Wnt4 Enhances Murine Hematopoietic Progenitor Cell Expansion Through a Planar Cell Polarity-Like Pathway

Background: While the role of canonical (b-catenin-mediated) Wnt signaling in hematolymphopoiesis has been studied extensively, little is known of the potential importance of non-canonical Wnt signals in hematopoietic cells. Wnt4 is one of the Wnt proteins that can elicit non-canonical pathways. We have previously shown that retroviral overexpression of Wnt4 by hematopoietic cells increased thymic cellularity as well as the frequency of early thymic progenitors and bone marrow hematopoietic progenitor cells (HPCs). However, the molecular pathways responsible for its effect in HPCs are not known. Methodology/Principal Findings: Here we report that Wnt4 stimulation resulted in the activation of the small GTPase Rac1 as well as Jnk kinases in an HPC cell line. Jnk activity was necessary, while b-catenin was dispensable, for the Wnt4-mediated expansion of primary fetal liver HPCs in culture. Furthermore, Jnk2-deficient and Wnt4 hemizygous mice presented lower numbers of HPCs in their bone marrow, and Jnk2-deficient HPCs showed increased rates of apoptosis. Wnt4 also improved HPC activity in a competitive reconstitution model in a cell-autonomous, Jnk2-dependent manner. Lastly, we identified Fz6 as a receptor for Wnt4 in immature HPCs and showed that the absence of Wnt4 led to a decreased expression of four polarity complex genes. Conclusions/Significance: Our results establish a functional role for non-canonical Wnt signaling in hematopoiesis throug

Genome-Wide Interrogation of Mammalian Stem Cell Fate Determinants by Nested Chromosome Deletions

Understanding the function of important DNA elements in mammalian stem cell genomes would be enhanced by the availability of deletion collections in which segmental haploidies are precisely characterized. Using a modified Cre-loxP–based system, we now report the creation and characterization of a collection of ∼1,300 independent embryonic stem cell (ESC) clones enriched for nested chromosomal deletions. Mapping experiments indicate that this collection spans over 25% of the mouse genome with good representative coverage of protein-coding genes, regulatory RNAs, and other non-coding sequences. This collection of clones was screened for in vitro defects in differentiation of ESC into embryoid bodies (EB). Several putative novel haploinsufficient regions, critical for EB development, were identified. Functional characterization of one of these regions, through BAC complementation, identified the ribosomal gene Rps14 as a novel haploinsufficient determinant of embryoid body formation. This new library of chromosomal deletions in ESC (DelES: http://bioinfo.iric.ca/deles) will serve as a unique resource for elucidation of novel protein-coding and non-coding regulators of ESC activity

The role of erythropoietin and erythropoietin receptor in regulation of hemopoiesis

To analyze the molecular mechanisms by which erythropoietin (Epo) can stimulate proliferation and differentiation of hemopoietic cells, I studied the effects of quantitative and qualitative alterations in the expression of erythropoietin receptors (EpoRs) in hemopoietic cell lines and in the primary bone marrow cells using retrovirus mediated gene transfer to engineer high level expresson of normal and mutant EpoRs in these cells, lnterleukin-3 (IL-3)- dependent murine bone marrow derived Ba/F3 cells engineered to express normal EpoR increased their levels of β-globin mRNA in response to Epo, and this partial differentiation correlated with a marked Epo-induced growth delay, indicating that the transduced EpoR was capable of inducing a distinct set of intracellular events. The tyrosine kinase inhibitor genistein blocked both Epoinduced β-globin mRNA accumulation and proliferation in this model system. In contrast, inhibition of protein kinase C by Compound 3 suppressed only Epo-induced differentiation without affecting proliferation, indicating that the proliferative and differentiation functions of the EpoR can be uncoupled. Mutant EpoRs lacking all intracellular tyrosines were compromised only in proliferative signaling, implying that tyrosine phosphorylation of the EpoR itself is not required for its differentiation function. With IL-3 and Epo costimulation, IL-3 signaling appeared to be dominant, since no increase in β-globin mRNA occurred. Chimeric EpoRs comprising the extracellular domain of the EpoR and the transmembrane and cytoplasmic region of IL-3-R-βIL-3 were capable of inducing β-globin mRNA accumulation, suggesting the existence of a second EpoR subunit responsible for differentiation or that the a subunit of the IL-3 R prevents it. Arguing against the former, a truncated EpoR lacking an intracellular domain possessed no biological activity. Chimeric EpoRs comprising the extracellular domain of the EpoR and the transmembrane and intracellular domains of the IL-3R a subunit were, however, capable of transmitting the Epo-induced mitogenic signal but failed to stimulate accumulation of β-globin mRNA. Moreover, coexpression of EpoR/IL-3Ra with EpoR/IL-3R β-IL-3 suppressed β-globin mRNA accumulation, which implicated an active role for the IL-3-Ra subunit in inhibiting EpoR-specific differentiating signals. Epo also exhibited a marked effect on proliferation of EpoR-transduced primary mouse bone marrow cells. Epo alone supported proliferation of EpoRtransduced CFU-GM and CFU-GEMM in semi-solid and suspension cultures, indicating that Epo was capable of replacing other cytokines normally required for the in vitro proliferation of non-erythroid and multipotent clonogenic progenitors. No Epo-induced proliferation of control cells could be detected in cultures containing high numbers of irradiated EpoR-transduced cells, indicating that Epo stimulated proliferation directly, through activation of the transduced EpoR, and arguing against the possibility of Epo-induced secretion of growth factor(s) within the population of the EpoR-transduced cells. To study effects of ectopic EpoR expression on proliferation of stem cells in vivo, EpoR- and neo-transduced bone marrow cells were transplanted into lethally irradiated mice. Recipients of the EpoR-transduced bone marrow developed within 6-14 weeks severe anemia, leukocytosis characterized by accumulation of undifferentiated blasts, and had significantly increased numbers of all clonogenic progenitor classes, consistent with development of myeloproliferative disease. Bone marrow and spleen cells recovered from the affected mice expressed high levels of surface EpoRs and proliferated in response to Epo, but not in the absence of growth factors, supporting a link between the Epo-induced deregulation in proliferation of the EpoR transduced stem cells and development of neoplasia. Together, the data presented in this thesis provide evidence that EpoRs may influence both proliferative and differentiative decisions of hemopoietic cells subject to their ability to interact with different signalling intermediates.Medicine, Faculty ofMedical Genetics, Department ofGraduat

Analysis of HSC activity and compensatory Hox gene expression profile in Hoxb cluster mutant fetal liver cells

Overexpression of Hoxb4 in bone marrow cells promotes expansion of hematopoietic stem cell (HSC) populations in vivo and in vitro, indicating that this homeoprotein can activate the genetic program that determines self-renewal. However, this function cannot be solely attributed to Hoxb4 because Hoxb4-/- mice are viable and have an apparently normal HSC number. Quantitative polymerase chain reaction analysis showed that Hoxb4-/- c-Kit+ fetal liver cells expressed moderately higher levels of several Hoxb cluster genes than control cells, raising the possibility that normal HSC activity in Hoxb4-/- mice is due to a compensatory up-regulation of other Hoxb genes. In this study, we investigated the competitive repopulation potential of HSCs lacking Hoxb4 alone, or in conjunction with 8 other Hoxb genes. Our results show that Hoxb4-/- and Hoxb1-b9 -/- fetal liver cells retain full competitive repopulation potential and the ability to regenerate all myeloid and lymphoid lineages. Quantitative Hox gene expression profiling in purified c-Kit+ Hoxb1-b9-/- fetal liver cells revealed an interaction between the Hoxa, b, and c clusters with variation in expression levels of Hoxa4,-a11, and -c4.Together, these studies show a complex network of genetic interactions between several Hox genes in primitive hematopoietic cells and demonstrate that HSCs lacking up to 30% of the active Hox genes remain fully competent. (Blood. 2006;108:116-122

A mutant allele of the Swi/Snf member BAF250a determines the pool size of fetal liver hemopoietic stem cell populations

It is believed that hemopoietic stem cells (HSC), which colonize the fetal liver (FL) rapidly, expand to establish a supply of HSCs adequate for maintenance of hemopoiesis throughout life. Accordingly, FL HSCs are actively cycling as opposed to their predominantly quiescent bone marrow counterparts, suggesting that the FL microenvironment provides unique signals that support HSC proliferation and self-renewal. We now report the generation and characterization of mice with a mutant allele of Baf250a lacking exons 2 and 3. Baf250aE2E3/E2E3 mice are viable until E19.5, but do not survive beyond birth. Most interestingly, FL HSC numbers are markedly higher in these mice than in control littermates, thus raising the possibility that Baf250a determines the HSC pool size in vivo. Limit dilution experiments indicate that the activity of Baf250aE2E3/E2E3 HSC is equivalent to that of the wild-type counterparts. The Baf250aE2E3/E2E3 FL-derived stroma, in contrast, exhibits a hemopoiesis-supporting potential superior to the developmentally matched controls. To our knowledge, this demonstration is the first that a mechanism operating in a cell nonautonomous manner canexpand the pool size of the fetal HSC populations

RNAi screen identifies Jarid1b as a major regulator of mouse HSC activity

Histone methylation is a dynamic and reversible process proposed to directly impact on stem cell fate. The Jumonji (JmjC) domain–containing family of demethylases comprises 27 members that target mono-, di-, and trimethylated lysine residues of histone (or nonhistone) proteins. To evaluate their role in regulation of hematopoietic stem cell (HSC) behavior, we performed an in vivo RNAi-based functional screen and demonstrated that Jarid1b and Jhdm1f play opposing roles in regulation of HSC activity. Decrease in Jarid1b levels correlated with an in vitro expansion of HSCs with preserved long-term in vivo lymphomyeloid differentiation potential. Through RNA sequencing analysis, Jarid1b knockdown was associated with increased expression levels of several HSC regulators (Hoxa7, Hoxa9, Hoxa10, Hes1, Gata2) and reduced levels of differentiation-associated genes. shRNA against Jhdmlf, in contrast, impaired hematopoietic reconstitution of bone marrow cells. Together, our studies identified Jarid1b as a negative regulator of HSC activity and Jhdmlf as a positive regulator of HSC activity