It's early February, 2001.  The preeminent journals, Science and Nature have just published the first map of the human genome.  There is a massive buzz in both the scientific and the non-scientific communities about the promise genetics will have to imporve the human condition.   What better time for neonatologist and lung development researcher, David Warburton to open his seminar with a joke.  "Mother always reminded us to watch our flies.  Little did we know how right she was," he quipped.

Excuse me?

Warburton joked of flies, fruit flies that is, because the release of the human genome map revealed that men and flies have more in common than would be evident by just looking at the two side by side.  Human beings, it turns out, have far fewer genes than scientists expected, just about three times more than the lowly fruit fly.  The lower than expected number of genes means that molecular regulation may differ greatly amongst these genes from creature to creature or even within different systems of the same organism.  Still, large numbers of these genes bear striking similarity to one another both in form and function when the sequences are compared between vastly different species. The genes that are maintained in common across phyla control such elemental cellular processes as cell proliferation, cell death and cell maturation.  One particular conserved gene may have utility in shaping development of multiple organs such as the lung, teeth, bone, and muscle in higher vertebrates.  Of equal import, this same gene may exert similar effects over an analogous structure in an organism as seemingly simple and different as the fly.

The multi-organism and multi-system conservation that exists helps explain why a group that studies lung development can exist not only peacefully, but productively and highly cooperatively in a center that focuses on the development of craniofacial structures. As Warburton points out, "There really wouldn't have been a lung group without the CCMB." 

But how did Warburton end up in a research unit of the Dental School in the first place?  "Much of intellectual impetus was from Hal Slavkin," says Warburton, "He could train and inspire even quite senior people like myself."  Slavkin, who initiated the CCMB, brought Warburton into the fray because of what he believed were their common interests.  "We had a mutual interest in epithelial-mesenchymal interactions, explains Warburton.  A number of vertebrate organs start as simple tissue layers like the epithelium and mesenchyme.  The embryonic tissue layers instruct one another, grow, assume particular shapes and elaborate tissue-specific proteins that eventually result in the formation of a unique organ.

Hal was working on the face and mandible and had developed, with Pablo Bringas and Tina Jaskoll, a model for studying organogenesis," explains Warburton.  The organ explant system those three had developed proved to be an instrumental tool to all the groups that have worked in the Center, including Warburton's.  It’s utility lies in the fact that cultured embryonic organs will continue to develop outside the womb. Once in culture, the tissues can be evaluated for their developmental response to exogenous chemicals, proteins or genes. Since development of the lung embryologically begins in the floor of the mouth the potential to exploit an in vitro model system developed for craniofacial purposes was very high.

Warburton and colleagues made use of the organ culture system nearly from the outset..  The group started with a quite fundamental question.  What it comes down to, explains Warburton, is that, We want to understand how you get a lung in the first place.  That broad-based point of inquiry begs a series of simpler, experimentally approachable questions.  As Warburton asks, What molecules are involved?  What are the signaling pathways that drive those processes?  We want to know what the impact of premature delivery and indeed injury is on those signaling processes. How does the lung normally repair from an injury such as hyperoxia?  What are the signaling mechanisms involved in repair and are they different from those found during development?

Warburton and his colleagues started with the simpler questions.  Ten years ago some were not scientifically or technologically simple to address.  "Our first finding was that there were actually growth factors in the lung.  When we started, the dogma was that growth factors had nothing to do with morphogenesis because they weren't measurable.  Then PCR technology came along and we were able to look and see that all the growth factors were there," explains Warburton.  Researchers in Warburton's group utilized the culture system to dissect apart the growth factor pathways.  They started with epidermal growth factor (EGF) and progressed to transforming growth factor-b (TGF-b) and fibroblast growth factor (FGF).  For each growth factor they demonstrated an involvement with the branching of the embryonic lung, either accelerating or slowing the process.

Individual members of the lung group have subsequently delved deeper into the more subtle aspects of growth factor regulation and lung development.  This team of CCMB investigators has developed a collaborative program assessing different parameters of lung morphogenesis and maturation and utilizing different models to accomplish their goals.  But while the tools may be getting more sophisticated and the list of genetic players more lengthy, the same motivating question remains.  Just what makes a lung?

With these thoughts and a bit of history, Warburton has set the stage for the accomplishments of the lung developmental biology group in the CCMB.  Let the players, the basic scientists and physicians of the lung group demonstrate what they’re thinking and doing in the lab to address some of the questions that linger.  The common genes and genetic pathways have allowed the cell and molecular biology achievements in the lung group to be extended to better understand numerous processes in craniofacial development.

Jing-Song Zhao- Beyond branching 

CCMB researcher, Dr. Jing-Song Zhao and his lung group colleagues have concentrated on examining the TGF-b growth factor family.  Using the lung organ explant system, they've studied all the variants of the TGF-b ligand and each of the cell surface receptors that transmit the TGF-b signal into the cell.  Their experiments have demonstrated how each of these factors contributes to the branching morphogenesis of the early embryonic lung.  If the cell were a box with an input and an output, these studies, have revealed a select number of lung development outputs, including branching morphogenesis and expression of a select subset of maturity gene markers.  The growth factor studies were specifically designed to examine the signaling pathway in isolation and exclude what went on inside the cell as well as around it.  Having conducted these experiments, members of the lung group, including Zhao, itched to move into previously unexplored areas.

In his most recent work, Zhao has analyzed TACE gene effects on lung development.  TACE acts outside the cell to activate a positive regulator of lung development called TNF-a.  It's a molecular scissors of sorts that liberates TNF-a from the cell surface and allows TNF-a to participate in the activation of a specific signal transduction pathway.  In TACE deficient transgenic mice, the liberation of TNF-a is diminished and lung branching morphogenesis is decreased.  There was something else of note that was perturbed in the TACE knockout mice.  These mice had substantially decreased blood vessel formation within the lung. 

"So the hypothesis," explains Zhao, "is that there is some interaction between the branching morphogenesis and vasculogenesis in early lung development.  People say it's a chicken-egg issue. Which comes first, blood vessel formation or branching?"  Though the structures likely develop simultaneously, Zhao suggests that there must be either positive or negative interaction between the developing air sacs and the emerging blood vessels.  Disturbing one necessarily disturbs the other. 

To study this developmental interaction, Zhao proposes to utilize a combination of transgenic mice.  The first mouse, a flk-1/LacZ transgenic mouse, has been engineered to express a blue stain wherever and whenever the blood vessels develop.  The second is the TACE knockout mouse.   As Zhao explains, "We want to cross the flk-1/LacZ mouse with the TACE knock-out.  This way we can trace what happens to the vasculature when there's no TACE and evaluate the relationship between vasculogenesis and lung branching morphogenesis."  Zhao hopes to characterize the nature of this defect, to determine whether vasculature formation is impaired or delayed in the TACE mice. 

The flk-1/LacZ mouse has the capability to serve as a powerful marker to examine other knockout mice.  Any genetic knockout that produces a defect in lung branching morphogenesis could be reevaluated for the coincident effects on blood vessel formation.  In some of the lung branching mutants, for example, the defect can be partially rescued with the addition of exogenous factors like EGF or TNF-a.  "We'd like to see if we can rescue the vasculature as well.  That will help address the fundamental relationship between the vasculogenesis and branching morphogenesis in early lung development, says Zhao.

Denise Tefft- Peeking inside the black box

Craniofacial Biology graduate student, Denise Tefft studying with David Warburton, cloned a mouse gene with a peculiar name and an important function in embryonic lung development.  The gene's name is Sprouty and like its Drosophila counterpart, which was characterized first, the Sprouty gene instructs cells of the early lung to stop dividing at branch points.  Essentially, Sprouty controls how finely divided the highly branched lung structure will become during development.   

The high degree of conservation of gene function in respiratory organogenesis among flies, mice and people is quite remarkable, explains David Warburton, "In the fly, the respiratory tubules, called trachea, are organized by the fibroblast growth factor (FGF) signaling system.  That process is negatively regulated by Sprouty so that when the gene is removed, excess branches form."  Warburton, Tefft and their international collaborators have discovered that the mouse version of Sprouty also works through the FGF signaling pathway.

Having completed descriptive lung explant experiments with murine Sprouty, Tefft has decided to take her work to a different level, that of the cell.  The descriptive work has been useful because it demonstrates that perturbing the levels of Sprouty gene product produces a notable phenotype in the developing lung.  Unfortunately, those experiments don't speak to what Sprouty is doing inside individual cells to alter the degree of branching in the entire organ.  The limitations of the organ culture model altered the experimental plan as Tefft describes, "We've gone to cell culture to study the biochemistry of murine Sprouty-2 in response to FGF10 stimulus."

Tefft's analysis of Sprouty's amino acid sequence revealed two interesting sites within the protein, one a tyrosine phosphorylation site, the other a mystyrlation (sp?) site.  Each of these special amino acid sites serves as an activation site that when stimulated can have a chemical moiety added to it.  Once chemically modified, or phosphorylated, the protein can aid in signal propagation into the cell nucleus.  Tefft used FGF10 to stimulate the cells to see if murine Sprouty was phosphorylated and thus capable of interacting in potential signal transduction.  It was.  She then mutated the tyrosine phosphorylation site in the Sprouty protein and reintroduced it into the cells.  "I'm trying to figure out the story behind this because when I eliminate the Sprouty phosphorylation site something interesting happens.  The non-stimulated cells are phosphorylated whereas the stimulated cells have decreased phosphorylation," says Tefft. 

Tefft is still performing experiments to sort out her findings but the evidence thus far points to a second, phosphorylation-related protein called Shp-2.  Shp-2 functions to remove phosphate moieties from phosphorylated proteins like Sprouty.  Tefft’s FGF stimulated cells have decreased Shp-2 association, while the non-stimulated cells have an increase in Shp-2 association.  So although the FGF stimulation serves to increase the phosphorylation of Sprouty’s tyrosine site, it is also associated with decreased Shp-2 association.  Right now, I think that the Shp-2 phosphatase has moved off allowing for the phosphorylation of Sprouty. In the Sprouty tyrosine mutant, it could be that the tyrosine site is a regulator for the phosphatase activity.  When the tyrosine site isn't there’s the regulation is gone, says Tefft.

What do these basic molecular biology findings suggest about Sprouty’s general function?  Results from Tefft’s experiments all point to the notion that Sprouty plays an important role in transducing growth factor signals to the nucleus.  Tefft is still in the process of confirming what these multiple Sprouty interactions mean to the overall function of the cell.  Do these proteins interactions and modifications slow cell proliferation or enhance the selective reduction of particular cells?  Tefft, who is weeks away from defending her Ph.D. dissertation, remains intrigued by these questions and will stay at CCMB as a post-doc with Warburton so that she can continue with the Sprouty project.

Carol Wuenschell- Nicotine, nicotinic receptors and lung development 

Researcher Dr. Carol Wuenschell came to the CCMB in the early 90s as a post-doctoral research fellow with David Warburton.  She was a neurobiologist by training but adapted herself to the field of lung biology by redefining her interests to focus on a special population of ill-studied cells found in the lung, the neuroendocrine cells.  Though she started out as a post-doc with Warburton, he soon encouraged her to apply for grant money and seek a junior faculty position within the CCMB. 

Wuenschell's first grant, from the California Tobacco Related Disease Program, addressed the role of nicotine on the developing lung.  "What I did first was to show that nicotine itself has a direct effect on the fetal lung tissue in culture.   Prior to that time it wasn't possible to show that you weren't just having some global effect on the lung through fetal hypoxia, or some fetal maternal hormonal phenomena.  There are many constituents in tobacco smoke, but nicotine alone can directly effect the development of the lung in the absence of the rest of the animal," explains Wuenschell. 

The effect Wuenschell had observed in her nicotine lung explant experiments was quizzical.  Nicotine actually stimulated growth of the embryonic lung explants.  Frankly, says Wuenschell, "It sounds counterintuitive."  On the face of it, increased branching sounds as if it would be beneficial to the developing lung.  But as Wuenschell explains, perturbing development in any way can cause unanticipated and detrimental effects.  "We're only looking at one thing that we can measure in culture," she points out, "You could hypothesize that if the lung develops faster but also terminates sooner, and possibly smaller, that it would have smaller airways."  Such an interpretation would be reasonable in light of a number of clinical reports and scientific observations.  Researchers have shown that infants born to mother's who smoke during pregnancy often have decreased lung capacity, increased incidence of lung infection, SIDS, and transient wheezing that resolves as the child grows.     

As a result of her initial findings, Wuenschell and her research group have concentrated their recent efforts on analysis of the nicotinic receptors.  "Obviously there must be nicotinic receptors for the nicotine to have some effect.  But there's very little known about the nicotinic receptors in the lung."  Wuenschell would ultimately like to show that nicotinic receptor activation has a function in lung development. 

Thus far, no one knows exactly which lung cells express the nicotinic receptors, much less what these cells do once their nicotinic receptors are signaled.  More and more, I'm seeing reports of nicotinic receptors in unexpected places, in skin cells, oral epithelia, immune cells, bone marrow, and the bronchial epithelia.  It's becoming clear that they're not just restricted to neurons and a few immediate targets of neurons, explains Wuenschell.  There's going to be a whole story whether all of these cells are being signaled by acetylcholine from the nervous system or whether cells, other than neurons, are producing that signaling molecule or there's another signaling molecule altogether," she adds.

  Wuenschell's characterization of the nicotinic receptors in the lung has been complicated by the complexity of the receptor itself.  At present, ten genes that code for nicotinic receptor subunits are known.  These subunits are arranged in pentameric fashion, providing the potential for numerous different assemblies.  "We've been looking at the basics of what's there by PCR screening and we're trying to chart where they're localized within the developing lung. At least 5, perhaps as many as 8, of the subunits are present," explains Wuenschell.     

Discovering which cells in the lung are expressing a particular configuration of nicotinic receptors will be an accomplishment for the Wuenschell group.  But that still leaves open the question of how these cells signal with nicotinic receptors when some aren't even classically innervated and specialized to process this signal.  It again begs the question, what signaling molecule is being used to stimulate nicotinic receptors on these cells and once signaled, how do the cells respond? 

Wuenschell is exploring known signaling routes in hopes of discovering how the nicotinic receptor positive cells within the lung are processing stimulation.  "We're definitely looking at calcium mediated events and we're also interested in looking at phosphorylation types of signaling because that could also be occurring downstream," explains Wuenschell.  With the first option, Wuenschell is loading embryonic lung explants with calcium-dependent fluorescent dyes and imaging the lungs with a confocal microscope to visualize how the different cells within the lung process calcium.  Her group is attempting to work out conditions for the procedure at present.  "We're not certain that we can image through the entire lung bud because of its thickness, she says,  We didn't see any changes with our first effort so we're considering dissociating the cells and doing primary culture and identifying them." 

As Wuenschell herself admits, "There's a lot of groundwork to be done first."  When asked what role she speculates nicotinic receptors play in the development of the lung, she answers realistically, "As a developmental biologist I'd love to see that these receptors have some sort of function in development in order to signal between various cell types.  But I'm perfectly willing to accept that that might not be the case because there are so many things that they can be important for, i.e. lung cancer, asthma, SIDS.

Matt Lee- To each a season, even Shc/The stoichiometry of Shc

Neonatologist Matt Lee started as a research fellow in David Warburton’s research group during the early 90s.  He arrived at the CCMB, a well-trained clinician, who had a keen interest to discover more about the basic science behind the process of lung development.   An understanding of the scientific principles that govern cellular proliferation and organization of the developing lung, he rationalized, might someday lead to the development of therapies for premature infants born with immature lungs.  Premature infants, he describes, are at an especially high risk for developing pulmonary fibroproliferation, a thick, rigid, scar tissue in the lungs that compromises blood flow and respiration. 

Lee's first research efforts focused on how growth factors, such as EGF and TGF-b, affected embryonic lung proliferation and maturation.  But as Lee secured his own funding and became an independent investigator, he found his research program was centering on the next step of the growth factor pathway, that of signal transduction.  "We started looking at the signaling pathways that control cell proliferation and what components in these pathways were different in pre-term as opposed to full-term lungs, explained Lee. 

Lee discovered that one such family of signaling proteins, the Shc proteins, appeared to be differentially regulated in the lung during gestation.  Of the three members of the Shc family, Shc-66's expression was of particular interest.  Shc66 was abundant in fetal lungs and virtually absent once the lungs had reached full maturity.  Even more telling explains Lee is that,  "Shc-66 is found in the cells that grow uncontrollably and cause the problems.  Shc is also a putative component of a major growth pathway that could be telling these cells to grow like crazy."

The Shc proteins have been implicated to be involved in a number of basic cellular activities as diverse as cell division, cell maturation and cell death (apoptosis).  Shc-66 and its two highly related Shc family members, Shc-52 and Shc-42, can be activated by more than one signal transduction pathway thus enabling these diverse cellular activities to take place. As Lee describes, Shc proteins were originally described as being in the pathway of the TGF-b activated, tyrosine-kinase receptor cascade. More recently, scientists have demonstrated that the Shc proteins can be activated by a variety of means including: integrin receptors, cytosolic tyrosine kinases and even by calcium modulated kinases.  It seems the Shc proteins act as signal integrators in numerous, responsive signaling pathways, adds Lee. 

Each Shc protein is proposed to perform a unique function.  As Lee explains, If one Shc isoform is highly expressed early and it’s expression crashes when the baby is born, the expression of that particular isoform may be modulating contributions to different signaling pathways.  The relative expression of each isoform can govern the relative contribution to these separate signaling pathways."

In terms of the 'big picture', Lee explains that the Shc proteins serve as a good example of how cells can respond differently to growth factors and particular signal transduction proteins depending on their developmental stage.  With the Shc proteins for instance, the stoichiometry of the different isoforms changes dramatically from fetal stages to the adult.  Such changes might facilitate the activation or repression of some signal transduction pathways over others.  Depending on the paths taken, cells might gain the propensity to divide and grow or conversely, to stop and mature. 

Cells utilize parallel processes that are difficult to understand, says Lee.  So while much of science has been traditionally reductionist, isolating individual variables, one gene, one outcome, for simplicity’s sake, cells go on expressing multiple genes and processing dozens of inputs simultaneously.  It is not the cell’s fault, as Lee explains, that people are generally linear thinkers. 

Experimental methodologies and scientific emphasis may have changed over the years that David Warburton and members of the lung team have worked on the basic questions of lung development.  What has not changed substantially is the fact that the CCMB remains a positive, nurturing environment for those who work in it.  "The CCMB is a well-equipped center that houses a large number of people who are interested in developmental biology, epithelial-mesenchymal tissue interactions, growth factor pathways, etcetera," explains Warburton, "For those of the lung team that work here, including myself, it's a supportive environment scientifically and psychologically.  As a result of our collaborations, I think we nearly double our productivity from what we could do on our own.  And I like to think we're able to contribute back to the CCMB as well."   Lung development begins in the oral cavity and while the lungs are not retained in the head during fetal development, the lung research group has contributed scientific and intellectual diversity that has been a major asset to all the scientists conducting programs of research in the USCSD Center for Craniofacial Molecular Biology.