Embryonic Senescence and Laminopathies in a Progeroid Zebrafish Model

Abstract

BackgroundMutations that disrupt the conversion of prelamin A to mature lamin A cause the rare genetic disorder Hutchinson-Gilford progeria syndrome and a group of laminopathies. Our understanding of how A-type lamins function in vivo during early vertebrate development through aging remains limited, and would benefit from a suitable experimental model. The zebrafish has proven to be a tractable model organism for studying both development and aging at the molecular genetic level. Zebrafish show an array of senescence symptoms resembling those in humans, which can be targeted to specific aging pathways conserved in vertebrates. However, no zebrafish models bearing human premature senescence currently exist.Principal FindingsWe describe the induction of embryonic senescence and laminopathies in zebrafish harboring disturbed expressions of the lamin A gene (LMNA). Impairments in these fish arise in the skin, muscle and adipose tissue, and sometimes in the cartilage. Reduced function of lamin A/C by translational blocking of the LMNA gene induced apoptosis, cell-cycle arrest, and craniofacial abnormalities/cartilage defects. By contrast, induced cryptic splicing of LMNA, which generates the deletion of 8 amino acid residues lamin A (zlamin A-Δ8), showed embryonic senescence and S-phase accumulation/arrest. Interestingly, the abnormal muscle and lipodystrophic phenotypes were common in both cases. Hence, both decrease-of-function of lamin A/C and gain-of-function of aberrant lamin A protein induced laminopathies that are associated with mesenchymal cell lineages during zebrafish early development. Visualization of individual cells expressing zebrafish progerin (zProgerin/zlamin A-Δ37) fused to green fluorescent protein further revealed misshapen nuclear membrane. A farnesyltransferase inhibitor reduced these nuclear abnormalities and significantly prevented embryonic senescence and muscle fiber damage induced by zProgerin. Importantly, the adult Progerin fish survived and remained fertile with relatively mild phenotypes only, but had shortened lifespan with obvious distortion of body shape.ConclusionWe generated new zebrafish models for a human premature aging disorder, and further demonstrated the utility for studying laminopathies. Premature aging could also be modeled in zebrafish embryos. This genetic model may thus provide a new platform for future drug screening as well as genetic analyses aimed at identifying modifier genes that influence not only progeria and laminopathies but also other age-associated human diseases common in vertebrates.

Autolysosome biogenesis and developmental senescence are regulated by both Spns1 and v-ATPase

“…4 Furthermore, we have established the senescence-associated Glb1/b-galactosidase (SA-Glb1) assay, which is commonly used to monitor senescence in mammalian cells, in zebrafish embryos and larvae as well as adults. 4,7,[15][16][17] The appearance of yolk opaqueness accompanied by increased SA-Glb1 activity in spns1-mutant fish embryos or larvae supports the notion that the interruption of the intrinsic nutrient supply, supposedly from autophagydependent catabolism of the yolk in zebrafish embryos and larvae, 18 may lead to profound energetic exhaustion under the aberrant autolysosomal condition resulting from Spns1 deficiency. As reported previously, we found that a specific inhibitor of the v-ATPase, bafilomycin A 1 (BafA), and several other U.S. Food and Drug Administration-approved proton-pump inhibitors such as omeprazole, lansoprazole and pantoprazole could significantly suppress the phenotypes induced by Spns1 deficiency ( Fig.…”

“…4,7,15 All animals were photographed under the same conditions using reflected light with a macro microscope, AZ100 (Nikon, Tokyo, Japan). SA-Glb1 activity in each animal was quantified using a selection tool in Adobe Photoshop software for a color range that was chosen using 25 additive color selections of regions that showed visual SA-Glb1 staining.…”

De novo SOX11 mutations cause Coffin–Siris syndrome

“…To detect apoptotic cells in live embryos, embryos at 30 hpf were manually dechorionated and incubated in acridine orange (2 mg ml À 1 in egg water) at 28°C for 1 h. After washing with egg water six times for 10 min each, embryos were anaesthetized with tricaine, mounted in 2% methylcellulose and examined by confocal microscopy. Apoptotic cells were also examined by the TUNEL assay, as previously described 26 . Embryos at 30 hpf, were fixed overnight in 4% PFA with PBS at 4°C and stored in 100% methanol at À 20°C.…”

The search for evolutionary developmental origins of aging in zebrafish: A novel intersection of developmental and senescence biology in the zebrafish model system

“…To assess human disease associated with accelerated aging symptoms, we recently developed new zebrafish models for a human progeria and further demonstrated their utility for studying laminopathies (Koshimizu et al, 2011). Mutations that disrupt the conversion of prelamin A to Encodes a small nuclear ribonucleoprotein SNRPD1 that belongs to the spliceosomal snRNP core protein family.…”

“…We anticipated that zebrafish could show an array of senescence symptoms resulting from deteriorations of the lamin A-specific pathway conserved in vertebrates. We attempted to generate zebrafish models of HGPS and laminopathies by disturbing the expressions of the lamin A gene (LMNA) (Koshimizu et al, 2011). Impairments in the compromised fish arise in the skin, muscle, and adipose tissue, and sometimes in the cartilage.…”