This laboratory is investigating molecular processes required for establishing a terminally differentiated organism from a homogeneous population of totipotent cells and is defining signal transduction pathways that specify developmental cell fates and pattern formation. By characterizing receptor-mediated cascades and nuclear targets, our research serves to identify mechanisms that are basic to multicellular differentiation.
Organizing the embryonic body plan is a major requisite governing early metazoan development, with morphogen signaling central to establishing and specifying cell fates. One of these pathways involves Wnt/wingless signaling and includes 7-transmembrane (7-TM) target receptors and a downstream effector, the protein kinase, GSK3. In Dictyostelium, stimulation of a family of 7-TM receptors by its ligand, the secreted chemoattractant/morphogen, cAMP, also establishes a fundamental developmental organization, the anterior/posterior axis. We have shown that different cAMP receptor subtypes act antagonistically to promote or to inhibit cell-specific differentiation and axis formation. These antagonistic pathways converge at GSK3 in the context of cell fate determination. The latter inhibitory pathway parallels Wnt/wingless signaling through the 7-span frizzled receptors to control GSK3 activity and axes formation in Drosophila, Xenopus, and other metazoa. Separately, we have identified a novel tyrosine kinase, ZAK1, that is required for receptor-mediated activation of GSK3 during development, and have shown that recombinant ZAK1 will phosphorylate and consequently activate mammalian GSK3 in vitro. Remarkably, this 7-TM receptor-dependent activation of GSK3 may act independently of G proteins. These studies may reveal new mechanisms involving protein tyrosine kinases that are critical for cell fate specification or tumor suppression in other systems.
Other receptor-mediated pathways in Dictyostelium have an absolute requirement for G protein signaling. During early development, a pulsatile release of cAMP directs chemotatic migration and induces gene expression. The cAMP signal is transduced through the membrane by a receptor/G protein coupled pathway that regulates adenylyl cyclase (AC). AC is transiently activated by a cAMP signal, but rapidly adapts to a persistent cAMP signal. While G-beta is implicated in AC activation, the mechanism for adaptation of the response to cAMP remains unknown. We have identified a novel G-alpha (Ga9) in Dictyostelium that may participate in the adaptation pathway, and the Ga9-nulls are hypersensitive to the newly identified secreted factor, APF, that potentiates the chemotatic response to cAMP. APF was purified to homogeneity, and two proteins were individually isolated and are being sequenced. These observations suggest that Ga9 is part of an inhibitory signaling network that directs cell movement and senses cell density.
We also have a very strong interest in understanding how signaling circuits ultimately regulate cell-specific patterns of gene expression. We have analyzed several developmentally regulated promoters and have purified CRTF, a novel Zn-finger transcription factor, isolated its gene, and created null strains. CRTF is required for early development, and data suggest that CRTF activity is dependent upon G protein signaling through the cAMP receptors. Studies are in progress to understand structure/function relationships of CRTF activation with regard to developmentally regulated gene expression.
Finally, we maintain an active collaboration to understand molecular mechanisms required for lipogenic and lipolytic action in mammalian adiopocytes. Primary focus has centered on the lipid droplet-associated proteins, Perilipin and ADRP, and on HSL, the hormone sensitive lipase, using knockout and transgenic mouse models.
Multiple signaling pathways that ultimately regulate chemotaxis can be attenuated by a single heterotrimeric Ga protein in Dictyostelium discoideum. Shown here is a composite image of Dictyostelium cells moving toward a pipette (gray) that is emitting a chemoattractant. Relative to wild-type cells (blue), Ga9-null cells (green) are hyperpolarized and rarely produce lateral pseudopods, indicative of a loss of a negative regulator of chemotaxis. Cells expressing constitutively activated Ga9 (red) display the expected opposite phenotype, are poorly polarized, and produce numerous lateral pseudopods. In the background, a population of Ga9-null cells chemotax to form aggregation centers during development. (For details, see Brzostowski et al., Genes Dev. 2004 18: 805-815).