September 18, 2003
Zebrafish Studies Provide Insight into Blood-Cell Formation
Researchers tracking down the cause of anemia in mutant zebrafish
embryos have discovered a protein that guides the creation of new blood
cells.
The researchers, led by Howard Hughes Medical Institute (HHMI)
investigator Leonard I.
Zon and HHMI associate Alan J. Davidson at Children's Hospital,
Boston, published their findings in the September 18, 2003, issue of
the journal Nature. Zon and Davidson collaborated with
researchers at Dana-Farber Cancer Institute, the Whitehead Institute
for Biomedical Research at MIT, the Max Planck Institute for
Developmental Biology in Germany and the University of Rochester.
“So it seems as if there are specific hox genes that actually regulate blood cells, and others that aren’t so important.”
Leonard I. Zon
In the initial discovery that sparked the study, researchers
observed that a zebrafish embryo with a mutation called kugelig
(kgg) developed a lethal anemia in addition to having a
malformed tail.
“After evaluating the kgg mutant for blood-forming stem
cells, we realized that its mutation seemed to affect the genetic
program for creating blood cells,” said Zon. “The program
for making blood vessels looked perfectly normal, but there seemed to
be some specificity there that made us want to explore this mutant
further.” Stem cells are immature progenitor cells that give rise
to more specialized cells that form tissues and organs.
The researchers traced the genetic defect in the kgg mutant
zebrafish to a gene called cdx4. This gene is a member of a
family of “caudal” genes that other researchers had
shown were regulators of a key suite of genes called hox genes
that control development. Hox genes are known to govern the
pattern of body formation in animals, but “nobody had thought
about this combination of caudal genes and hox genes as
regulating stem cells, and in particular blood stem cells,” said
Zon.
When the researchers eliminated cdx4 from normal zebrafish,
they observed the same defects that they saw in the kgg mutants.
Conversely, injecting the normal cdx4 gene into kgg
mutants “rescued” them from the defect.
To see whether the cdx4 gene actually controlled hox
genes, the researchers overexpressed various members of the hox
gene family into the mutant zebrafish. The researchers found that some
hox genes, but not others, restored blood formation in the
kgg zebrafish.
“So it seems as if there are specific hox genes that
actually regulate blood cells, and others that aren't so
important,” said Zon. “By doing those sets of experiments,
we were able to say definitively that cdx4 controlled some
hox genes, and that it regulated blood development.”
In further tests to define the regulatory role of cdx4, the
researchers overexpressed cdx4 in normal zebrafish. “We
found that the middle part of the embryo, or mesoderm, which does not
normally consist of blood cells, converted to blood cells,” said
Zon. “So, we demonstrated that overexpressing cdx4 changed
hox gene expression, and also altered the fate of this mesoderm
to actually become blood-forming,” he said.
“That was a real surprise, because for years we had been
looking for some regulatory factor that would actually change the
middle part of the embryo into blood, altering its fate, but we hadn't
been able to find such a component,” he said.
To extend their findings to mammals, the researchers studied the
Cdx4/Hox machinery in mouse embryonic blood-forming stem cells.
They found that overexpressing Cdx4 in the mouse embryonic stem
cells both altered the expression of mouse Hox genes and caused
a pronounced expansion in the numbers of hematopoietic progenitor
cells.
According to Zon, the group's findings not only increase
understanding of the embryonic blood-forming machinery, but they could
also help reveal how it can go awry in human leukemias.
“We know that a human cousin of cdx4, called
CDX2, produces leukemia when it fuses to a gene called
TEL,” said Zon. He and his colleagues believe that this
fusion disrupts the normal HOX-regulating function of the cell
and transforms it into a leukemic cell. Similarly, he said, the gene
MLL, whose involvement in abnormal fusion proteins has been
implicated in leukemia, might also be related to the cdx4/hox
signaling machinery.
“So, we believe there is a subset of leukemias that are caused
by MLL fusions, CDX fusions, and even HOX
fusions,” said Zon. “Now, with this zebrafish system, we
can really begin to understand the role that the specific hox
and cdx genes play during normal hematopoietic development. We
can introduce the genes one by one into the kgg mutant and test
whether and how they participate in such development.”
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