CSF-1–induced Src signaling can instruct monocytic lineage choice

Abstract

Controlled regulation of lineage decisions is imperative for hematopoiesis. Yet, the molecular mechanisms underlying hematopoietic lineage choices are poorly defined. Colony-stimulating factor 1 (CSF-1), the cytokine acting as the principal regulator of monocyte/macrophage (M) development, has been shown to be able to instruct the lineage choice of uncommitted granulocyte M (GM) progenitors toward an M fate. However, the intracellular signaling pathways involved are unknown. CSF-1 activates a multitude of signaling pathways resulting in a pleiotropic cellular response. The precise role of individual pathways within this complex and redundant signaling network is dependent on cellular context, and is not well understood. Here, we address which CSF-1-activated pathways are involved in transmitting the lineage-instructive signal in primary bone marrow-derived GM progenitors. Although its loss is compensated for by alternative signaling activation mechanisms, Src family kinase (SFK) signaling is sufficient to transmit the CSF-1 lineage instructive signal. Moreover, c-Src activity is sufficient to drive M fate, even in nonmyeloid cells.

Pitfalls and requirements in quantifying asymmetric mitotic segregation

“…Continuous quantitative long-term single-cell imaging is one way to address this issue and seems predestined to connect asymmetric inheritance with future daughter cell behaviors. 7,40 This technology has already contributed to answering other longstanding questions [41][42][43][44][45][46][47][48][49][50][51] and has been used to quantify fluorescence reporter expression continuously over time, [52][53][54][55][56][57][58] a prerequisite to demonstrate the functional relevance of asymmetrically inherited factors. Using the here-described protocols to reliably detect mitotic hematopoietic cells in fixed samples and live-cell imaging, we just published that the asymmetric inheritance of the cellular degradative machinery, including lysosomes, autophagosomes, mitophagosomes, and NUMB, can predict the fates of daughter cells.…”

Understanding cell fate control by continuous single-cell quantification

“…Although cell motility can be restricted by microstructures, extracellular matrix coatings, or stroma cocultures, these solutions have their own drawbacks. [55][56][57] Simple coating with anti-CD43 or anti-CD44 has recently been described to drastically reduce hematopoietic cell motility in vitro without affecting cell behavior. 58 On this coating, cells do not leave the field of view anymore and form 2-dimensional colonies.…”

“…Combined loss-and gain-of-function experiments with single-cell tracking demonstrated that Src signaling is sufficient for M-CSF-instructed differentiation of primary GMPs into macrophages. 57 Mechanism of myeloid lineage decisions: the PU.1/GATA-1 switch paradigm…”

Mouse and human HSPC immobilization in liquid culture by CD43- or CD44-antibody coating

“…Time-lapse imaging and in-culture antibody staining was performed as described 12,16,17 in either 384 plates (Greiner) or IBIDI VI 0.4 slides coated at indicated concentrations at 4°C overnight. Cells were segmented and quantified as described.…”