Quantitative Phenotyping as an Efficient Means to Estimate C-Cell Number in a Knock-in Mouse Model of MEN2B

Abstract

Over the last two decades we have witnessed the generation of hundreds, if not thousands, of lines of genetically altered mice, large numbers of which are being produced in order to model human disease. Given that their creation is still rather technically demanding and labour intensive, the time taken analysing the resultant phenotypes should be such that the maximal amount of information can be gleaned efficiently in an unbiased manner so as to be as close to the 'true' value as possible. In an attempt to characterise a cell-specific phenotype in a genetically defined knock-in mouse model of multiple endocrine neoplasia type 2B (MEN2B) we used a modern, unbiased, stereological approach called the optical fractionator to estimate total cell number in 3-D space. By applying a sampling technique to tissue blocks in a systematic random uniform manner, we demonstrate that the total number of calcitonin-immunoreactive C-cells in the thyroid glands of littermate mice harbouring activating mutations in one or both alleles of ret does not vary significantly (p = 0.46) from an unbiased estimate of 23,000 in wild-type controls; likewise, neither does mean thyroid volume (p = 0.78) when estimated using Cavalieri's principle. We demonstrate that the variation associated with the quantitative phenotyping method is negligible. Using this efficient, unbiased stereological method our results provide new insights into cell number and positioning with consequences for both normal and disease states. In summary, this unbiased stereological technique is conceptually simple, can be applied efficiently, and is pertinent to quantitating a wide variety of cell phenotypes thereby bridging specialisation boundaries. We propose the adoption of this technique to mouse experimental geneticists and recommend its horizontal transmission across all fields within experimental biology.