Title: An Integrated Gene Regulatory Network Controls Stem Cell Proliferation in Teeth

Deanna Jacobson

BIOL 1615-0111

November 14, 2013

 

 

Introduction

            Epithelial stem cells are involved in the regeneration of tissues.  Stem cells inhabit a specific niche, or natural environment, to control their own differentiation and continuous regeneration. The incisors of a mouse contain stem cells that help stimulate growth throughout the mammal’s life. The stem cells are located at the ends of the roots known as the cervical loop. Continuing from the cervical loop to the enamel of the tooth, small ameloblasts can be found on the surface. Experiments have been conducted to identify molecules that help control the reproduction of stem cells in the cervical loop and the effects of increasing the amount of those molecules in tooth germ embryos.

Methods & Materials

            Mice containing K-14 Follistatin were produced and genotyped in preparation for the experiment.  This was done by injecting pregnant mice with 1.5ml/ 100g body weight BrdU solution. BrdU is used to detect proliferating, or reproducing, stem cells. The mice were killed after two hours. In the last two hours of the in vitro culture experiment, 10μM of BrdU was added. Tooth germs from the incisors of the prepared mice embryos were extracted and cultured for later dissection. Affi-Gel agarose beads incubated in Activin A, were inserted into the incisor embryos. More beads were divided up and soaked in different protein solutions for 45 minutes at 37 degrees Celsius. Using forceps, the prepared beads were implanted into the prepared tooth germ embryos. After the completed of this process, the prepped incisor explants were secured with ice cold 100% Methanol for 2 minutes, and then placed in 4% paraformaldehyde (PFA) overnight. In final preparation of the tooth incisor embryos, the explants were processed through in situ hybridization for RNA. In situ hybridization involves probing to inject specific molecules. In this experiment the following probes were used: Follistatin, rat Fgf10 Bmp2, Bmp4, Bmp7, Fgf3, Activin βA, Alk3, and Alk4. The process results in a hybridization of the prepped incisor sample. 

Results

            It was observed the growth of the incisors in the presence of K14 Follistatin decelerated. Based on the results it appears the molecule Follistatin affects the epithelial stem cells production, which inhibits growth of incisors in mice. This observation was tested using BrdU experiments to help identify the location of reproducing stem cells. In mice, activity of proliferating epithelial stem cells occurred most frequently in the inner labial of the dental cervical loop epithelium. A minimal amount of stem cells was observed at the core of the cervical loop.        

            In mice containing samples of Fgf3 and Fgf10, development of incisors was observed. The Fgf3 hybrid produced a defective enamel structure in the molars of the teeth. The enamel layer was very thin and caused the wearing down of the teeth. In Fgf10 samples, the lower incisors appeared white, were very thin, and continued to break more frequently.

            The BMPs sampled mice were observed with an increase of stem cells reproduction in both the lingual and labial cervical loops. The incisors developed were overgrown and dense.

            Furthermore, Activin sampled mice were examined and appeared to neither inhibit or accelerate stem cell production in the cervical loop of the dental epithelium.

Discussion

            The results of the conducted experiments using Follistatin, FGF, BMP, and Activin indicate a relationship between the proliferation of epithelial stem cells. Cell signaling regulates the production of stem cells, resulting in continuous growth of mice incisors. Potential growth and proliferation activity were recorded to identify the effects of inserted molecules. In the presence of Follistatin, growth of the mice incisors slowed, whereas BMP sampled mice developed overgrown incisors. Using the results from this experiment increases understanding of proliferating stem cells and their potential.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cited Source

 Wang X-P, Suomalainen M, Felszeghy S, Zelarayan LC, Alonso MT, et al. (2007) An Integrated Gene Regulatory Network Controls Stem Cell Proliferation in Teeth. PLoS Biol 5(6): e159. doi:10.1371/journal.pbio.0050159

http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.0050159#s2

 

 

 

 

 

 

 

 

 

 

 

 

 
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