Morphological phenotypes and functional capabilities of submandibular parenchymal cells of the ferret investigated by protein, mucosubstance and enzyme histochemistry

Submandibular glands obtained post-mortem from mature ferrets of both sexes were examined with the use of light microscopical histochemical methods for proteins, mucosubstances and enzymes associated with cell functions or organelles. Demilunar cells showed carboxylated mucosubstances that were mainly non-sulphated, and diffuse activity for peroxidase, E600-sensitive esterase and acid phosphatase. Thiol groups were also detected in these cells. Central acinar cells showed sulphated mucosubstances, disulphides and reticular staining for thiamine pyrophosphatase. Intercalary ducts showed diffuse activity for NADH and NAD(P)H dehydrogenases. Striated ducts contained protein, tryptophan, disulphides, neutral mucosubstances and E600-sensitive esterase periluminally. Basally, the striated ductal cells showed variable activity for peroxidase, cytochrome oxidase, succinate dehydrogenase and acid phosphatase. Basolateral plasma membranes of these cells exhibited ouabain-sensitive Na,K-ATPase activity. The collecting ducts were characterized by variable periluminal staining for acid phosphatase, -glucuronidase, acid -galactosidase and E600-resistant esterase. The results suggest that the histological appearances of the acini of the submandibular gland of the ferret are dependent on the synthesis of secretory acid glycoproteins, that the striated ducts are involved with the secretion of tryptophan-rich product comprising neutral glycoproteins and showing esterase activity and with marked transport of ions and that the collecting ducts are involved with absorption.     

Neural crest induction by paraxial mesoderm in Xenopus embryos requires FGF signals

At the border of the neural plate, the induction of the neural crest can be achieved by interactions with the epidermis, or with the underlying mesoderm. Wnt signals are required for the inducing activity of the epidermis in chick and amphibian embryos. Here, we analyze the molecular mechanisms of neural crest induction by the mesoderm in Xenopus embryos. Using a recombination assay, we show that prospective paraxial mesoderm induces a panel of neural crest markers (Slug, FoxD3, Zic5 and Sox9), whereas the future axial mesoderm only induces a subset of these genes. This induction is blocked by a dominant negative (dn) form of FGFR1. However, neither dnFGFR4a nor inhibition of Wnt signaling prevents neural crest induction in this system. Among the FGFs, FGF8 is strongly expressed by the paraxial mesoderm. FGF8 is sufficient to induce the neural crest markers FoxD3, Sox9 and Zic5 transiently in the animal cap assay. In vivo, FGF8 injections also expand the Slug expression domain. This suggests that FGF8 can initiate neural crest formation and cooperates with other DLMZ-derived factors to maintain and complete neural crest induction. In contrast to Wnts, eFGF or bFGF, FGF8 elicits neural crest induction in the absence of mesoderm induction and without a requirement for BMP antagonists. In vivo, it is difficult to dissociate the roles of FGF and WNT factors in mesoderm induction and neural patterning. We show that, in most cases, effects on neural crest formation were parallel to altered mesoderm or neural development. However, neural and neural crest patterning can be dissociated experimentally using different dominant-negative manipulations: while Nfz8 blocks both posterior neural plate formation and neural crest formation, dnFGFR4a blocks neural patterning without blocking neural crest formation. These results suggest that different signal transduction mechanisms may be used in neural crest induction, and anteroposterior neural patterning.     

The structure and function of MCM from archaeal M. Thermoautotrophicum

Eukaryotic chromosomal DNA is licensed for replication precisely once in each cell cycle. The mini-chromosome maintenance (MCM) complex plays a role in this replication licensing. We have determined the structure of a fragment of MCM from Methanobacterium thermoautotrophicum (mtMCM), a model system for eukaryotic MCM. The structure reveals a novel dodecameric architecture with a remarkably long central channel. The channel surface has an unusually high positive charge and binds DNA. We also show that the structure of the N-terminal fragment is conserved for all MCMs proteins despite highly divergent sequences, suggesting a common architecture for a similar task: gripping/remodeling DNA and regulating MCM activity. An mtMCM mutant protein equivalent to a yeast MCM5 (CDC46) protein with the bob1 mutation at its N terminus has only subtle structural changes, suggesting a Cdc7-bypass mechanism by Bob1 in yeast. Yeast bypass experiments using MCM5 mutant proteins support the hypothesis for the bypass mechanism.     

Effect of cell confluence on production of cloned mice using an inbred embryonic stem cell line

Mice have been successfully cloned from both somatic cells and hybrid embryonic stem (ES) cells. Heterozygosity of the donor ES cell genome has been suggested as a crucial factor for long-term survival of cloned mice. In the present study, an inbred ES cell line, HM-1 (129/Ola), and a well-tested ES cell line, R1 (129/Sv x 129/Sv-CP), were used as donor cells to evaluate the developmental potential of nuclear transfer embryos. We found that ES cell confluence dramatically affects the developmental potential of reconstructed embryos. With the ES cell line HM-1 and 80-90% confluence, 49% of reconstructed embryos developed to the morula/blastocyst stage, 9% of these embryos developed to live pups when transferred to the surrogate mothers, and 5 of 18 live pups survived to adulthood. By contrast, at 60-70% confluence, only 22% of embryos developed to the morula/blastocyst stage, and after transfer, only a single fetus reached term. Consistent with previous reports, the nuclei of R1 ES cells were also shown to direct development to term, but no live pups were derived from cells at later passages (>20). Our results show that the developmental potential of reconstructed embryos is determined by both cell confluence and cell passage. These results also demonstrate that the inbred ES cell line, HM-1, can be used to produce viable cloned mice, although less efficiently than most heterozygous ES cell lines.     

In Vivo Analysis of Voltage-Dependent Calcium Channels

The molecular cloning of calcium channel subunits has identified an unexpectedly large number of genes and splicing variants, many of whichhave complex expression patterns: a central problem of calcium channel biology is to understand the functional significance of this genetic complexity. The genetic analysis of voltage-dependent calcium channels (VDCCs) provides an approach to defining channel function that is complimentary to pharmacological, electrophysiological, and other molecular methods. By discovering or creating alleles of VDCC genes, one can gain an understanding of the VDCC function at the whole animal level. Of particular interest are mutations in the alpha1 genes that encode the pore forming subunits, as they define the specific channel subtypes. In fact, a variety of calcium channelopathies and targeted mutations have been described for these genes in the last 6 years. The mutant alleles described below illustrate how phenotype analysis of these alleles has uncovered very specific functional roles that can be localized to specific synapses or cells.     

The Arabidopsis CLV3-like (CLE) genes are expressed in diverse tissues and encode secreted proteins

Members of the receptor-like kinase gene family play crucial regulatory roles in many aspects of plant development, but the ligands to which they bind are largely unknown. In Arabidopsis, the receptor kinase CLAVATA1 (CLV1) binds to the small secreted polypeptide CLV3, and three proteins act as key elements of a signal transduction pathway that regulates shoot apical meristem maintenance. To better understand the signal transduction mechanisms involving small polypeptides, we are studying 25 Arabidopsis CLV3/ESR (CLE) proteins that share a conserved C-terminal domain with CLV3 and three maize ESR proteins. Members of the CLE gene family were identified in database searches and only a few are known to be expressed. We have identified an additional member of the CLE gene family in Arabidopsis, which is more similar in gene structure to CLV3 than the other CLE genes. Phylogenetic analysis reveals that few of the putative CLE gene products are closely related, suggesting there may be little functional overlap between them. We show that 24 of the 25 Arabidopsis CLE genes are transcribed in one or more tissues during development, indicating that they do encode functional products. Many are widely expressed, but others are restricted to one or a few tissue types. We have also determined the sub-cellular localization of several CLE proteins, and find that they are exported to the plasma membrane or extracellular space. Our results suggest that the Arabidopsis CLE proteins, like CLV3, may function as secreted signaling molecules that act in diverse pathways during growth and development.     

Equivalence relationships between stage-structured population models

Matrix population models are widely applied in conservation ecology to help predict future population trends and guide conservation effort. Researchers must decide upon an appropriate level of model complexity, yet there is little theoretical work to guide such decisions. In this paper we present an analysis of a stage-structured model, and prove that the model's structure can be simplified and parameterised in such a way that the long-term growth rate, the stable-stage distribution and the generation time are all invariant to the simplification. We further show that for certain structures of model the simplified models require less effort in data collection. We also discuss features of the models which are not invariant to the simplification and the implications of our results for the selection of an appropriate model. We illustrate the ideas using a population model for short-tailed shearwaters (Puffinus tenuirostris). In this example, model simplification can increase parameter elasticity, indicating that an intermediate level of complexity is likely to be preferred.