By David N. Leff

A woman who sustains a spontaneous abortion during the first month or so after intercourse may not even know she was pregnant.

An embryo may be stillborn when the genes and growth factors that direct its development detect an error of embryogenesis sufficiently serious to warrant terminating the prenatal processes. One such occasionally mutated gene is pitx2, which accounts for a rare and obscure disease called Rieger's syndrome. The gene's job is to specify the physical left-hand/right-hand layout of visceral organs in the growing embryo.

"To generate pitx2 knockout mice," explained developmental biologist Juan Carlos Izpisza Belmonte, "we removed the two copies of the gene [one from each parent], leaving them homozygous, and that is when we see those problems. What happened with those mice," he recounted, "was that they were not born. They died during embryogenesis when we removed the two gene copies.

"Humans with Rieger's syndrome," he went on, "have only one mutated copy of the pitx2 gene, with the other a working copy. So they are heterozygotes, with no clear left-right problem. Of course, you don't see the humans that are not alive, but stillborn. Those homozygotes, who have both gene copies mutated, will not make it."

There is no precise estimate as to the prevalence of Rieger's syndrome, but Izpisza Belmonte, who is at the Salk Institute for Biological Studies in La Jolla, Calif., noted, "One in every 8,000 to 10,000 people have a problem with left-right organ placement." (See BioWorld Today, Oct. 30, 1997, p. 1.)

Rieger's patients are spared those challenges, but they suffer from others, especially severe eye malformations with glaucoma, faulty tooth growth and unilateral craniofacial distortion.

While the naked human body looks to the naked eye perfectly symmetrical from right to left, underneath its skin the visceral organs nestle in decidedly asymmetrical locations. The left-leaning heart is obvious, as are the intestines, stomach, liver and other organs. Human lungs, for example, have three right-hand lobes, two on the left.

A Heart In The Wrong Place

These internal architectural features, common to most humans on earth, don't just happen. A cascade of genes and growth factors, culminating in pitx2, guides each organ to its assigned place and position in the scheme of things. Consider the embryonic heart. "Usually in vertebrates," observed molecular geneticist James Martin of Texas A&M University in Houston, "the heart starts off as a straight tube, and then it loops or curls to the right, and forms an S-shaped structure. After these initial events occur, this leads to later morphogenic events ending in a left-situated mature heart.

"The pitx2 in the heart," Martin continued," initiates that loop correctly, out to the right all the way. But the mutant gene product doesn't perform the later steps in a correct fashion. The heart sort of sticks out to the right of the body, and stays there. And it's quite malformed. The valves don't perform correctly. All things for maintaining a four-chambered heart are abnormal. The homozygotes die.

"The heterozygote hearts are normal in humans with Rieger's," he continued, "whereas the phenotypes we observe are eye, tooth and umbilical abnormalities. A half dose of the gene is enough for normal cardiac development, but not enough for normal eye development." Martin is senior author of a paper in today's Nature, dated Sept. 16, 1999, titled, "Function of Rieger's syndrome gene in left-right asymmetry and craniofacial development."

The cascade of early embryonic gene and growth factor guidance, culminating in the pitx2 organ-siting activity, starts in the early days after egg fertilization with a small bump called Hensen's node, on one of the three embryonic cell layers. "It's called the Organizer," observed developmental biologist Clifford Tabin, of Harvard Medical School in Boston, "because it sets up some localized asymmetric gene expression. In broad outline," he said, "what you've got is little signals near the node, secreted proteins that in a cascade lead to broad expression patterns of signals on the left or right. These turn on transcription factors, which give the cells different properties so that when the heart tube forms, it bends one way and not the other."

Along the pathway from Organizer node to pitx2 gene, Salk's Izpisza Belmonte has discovered a new gene he dubbed Caronte. It's announced in today's Nature, dated Sept. 16, 1999, and titled: "The novel Cer-like protein Caronte mediates the establishment of embryonic left-right asymmetry."

Tabin, a close though independent associate of Izpisza Belmonte, has a companion paper in the current issue of Cell, dated Sept. 3, 1999, bearing the title: "Antagonistic signaling by Caronte, a novel Cerberus-related gene, establishes left-right asymmetric gene expression."

A Leaf From Greek Mythology

Izpisza Belmonte told BioWorld Today, "Caronte helps to transmit the information from one group of cells to the cells that are going to make the organs later on. It tells them where to place them, for instance the heart on the left, the spleen on the right, or whatever. This information is encoded very early in the embryo. But how it is transferred from these early cells later on to those that make the organs was not known until now. Caronte is a gene involved in that process, transferring the message from one group of cells to the others.

"Hence the name, in Spanish, of Caronte - in English, Charon - the boatman who transferred the dead souls from one side of the river Styx to the other - and presented them to Cerberus, the three-headed dog who guarded the gates to Hades. The first gene of this family," he continued, "was called Cerberus, because when you put an excess of that gene into a frog embryo, you get a frog with three heads. Then we found that both genes belong to the same family, so we had to go with the same mythological family.

"We call Carontes a realizator gene," Izpisza Belmonte continued, "because it tells pitx2, 'You have to be activated here,' and then pitx2 answers, 'Okay, now I'll do my job,' which is to start locating the heart on the left and not on the right, for instance."