The Role of Sprouty Family Members in Hematopiesis in Zebrafish and Mammals.

In an effort to identify novel regulators of hematopoiesis, we identified a Sprouty family member, SPRY1, significantly enriched (>1.5 fold, p-value < 0.03) in human hematopoietic stem cells (HSC) compared to more committed hematopoietic progenitor cells (HPC) using Affymetrix-based global gene expression profiling (see Eckfeldt et al. abstract). Predominantly FGF, but also VEGF, and EGF signaling induces expression of Sprouty family members (SPRY1-4). SPRY proteins form heterodimers with other Sprouty members along with homodimers. They and are intracellular antagonists and agonists of receptor tyrosine kinase signaling via the RAS/MAPK pathway. SPRY proteins are best characterized as antagonists of the FGF, EGF, and VEGF pathways. As part of a high-throughput functional analysis of the genes identified by human gene array in zebrafish, we evaluated the role of the only identified zebrafish homologue for Sprouty, SPRY4, in zebrafish hematopoiesis. Using morpholino (MO) antisense technology we knocked-down expression of zebrafish SPRY4 during early zebrafish development in a double transgenic GATA1:dsRed/Fli1:eGFP zebrafish line. The SPRY4 morpholino injected fish (SPRY4[superscript]MO[/superscript]) showed an almost complete loss of blood with normal vasculature, which was confirmed by injection of a second unique morpholino designed against SPRY4. [italics] In situ [/italics]hybridization of the hematopoietic markers SCL, GATA1 and Ikaros were all significantly reduced or absent at multiple developmental stages in the SPRY4[superscript]MO[/superscript] embryo. Together with the normal development of the vasculature, this data indicates SPRY4 acts early in hematopoiesis and after the specification of the hemangioblast. [italics] In situ[/italics] hybridization for SPRY4 demonstrated expression in the otic vesicle, tail bud and lateral plate mesoderm, the first site of hematopoietic stem cells. Coinjection of SPRY morpholino and low doses of VEGF morpholino did not rescue the SPRY blood phenotype, indicating that the blood phenotype is not likely through the VEGF pathway. Previous gain-of-function experiments injecting SPRY4 mRNA showed an expansion of hematopoietic progenitors (Furthauer et al. Development 2001), a phenotype we confirmed with overexpression of SPRY4 cDNA. Experiments are ongoing to overexpress human SPRY1 or zebrafish SPRY4 cDNAs in mammalian cells to determine if the hematopoietic phenotype correlates with the zebrafish data. Additional genes differentially expressed in human HSC and HPC evaluated via the high throughput zebrafish screen will also be presented. This is the first description of functional validation of a gene from a human microarray in zebrafish, an essential step in obtaining meaningful in vivo data from global gene profiling studies.