Morphometric analyses of shape: The analysis software toolbox for quantification of craniofacial shape

Fetal alcohol spectrum disorders (FASD) are characterized by a varying set of physical, cognitive, and behavioral disabilities caused by prenatal ethanol exposure, including those to the facial skeleton. Ethanol-sensitive genetic loci contribute to this high degree of variation in FASD, which complicates analyses of facial shape. We have previously shown that we can analyze these changes in facial shape from gene-ethanol interactions in zebrafish. Zebrafish are an ideal model to analyze this variation for several reasons; 1. 70% of genes are orthologs between humans and zebrafish; 2. external fertilization allows researchers to control timing and dosage of ethanol treatments; 3. the structure of their facial skeletal is conserved with vertebrates; 4. translucent larvae enable direct viewing of changes to the craniofacial skeletal structure during development. However, analyzing the shape of the craniofacial skeleton can be difficult to fully assess through simple linear measures, as these do not capture overall changes in shape. In addition, changes in head size can complicate data interpretation. To address this, we undertook a morphometric approach, analyzing overall facial shape through principal component analyses via freeware software, TPSDigs2, MorphoJ, and PAST. The combination of this software allows for pairwise comparison of overall facial morphology. Here, we outline our approach and analysis of facial shape in ethanol-treated zebrafish mutants using these programs to conduct a series of complementary multivariate statistical analyses. SUMMARYThis work describes a protocol for quantifying craniofacial cartilage shape using free software (TPSDigs2, MorphoJ, and PAST) to measure changes in facial structure in zebrafish larvae.