Signal Emanating from the AP Organizer Determines AP Positions

Although dpp RNA expression in the A/P organizer is bounded by the primordia of the L3 and L4 veins, the encoded Dpp protein is a longrange diffusible signal that can travel significantly farther from its site of production than Hh. As a result of its localized production and diffusion, Dpp protein levels are highest in the central region of the wing disc (i.e., where it is synthesized between the L3 and L4 veins), intermediate in cells lying between the primordia of the L2 and L3 veins and between the L4 and L5 veins, and lowest or absent in the most anterior and posterior extremes of the wing that are farthest from the Dpp source. Because Dpp activates expression of different target genes in the wing disc depending on its concentration, it functions as a morphogen in the wing as well as in the embryo.

To illustrate how Dpp acts as a morphogen in the wing, we can consider two known target genes of Dpp signaling called spalt (sal) and op-timotor blind (omb). These genes are expressed in two broad nested domains centered over the A/P organizer. The sal expression domain is contained within the wider omb expression domain (Fig. 4.2C; see also Plate 3A for actual expression of sal relative to dpp). Activation of the sal and omb genes requires Dpp signaling because cells carrying crippled receptors for Dpp fail to express either gene. Gary Struhl at Columbia University invented a genetic method to test the idea that different doses of Dpp activate expression of sal versus omb. He devised a way to activate expression of the dpp gene randomly in small patches of cells in the wing disc through a mechanism that is independent of Hh protein signaling. Wings derived from such flies contain small islands of cells misexpressing the dpp gene in addition to the normal central stripe of dpp expression in the A/P organizer (Fig. 4.2C; see also Plate 3B, C for actual data). Struhl observed that concentric circles of sal and omb expression surrounded the small patches of cell misexpressing dpp. Most importantly, he found that omb was expressed in a larger circle than sal (Plate 3C, arrow). This result suggested that Dpp diffused out from the small patch of dpp expression where it was produced and activated expression of the sal and omb genes at different doses. Expression of the sal gene could only be induced by high Dpp concentrations in neighboring cells, which were close to the source of Dpp, whereas expression of omb could be induced by lower levels of Dpp at a greater distance from the A/P organizer.

As a graduate student and then a postdoc, Struhl performed a series of ingenious experiments to examine the role of homeotic genes and other A/P patterning genes in the fly embryo. One of the many interesting observations that Struhl made during this very productive period was defining the default segmental state obtained when all homeotic genes are expressed throughout the developing embryo. He found that in this situation all segments developed as the most posterior abdominal segment. This result is the opposite of that caused by eliminating homeotic gene function, in which segments adopt anterior identities (e.g., every segment looks like an antennal segment in beetles lacking all homeotic genes). These and other experiments, such as those in which Struhl compared the reciprocal sets of defects in loss-of-function versus misexpression

Gary Struhl (1954- )

mutants of the Antennapedia gene, ultimately led him to realize that comparing the consequences of ectopic activity with loss of activity of such genes should be a powerful general strategy for demonstrating that the product of a patterning gene confers spatial information and could also provide insights into the nature of that information.

Gary Struhl was born in Brooklyn, New York. He obtained bachelor's and master's degrees from the Massachusetts Institute of Technology and moved to Cambridge, England, for his graduate studies, where he worked with Peter Lawrence at the University of Cambridge to study the regulatory relationship between homeotic genes. Following his graduate studies, Struhl worked briefly with Nüsslein-Volhard and Wieschaus in Tübingen on their screen for embryonic patterning mutants, which galvanized his interest in the segment-polarity genes and embryonic patterning as ways for getting at morphogens and gradients, concepts he was already well aware of from being a student of Lawrence. Struhl then moved to Harvard, where he continued his work independently in Tom Maniatis's lab on anterior-posterior patterning in flies. After completing these studies, he accepted a faculty position at Columbia University Medical School in New York, where he currently resides.

Beginning in the late 1980s, Struhl became frustrated by the fact that long-range organizing events—in which he was, and remains, most interested—were occurring under the unusual circumstance of the early fruit fly embryo in which cells were not fully enclosed by membranes. He realized that it would be important to understand how spatial information is generated and interpreted in cell populations and decided that the best approach, at least for him, would be to develop methods that would allow potential signaling molecules, or components of their receptor systems, to be activated or inactivated at will during imaginal disc development. This need, and his background in comparing loss-of-function and misexpression mutants, led Struhl and his postdoctoral collaborator Konrad Basler to develop a method called the Flp-out technique, in which genes are ectopically expressed in isolated patches of cells. (This method is the converse of mutant clone analysis pioneered by García-Bellido, in which patches of cells are produced that lack the function of a gene of interest.) This ingenious Flp-out method has proven very successful for studying the organizing roles of secreted signals like Dpp and Hh, which have been a major focus of Struhl's work over the past several years. It is worth noting that the grade school adage "success is 1% inspiration and 99% perspiration" often applies to scientific success as well. Struhl recalls that this was true regarding development of the Flp-out technique. "The Flp-out method, unfortunately, did not come easily—the Struhl and Basler paper of 1993 represents the culmination of around 4 years of work—but it was well worth the investment."

Although the existence of gradients and morphogens was not unanticipated when Struhl performed his Flp-out experiments, it was not clear what molecules were the morphogens, and more importantly, whether any molecules really behaved like hypothetical morphogens. Two general types of models had been proposed, and they can be traced back to Boveri and Spemann. In the first model (the one Struhl has demonstrated to act in the wing), a mor-phogen acts as a long-range signal to activate expression of different genes in a concentration-dependent fashion, whereas the second model proposed that morphogens act through a series of short-range inductive events that tend to peter out. Regarding this point, Struhl comments, "The Flp-out method was critical for doing this in Drosophila (fruit fly), as was the recognition that a way to discriminate between morphogen and sequential inductive models was to compare the effects of manipulating ligand production with signal transducing activity."

A predominant theme in Struhl's work is that he often invents a clever genetic method (e.g., the Flp-out technique) to answer an important question. Regarding this style of experimentation, Struhl remarks, "I get my kicks out of trying to understand things I cannot see or touch—and in particular by setting genetic traps for obtaining information about how things work in vivo. I can't explain why I find this so challenging and engrossing, but this has always been the case since I began research as an undergraduate. In terms of subject matter, I am indebted to Peter Lawrence for making me aware of the general problem of understanding how cells know where they are and how they use such spatial information to decide what to do.

This is a fundamental problem of animal development, and it is one which is particularly amenable to the sort of approach I like to take. It is also a broad problem—there are many kinds of spatial information and many different contexts in which it is generated and used. As a consequence, my work tends to cover a broad range of patterning problems which has the advantage of allowing unexpected connections to emerge, but the disadvantage that it is difficult to think creatively about more than a few problems at a time." Although Struhl has tackled a broad range of patterning problems, he believes that there are many viable approaches to science and that style depends much on individual temperament, approach, and scientific question. He notes "There are many paths through the forest."

Struhl is somewhat unusual among lab heads in that he works at the bench himself much of the time (Ed Lewis and Eric Wieschaus are notable examples of this hands-on approach). Struhl comments "The most fulfilling experiences I have had in science have been the moments of actual discovery. The experience is not quite the same when someone else, even under my guidance, or in collaboration, makes the discovery. This is one of my main motivations for continuing to work at the bench."

It is interesting to consider Struhl's penchant for inventing genetic traps in light of his family background. His father is an entrepreneur/inventor who prospered in business by thinking about unrequited needs that average people or retailers might have and then creating products to fill those needs. In pondering whether this Rugrats-type household had any effect on him or his brother Kevin (also a talented geneticist) as children, Struhl comments "...there is a loose analogy with identifying missing links in a scientific problem and designing strategies to resolve the problem. My parents certainly value 'sciences' in a general sense " Perhaps the most intriguing Struhl family parallel is the remarkable similarity between the approaches of the two Struhl brothers. Struhl remarks, "What is actually more strange to me is that Kevin and I both generally develop in vivo, genetic strategies to address biological problems, which is not the tactic most people use. And what makes it strange is that Kevin and I arrived in our present areas by opposite routes, in his case via Mathematics, then Chemistry, then Molecular Biology, and in my case via Natural History then Biology. We both did go to MIT (two years apart), and we both did attend a few of the same interesting and challenging classes in phage and bacterial genetics, one in particular given by Ethan Signer. So perhaps that is where we both got interested in using genetic strategies for understanding biological problems." Or, could scientific style just be genetic?

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