Zebra "Fishing" the Role of Granulin in Hematopoiesis

Granulin (GRN) is a multifunctional protein with anti-inflammatory properties and involved in neurological diseases and tumorigenesis. It contains several cysteine-rich motifs that are unique to this protein, which are conserved from sponges to humans indicating their ancient evolutionary origin. Despite being highly expressed by certain hematopoietic cell lineages, the role that GRN plays in hematopoiesis has reminded elusive. The multifunctional nature of this protein, together with its wide expression in all mammalian cell types has challenged the characterization of its functional role in hematopoiesis due to its effects on other tissues. Therefore, we took advantage of the whole genomic duplication of the zebrafish (Danio rerio) and the high conservation of the cysteine-rich motifs among the zebrafish and human granulins to address this knowledge gap and explore their role in hematopoiesis in vivo.

The whole genome duplication that separated teleost fish from mammals resulted in two copies of the granulin gene in the zebrafish (Granulin a and Granulin b, Grna and Grnb respectively). This has allowed us an unprecedented view into the function of this protein in hematopoiesis. We show that like mammals, grnb transcripts are found in all cell types, including hematopoietic cells. In contrast, grna is restricted to hematopoietic cells, including myeloid populations. The distinct cell expression of grna and grnb suggests that, in the zebrafish, grna evolved to specifically function in hematopoiesis, while grnb may have taken on the rest of the biological roles assigned to the mammalian granulin.

The zebrafish is an animal model with unique advantages for in vivo studies. Its external development allows us to circumvent the challenges of in utero experimentation required using mammals, permitting the use of non-invasive imaging techniques to study developmental hematopoiesis. In addition, more than 70% of genes identified in the zebrafish are conserved in humans. These, together with its high conservation with the human hematopoietic system has led to a greater understanding and prevention of human hematologic diseases by using this elegant animal model. These unique advantages of the zebrafish, in addition to its genetic amenability allowed us to generate Grna and Grnb single mutants and identify their impact in the hematopoietic system in vivo. While the absence of Grnb did not affect the development of the hematopoietic system, lack of Grna led to decreased differentiation of myeloid precursors into neutrophils and macrophages. Therefore, Grna knockout allowed us to disrupt the hematopoietic function of granulin while keeping unaltered its function in the brain and other non-hematopoietic tissues. Although viable, adult Grna mutants developed kidney marrow (the fish analogous to the mammalian bone marrow) failure, with increased progenitors and decreased mature myeloid cells. Mechanistically, we found that pu.1, the main transcription factor that leads to myeloid differentiation, directly bound grna enhancers, upregulating its expression. We have demonstrated that Grna enhanced myeloid gene expression, and decreased gata1 expression thereby facilitating myeloid differentiation and inhibiting the erythroid genetic program. Finally, we show that these findings in the zebrafish are also conserved in humans.

Altogether, we have identified the hematopoietic role of granulin without disturbing its biological functions in other tissues. We have unveiled a powerful and novel master regulator for myeloid differentiation that could potentially be utilized for the treatment of hematological disorders such as neutropenia and leukemia.