Hematopoietic stem cells (HSCs) reside in a perivascular niche but the location remains controversial1. HSCs are rare and few can be found in thin tissue sections2,3 or upon live imaging4, making it difficult to comprehensively localize dividing and non-dividing HSCs. We discovered that α-catulinGFP/+ was expressed by only 0.02% of bone marrow hematopoietic cells, including virtually all HSCs. One in 3.5 α-catulin-GFP+c-kit+ cells gave long-term multilineage reconstitution of irradiated mice, indicating that α-catulin-GFP+c-kit+ cells contain HSCs with a purity comparable to the best markers available. We were able to optically clear the bone marrow to perform deep confocal imaging, making it possible to image thousands of α-catulin-GFP+c-kit+ cells and to digitally reconstruct large segments of bone marrow. The distribution of α-catulin-GFP+c-kit+ cells indicated that HSCs were more common in central marrow than near bone surfaces and in the diaphysis relative to the metaphysis. Nearly all HSCs contacted Leptin Receptor+ and Cxcl12high niche cells. Approximately 85% of HSCs were within 10μm of a sinusoidal blood vessel. Most HSCs were distant from arterioles, transition zone vessels, and bone surfaces. This was true of Ki-67+ dividing HSCs and Ki-67− non-dividing HSCs. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Leptin Receptor+Cxcl12high cells throughout the bone marrow.

The body wall muscle of a Drosophila larva is generated by fusion between founder cells and fusion-competent myoblasts (FCMs). Initially, a founder cell recognizes and fuses with one or two FCMs to form a muscle precursor, then the developing syncitia fuses with additional FCMs to form a muscle fiber. These interactions require members of the immunoglobulin superfamily (IgSF), with Kin-of-IrreC (Kirre) and Roughest (Rst) functioning redundantly in the founder cell and Sticks-and-stones (Sns) serving as their ligand in the FCMs. Previous studies have not resolved the role of Hibris (Hbs), a paralog of Sns, suggesting that it functions as a positive regulator of myoblast fusion and as a negative regulator that antagonizes the activity of Sns. The results herein resolve this issue, demonstrating that sns and hbs function redundantly in the formation of several muscle precursors, and that loss of one copy of sns enhances the myoblast fusion phenotype of hbs mutants. We further show that excess Hbs rescues some fusion in sns mutant embryos beyond precursor formation, consistent with its ability to drive myoblast fusion, but show using chimeric molecules that Hbs functions less efficiently than Sns. In conjunction with a physical association between Hbs and SNS in cis, these data account for the previously observed UAS-hbs overexpression phenotypes. Lastly, we demonstrate that either an Hbs or Sns cytodomain is essential for muscle precursor formation, and signaling from IgSF members found exclusively in the founder cells is not sufficient to direct precursor formation.

The larval body wall muscles of Drosophila melanogaster arise by fusion of founder myoblasts (FMs) and fusion-competent myoblasts (FCMs). Sticks-and-Stones (SNS) is expressed on the surface of all FCMs and mediates adhesion with FMs and developing syncytia. Intracellular components essential for myoblast fusion are then recruited to these adhesive contacts. In the studies herein, a functional analysis of the SNS cytodomain using the GAL4/UAS system identified sequences that direct myoblast fusion, presumably through recruitment of these intracellular components. An extensive series of deletion and site-directed mutations were evaluated for their ability to rescue the myoblast fusion defects of sns mutant embryos. Deletion studies revealed redundant functional domains within SNS. Surprisingly, highly conserved consensus sites for binding post-synaptic density-95/discs large/zonula occludens-1-domain-containing (PDZ) proteins and serines with a high probability of phosphorylation play no significant role in myoblast fusion. Biochemical studies establish that the SNS cytodomain is phosphorylated at multiple tyrosines and their site-directed mutagenesis compromises the ability of the corresponding transgenes to rescue myoblast fusion. Similar mutagenesis revealed a requirement for conserved proline-rich regions. This complexity and redundancy of multiple critical sequences within the SNS cytodomain suggest that it functions through a complex array of interactions that likely includes both phosphotyrosine-binding and SH3-domain-containing proteins.