Petrelli B , Oztürk A , Pind M , Ayele H , Fainsod A , Hicks GG .

             retinoic acid metabolic process
             retinoic acid receptor signaling pathway involved in neural plate anterior/posterior pattern formation

                fetal alcohol spectrum disorder

	FIGURE 1. Gsc:Cyp26A1-eGFP gene targeting design and mouse derivation. (A) The Gsc:Cyp26A1-eGFP cassette was targeted to exon 2 of the endogenous Gsc gene by homologous recombination. (B) Cyp26A1 was used to catabolize RA and all RA isoforms in cells expressing Gsc. eGFP is a co-expressed fluorescent marker used to identify these cells in in vitro and in vivo studies. The cassette was constructed with two cyclin T2A peptide-bond-skipping translation elements to translate both the Cyp26A1 and eGFP gene products as individual proteins when the Gsc promoter was activated. Neomycin is a mammalian selection marker for gene targeting, which was later removed in vivo by crossing with a Cre mouse. The G12 strain uses a T2A translational element for Cyp26A1. The H4 strain uses an IRES translational element for Cyp26A1. (C) All targeting steps in ES cells and C57BL/6N mice were sequence validated to ensure correct recombination events and intact functional elements. Gateway recombineering sites (green box) and primers (dark blue arrows), and loxP sites (red triangle) are indicated.
	FIGURE 2. Gsc:Cyp26A1-eGFP is expressed in embryoid bodies induced with activin A and fibroblast growth factor (FGF). (A) Embryoid body (EB) formation was used to generate definitive endoderm germ cells in vitro. Activin A and FGF-2 were used to induce Gsc gene expression, including Cyp26A1-eGFP expression from the targeted Gsc allele. Results clearly demonstrate Gsc:Cyp26A1-eGFP is inducible under these conditions by eGFP expression in Activin A treated cells (middle panels), but not in untreated or EBs generated from wild-type ES cells (left and right panels, respectively). ES cell clones used to generate EBs are identified (C2, H4, G12). (B) Sox17 immunocytochemistry was performed on embryoid bodies to confirm definitive endoderm cell induction (Sox 17). Nuclear staining (DAPI) and overlay (MERGE) are shown. Scale bars: 200 um (A); 40 um (B).
	FIGURE 3. Gsc+/Cyp26A1 E8.5 embryos have a reduction in RA activity/RARE-LacZ expression. (A–I) Gsc+/Cyp26A1 mice were crossed with RARE-LacZ+/+mice containing a transgene reporter for intracellular RA levels. Each row shows representative embryos from the same litter with WT, and Gsc+/Cyp26A1 embryos with mild expression change or severe pattern and expression changes. Gsc+/Cyp26A1/RARE-Lac-Z+/− embryos demonstrate a reduction in retinoic acid (RA) activity/RARE-LacZ expression (B, C, E, F, H, I lighter blue X-gal staining) in the frontonasal prominence region. WT embryos (Gsc+/+/RARE-Lac-Z+/−) develop proper frontonasal prominence formation and show normal RA levels (A, D, G). These data show Gsc+/Cyp26A1 embryos have reduced retinoic acid activity, specifically a change in RARE-LacZ expression in the frontonasal prominence. Gsc+/Cyp26A1/RARE-Lac-Z+/− embryos also show aberrations in RA dependent embryonic patterning, including changes in morphology of the frontonasal prominence region (H′, I′), compared to WT siblings (G′, neural crest cell derived lineage; black arrowhead). Severe malformations and changes in RARE-LacZ expression in the frontonasal prominence region occur in approximately 16% of Gsc+/Cyp26A1/RARE-Lac-Z+/− (C, F, I; Table 2). n = 10 litters, n = 30 Gsc+/Cyp26A1/RARE-Lac-Z+/− embryos and n = 48 WT/RARE-Lac-Z+/− embryos. Scale bars: 200 um (A–I).
	FIGURE 4. RARE-LacZ expression returns to normal in E9.5 Gsc+/Cyp26A1 embryos. (A, B) E9.5 Gsc+/Cyp26A1 embryos have a smaller head morphology compared to WT embryos, but have no distinct changes in RARE-LacZ patterning compared to WT. E10.5 and E11.5 (E) Gsc+/Cyp26A1 embryos do not show distinct changes in embryonic patterning, gross morphology or intensity of RARE-LacZ expression in the frontonasal prominence, anterior somites, or developing trunk, as WT embryos (D, F, respectively). E9.5 n = 16 embryos, E10.5 n = 14 embryos, E11.5 n = 15 embryos, n = 2 (litters) per timepoint. Scale Bars: 500um (A–D), 1 mm (E, F).
	FIGURE 6. Gsc+/Cyp26A1 E10.5 embryos have aberrant neural crest cell migration in the developing cranial nerves. Gsc+/Cyp26A1 E10.5 embryos show a dysregulated cranial nerve patterning in the developing face and branchial arches derived from the neural crest cell lineage (B, D). Gsc+/Cyp26A1 E10.5 embryos have decreased neural crest cell migration in cranial nerves V (black arrows), VII, VIII, X, XI, and specifically IX (red arrows). WT Littermates demonstrate proper cranial nerve patterning in the developing face and branchial arches; and the cranial nerves are migrating as expected (A, C); cranial nerves are identified by red Roman numerals). Notice that cranial nerve V does not innervate the optic vesicle in either of the Gsc+/Cyp26A1 embryos, but correctly innervates the optic vesicle (black asterisk) in WT littermate embryos. These results demonstrate that the Gsc+/Cyp26A1 model results in aberrant neural crest cell proliferation and migration. * = optic vesicle. Immunohistochemistry marker: Neurofilament-200 (NF-200) protein. Scale Bars: 500 um (A–D).
	FIGURE 7. Gsc+/Cyp26A1 E18.5 embryos have Fetal Alcohol Syndrome (FAS)-like craniofacial malformations. (A) Landmark craniofacial measurements used for E18.5 embryo facial analysis. (Adapted from Anthony et al., 2010; Lipinski et al., 2012). E18.5 embryo SEM frontal pictures were used for Philtrum-Lip Ratio (1), Bigonial Line (2), Whisker Pad (3), and Snout Area (4) quantitative measurements. (B) E18.5 embryo SEM side-view pictures were used for Midfacial (5), Lower facial (6), Neck to Edge of Mandible (7), and Side Snout Area (8) quantitative measurements. Inset indicates the snout area (orange highlight). (C, E) Gsc+/Cyp26A1 E18.5 embryos have a less defined maxillary process resulting in a larger philtrum/philtrum-lip ratio compared to WT littermates (red arrow length/yellow arrow length). For comparison, WT littermates E18.5 embryos have a more defined, normal protruding maxillary formation resulting in a smaller philtrum/philtrum lip ratio. (D, F) Gsc+/Cyp26A1 E18.5 embryos have a narrower bigonial line width (red dashed line, asterisk marks the comparative length of Gsc+/Cyp26A1 bigonial line width on a WT sibling). (D) Whisker pad length measurement can be seen by the dark blue line, asterisk marks the comparative length of Gsc+/Cyp26A1 whisker pad length on a WT sibling (Table 4). (B, F) The orange overlay defines a smaller snout area in Gsc+/Cyp26A1 E18.5 embryos compared to WT sibling that has a larger snout area (Panel D, Table 4). Representative analysis of the most significant measurements are shown and box plots show data from litter 38 and 41, see Table 4 for litter measurement details. Scale bar = 4 mm. A t-test was used to determine statistical significance. * = p < 0.05.
	FIGURE 8. Gsc+/Cyp26A1 mice have severe craniofacial malocclusions. Gsc+/Cyp26A1 mice (B) demonstrate a curvature in the pre-maxillary process when compared to WT littermates (A). Gsc+/Cyp26A1 mice (E), red arrow; (F), white arrow do not have a curvature of the mandibular component, the mandible is straight when compared to the skull as seen in WT littermates (C), red arrow; (D), white arrow. The curvature to the maxillary component can be seen to impact the natural grinding of the incisors, ultimately causing a severe malocclusion in Gsc+/Cyp26A1 mice (B, I = maxillary incisors); the incisors grind normally in WT littermate mice (A, I = maxillary incisors).
	FIGURE 9. Gsc+/Cyp26A1 mice have a wider cranium compared to WT littermates. P60 Gsc+/Cyp26A1 mice show a statistically significant wider cranium in all 4 major skull region width measurements: Nasal Bone (A), L-R Anterolateral Frontal Bone (B), Frontal Bone (C), and Intraparietal Bone Widths (D). Gsc+/Cyp26A1 mice do not demonstrate a statistically significant difference in the 4 major skull region length measurements: Nasal Bone (E), Frontal Bone (F), Parietal Bone (G) and Intraparietal Bone lengths (H). Gsc+/Cyp26A1 mice, n = 10; C57BL/6N mice, n = 5; WT with spontaneous malocclusions, n = 3. A t-test was used to determine statistical significance. *p < 0.05, **p < 0.01.
	Supplemental Figure S1 | Gsc+/Cyp26A1 P60 mice have large upper incisors, but do not have any length or height variations in the cranium or mandible regions. Box plots for all height and length craniofacial landmark features are shown, as indicated. Gsc+/Cyp26A1 P60 mice were statistically different for only one measurement: the upper incisor height (upper right panel, P < 0.001). Trending results in the inferior incisor axis, posterior cranial height and mandible axis were observed in Gsc+/Cyp26A1 mice. Gsc+/Cyp26A1 mice, n = 10; C57BL/6N mice, n = 4; WT with spontaneous malocclusions, n = 3. ***P < 0.001.

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