Identification of genes expressed during Xenopus laevis limb regeneration by using subtractive hybridization

Abstract

Suppression polymerase chain reaction-based subtractive hybridization was used to identify genes that are expressed during Xenopus laevis hindlimb regeneration. Subtractions were done by using RNAs extracted from the regeneration-competent stage (stage 53) and regeneration-incompetent stage (stage 59) of limb development. Forward and reverse subtractions were done between stage 53 7-day blastema and stage 53 contralateral limb (competent stage), stage 59 7-day pseudoblastema and stage 59 contralateral limb (incompetent stage), and stage 53 7-day blastema and stage 59 7-day pseudoblastema. Several thousand clones were analyzed from the various subtracted libraries, either by random selection and sequencing (1,920) or by screening subtracted cDNA clones (6,150), arrayed on nylon membranes, with tissue-specific probes. Several hundred clones were identified from the array screens whose expression levels were at least twofold higher in experimental tissue vs. control tissue (e.g., blastema vs. limb) and selected for sequencing. In addition, primers were designed to assay several of the randomly selected clones and used to assess the level of expression of these genes during regeneration and normal limb development. Approximately half of the selected clones were differentially expressed, as expected, including several that demonstrate blastema-specific enhancement of expression. Three distinct categories of expression were identified in our screens: (1) clones that are expressed in both regeneration-competent blastemas and -incompetent pseudoblastemas, (2) clones that are expressed at highest levels in regeneration-competent blastemas, and (3) clones that are expressed at highest levels in regeneration-incompetent pseudoblastemas. Characterizing the role of each of these three categories of genes will be important in furthering our understanding of the process of tissue regeneration. Developmental Dynamics 226:398 -409, 2003.

Mechanisms of urodele limb regeneration

“…Most of these genes fell into the categories of metabolism, cell physiological process, cell cycle regulation, and protein synthesis and transport. Subtractive hybridization was also used to compare transcript expression after amputation at a regeneration‐competent versus a regeneration‐deficient stage of Xenopus limb bud development (King et al., 2003). This study identified three categories of cDNA clones: clones expressed at both competent and deficient blastemas, clones with highest expression in regeneration‐competent blastemas, and clones with highest expression in regeneration‐deficient blastemas.…”

Beyond early development: Xenopus as an emerging model for the study of regenerative mechanisms

“…Subtractive hybridization was also used by King et al (2003) to identify regeneration specific genes in the hindlimb bud. By subtracting combinations of cDNA obtained from competent (st. 53) vs. incompetent (st. 59) 7 day blastemas, or control, unoperated limbs at the same stage, they identified genes that were preferentially expressed in regenerating limbs, nonregenerating limbs, or both, relative to uncut limbs.…”

Neural MMP‐28 expression precedes myelination during development and peripheral nerve repair

“…A full-length cDNA was subsequently isolated from a Xenopus stage 17 neurula whole-embryo cDNA library (King et al, 2003). The cDNA was sequenced and characterized and shown to be most highly homologous to MMP-28 (Fig.…”

“…As the original identification of XMMP-28 was as an up-regulated transcript in 7-day postamputation pseudoblastemas from regeneration incompetent stage 59 hindlimbs (King et al, 2003), it was of interest to determine whether XMMP-28 expression was altered during functional regeneration. Hindlimbs of regeneration competent stage 53 Xenopus laevis were amputated at the level of the prospective tibia/fibula to examine expression of XMMP-28 during blastema formation and the events leading to limb regeneration (Dent, 1962).…”

“…Recently, MMP-28 has been shown to be significantly up-regulated in cartilage tissue and in the synovium of osteoarthritic joints Momohara et al, 2004;Davidson et al, 2006). King et al (2003) showed that MMP-28 was associated with limb regeneration in the frog Xenopus laevis.…”