An RNAi screen identifies Msi2 and Prox1 as having opposite roles in the regulation of hematopoietic stem cell activity.
In this study, we describe an in vivo RNA interference functional genetics approach to evaluate the role of 20 different conserved polarity factors and fate determinants in mouse hematopoietic stem cell (HSC) activity. In total, this screen revealed three enhancers and one suppressor of HSC-derived reconstitution. Pard6a, Prkcz, and Msi2 shRNA-mediated depletion significantly impaired HSC repopulation. An in vitro promotion of differentiation was observed after the silencing of these genes, consistent with their function in regulating HSC self-renewal. Conversely, Prox1 knockdown led to in vivo accumulation of primitive and differentiated cells. HSC activity was also enhanced in vitro when Prox1 levels were experimentally reduced, identifying it as a potential antagonist of self-renewal. HSC engineered to overexpress Msi2 or Prox1 showed the reverse phenotype to those transduced with corresponding shRNA vectors. Gene expression profiling studies identified a number of known HSC and cell cycle regulators as potential downstream targets to Msi2 and Prox1.
Hope
Kristin J
KJ
Molecular Genetics of Stem Cells Laboratory, Institute of Research in Immunology and Cancer, University of Montreal, Montreal, Quebec H3C 3J7, Canada.
Cellot
Sonia
S
Ting
Stephen B
SB
MacRae
Tara
T
Mayotte
Nadine
N
Iscove
Norman N
NN
Sauvageau
Guy
G
D000818:Animals; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D020411:Oligonucleotide Array Sequence Analysis; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D000098551:Prospero-Related Homeobox 1 Protein; D000099221:Protein Kinase C zeta
D018398:Homeodomain Proteins; D011493:Protein Kinase C; D016601:RNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D000098551:Prospero-Related Homeobox 1 Protein; D000099221:Protein Kinase C zeta; C499059:Msi2h protein, mouse