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Ambra1 knockdown in zebrafish leads to incomplete development due to severe defects in organogenesis.

ABSTRACT: AMBRA1 is a positive regulator of the BECN1-dependent program of autophagy recently identified in mouse. In this study, we cloned the full-length cDNAs of ambra1a and ambra1b zebrafish paralogous genes. As in mouse, both Ambra1 proteins contain the characteristic WD40 repeat region. The transcripts of both genes are present as maternal RNAs in the eggs and display a gradual decline until 8 hpf, being replaced by zygotic mRNAs from 12 hpf onwards. After 24 hpf, the transcripts are mainly localized in the head, suggesting a possible role in brain development. To check their developmental roles, we adopted morpholino knockdown to block either translation (ATGMOs) or splicing (SPLICMOs). Treatment with ATGMOs causes severe embryonic malformations, as prelarvae could survive for only 3 and 4 days in ambra1a and b morphants, respectively. Treatment with SPLICMOs led to developmental defects only at a late stage, indicating the importance of maternally supplied ambra1 transcripts. Analysis of the levels of Lc3-II, an autophagosome-specific marker, in the presence of lysosome inhibitors evidenced a reduction in the rate of autophagosome formation in both MOs-injected embryos at 48 hpf, more pronounced in the case of ambra1a gene. Although some defects, such as body growth delay, curved shape and hemorrhagic pericardial cavity were present in both morphants, the occurrence of specific phenotypes, such as major abnormalities of brain development in ambra1a morphants, suggests the possible acquisition of specific functions by the two paralogous genes that are both required during development and do not compensate each other following knockdown.

SUBMITTER: Benato F

PROVIDER: S-EPMC3627665 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Ambra1 knockdown in zebrafish leads to incomplete development due to severe defects in organogenesis.

Benato Francesca F Skobo Tatjana T Gioacchini Giorgia G Moro Isabella I Ciccosanti Fabiola F Piacentini Mauro M Fimia Gian Maria GM Carnevali Oliana O Dalla Valle Luisa L

Autophagy 20130124 4

AMBRA1 is a positive regulator of the BECN1-dependent program of autophagy recently identified in mouse. In this study, we cloned the full-length cDNAs of ambra1a and ambra1b zebrafish paralogous genes. As in mouse, both Ambra1 proteins contain the characteristic WD40 repeat region. The transcripts of both genes are present as maternal RNAs in the eggs and display a gradual decline until 8 hpf, being replaced by zygotic mRNAs from 12 hpf onwards. After 24 hpf, the transcripts are mainly localize ...[more]

PMID: 23348054

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Project description:BackgroundProgranulin (PGRN) encoded by the GRN gene, is a secreted glycoprotein growth factor that has been implicated in many physiological and pathophysiological processes. PGRN haploinsufficiency caused by autosomal dominant mutations within the GRN gene leads to progressive neuronal atrophy in the form of frontotemporal lobar degeneration (FTLD). This form of the disease is associated with neuronal inclusions that bear the ubiquitinated TAR DNA Binding Protein-43 (TDP-43) molecular signature (FTLD-U). The neurotrophic properties of PGRN in vitro have recently been reported but the role of PGRN in neurons is not well understood. Here we document the neuronal expression and functions of PGRN in spinal cord motoneuron (MN) maturation and branching in vivo using zebrafish, a well established model of vertebrate embryonic development.ResultsWhole-mount in situ hybridization and immunohistochemical analyses of zebrafish embryos revealed that zfPGRN-A is expressed within the peripheral and central nervous systems including the caudal primary (CaP) MNs within the spinal cord. Knockdown of zfPGRN-A mRNA translation mediated by antisense morpholino oligonucleotides disrupted normal CaP MN development resulting in both truncated MNs and inappropriate early branching. Ectopic over-expression of zfPGRN-A mRNA resulted in increased MN branching and rescued the truncation defects brought about by knockdown of zfPGRN-A expression. The ability of PGRN to interact with established MN developmental pathways was tested. PGRN over-expression was found to reverse the truncation defect resulting from knockdown of Survival of motor neuron 1 (smn1). This is involved in small ribonucleoprotein biogenesis RNA processing, mutations of which cause Spinal Muscular Atrophy (SMA) in humans. It did not reverse the MN defects caused by interfering with the neuronal guidance pathway by knockdown of expression of NRP-1, a semaphorin co-receptor.ConclusionsExpression of PGRN within MNs and the observed phenotypes resulting from mRNA knockdown and over-expression are consistent with a role in the regulation of spinal cord MN development and branching. This study presents the first in vivo demonstration of the neurotrophic properties of PGRN and suggests possible future therapeutic applications in the treatment of neurodegenerative diseases.

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Ambra1 knockdown in zebrafish leads to incomplete development due to severe defects in organogenesis.

Publications

Ambra1 knockdown in zebrafish leads to incomplete development due to severe defects in organogenesis.

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