Dr. Robert Paulson's Lab
We are interested in how cells integrate the different signals they
receive and make correct developmental decisions. To explore this
question we are studying murine hematopoiesis. There are several
advantages to this model system. Hematopoietic differentiation relies
for the most part on extracellular signals (growth factors and
cytokines) to stimulate proliferation, survival, and maturation of
progenitors, which makes it an ideal system to explore the how different
signaling pathways interact during differentiation. Our approach is to
combine genetic analysis of mouse mutants that have hematopoietic
defects with biochemical analysis of cell signaling in primary
progenitor cell cultures. The mouse mutants we are studying are not
targeted mutations, but rather spontaneous or mutagen induced mutations
that were identified by their hematopoietic phenotypes. Thus the
characterization of the genes mutated at these loci will identify new
genes that play key roles in the regulation of hematopoiesis. In the
laboratory we have been concentrating on the analysis of three mutations
that affect erythropoiesis, the flexed-tail, (f), Friend virus
susceptibility gene 2, (Fv2) and Polycythemia, (Pcm).
flexed-tail (f), a
regulator of expansive erythropoiesis in the fetal liver and adult
Erythropoiesis in the adult bone marrow is primarily
homeostatic and produces new erythrocytes at a constant rate throughout
life. The situation is radically different in the fetal liver during
embryogenesis and in the adult spleen in response to acute anemia. At
these times, rapid expansive erythropoiesis occurs. Because of the
similarities between fetal liver and spleen erythropoiesis, it has been
hypothesized that they are regulated by similar mechanisms. This
connection between fetal liver and spleen erythropoiesis is evident in
flexed-tail (f) mutant mice. f/f mice exhibit a severe fetal anemia,
that resolves two weeks after birth. Adult f/f mice exhibit normal
steady state blood values and exhibit no defects in bone marrow
erythropoiesis. However, f/f mice are unable to rapidly respond to
acute anemia. Thus f/f mice have a defect in the ability to rapidly
produce erythrocytes at times of great need. We have cloned the f locus
and shown that it encodes the transcription factor Madh5 or Smad5. Our
work shows that f/f mice have defect in the expansion of a novel stress
erythroid progenitor in the spleen during the response to acute
anemia. The receptors for BMP2, 4 and 7 require Smad5 to signal, which
implicates BMP signaling in the expansion of these stress progenitors.
We observed that expression of BMP4 is induced in the spleen just prior
to the expansion of the stress progenitors and treatment of spleen cells
from non-anemic mice with BMP4 can convert these cells to stress
progenitors. Our analysis also showed that BMP4 signaling through Smad5
actually acts on an earlier progenitor cell, the BMP4 responsive
(BMP4R) cell, converting it into a stress erythroid progenitor. Our
current work has focused on the characterization of the BMP4R cells and
stress erythroid progenitors, the regulation of BMP4 expression and the
analysis of the role of BMP4/Smad5 signaling in fetal liver
Fv2/Stk, a host gene involved in Friend virus induced erythroleukemia
Friend virus induces erythroleukemia in adult mice. The disease progresses through a characteristic two stage progression.
During the initial stage early erythroid progenitors are infected and a
polyclonal expansion of infected cells is observed in the spleen.
During the latter stages of the disease infected cells acquire new
mutations and a clonal leukemia is observed. Given that Friend virus is
specific for the erythroid lineage and proceeds through these
characteristic steps, several genes have been identified that affect the
progression of Friend erythroleukemia. We are characterizing these
“host genes” in order to learn more about the pathogenesis of leukemia.
Because Friend virus co-opts the normal erythroid machinery, study of
these genes will also give us insight into their role in regulating
erythropoiesis. Our study of Friend virus has focused on three host
genes, Fv2/Stk, Kit and flexed-tail/Madh5.
Fv2 encodes a naturally occurring truncated form of the Stk receptor tyrosine kinase referred to as SF-Stk. We have shown that SF-Stk forms a complex with the Friend virus envelope glycoprotein gp55 and the Epo receptor. It is this complex that drives the polyclonal expansion of infected cells during the initial phase of the disease. SF-Stk kinase activity is essential for this process and our present work has focused on analyzing the signaling pathways downstream of SF-Stk in Friend virus infected cells.
Kit is the receptor for Stem cell factor (SCF) and is encoded by the Dominant white spotting (W) locus. W mutant mice are resistant to Friend virus and our analysis suggests that the defect in W mice is due to an inability of the infection to spread from the bone marrow to the spleen. Work on W mutant mice has shown that the mice have normal numbers of targets in the bone marrow but infected cells do not expand in the spleen. Further analysis of this defect will examine whether the defect is in the migration/homing of infected cells to spleen or in the ability of infected cells to expand in the spleen in the absence of a Kit/SCF signal.
The murine flexed-tail locus encodes the Madh5 transcription factor. Early work on Friend virus showed that f/f mice were resistant Friend virus. We have characterized the resistance of f/f mice and have shown that these mice fail to express SF-Stk in the bone marrow. This result suggests that BMP4 may control SF-Stk expression. We have also observed that acute anemia can induce Friend virus target cells in the spleen of Fv2 resistant mice. These results suggest that the BMP4 dependent stress progenitors, which expand in the spleens of anemic mice, might be the target Friend virus in the spleen. However it is not clear whether target cells in the bone marrow are the same as target cells in the spleen and we are addressing that question.