Morphometric Analyses of Shape: The Analysis Software Toolbox for Craniofacial Shape Quantification in Zebrafish

Summary

This 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.

Abstract

Fetal alcohol spectrum disorders (FASD) are characterized by a varying set of physical, cognitive, and behavioral disabilities caused by prenatal ethanol exposure, including those affecting 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; (i) 70% of genes are orthologs between humans and zebrafish; (ii) external fertilization allows researchers to control timing and dosage of ethanol treatments; (iii) the structure of their facial skeletal is conserved with vertebrates; (iv) 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.

Introduction

Fetal Alcohol Spectrum Disorders (FASD) are characterized by a broad range of developmental defects, including behavioral, neurological, and physical^1,2. Included in these physical defects are craniofacial defects, such as jaw hypoplasia^3,4,5,6. While timing and dosage of ethanol exposure contribute to the complex etiology of FASD, genetic contribution plays a significant role in FASD etiology^6,7,....

Protocol

All zebrafish larvae used in this procedure were raised and bred following established IACUC protocols approved by the University of Louisville²³.

NOTE: The zebrafish strain bmp7a^ty68a24 and wild-type siblings (all in the AB background) were used in this study, with adult fish maintained at 28.5 °C with a 14/10-h light / dark cycle. Use the appropriate lines per the desired experimental outcomes. Use water that was sterilized by reverse osmosis. Sex as a biological variable does not apply to the studied development stages, as sex is first detectable in zebrafish around....

Results

To identify ethanol-induced facial shape changes in zebrafish, we combined freely available software applications to generate and quantify morphological data of the facial skeleton. Embryos from bmp7a heterozygous adult carrier fish were generated and treated with ethanol from 6-24 hpf. Larvae from these crosses were fixed at 5 dpf, and facial cartilages were stained with Alcian Blue. Images of the viscerocranium were taken for each larva in each genotype and treatment group (Figure 1A

Discussion

FASD is characterized by a wide range of developmental defects, including craniofacial defects such as jaw hypoplasia. Zebrafish are a strong model for FASD due to their genetic conservation with humans, high fecundity, translucent larvae, and external fertilization. Zebrafish have been used for decades to study both the formation of the craniofacial skeleton and the impact of ethanol on development ^6,12,13,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
tpsUtil, tpsDig2, MorphoJ	https://sbmorphometrics.org		Freeware Software - may require administrative permissions to download
PAST	https://www.nhm.uio.no/english/research/resources/past/		Freeware Software - may require administrative permissions to download
Compound Brightfield Microscope	Olympus	BX53