A zebrafish model of Granulin deficiency reveals essential roles in myeloid cell differentiation

Granulin (GRN) is a pleiotropic protein involved in inflammation, wound healing, neurodegenerative disease, and tumorigenesis. These roles in human health have prompted research efforts to utilize Granulin in the treatment of rheumatoid arthritis, frontotemporal dementia, and to enhance wound healing. How granulin contributes to each of these diverse biological functions, however, remains largely unknown. Here, we have uncovered a new role for granulin during myeloid cell differentiation. Using a zebrafish model of granulin deficiency, we reveal that myeloid progenitors are unable to terminally differentiate into neutrophils and macrophages in the absence of granulin a (grna), and fail to express the myeloid genes cebpa, rgs2, lyz, mpx, mpeg1, mfap4, and apoeb. Pathology studies in combination with RNA-sequencing show that in addition to facilitating myeloid cell differentiation, granulin actively inhibits the erythroid program. Moreover, grna deficient myeloid progenitors are incapable of triggering a myelopoiesis emergency response, resulting in decreased recruitment of macrophages to the wound and therefore abnormal healing showing aberrant collagen depositions. Mechanistically, we have performed CUT&RUN for the first time in zebrafish, and identified that Pu.1 directly binds grna enhancers, triggering its expression. Similarly, mammalian granulin is also upregulated in myeloid cells, and its expression is controlled by the myeloid transcription factors PU.1 and IRF8, demonstrating a conserved regulatory mechanism among the zebrafish and mammalian genes. Altogether, our findings uncover a previously unrecognized role for granulin during myeloid cell differentiation, opening a new field of study that will help elucidate how granulin impacts inflammation, wound healing, tumor progression, and neurodegenerative disease. Adult grna-/- and grna+/+ control fish (three fish per condition) were subjected to cardiocentesis and kidney dissection as described here. RNA was isolated with RNeasy (Qiagen) following the manufacturer instructions. Total RNA was assessed for quality using an Agilent Tapestation 4200, and samples with an RNA Integrity Number (RIN) greater than 8.0 were used to generate RNA sequencing libraries using the TruSeq Stranded mRNA Sample Prep (Illumina, San Diego, CA). Samples were processed following manufacturer’s instructions, starting with 50 ng of RNA and modifying RNA shear time to five minutes. Resulting libraries were multiplexed and sequenced with 75 basepair (bp) single reads (SR75) to a depth of approximately 20 million reads per sample on an Illumina HiSeq 4000. Samples were demuxltiplexed using bcl2fastq v2.20 Conversion Software (Illumina, San Diego, CA).