage of 48 hpf embryos in which the heart is organized as a linear tube. Moreover, WMISH of pitx2 transcripts, which at the 21-somite stage are expressed on the left plate mesoderm, reveals both bilateral or reversed expression of this gene in both ambra1 morphants indicating that ambra1 silencing interferes on heart laterality.

3-dpf WT and morphants purified hearts were used for expression analysis of developmental heart markers by means of qPCR. This analysis shows a differential expression not only between WT and morphants hearts but also between ambra1a and ambra1b morphants. In fact, while atrial and ventricular myosin heavy chain, as well as gata5 transcripts present a general down regulation in both types of morphants, the expression of other cardiac markers, such as nkx2.5 and hand2, is up regulated only in ambra1b morphants. Although both ambra1a and ambra1b genes appear to be involved in heart development, the different expression pattern suggests the possible acquisition of specific functions by the two paralogous genes.



ZFIN Genetics Index
1. ambra1a
2. ambra1b