Quantification of Orofacial Phenotypes in Xenopus

Summary

A method to quantify the orofacial size and shape of Xenopus laevis embryos has been developed. In this protocol, traditional size measurements are combined with geometric morphometrics to allow for more sophisticated analyses of orofacial development and defects.

Abstract

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial development will allow for more rigorous analysis of orofacial phenotypes upon abrogation with substances that can genetically or molecularly manipulate gene expression or protein function. Using two dimensional images of the embryonic heads, traditional size dimensions-such as orofacial width, height and area- are measured. In addition, a roundness measure of the embryonic mouth opening is used to describe the shape of the mouth. Geometric morphometrics of these two dimensional images is also performed to provide a more sophisticated view of changes in the shape of the orofacial region. Landmarks are assigned to specific points in the orofacial region and coordinates are created. A principle component analysis is used to reduce landmark coordinates to principle components that then discriminate the treatment groups. These results are displayed as a scatter plot in which individuals with similar orofacial shapes cluster together. It is also useful to perform a discriminant function analysis, which statistically compares the positions of the landmarks between two treatment groups. This analysis is displayed on a transformation grid where changes in landmark position are viewed as vectors. A grid is superimposed on these vectors so that a warping pattern is displayed to show where significant landmark positions have changed. Shape changes in the discriminant function analysis are based on a statistical measure, and therefore can be evaluated by a p-value. This analysis is simple and accessible, requiring only a stereoscope and freeware software, and thus will be a valuable research and teaching resource.

Introduction

Among the most prevalent and devastating types of human birth defects are those affecting the mouth and face, such as orofacial clefts¹. Children with malformed orofacial structures undergo multiple surgeries throughout their lifetime and struggle with facial disfigurements, speech, hearing and eating problems. Therefore, facilitating new research in cranio- and orofacial development is paramount to prevention and treatment of these types of birth defects in humans. Xenopus laevis has emerged as a new tool for dissecting the mechanisms governing craniofacial development (some examples include^2,3,4-11). Therefore, a quantitative method to....

Protocol

All experiments performed using Xenopus laevis have been approved by IACUC (protocol #AD20261) .

1. Preparing Reagents and Required Materials

1. Reagents:
   1. Make 1 L of 10x MBS (Modified Barth’s Saline) solution²². Add NaCl (880 mM), KCl (10 mM), MgSO₄ (10 mM), HEPES (50 mM, pH 7.8), and NaHCO₃ (25 mM) to 1 L of distilled water. Adjust pH to 7.8 with NaOH.
   2. Make 1 L of 1x MBS by diluting 100 ml of 10x MBS solution in 900 ml of distilled water. Add 0.7 ml of 1 M CaCl₂ solution.
   3. Make 1 L of 0.1x MBS by diluting 100 ml of 1x MBS solution in 900 m....

Results

Here, a quantitative analysis of orofacial size and shape was demonstrated to compare embryos treated with a retinoic acid receptor inhibitor (RAR inhibitor) to untreated controls. Embryos were treated with a 1 μM concentration of this chemical inhibitor from stage 24 to 30 (26-35 hpf), washed out, and fixed at stage 42 (82 hpf). They were then processed and analyzed as described in the protocol. Results are original data, but consistent with observations in previous publications^2,3. Control embryos were treat.......

Discussion

Xenopus laevis has become a useful tool for dissecting the developmental mechanisms underlying orofacial development; however, there are currently no protocols describing size and shape changes of this region in frogs. The method described here will contribute significantly to the field of orofacial development by allowing for more rigorous quantification of orofacial phenotypes in Xenopus and other vertebrates.

The first, most critical aspect of properly executing this proto.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dissecting microscope	Zeiss		fitted with AxioCamICC1 camera
Dumont #5 Inox forceps	Fine Science Tools	11251-10
Sterile, disposable scalpel	Sklar	06-2015
24-well plate	Fisher Scientific	087721
Standard Disposable transfer pipettes	Fisher Scientific	13-711-7M
150 mm x 15 mm Petri dishes	Falcon	351058
Incubators	Ectotherm		set to 15 °C or 20 °C
Modeling Clay	Premo, or other non-toxic modeling clay		in black or white
Straight teasing needle	Thermo Scientific	19010
Capillary Tubing (for needles)	FHC	30-30-1	Borosil 1.0 mm OD x 0.5 mm ID/Fiber, 100 mm each
Needle Puller, Model P-97	Sutter Instrument Co.	Needle Puller: P-97 Flaming/ Bown micropipette puller Filament: FB300B	For filaments, use Sutter 3.00 mm square box filaments, 3.0 mm wide.
Pipettemen	Gilson	F144802, F123600, F123602
BMS-453	Tocris	3409
DMSO	American Bioanalytical	AB00435-01000
Cysteine	Sigma-Aldrich	52-90-4
Paraformaldehyde powder	Sigma-Aldrich	158127
Petri dishes	Falcom	353003, 351058	100 mm diameter and 150 mm in diameter
100% Ethanol	VWR	89125-170