Deflagellation of Chlamydomonas reinhardtii follows a rapid transitory accumulation of inositol 1,4,5-trisphosphate and requires Ca2+ entry.

C. reinhardtii sheds its flagella in response to acidification. Previously, we showed correlations between pH shock, deflagellation, and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production, but 100% of cells deflagellated by 5 s, which was the earliest that Ins(1,4,5)P3 accumulation could be accurately measured by techniques available to us at that time (Quarmby, L. M., Y. G. Yueh, J. L. Cheshire, L. R. Keller, W. J. Snell, and R. C. Crain. J. Cell Biol. 1992. 116:737-744). To learn about the causal relationship between Ins(1,4,5)P3 accumulation and deflagellation, we extended these studies to early times using a continuous-flow rapid-quench device. Within 1 s of acidification to pH 4.3-4.5, 100% of cells deflagellated. A transient peak of Ins(1,4,5)P3 was observed 250-350 ms after pH shock, preceding deflagellation. Preincubation with 10 microM neomycin, which prevents hydrolysis of phosphatidylinositol 4,5-bisphosphate, inhibited both the transient production of Ins(1,4,5)P3 and the subsequent deflagellation. The nonspecific Ca2+ channel blockers La3+ and Cd2+ prevented flagellar excision induced by mastoparan without inhibiting rapid Ins(1,4,5)P3 production. Likewise, the Ins(1,4,5)P3-gated channel inhibitors ruthenium red and heparin blocked deflagellation in response to mastoparan. These studies were extended to mutants defective in flagellar excision. Fa-1, a mutant defective in flagellar structure, produced Ins(1,4,5)P3 but failed to deflagellate. These results support a model in which acid pH activates a putative cellular receptor leading to G-protein dependent activation of phospholipase C and accumulation of Ins(1,4,5)P3. These events are upstream of Ins(1,4,5)P3-dependent Ca2+ entry from the medium, and of deflagellation.

Inositol phospholipid metabolism may trigger flagellar excision in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii cells shed their flagella in response to environmental stress. Under favorable conditions, flagella are quickly regrown. To learn more about the signals that trigger flagellar excision and regrowth we have investigated inositol phospholipid metabolites, molecules implicated in signal transduction in several other systems. After deflagellation by low pH or mastoparan, a potent activator of G proteins, there was a rapid increase in levels of inositol 1,4,5-trisphosphate measured by use of receptor-binding assays and HPLC. This increase was concomitant with a decrease in levels of phosphatidylinositol 4,5-bisphosphate and was followed by an increase in phosphatidic acid, results consistent with activation of phospholipase C and diacylglycerol kinase. Additional experiments suggest that this activated phospholipase C is not important for flagellar regrowth but plays a role in informing the excision apparatus of the environmental stress. Addition of neomycin (an inhibitor of phospholipase C) before exposure of cells to low pH or mastoparan prevented the increase in inositol 1,4,5-trisphosphate and also prevented deflagellation. Addition of neomycin after deflagellation blocked increases in inositol 1,4,5-trisphosphate that normally followed deflagellation, but did not block flagellar assembly. Furthermore, a flagellar excision-defective mutant, fa-1, did not shed its flagella in response to low pH or mastoparan, yet both of these agents activated phospholipase C in these cells. The results suggest that activation of phospholipase C, possibly via a G protein, is a proximal step in the signal transduction pathway inducing deflagellation in Chlamydomonas.

Transactivation of Hox gene expression in a VP16-dependent binary transgenic mouse system.

Mice with aberrant expression of Hox genes have provided valuable insight into the role of Hox class transcription factors in patterning the developing skeleton and the nervous system. However, a recurrent problem is the lethality of mice expressing a Hox-transgene. To circumvent premature death frequently associated with transgenes that interfere with development, we have established a binary transgenic mouse system. Transactivator mice harbor the VP16 gene regulated by a promoter of interest while transresponder mice contain the VP16-responsive immediate early (IE) promoter linked to the gene to be expressed [G.W. Byrne, F.H. Ruddle, Multiplex gene regulation: a two-tiered approach to transgene regulation in transgenic mice, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 5473-5477]. Here, we report the generation of transresponder mouse strains that harbor murine homeobox genes linked to the IE promoter. We provide evidence that these transgenes are transcriptionally activated in progeny that inherit both a transactivator and transresponder transgene. By microdissection of mouse embryos and reverse transcription polymerase chain reaction (RT-PCR) analysis, we demonstrate that the expression of the Hox-transgenes is restricted to those regions of the mouse embryos where VP16 is present. The ability to activate stable Hox-transgenes in a reproducible fashion now permits a detailed in vivo dissection of the molecular mechanisms that lead to developmental abnormalities caused by deregulated Hox-gene expression.

Evidence for regulation of cartilage differentiation by the homeobox gene Hoxc-8.

Homeobox genes of the Hox class are required for proper patterning of skeletal elements, but how they regulate the differentiation of specific tissues is unclear. We show here that overexpression of a Hoxc-8 transgene causes cartilage defects whose severity depends on transgene dosage. The abnormal cartilage is characterized by an accumulation of proliferating chondrocytes and reduced maturation. Since Hoxc-8 is normally expressed in chondrocytes, these results suggest that Hoxc-8 continues to regulate skeletal development well beyond pattern formation in a tissue-specific manner, presumably by controlling the progression of cells along the chondrocyte differentiation pathway. The comparison to Hoxd-4 and Isl-1 indicates that this role in chondrogenesis is specific to proteins of the Hox class. Their capacity for regulation of cartilage differentiation suggests that Hox genes could also be involved in human chondrodysplasias or other cartilage disorders.

Herpes simplex virus transcriptional activator VP16 is detrimental to preimplantation development in mice.

The herpes simplex virus transactivator protein VP16 is frequently used to regulate gene expression in several experimental systems, including transgenic mice. It has been suggested that high levels of VP16 expression in mice may be lethal. In order to systematically address this issue, we linked the VP16 gene to promoters that are active early and in a variety of tissues throughout development, such as the human beta-actin promoter or the rat nestin gene enhancer. VP16 expression was assayed using a LacZ reporter gene linked to a VP16-responsive immediate early gene promoter. We show here that expression of VP16 at high levels is detrimental to pre-implantation development. By culturing embryos in vitro, we demonstrate that this effect is exerted at the transition from the 2-cell to the 4-cell stage, reducing survival to the blastocyst stage dramatically. On the other hand, transgenic mice expressing VP16 transgenes at postimplantation stages are viable. These results suggest a differential sensitivity to VP16 expression in different cell types and stages of development. The reduction of embryo survival by VP16 implicates herpes virus infection as a potential cause of infertility.

Phospholipase C activation during elicitation of the oxidative burst in cultured plant cells.

Although phospholipase C hydrolysis of polyphosphoinositides constitutes one of the major second messenger pathways in animal cells, its participation in signal transduction in higher plants has not been established. To determine whether activation of phosphatidylinositol-directed phospholipase C might be involved in signaling the elicitor-induced oxidative burst in plants, suspension-cultured soybean cells were treated with two stimulants of the H2O2 burst and examined for polyphosphoinositide turnover. Both polygalacturonic acid elicitor and the G protein activator, mastoparan, promoted a transient increase in inositol 1,4,5-trisphosphate (IP3) content that exceeded basal IP3 levels (0.9 +/- 0.4 pmol of IP3/10(6) cells, n = 28) by 2.6- and 7-fold, respectively. In each case, intracellular IP3 content reached a maximum at 1 min post-stimulation and declined to near basal levels during the subsequent 5-10 min. Neomycin sulfate, an inhibitor of polyphosphoinositide hydrolysis, blocked the IP3 transient, and Mas-17, an inactive analogue of mastoparan, induced no change in IP3. Thin layer chromatography of lipid extracts of the soybean cells corroborated the above results by revealing a rapid decrease in phosphatidyl-inositol monophosphate and phosphatidylinositol 4,5-bisphosphate following polygalacturonic acid elicitor and mastoparan (but not Mas-17) stimulation. Since the rise in IP3 preceded H2O2 production and since neomycin sulfate inhibited the appearance of both, we hypothesize that phospholipase C activation might constitute one pathway by which elicitors trigger the soybean oxidative burst.