Understanding the functional relationship between intracellular factors and
extracellular signals is required for reconstructing gene regulatory networks
(GRN) involved in complex biological processes. One of the best-studied
bilaterian GRNs describes endomesoderm specification and predicts that both
mesoderm and endoderm arose from a common GRN early in animal evolution.
Compelling molecular, genomic, developmental, and evolutionary evidence supports
the hypothesis that the bifunctional gastrodermis of the cnidarian-bilaterian
ancestor is derived from the same evolutionary precursor of both endodermal and
mesodermal germ layers in all other triploblastic bilaterian animals. We have
begun to establish the framework of a provisional cnidarian
“endomesodermal” gene regulatory network in the sea anemone,
Nematostella vectensis, by using a genome-wide microarray
analysis on embryos in which the canonical Wnt/ß-catenin pathway was
ectopically targeted for activation by two distinct pharmaceutical agents
(lithium chloride and 1-azakenpaullone) to identify potential targets of
endomesoderm specification. We characterized 51 endomesodermally expressed
transcription factors and signaling molecule genes (including 18 newly
identified) with fine-scale temporal (qPCR) and spatial (in
situ) analysis to define distinct co-expression domains within the
animal plate of the embryo and clustered genes based on their earliest zygotic
expression. Finally, we determined the input of the canonical
Wnt/ß-catenin pathway into the cnidarian endomesodermal GRN using
morpholino and mRNA overexpression experiments to show that NvTcf/canonical Wnt
signaling is required to pattern both the future endomesodermal and ectodermal
domains prior to gastrulation, and that both BMP and FGF (but not Notch)
pathways play important roles in germ layer specification in this animal. We
show both evolutionary conserved as well as profound differences in
endomesodermal GRN structure compared to bilaterians that may provide
fundamental insight into how GRN subcircuits have been adopted, rewired, or
co-opted in various animal lineages that give rise to specialized endomesodermal
cell types.
Backbone N-methylation and macrocyclization improve the pharmacological properties of peptides by enhancing their proteolytic stability, membrane permeability and target selectivity. Borosins are backbone N-methylated peptide macrocycles derived from a precursor protein which contains a peptide α-N-methyltransferase domain autocatalytically modifying the core peptide located at its C-terminus. Founding members of borosins are the omphalotins from the mushroom Omphalotus olearius (omphalotins A-I) with nine out of 12 L-amino acids being backbone N-methylated. The omphalotin biosynthetic gene cluster codes for the precursor protein OphMA, the protease prolyloligopeptidase OphP and other proteins that are likely to be involved in other post-translational modifications of the peptide. Mining of available fungal genome sequences revealed the existence of highly homologous gene clusters in the basidiomycetes Lentinula edodes and Dendrothele bispora. The respective borosins, referred to as lentinulins and dendrothelins are naturally produced by L. edodes and D. bispora as shown by analysis of respective mycelial extracts. We produced all three homologous peptide natural products by coexpression of OphMA hybrid proteins and OphP in the yeast Pichia pastoris. The recombinant peptides differ in their nematotoxic activity against the plant pathogen Meloidogyne incognita. Our findings pave the way for the production of borosin peptide natural products and their potential application as novel biopharmaceuticals and biopesticides.
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