Abnormal lens morphogenesis and ectopic lens formation in the absence of beta-catenin function

beta-Catenin plays a key role in cadherin-mediated cell adhesion as well as in canonical Wnt signaling. To study the role of beta-catenin during eye development, we used conditional Cre/loxP system in mouse to inactivate beta-catenin in developing lens and retina. Inactivation of beta-catenin does not suppress lens fate, but instead results in abnormal morphogenesis of the lens. Using BAT-gal reporter mice, we show that beta-catenin-mediated Wnt signaling is notably absent from lens and neuroretina throughout eye development. The observed defect is therefore likely due to the cytoskeletal role of beta-catenin, and is accompanied by impaired epithelial cell adhesion. In contrast, inactivation of beta-catenin in the nasal ectoderm, an area with active Wnt signaling, results in formation of crystallin-positive ectopic lentoid bodies. These data suggest that, outside of the normal lens, beta-catenin functions as a coactivator of canonical Wnt signaling to suppress lens fate.     

The effect of ammonia on stalk cell formation in submerged monolayers of Dictyostelium discoideum

It has been shown that ammonia inhibits stalk cell formation in monolayers of V12M2, and it was suggested that this inhibition was due to an antagonism of the differentiation-inducing factor (DIF) (Gross, J.D. et al., Nature, 303, 244-246, 1983). However, the results presented here indicate that ammonia inhibition is independent of DIF concentration, and that it occurs well in advance of the period of DIF requirement. Ammonia completely inhibits DIF accumulation and inhibits stalk cell differentiation, but there is no inhibition of prespore cell formation. These results imply the existence of an early ammonia-sensitive event that influences terminal cell type differentiation.     

A possible role for DIF-2 in the formation of stalk cells during Dictyostelium development.

The differentiation inducing factor (DIF) is essential for stalk cell formation in monolayers of Dictyostelium discoideum and is necessary for the expression of several prestalk cell-specific genes. DIF activity has been fractionated into a major species, designated DIF-1, and several minor species, including DIF-2. Although DIF-1 is an excellent inducer of stalk cell formation from vegetative cells, it is a poor inducer of stalk cell formation from prestalk cells. In contrast, DIF-2 is more active for the conversion of prestalk cells into stalk cells, than for the conversion of vegetative cells to stalk cells. The same results were obtained regardless of whether chemically synthesized or naturally occurring components were utilized. In addition, stalk cell formation was three- to fourfold higher when vegetative cells were incubated with DIF-1 for a suboptimal period and then subsequently incubated with DIF-2, than when cells were incubated with DIF-2 first and then subsequently with DIF-1. These results indicate a distinct role for DIF-2 during stalk cell formation and suggest the possibility that DIF-1 and DIF-2 act sequentially.     

Loss of Dictyostelium HSPC300 causes a scar-like phenotype and loss of SCAR protein

Background  

SCAR/WAVE proteins couple signalling to actin polymerization, and are thus fundamental to the formation of pseudopods and lamellipods. They are controlled as part of a five-membered complex that includes the tiny HSPC300 protein. It is not known why SCAR/WAVE is found in such a large assembly, but in Dictyostelium the four larger subunits have different, clearly delineated functions.     

Monoclonal antibodies specific for stalk differentiation in Dictyostelium discoideum

We have produced two monoclonal antibodies specific to the stalk cells of Dictyostelium discoideum fruiting bodies. Both monoclonal antibodies react with high molecular weight proteins previously found to be stalk-specific by two-dimensional gel analysis. One antibody (JAb 1) is specific for a single protein of apparent molecular weight 310 000 which first appears when overt stalk differentiation begins at 20 h. The other monoclonal antibody (JAb 2) is also stalk-specific, though earlier in development it binds to proteins extracted from both prestalk and prespore cells of the migrating slug. It reacts with two proteins in stalks, one of apparent molecular weight 430 000 which is first detected during tip formation at 12 h and a lower molecular weight protein (310 000) detected from 20 h. Although several markers are available for the investigation of prespore/spore differentiation there is a distinct lack of suitable prestalk/stalk markers. The monoclonal antibodies described here are highly specific stalk markers and should prove useful in the study of cell proportioning and terminal differentiation.     

Caulobacter crescentus requires RodA and MreB for stalk synthesis and prevention of ectopic pole formation

Caulobacter crescentus cells treated with amdinocillin, an antibiotic which specifically inhibits the cell elongation transpeptidase penicillin binding protein 2 in Escherichia coli, exhibit defects in stalk elongation and morphology, indicating that stalk synthesis may be a specialized form of cell elongation. In order to investigate this possibility further, we examined the roles of two other proteins important for cell elongation, RodA and MreB. We show that, in C. crescentus, the rodA gene is essential and that RodA depletion leads to a loss of control over stalk and cell body diameter and a stalk elongation defect. In addition, we demonstrate that MreB depletion leads to a stalk elongation defect and conclude that stalk elongation is a more constrained form of cell elongation. Our results strongly suggest that MreB by itself does not determine the diameter of the cell body or stalk. Finally, we show that cells recovering from MreB depletion exhibit a strong budding and branching cell body phenotype and possess ectopic poles, as evidenced by the presence of multiple, misplaced, and sometimes highly branched stalks at the ends of these buds and branches. This phenotype is also seen to a lesser extent in cells recovering from RodA depletion and amdinocillin treatment. We conclude that MreB, RodA, and the target(s) of amdinocillin all contribute to the maintenance of cellular polarity in C. crescentus.     

Pyridoxal kinase knockout of Dictyostelium complemented by the human homologue

The gene (pykA) encoding pyridoxal kinase which converts pyridoxal (vitamin B(6)) to pyridoxal phosphate was isolated from Dictyostelium discoideum using insertional mutagenesis. Cells of a pykA gene knockout grew poorly in axenic medium with low yield but growth was restored by the addition of pyridoxal phosphate. Sequencing indicated a gene, with one intron, encoding a predicted protein of 301 amino acids that was 42% identical in amino acid sequence to human pyridoxal kinase. After expression of the wild-type gene in Escherichia coli, the purified PykA protein product was shown to have pyridoxal kinase enzymatic activity with a K(m) of 8.7 microM for pyridoxal. Transformation of the Dictyostelium knockout mutant with the human pyridoxal kinase gene gave almost the same level of complementation as that seen using transformation with the wild-type Dictyostelium gene. Phylogenetic analysis indicated that the Dictyostelium amino acid sequence was closer to human pyridoxal kinase than to pyridoxal kinases of lower eukaryotes.     

A retinoblastoma ortholog controls stalk/spore preference in Dictyostelium

We describe rblA, the Dictyostelium ortholog of the retinoblastoma susceptibility gene Rb. In the growth phase, rblA expression is correlated with several factors that lead to 'preference' for the spore pathway. During multicellular development, expression increases 200-fold in differentiating spores. rblA-null strains differentiate stalk cells and spores normally, but in chimeras with wild type, the mutant shows a strong preference for the stalk pathway. rblA-null cells are hypersensitive to the stalk morphogen DIF, suggesting that rblA normally suppresses the DIF response in cells destined for the spore pathway. rblA overexpression during growth leads to G1 arrest, but as growing Dictyostelium are overwhelmingly in G2 phase, rblA does not seem to be important in the normal cell cycle. rblA-null cells show reduced cell size and a premature growth-development transition; the latter appears anomalous but may reflect selection pressures acting on social ameba.     

Developmental regulation of a stalk cell differentiation-inducing factor in Dictyostelium discoideum

Previous work has shown that cells developing at high density release a low-molecular-weight factor that can induce isolated Dictyostelium discoideum amoebae of strain V12M2 to differentiate into stalk cells in the presence of cyclic AMP. We now show that this differentiation-inducing factor, called DIF, can be extracted from cells during normal development and that its production is strongly developmentally regulated. DIF is not detectable in vegetative cells but rises dramatically after aggregation to reach a peak during slug migration. DIF levels are very low in two mutants defective in aggregation. The postaggregative synthesis of DIF is stimulated by the addition of extracellular cyclic AMP. We propose that DIF is a morphogen controlling prestalk cell differentiation.     

Dictyopyrones, novel alpha-pyronoids isolated from Dictyostelium spp., promote stalk cell differentiation in Dictyostelium discoideum

Dictyopyrones A and B (DpnA and B), whose function(s) is not known, were isolated from fruiting bodies of Dictyostelium discoideum. In the present study, to assess their function(s), we examined the effects of Dpns on in vitro cell differentiation in D. discoideum monolayer cultures with cAMP. Dpns at 1-20 microM promoted stalk cell formation to some extent in the wild-type strain V12M2. Although Dpns by themselves could hardly induce stalk cell formation in a differentiation-inducing factor (DIF)-deficient strain HM44, both of them dose-dependently promoted DIF-1-dependent stalk cell formation in the strain. In the sporogenous strain HM18, Dpns at 1-20 microM suppressed spore formation and promoted stalk cell formation in a dose-dependent manner. Analogs of Dpns were less effective in affecting cell differentiation in both HM44 and HM18 cells, indicating that the activity of Dpns should be chemical structure specific. It was also shown that DpnA at 2-20 microM dose-dependently suppressed spore formation induced with 8-bromo cAMP and promoted stalk cell formation in V12M2 cells. Interestingly, it was shown by the use of RT-PCR that DpnA at 10 microM slightly promoted both prespore- and prestalk-specific gene expressions in an early phase of V12M2 and HM18 in vitro differentiation. The present results suggest that Dpns may have functions (1) to promote both prespore and prestalk cell differentiation in an early stage of development and (2) to suppress spore formation and promote stalk cell formation in a later stage of development in D. discoideum.     

A Dictyostelium morphogen that is essential for stalk cell formation is generated by a subpopulation of prestalk cells

The stalk cell differentiation inducing factor (DIF) has the properties required of a morphogen responsible for pattern regulation during the pseudoplasmodial stage of Dictyostelium development. It induces prestalk cell formation and inhibits prespore cell formation, but there is as yet no strong evidence for a morphogenetic gradient of DIF. We have measured DIF accumulation by monolayers of isolated prestalk and prespore cells in an attempt to provide evidence for such a gradient. DIF is accumulated in the largest quantities by a subpopulation of prestalk cells that specifically express the DIF-inducible genes pDd56 and pDd26. Since it has been shown recently that cells that express pDd56 are localized in the central core of the prestalk cell region of the pseudoplasmodia, our current results suggest a morphogenetic gradient generated by this region.     

Signalling pathways that direct prestalk and stalk cell differentiation in Dictyostelium

Prestalk cell differentiation in Dictyostelium is induced by DIF and two DIF-induced genes, ecmA and ecmB, have revealed the existence of multiple prestalk and stalk cell sub-types. These different sub-types are defined by the pattern of expression of subfragments derived from the ecmA and ecmB promoters. These markers have been utilised in three ways; for fate mapping in vivo, to investigate the molecular mechanisms underlying DIF signalling and to explore the relative requirement for DIF and other signalling molecules for prestalk and stalk cell differentiation in vitro. The heterogeneity of the prestalk and stalk populations seems to be reflected in differences in the cell signalling pathways that they utilise.     

The induction of stalk cell differentiation in submerged monolayers of Dictyostelium discoideum

Abstract. Differentiation of Dictyostelium discoideum cells in submerged monolayers was studied and compared with in vivo development. The accumulation patterns of three developmentally regulated enzymes in cells of strain V12M2 differentiating in vivo on Millipore Filters or in vitro in monolayers at high cell-densities were found to be similar. Moreover, stalk cell formation occurred at approximately the same time in high or low cell density monolayers as it did during normal differentiation. These observations suggest that the timing of differentiation in vitro and in vivo is similar.
In vitro stalk cell formation requires exogenous cyclic AMP, and in its absence, the accumulation patterns of the three developmentally regulated enzymes are alterd. At low cell densities, in vitro stalk cell induction also requires a differentiation-inducing factor (DIF). The addition or removal of cyclic AMP or DIF during development under these conditions revealed the sequence of these two requirements. Cyclic AMP is not required for stalk cell induction for the first 8 hours of incubation, but thereafter, a gradually increasing proportion of cells are induced by cyclic AMP. After a brief delay there is a period of induction by DIF, and this period corresponds approximately to the period of DIF accumulation during in vivo development. The two induction events are clearly separate, in that each inducer can act in the absence of the other, as long as cyclic AMP induction precedes DIF induction. Cyclic AMP is only required at a concentration of 40 μM when added 8 hours after the beginning of the differentiation period.     

Multiple forms of acid phosphatase and their differential secretion during stalk formation under submerged monolayer incubation of Dictyostelium discoideum

The electrophoretic pattern of intracellular and secreted acid phosphatases (AcPases) in Dictyostelium discoideum was examined during incubation of the cells as a submerged monolayer. Three distinct forms of the enzyme were observed in the cell during differentiation; one was detected throughout development (AcPase 1), whereas the others including AcPase 2 were stage-specific. AcPase 1 was released in the medium predominantly in early development and AcPase 2, a prestalk specific form, was secreted during stalk formation. When cells were incubated under conditions where stalk cells did not form, only AcPase 1 was recognized both in the cell and in the medium.     

Cyclic AMP is an inhibitor of stalk cell differentiation in Dictyostelium discoideum

Cyclic AMP and DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) together induce stalk cell differentiation in vitro in Dictyostelium discoideum strain V12M2. The induction can proceed in two stages: in the first, cyclic AMP brings cells to a DIF-responsive state; in the second, DIF-1 alone can induce stalk cell formation. We report here that during the DIF-1-dependent stage, cyclic AMP is a potent inhibitor of stalk cell differentiation. Addition of cyclic AMP at this stage to V12M2 cells appreciably delays, but does not prevent, stalk cell formation. In contrast, stalk cell differentiation in the more common strain NC4 is completely suppressed by the continued presence of cyclic AMP. This fact explains earlier failures to induce stalk cells in vitro in NC4. We now consistently obtain efficient stalk cell induction in NC4 by removing cyclic AMP in the DIF-1-dependent stage. Cyclic AMP also inhibits the production of a stalk-specific protein (ST310) in both NC4 and a V12M2 derivative. Adenosine, a known antagonist of cyclic AMP action, does not relieve this inhibition by cyclic AMP and does not itself promote stalk cell formation. Finally, stalk cell differentiation of NC4 cells at low density appears to require factors in addition to cyclic AMP and DIF-1, but their nature is not yet known. The inhibition of stalk cell differentiation by cyclic AMP may be important in establishing the prestalk/prespore pattern during normal development, and in preventing the maturation of prestalk into stalk cells until culmination.