Starvation Stress in the Sbds -Null Zebrafish: Implications for Human Shwachman-Diamond Syndrome

Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency, bone marrow dysfunction, and an increase risk of acute myeloid leukemia. Patients with SDS exhibit abnormalities in other organs, including bone, liver and immunologic and endocrine systems. Almost all cases of SDS result from biallelic mutations in the SBDS gene¹. SBDS is involved in ribosomal biogenesis and stabilization of mitotic spindle microtubules^2,3. Recently, it has also been shown a reduction cell proliferation and mTOR and STAT3 expression, both with an important role in cellular growth and tumorigenesis, in SDS derived leukocytes.⁴

Genetic ablation of Sbds results in early embryonic lethality (ED 7.5) in mice⁵. Zebrafish provide an attractive, alternative model organism to study SDS. Genome analysis of zebrafish (Danio rerio) revealed the presence of a single sbds gene, encoding a protein 90% identical to the human orthologue.

We created sbds zebrafish knockout strains that phenocopies the human Shwachman-Diamond Syndrome. Survival rate of mutants was normal at 15 days post fertilization (dpf) (100%) but decline significantly at 21 dpf (4%). Western blot analysis showed a progressive decrease of Sbds during the first 8 dpf, with total absence at 10 dpf. These results suggest that larval viability is due to maternally deposited sbds transcript and corresponding protein, which was confirmed by RT-qPCR and immunoblot at earliest developmental stages. Zebrafish sbds mutants showed neutropenia at 5 and 15 days post fertilization (dpf) but normal macrophage number and hemoglobinization. Pancreas, liver, digestive tract, and eye showed histologic evidence of atrophy at 21 dpf. Moreover, we observed an important reduction in the intestinal epithelium at 15 dpf.

To further characterize effects of Sbds loss, we tested expression of genes involved in lipid metabolism (acc, ppar α , ppar γ and srbp1) and pancreatic transcription factors (ptf1a and prox1) by qPCR. Our results showed that they were not affected at 10 dpf but upregulated at 18 dpf. After staining 15 dpf larvae with oil red we observed, in the mutants compared to their sibling wild type or heterozygous, significantly reduced accumulation of lipids. In addition, we found an increase of expression of genes involved in autophagy (atg5, atg7 and mTOR), and upregulation of casp9 and tp53 expression.With these results we thought that reduced viability was due to a problem in nutrient absorption and therefore starvation. We carried out two starvation experiments, a diet restriction from 10 to 15 dpf, and from 15 to 21 dpf. Unlike sbds mutants, mortality rates were not affected comparing to control groups in any of these experiments. However, genes related to lipid metabolism and autophagy were upregulated and histology showed a reduction in intestinal epithelium as well. Moreover, oil red staining showed lower lipid accumulation in starved fish as we observed in sbds mutants.

With these results, we conclude sbds -null zebrafish showed signs of malnutrition and an effect in fatty acid-metabolism gene expression as seen in starved fish. Our results make it a highly relevant model to investigate new mechanistic hypotheses and develop new small molecule therapies for Shwachman-Diamond syndrome.

1. Boocock GR, Morrison JA, Popovic M, et al. Mutations in SBDS are associated with Shwachman-Diamond syndrome. Nat Genet . 2003;33(1):97-101.

2. Austin KM, Gupta ML, Jr., Coats SA, et al. Mitotic spindle destabilization and genomic instability in Shwachman-Diamond syndrome. J Clin Invest . 2008;118(4):1511-1518.

3. Finch AJ, Hilcenko C, Basse N, et al. Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome. Genes Dev . 2011;25(9):917-929.

4. Bezzerri V, Vella A, Calcaterra E, et al. New insights into the Shwachman-Diamond Syndrome-related haematological disorder: hyper-activation of mTOR and STAT3 in leukocytes. 2016;6:33165.

5. Zhang S, Shi M, Hui CC, Rommens JM. Loss of the mouse ortholog of the Shwachman-Diamond syndrome gene (Sbds) results in early embryonic lethality. Mol Cell Biol . 2006;26(17):6656-6663.