XB-ART-51800Semin Cell Dev Biol March 1, 2016; 51 54-63.
Using frogs faces to dissect the mechanisms underlying human orofacial defects.
In this review I discuss how Xenopus laevis is an effective model to dissect the mechanisms underlying orofacial defects. This species has been particularly useful in studying the understudied structures of the developing face including the embryonic mouth and primary palate. The embryonic mouth is the first opening between the foregut and the environment and is critical for adult mouth development. The final step in embryonic mouth formation is the perforation of a thin layer of tissue covering the digestive tube called the buccopharyngeal membrane. When this tissue does not perforate in humans it can pose serious health risks for the fetus and child. The primary palate forms just dorsal to the embryonic mouth and in non-amniotes it functions as the roof of the adult mouth. Defects in the primary palate result in a median oral cleft that appears similar across the vertebrates. In humans, these median clefts are often severe and surgically difficult to repair. Xenopus has several qualities that make it advantageous for craniofacial research. The free living embryo has an easily accessible face and we have also developed several new tools to analyze the development of the region. Further, Xenopus is readily amenable to chemical screens allowing us to uncover novel gene-environment interactions during orofacial development, as well as to define underlying mechanisms governing such interactions. In conclusion, we are utilizing Xenopus in new and innovative ways to contribute to craniofacial research.
PubMed ID: 26778163
PMC ID: PMC4798872
Article link: Semin Cell Dev Biol
Genes referenced: actb cdh1 fn1 grap2
GO keywords: retinoic acid receptor activity
Disease Ontology terms: orofacial cleft
OMIMs: SMITH-MAGENIS SYNDROME; SMS
Article Images: [+] show captions
|Fig. 1. Xenopus techniques for orofacial research. (A) Frontal and lateral views of Xenopus at stage 40. (B) Schematic showing the steps in performing a face transplant.Orofacial tissue is removed from a donor injected embryo and transplanted to the same region of sibling un-injected embryo. (C) Schematic showing the steps in performinga face explant. Tissue is excised from the head and plated on a fibronectin coated glass bottom dish. (D) Schematic showing a representative example of morphometricanalysis of the orofacial region in Xenopus. Landmarks are assigned coordinates which are then used to perform a canonical variate analysis. This analysis can statisticallyseparate landmark locations from a normal and morphant (or chemically treated) embryo which can then be presented graphically or on a transformation grid.|
|Fig. 2. Buccopharyngeal membrane rupture. (A) Sagital section through the middle of the head showing the buccopharyngeal membrane. Cells are labeled with phalloidin(green) and the red is autofluorescence. Phalloin labels F-actin which is located along membranes of cells. scale bar = 33 m. (B) Frontal view of a face as the buccopharyngealmembrane perforates or ruptures. scale bar = 130 m. (C–E) Frontal views of phalloidin labeled buccopharyngeal membranes before (C) and during (D,E) perforation. scalebar = 80 m. F Schematic of embryonic faces at stage 40, showing the stages of buccopharyngeal membrane rupture and presenting the hypothesis that biomechanical forcesare important for this process. Abbreviations: BM; buccopharyngeal membrane.|
|Fig. 3. Overview of orofacial development and primary palate anatomy. (A) Schematic of the human fetal face at 10 weeks of development showing major anatomicalfeatures. The colored domains correspond to the facial prominences in (C) during embryonic development. (B) Schematic of the adult palate showing the location of theprimary verses secondary palate. (C) Schematic of the face of a 4–5 week human fetus showing the location of the facial prominences.|
|Fig. 4. Retinoic acid and median clefts. (A,B) Frontal views of the stage 43–44 face of (A) control embryo and (B) an embryo treated with a retinoic acid receptor inhibitor(from stage 24–32). The mouth is outlined with red dots. scale bars = 225 m. (C, D) Transverse sections through the face at stage 43–44 where E-cadherin is labeled (red)and F-actin is labeled using phalloidin (green). E-cadherin marks epithelium of the oral cavity and phalloidin shows outlines of cells and muscle for context (see Ref.  fordetails on this labeling). scale bars = 120 m. (E) Schematic showing our hypothesis of the role of RA signaling in regulating primary palate and midface development. RAR is expressed in the early face and regulates homeobox genes, cell cycle regulators and transcriptional regulators to modulate both growth and differentiation.|
|Fig. 5. Retinoic acid-folate interaction. (A) Schematic showing the experimental design to test for an interaction between folate and retinoic acid using inhibitors. (B)Schematics of embryonic faces summarizing the results of the experiment outlined in A. Low concentration of inhibitors on their own had no effect on facial morphology.However, when these inhibitors were combined, a median cleft could be observed.|