In a surprising finding that underscores the difficulty in understanding how genes guide the developing mammalian embryo, an international team of scientists has found that the same master gene that controls development of all of the body's blood cells is later activated in other cells of the embryo to form the genitourinary tract.
Disorders in the gene's function, particularly in the genitourinary tract, where it was not previously known to act, may underlie some human diseases.
The researchers, from Northwestern University, Harvard Medical School and the University of Tsukuba in Japan, claim that the finding points to one obvious pitfall to a powerful experimental technique in mice that scientists have used for a decade to try to unravel the genetics of mammalian fetal development: Simply, if a single gene has more than one vital function, the first lethal defect will abort the embryo before the second defect manifests.
"The mouse embryo may be lost early in gestation before the second defect can be revealed," says J. Douglas Engel, Owen L. Coon Professor of Biochemistry, Molecular Biology and Cell Biology at Northwestern, who directed the research. "We may miss additional roles for a gene we're studying, unless we can somehow circumvent the embryologically disasterous effects of the first missing function."
Engel and his colleagues found a way to circumvent early abortion due to a missing function from a known vital gene, unmasking a previously unknown second role later in fetal development. The results are reported in the Nov. 16 issue of the journal of the European Molecular Biology Organization.
Engel and his co-workers were studying a master signal molecule called GATA-2 that turns on other genes necessary to set embryonic precursor cells along the pathway that will give rise to all the various types of blood cells in a newborn mouse. By using a now-common technique called gene targeting, they created mice that were missing one of their two copies of the gene for GATA-2. When two such mice are bred, one-fourth of the resulting embryos will lack both copies of the GATA-2 gene.
"These gene-knockout mice die early in embryogenesis," Engel said. The GATA-2 defect is unknown in humans, he said, presumably because it results in very early miscarriage. Mice and humans are very similar in the genetic mechanisms of their embryonic development.
In an effort to understand the signals that cause GATA-2 to turn on in the embryonic tissues that eventually produce blood cells, the researchers constructed a huge piece of DNA called an artificial chromosome. This artificial chromosome contains the GATA-2 gene plus long stretches of the DNA that brackets it in the normal chromosome. When the artificial chromosome was injected into mouse embryos that lacked GATA-2, blood cell formation was restored, or "rescued," and live mice were born.
Surprisingly, however, despite a normal blood forming system, all the mice died shortly after birth. Post-mortem examination revealed that they had malformations of the kidneys and bladder and other genitourinary structures.
"When we went back and looked at normal mice, we found that GATA-2 is expressed in a lot of the organs in the developing urogenital system," Engel said. GATA-2 had been thought to be important only for blood cell formation.
"And what was really surprising," he continued, "was that in the knockout mutants, the artificial chromosome, with GATA-2 and that enormous length of flanking DNA, was not sufficient to rescue the urogenital system."
The researchers conclude that GATA-2 expression in developing genitourinary tissues must be governed by a regulatory DNA sequence a vast distance away from the GATA-2 gene itself.
"The latter surprise is going to be disturbing to the scientific community, because everybody thinks that you control genes with relatively small pieces of DNA located right next to the gene," Engel said. "As far as I know, these are clearly the most distant regulatory elements that have been identified in any structural gene. It's a further demonstration of why we need to map and sequence the entire genome if we hope to understand how these elements work."
Derangements of GATA-2 expression could underlie any of a wide range of human conditions. The researchers note that developmental abnormalities of the genitourinary system are estimated to affect 10 percent of the population, accounting for one-third of all congenital malformations and 40 percent of kidney diseases.
The researchers also note that the general approach of trying to replace a missing function in mice that have had a gene knocked out may reveal unknown functions of that gene at later developmental stages.
Other authors on the EMBO Journal paper include graduate student Yinghui Zhou and postdoctoral fellows Kim-Chew Lim and Ko Onodera, all of Northwestern; Stuart H. Orkin, Leland Fikes Professor of Pediatric Medicine, and postdoctoral researcher Fong-Ying Tsai of Harvard Medical School; and Masayuki Yamamoto, professor of molecular and developmental biology, and postdoctoral researchers Satoru Takahashi, Naoko Minegishi and Jun Ohta of the University of Tsukuba.
The research was funded by the National Institute of General Medical Sciences, one of the National Institutes of Health.
Journal
The EMBO Journal