Abstract
The goal of our study was the identification of up-regulated genes during axolotl (Ambystoma mexicanum) hindlimb regeneration 4 days after amputation using suppression subtractive hybridization (SSH). Approximately 400 clones that harbored upregulated genes in regenerating blastema tissue were selected for sequence analysis. A BLAST homology search against NCBI non-redundant database and an ambystoma EST database revealed 102 clones that showed homology to known sequences in GenBank with annotated function, 31 were known genes without known function, 74 were novel and 72 belonged to mitochondrial sequences. Differential expression of Hmox1, Orc4L, Pls3, Fen-1, Mcm7 and Mmp3/10a was confirmed using qRT-PCR analysis. Among all genes, only Mmp3/10a has been previously described as involved in limb regeneration. Other important identified genes belong to the group of cell cycle regulators (Orc4L, Nasp, Skp1A and Mcm7, the latter being a possible proliferative marker), those involved in protein synthesis and transport (Sec63, Srp72, Sara2) and V-ATPase pump. The novel genes we identified might be important for the process of blastema formation and the onset of cell proliferation in a regenerating limb.
Resumen
El objetivo del estudio es la identificación de genes sobreexpresados durante la regeneración de las extremidades en el ajolote (Ambystoma mexicanum), cuatro días después de su amputación, mediante hibridación sustractiva supresora (SSH). Aproximadamente 400 clones que contenían genes sobreexpresados en tejido de blastema en regeneración fueron seleccionados para su análisis por secuenciación. Las búsquedas de homología con BLAST en la base de datos no redundante del NCBI y en una base de datos de secuencias de expres’ón (EST) de ajolote revelaron 102 clones que muestran homología con secuencias conocidas y de función definida, 31 que corresponden a secuencias conocidas pero sin función determinada, 74 secuencias son novedosas y 72 corresponden a secuencias mitocondriales. La expresión diferencial de los genes Hmox1, Orc4L, Pls3, Fen-1, Mcm7 y Mmp3/10a fue confirmada mediante análisis por PCR cuantitativa a tiempo real. De todos los genes identificados, solamente Mmp3/10a ha sido anteriormente relacionado con procesos regenerativos de extremidades. Otros genes importantes identificados pertenecen al grupo de reguladores del ciclo celular (Orc4L, Nasp, Skp1A y Mcm7, siendo este último un probable marcador de proliferación), genes involucrados en la síntesis y el transporte de proteínas (Sec63, Srp72, Sara2) y la bomba V-ATPasa. Los genes nuevos identificados podrían ser de importancia en el proceso de formación del blastema y en la iniciación de la proliferación celular de miembros en regeneración.
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Gorsic, M., Majdic, G. & Komel, R. Identification of differentially expressed genes in 4-day axolotl limb blastema by suppression subtractive hybridization. J. Physiol. Biochem. 64, 37–50 (2008). https://doi.org/10.1007/BF03168233
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DOI: https://doi.org/10.1007/BF03168233