Thymosin β4 dynamics during chicken enteroid development.

The sheared avian intestinal villus-crypts exhibit high tendency to self-repair and develop enteroids in culture. Presuming that this transition process involves differential biomolecular changes, we employed matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) to find whether there were differences in the spectral profiles of sheared villi versus the enteroids, assessed in the mass range of 2-18 kDa. The results showed substantial differences in the intensities of the spectral peaks, one particularly corresponding to the mass of 4963 Da, which was significantly low in the sheared villus-crypts compared with the enteroids. Based on our previous results with other avian tissues and further molecular characterization by LC-ESI-IT-TOF-MS, and multiple reaction monitoring (MRM), the peak was identified to be thymosin β4 (Tβ4), a ubiquitously occurring regulatory peptide implicated in wound healing process. The identity of the peptide was further confirmed by immunohistochemistry which showed it to be present in a very low levels in the sheared villi but replete in the enteroids. Since Tβ4 sequesters G-actin preventing its polymerization to F-actin, we compared the changes in F-actin by its immunohistochemical localization that showed no significant differences between the sheared villi and enteroids. We propose that depletion of Tβ4 likely precedes villous reparation process. The possible mechanism for the differences in Tβ4 profile in relation to the healing of the villus-crypts to developing enteroids is discussed.