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1.
Figure 8.

Figure 8. From: 22q11 Gene dosage establishes an adaptive range for sonic hedgehog and retinoic acid signaling during early development.

Genetic alteration of RA signaling selectively modifies cranial and cardiac phenotypes in LgDel embryos. (A) RA regulated RA signaling genes are diminished in LgDel and LgDel:Raldh2+/−embryos. Asterisks indicate RA-regulated or 22q11 genes whose expression is significantly altered from WT levels in Raldh2+/−, LgDel and Raldh2+/−:LgDel compound embryos. There is no apparent interaction between the two genotypes for further change in RA-regulated genes. (B) Morphogenetic phenotypes, including smaller limb buds (lb) and branchial arches (ba), diminished frontonasal processes (fnp), and exencephaly (arrowheads) are seen in compound LgDel:Raldh2+/− embryos. (C) Confocal image and accompanying cartoon of PAAs in LgDel:Raldh2+/− embryos show PAA4 absence. (D) Tbx1+/−:Raldh2+/− compound mutant embryos have no observable gross phenotypes. (E) PAA morphology in Tbx1+/−:Raldh2+/− compound mutants.

Thomas M. Maynard, et al. Hum Mol Genet. 2013 January 15;22(2):300-312.
2.
Figure 9.

Figure 9. From: 22q11 Gene dosage establishes an adaptive range for sonic hedgehog and retinoic acid signaling during early development.

Diminished dosage of 22q11 genes constricts an adaptive range for optimal morphogenetic signaling. First row: Optimal adaptive range for morphogenetic signaling. In this model, signaling via Shh, RA (or, potentially, other morphogenetic signals that act at M/E sites) might fall a considerable amount below or above an optimal level (100%) before phenotypic consequences are seen. Second row: Adaptive range for Shh signaling buffers against phenotypic changes when Shh signaling is reduced by as much as 86% in WT embryos. Third row: Diminished 22q11 gene expression constricts the adaptive range of Shh signaling and modifies levels of aberrant signaling that can be tolerated without phenotypic change. Fourth row: The adaptive range for RA signaling in WT extends from ∼80 to 173% WT signaling levels, based upon our data. Fifth row: The adaptive range for RA signaling is constricted when 22q11 gene expression is diminished, and novel or more severe phenotypes are seen.

Thomas M. Maynard, et al. Hum Mol Genet. 2013 January 15;22(2):300-312.
3.
Figure 7.

Figure 7. From: 22q11 Gene dosage establishes an adaptive range for sonic hedgehog and retinoic acid signaling during early development.

Diminished dosage of 22q11 genes sensitizes embryos to sub-teratogenic RA exposure. (A) RA-exposed LgDel (top half) are substantially more dysmorphic and smaller in size than WT littermates (bottom half; 15 LgDel and 19 WT littermate embryos from four litters). (B) Confocal images, and accompanying tracings, of PECAM-labeled PAAs in untreated WT, LgDel and Tbx1 embryos show the normal pattern (WT and WT + RA) and varying degrees of dysmorphology in LgDel and Tbx1+/− as well as LgDel and Tbx1+/− treated with RA. (C) Distinct expression changes in RA signaling-related genes in LgDel embryos. Brackets and asterisks indicate significant differences in expression levels between the four groups (WT – and + RA; LgDel – and + RA). (D) Increased RA between E8 and E10 does not significantly alter 22q11 gene expression beyond 50% in E10.5 LgDel embryos.

Thomas M. Maynard, et al. Hum Mol Genet. 2013 January 15;22(2):300-312.
4.
Figure 5.

Figure 5. From: 22q11 Gene dosage establishes an adaptive range for sonic hedgehog and retinoic acid signaling during early development.

Response of WT, LgDel and Tbx1+/− embryos to diminished Shh signaling due to 48 h cyclopamine exposure. (A) Consequences of cyclopamine-mediated decline of Shh signaling in LgDel versus WT littermates. LgDel embryos have far more severe and frequent morphogenetic anomalies at sites of M/E interaction (fb, ba, aah, lb). Numbers of embryos with similar severity of disrupted development are shown in brackets next to each example. (B) Transcriptional responses of four Shh-related signaling genes whose expression is modulated by altered Shh signaling (significant differences indicated with brackets and asterisks). (C) In LgDel embryos, cyclopamine does not significantly diminish expression of most 22q11 genes, with the exception of Zdhhc8, beyond the 50% decrement seen in LgDel alone. (D) Consequences of cyclopamine-mediated decline in Shh signaling for Tbx1+/− versus WT littermate embryos. Although there is some variation in size, there are no noticeable changes in differentiation of non-axial structures.

Thomas M. Maynard, et al. Hum Mol Genet. 2013 January 15;22(2):300-312.
5.
Figure 1.

Figure 1. From: 22q11 Gene dosage establishes an adaptive range for sonic hedgehog and retinoic acid signaling during early development.

Divergent expression levels of Shh, RA, Fgf and Bmp-related signaling genes assessed by qPCR in whole E10.5 LgDel and Tbx1+/− and WT littermate control embryos. (A) Among 40 candidates, there is significantly diminished expression (relative to WT littermates) of seven Shh-related genes (solid yellow bars; see text for ‘n’ and P-values), with two additional genes showing a trend toward significant decline (hatched yellow bars). There are RA signaling genes that decline significantly (solid purple bars), plus one additional significant trend (hatched purple bar). One Fgf-related declines significantly (solid red bar), and another shows a significant trend (hatched red bar). Six Bmp-related genes decline significantly (solid blue bars). (B) Signaling gene expression in E10.5 Tbx1+/− embryos does not mirror LgDel changes. There is modest but statistically significant diminished expression of two RA-related genes (solid purple bars), only one of which (Rarβ) also declines significantly in the LgDel.

Thomas M. Maynard, et al. Hum Mol Genet. 2013 January 15;22(2):300-312.
6.
Figure 3.

Figure 3. From: 22q11 Gene dosage establishes an adaptive range for sonic hedgehog and retinoic acid signaling during early development.

Diminished RA signaling in the head/forebrain and heart/aortic arches in LgDel, but not Tbx1+/− E10.5 embryos. Embryos in (A) and (D) carry DR5-RARE:βgal RA reporter transgene. (A) βgal labeling in the forebrain (fb) and eye (e; top panels) and the heart/aortic arches (H/AA; dotted lines) in E10.5 WT and LgDel embryos. βgal labeling is diminished in the forebrain/head (compare arrowheads, top right) and aortic arches (compare arrowheads, bottom right) of LgDel versus WT (9 WT and 6 LgDel embryos analyzed). (B) Diminished βgal activity in dissected LgDel head and heart based upon soluble βgal assays (ONPG). (C) Rarβ expression declines significantly in the forebrain/head in LgDel embryos, while both Raldh2 and Rarβ expression decline in the heart (solid purple bars). (D) No visible differences in cranial or cardiac βgal labeling in Tbx1+/− versus WT embryos. (E) βgal activity (ONPG assay) is statistically equivalent in Tbx1+/− and WT littermates (F) Expression levels of Raldh2 and Rarβ in the head and heart are statistically indistinguishable in Tbx1+/− and WT littermates.

Thomas M. Maynard, et al. Hum Mol Genet. 2013 January 15;22(2):300-312.
7.
Figure 4.

Figure 4. From: 22q11 Gene dosage establishes an adaptive range for sonic hedgehog and retinoic acid signaling during early development.

Morphogenetic signals regulate levels of 22q11 gene expression. Representative E10.5 whole embryo in situ hybridization (ISH) shows expression patterns and intensity for four 22q11 genes in WT embryos (left) and WT embryos treated pharmacologically to diminish Shh (cyclopamine), RA (DEAB), Fgf (FgfRi) or Bmp (dorsomorphin) signaling. For each gene WT control and WT treated embryos were hybridized and reacted for labeling in the same vials to insure absolutely identical conditions. Sites of mesenchymal/epithelial (M/E) interaction and 22q11DS phenotypes are indicated in the WT control embryo, top left: fb, forebrain; ba, branchial arches; h, heart; lb, limb bud. Black arrowheads indicate instances when expression levels change at some M/E 22q11DS phenotypic sites but not others. At right, mRNA levels in whole WT embryos treated pharmacologically with cyclopamine, DEAB, FgfRi or dorsomorphin have been measured using qPCR for each gene whose expression is localized using ISH. Asterisks indicate statistically significant differences.

Thomas M. Maynard, et al. Hum Mol Genet. 2013 January 15;22(2):300-312.
8.
Figure 6.

Figure 6. From: 22q11 Gene dosage establishes an adaptive range for sonic hedgehog and retinoic acid signaling during early development.

Genetic alteration of Shh signaling selectively modifies gene expression and cardiac phenotypes in LgDel embryos. (A) Lower magnification Z-stack confocal images showing the heart and pharyngeal arch arteries (PAA) in E10.5 WT and LgDel embryos stained for the cell-adhesion molecule PECAM/CD31, and imaged whole. The primary LgDel phenotype is a hypoplastic PAA4 (compare at double arrowheads). (B) Changes in expression levels of Shh-regulated genes in E10.5 Shh+/− embryos. Solid yellow bars indicate significant changes. (C) Changes in a broader range of Shh-regulated genes in E10.5 Gli3+/Xtj (left hand bar in each pair) and Gli3Xtj/Xtj (right hand bar in each pair) embryos. Hatched (Gli3+/Xtj) and solid (Gli3Xtj/Xtj) yellow bars indicate significant changes. (D) Higher magnification confocal images of PECAM labeled PAAs in Gli3Xtj/Xtj, LgDel:Gli3+/Xtj (middle) and LgDel:Gli3Xtj/Xtj (right). The PAAs in the Gli3Xtj/Xtj are comparable to WT. Compound mutants have increasingly severe and more frequent PAA4 and PAA6 hypoplasia not seen in LgDel or Gli3+/Xtj or Gli3Xtj/Xtj mutant embryos (Table 1). The tracings in the lower panels highlight primary phenotypic changes.

Thomas M. Maynard, et al. Hum Mol Genet. 2013 January 15;22(2):300-312.
9.
Figure 2.

Figure 2. From: 22q11 Gene dosage establishes an adaptive range for sonic hedgehog and retinoic acid signaling during early development.

Shh signaling is increased in the developing heart in LgDel, but not Tbx1+/− E10.5 embryos. Embryos in (A) and (D) carry a β-galactosidase (βgal) reporter under the control of the endogenous Ptch2 promoter. (A) Comparison of Ptch2: βgal labeling in the head/forebrain (fb, including the eye, e; top panels; dotted line indicates site of dissection) and heart/aortic arches (H/AA; dotted lines indicated site of dissection) of E10.5 WT (left) and LgDel embryos (right). βgal labeling in the nascent heart and aortic arches (arrowheads) is less extensive in WT than LgDel embryos (8 LgDel, 10 WT embryos analyzed). There is no apparent change in the forebrain/head. Dotted lines in left hand panels indicate regions dissected for quantitative measurements. (B) Increased Ptch2 promoter activation in the heart and aortic arches of the LgDel embryos, without change in the head/forebrain, assessed by soluble βgal levels (ONPG assay, see Materials and Methods). (C) Increased Ptch2 and Shh mRNA levels in the LgDel heart and aortic arches (H/AA; solid yellow bars; LgDel values plotted as fold change from WT values = 1), but not in the head/forebrain. (D) No visible difference in cranial or cardiac βgal labeling in WT and Tbx1+/− E10.5 embryos. (E) Equivalent levels of βgal activity in the head/forebrain and heart/aortic arches of WT and Tbx1+/− embryos. (F) No significant change in Shh or Ptch2 expression in either head or heart of Tbx1+/− embryos.

Thomas M. Maynard, et al. Hum Mol Genet. 2013 January 15;22(2):300-312.

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