A Zebrafish Model of Dnajc21 Deficiency Identifies Essential Roles in Granulocyte Differentiation and Metabolic Regulation

Shwachman-Diamond syndrome (SDS) is a rare inherited bone marrow failure syndrome (IBMFS) characterized by neutropenia and exocrine pancreatic insufficiency. It is caused by mutations in genes required for ribosome subunit maturation such as SBDS, DNAJC21 and EFL1. The cumulative risk of developing myelodysplastic syndrome and/or acute myeloid leukemia for SDS patients is 36% by 30 years of age. As IBMFS are rare and large numbers of primary human samples are not readily available for mechanistic studies, we employed the zebrafish (Danio rerio), given their highly conserved hematopoietic program and ease of genetic manipulation. In contrast to SBDS, no in vivo models of DNAJC21 mutations have been published to date.

Using CRISPR-Cas9 genomic editing, we generated mutations in the zebrafish dnajc21 gene. Homozygous mutant larvae recapitulated key features of SDS such as reduced growth and poor neutrophil counts as assessed by Sudan Black staining. Using whole-mount in situ hybridization, we observed reduced l-plastin⁺ leukocytes with no changes in upstream myb⁺/cebpa⁺ hematopoietic stem and progenitor cells and spi1b⁺ common myeloid progenitors, suggesting a potential differentiation block. In contrast, Dnajc21 loss did not affect erythrocyte specification. Flow cytometry of mutant whole kidney marrows (WKMs, equivalent to human bone marrow) revealed normal numbers of erythrocytes and lymphocytes but reduced myeloid cells accompanied by a trend towards increased progenitors. Further, we found reduced levels of phosphohistone-H3 (pH3) in the mutant larvae, suggesting hypoproliferation. Bulk RNA sequencing of whole larvae at 48 hours post-fertilization identified xenobiotic metabolism, regulation of endopeptidase activity and immune-related processes as key downregulated pathways in the mutants. Upregulated pathways included platelet-derived growth factor receptor signaling, cell adhesion and lysosomal transport. In addition, several genes involved in carbohydrate metabolism such as g6pd, pck1 and ganc were dysregulated. Further, using Oil Red O staining, we observed a reduced accumulation of neutral lipids in the mutants.

Leukemic transformation in SDS is driven by the acquisition of mutations in additional genes, including the tumor suppressor, TP53. To model SDS-AML transformation, we crossed the dnajc21 mutants with a zebrafish line carrying a tp53^R217H/R217H gain-of-function mutation that confers anti-apoptotic phenotypes. Embryonic pH3⁺ cell counts were restored and WKM myeloid cells and hematopoietic progenitors were increased in dnajc21^-/-/tp53^R217H/R217H fish. Finally, using liquid chromatography-mass spectrometry, we show that Dnajc21 is involved in the regulation of certain metabolite and lipid species including hexosyl-ceramides and sphingomyelins, which may contribute to SDS pathophysiology. Our findings suggest that Dnajc21 is required for normal granulocyte differentiation and cell proliferation. We also identified previously unrecognized roles for Dnajc21 in regulating cellular metabolism. We propose that the zebrafish models described here readily serve as in vivo platforms to identify therapeutic compounds that restore normal hematopoiesis and prevent leukemic transformation in SDS.

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