Allorecognition proteins in an invertebrate exhibit homophilic interactions.

Sessile colonial invertebrates-animals such as sponges, corals, bryozoans, and ascidians-can distinguish between their own tissues and those of conspecifics upon contact [1]. This ability, called allorecognition, mediates spatial competition and can prevent stem cell parasitism by ensuring that colonies only fuse with self or close kin. In every taxon studied to date, allorecognition is controlled by one or more highly polymorphic genes [2-8]. However, in no case is it understood how the proteins encoded by these genes discriminate self from non-self. In the cnidarian Hydractinia symbiolongicarpus, allorecognition is controlled by at least two highly polymorphic allorecognition genes, Alr1 and Alr2 [3, 5, 9-12]. Sequence variation at each gene predicts allorecognition in laboratory strains such that colonies reject if they do not share a common allele at either locus, fuse temporarily if they share an allele at only one locus, or fuse permanently if they share an allele at both genes [5, 9]. Here, we show that the gene products of Alr1 and Alr2 (Alr1 and Alr2) are self-ligands with extraordinary specificity. Using an in vitro cell aggregation assay, we found that Alr1 and Alr2 bind to themselves homophilically across opposing cell membranes. For both proteins, each isoform bound only to itself or to an isoform of nearly identical sequence. These results provide a mechanistic explanation for the exquisite specificity of Hydractinia allorecognition. Our results also indicate that hydroids have evolved a molecular strategy of self-recognition that is unique among characterized allorecognition systems within and outside invertebrates.

Analyzing cell physiology in C. elegans with fluorescent ratiometric reporters.

Ratiometric fluorescent reporters have recently emerged a new technique to non-invasively measure aspects of cell physiology such as redox status, calcium levels, energy production, and NADH levels. These reporters consist of either a single or pair of fluorophores along with specific modifications, such as the addition of a protein domain which binds to a metabolite of interest, thereby producing gradual alterations in fluorescence in response to changes in the measured parameter. Measurement of the changes in fluorescence produces a quantitative read-out of the cellular environment. While these reporters were initially developed to easily visualize and track changes in cultured cells, several groups have adapted these reporters to use in Caenorhabditis elegans which opens a new avenue through which to explore cell physiology during development or aging, in response to changes in external environment, or in response to genetic manipulation. These reporters have the advantage of being easily targeted to any part of the worm, and because C. elegans is transparent both the reporters and changes in their fluorescence can be clearly observed in vivo. Here we discuss the application of ratiometric reporters to C. elegans, and outline a method to quantitatively measure changes in intracellular peroxide levels using the HyPer ratiometric reporter. However, these principles can be applied to alternate ratiometric reporters which are designed to measure either other chemical species or other cellular parameters.

Characterization of the nutritional endoderm in the direct developing frog Eleutherodactylus coqui.

Unlike Xenopus laevis, Eleutherodactylus coqui develops without a tadpole. The yolk-rich vegetal region of the embryo forms a transient nutritive tissue, the nutritional endoderm (NE). The definitive endoderm (DE) in E. coqui comes from cells closer to the animal pole in contrast to its vegetal origin in X. laevis. RNA important for initiating the endoderm specification network is absent in presumptive NE cells, raising the question whether signaling occurs in them. We explored the nature of NE and asked how differences between NE and DE cells arise. We identified differences between NE and DE that first become evident at gastrula, when NE cells become multinucleated. Nuclear ß-catenin, an essential cofactor of sox 17, important for endoderm formation in X. laevis, is present in NE and DE at gastrula but remains in NE long after it is not seen in DE. We cloned E. coqui homologs of TGFßs activin b and derriere and provide evidence for their maternal expression. We also detected activin b and derriere RNAs in NE at gastrula and show that NE possesses some mesoderm-inducing activity, but it is delayed with respect to DE. Our findings indicate that altered development of NE begins at gastrula. RNAs important for mesendoderm induction and some mesoderm-inducing activity are present in NE.

Nutritional endoderm in a direct developing frog: a potential parallel to the evolution of the amniote egg.

The egg of the direct-developing frog, Eleutherodactylus coqui, has 20 x the volume as that of the model amphibian, Xenopus laevis. Increased egg size led to the origin of nutritional endoderm, a novel cell type that provides nutrition but does not differentiate into digestive tract tissues. As the E. coqui endoderm develops, a distinct boundary exists between differentiating intestinal cells and large yolky cells, which persists even when yolk platelets are depleted. The yolky cells do not become tissues of the digestive tract and are lost, as shown by histology and lineage tracing. EcSox17, an endodermal transcriptional factor, did not distinguish these two cell types, however. When cleavage of the yolky cells was inhibited, embryogenesis continued, indicating that some degree of incomplete cleavage can be tolerated. The presence of cellularized nutritional endoderm in E. coqui may parallel changes that occurred in the evolution of the amniote egg 360 million years ago.