Xenopus Laevis TGF-ß: Cloning And Characterization Of The Signaling Receptors

Abstract

The amphibian species Xenopus laevis, along with mouse and chicken is a very important model system, used widely to dissect the molecular intricacies of various aspects of vertebrate development. Study with Xenopus has clear advantages in terms of various technical considerations including the ease of handling early stage of embryos and due to the remarkable documentation of several early molecular events during development. The concept of inductive interactions between various cell types during early development was first revealed by the studies performed in Xenopus, and among the various factors proposed for mesoderm induction, the members of transforming growth factor-β (TGF- β) superfamily have been considered to be the most probable candidates. About forty different members of the TGF-β superfamily have been cloned and characterized from various organisms. The superfamily members like activins and BMPs have been studied extensively with respect to their functional role during development. While BMPs were assigned as candidates for inducing ventral mesoderm, activins oppose the role of BMPs by inducing dorsal mesoderm. Studies that helped in delineating their roles were performed using three approaches that utilized the ligands, receptors or down stream signaling components (Smads). All the three components were studied with respect to their endogenous expression pattern and effects of ectopic expressions of the wild type or dominant negative mutants. These approaches led to the accumulation of evidences supporting the importance of these signaling molecules. All the above mentioned studies were only possible due to the cloning and characterization of cDNAs of the various proteins involved in the signaling pathway including the ligands. TGF-β2 and 5 are the two isoforms of TGF-β cloned from the amphibian system. We have earlier cloned and characterized the promoter for TGF-β5 gene, which suggested possible regulation of this factor by tissue specific transcription factors. Messenger RNA in situ hybridization analysis to study the TGF-β5-expression pattern during Xenopus development, showed spatial and temporal expression pattern. The expression was confined to specific regions that include notochord, somites, and tail bud among others, in the various stages analyzed. This suggested a possible role for TGF-β5 in organogenesis during the amphibian development. To better understand the role of TGF-β in Xenopus development, studies to examine the specific receptor expression pattern for this growth factor is very essential. With the lack of any reports on cloning of TGF-β receptors from this system, the aim of the present study was to isolate and characterize the receptors for TGF-β from Xenopus laevis. PCR cloning using degenerate primers based on the conserved kinase domains of this class of receptors, coupled to library screenings enabled the identification of two novel receptor cDNAs of the TGF-β receptor superfamily. Characterization of the isolated cDNAs suggested that one of them codes for a type II receptor for TGF-β. Further the cDNAs were found to be ubiquitously expressed during development, as judged by RT-PCR analysis. The cloned cDNAs can now be employed as tools, to study the expression pattern by means of mRNA in situ hybridization, on the various developmental stage embryos and to perform studies using antisense and dominant negative mRNA injection experiments in vivo. Such studies will greatly assist in delineating the role of TGF-β ligands and receptors during amphibian development.