Karpinski BA , Maynard TM , Fralish MS , Nuwayhid S , Zohn IE , Moody SA , LaMantia AS .

	Fig. 1. Growth curves for LgDel and WT littermate males and females from P1 through P30. (A) Growth curves for male LgDel and WT littermates, weighed daily from P1 through P7, then weekly from P7 through P28 (x-axis). (B) Growth curve for female LgDel and WT littermates. (C) Normalized mean weights for male and female LgDel and WT littermates. The boxes represent standard errors of each mean (s.e.m.), and bars reflect standard deviations for each data point. Growth curves were compared by ANOVA (A,B), and mean normalized values compared using Mann-Whitney analysis (C).
	Fig. 2. P7 LgDel mice show signs of dysphagia, including nasal, ear and lung milk-aspiration inflammation or infection. Top row: aspiration-related protein aggregates adjacent to the turbinates of the olfactory epithelium (oe) in P7 WT and LgDel pups. The protein aggregates (asterisks), seen only in LgDel pups, contain murine milk protein (far right; fluorescent Nissl stain, green; immunolabel for milk protein, red) as well as neutrophils (anti-CD64, blue). Middle row: mucus aggregates are seen in the Eustachian tubes of LgDel P7 pups, accompanied by an apparent increase in the frequency of mucus-producing goblet cells (bracket/arrows, far right panel; PAS stain) at the boundary of the pharynx (Resp.; arrows) and Eustachian (Eust.; bracket) tube epithelium. Bottom row: LgDel lungs have more frequent evidence of inflammation and/or infection, including red blood cell aggregates (arrows, middle insert), macrophages (arrowheads, and inset, middle) and infiltration of murine milk protein (far right panel; green, fluorescent Nissl stain; red, milk protein immunolabel).
	Fig. 3. Altered palate and jaw morphology in LgDel mice. Top: quantitative real time PCR (qPCR) analysis of a subset of 22q11 deleted genes in microdissected rudiments of key orofacial structures for feeding and swallowing: BA1A, which includes the maxilla, which gives rise to the upper jaw, mouth structures and some muscles of mastication, and BA1B/BA2, which includes the mandible and hyoid, which gives rise to the lower jaw and some pharyngeal structures. These 22q11 genes were chosen based upon expression above a threshold level – 0.01% of Gapdh expression measured in the same sample – in a previous analysis of developing E10.5 embryos or the brain from E12.5 onward (Maynard et al., 2003). Expression was quantified. Middle: sections through the anterior and posterior palatal region in E13.5 WT and LgDel embryos demonstrating failure of palatal elevation in the LgDel (compare arrows). The palate has not fused in either the WT or LgDel at this age. WT palatal shelves are more frequently elevated (left arrows) than those in the LgDel (right arrows). Bottom left: lateral view of the mandibular process from a WT mouse, with reference points for measurements indicated as numbers 1–6. Bottom right: morphometry of point-to-point distances between cardinal locations shown at left indicates that mandibular growth is diminished in LgDel versus WT littermate mice (*P≤0.5; **P≤0.01, t-test; n=48 LgDel, 46 WT).
	Fig. 4. Altered gene expression, morphogenesis and patterning in the LgDel E9.5 anterior hindbrain. Top: qPCR analysis of 22q11 gene expression above threshold levels in the microdissected E9.5 hindbrain (r1 to r8). 16 out of 21 candidates are expressed above threshold. Note that Tbx1, a key 22q11 candidate gene for cardiovascular phenotypes, is not detected in the E9.5 hindbrain. Middle top: apparent pattern changes and dysmorphogenesis in the E9.5 anterior hindbrain. The distance between r4 and r1/2 in the WT, defined by Hoxb1 and En1 immunolabeling (left) is greater than that in the LgDel (right; compare white bars and arrow), and the dorsal margins of the neuroepithelium appear deformed (compare arrowheads). Middle bottom left: summary of qPCR analysis of expression of several genes that distinguish posterior (top) from anterior (bottom) rhombomeres in the E9.5 microdissected hindbrain (r1 to r8). Middle bottom right: scatterplots of expression levels in individual hindbrain samples of four RA-regulated genes – two found in posterior rhombomeres (Rarα, Rarβ), two with distinctly patterned expression in both anterior and posterior rhombomeres (Cyp26b1, Hoxa2). Bottom: (left) in the WT E9.5 hindbrain, Cyp26b1 is expressed at high levels in r5/6, lower levels in r3/4 and is barely detectable in r2. (Right) In the LgDel E9.5 hindbrain from an embryo hybridized concomitantly with the WT at left, Cyp26b1 is increased in r5/6, significantly stronger in r3/4 and is now clearly detectable in r2 (compare arrows, left and right). In addition, r5/6 boundaries have expanded in the LgDel (compare left brackets), and r2 to r4 have contracted (compare right brackets).
	Fig. 5. CN development is altered in E10.5 LgDel embryos. Top: 16 out of 21 22q11 deleted genes are expressed above threshold in CNg V, based upon qPCR analysis of microdissected WT E10.5 CNg V samples. Middle left: a representative E10.5 WT embryo labeled immunocytochemically for neurofilament protein shows the E10.5 WT hindbrain and associated cranial nerves (CN) and ganglia (CNg). In the LgDel (right), CNg V and VII appear more closely spaced (arrow), their motor roots less differentiated (asterisks), and, parallel to E9.5, the anterior hindbrain appears compressed. In addition, CNg IX and X appear fused in the LgDel (arrows). Bottom left panel: expression changes in LgDel CNg V determined by qPCR of microdissected ganglia from LgDel versus WT E10.5 embryos. Robo2 (black bar) is significantly increased (P≤0.03) and L1cam (gray bar) shows a significant trend toward increased expression (P≤0.06). Middle right: (left) a lateral view of typical WT axon trajectories and fasciculation as well as the position of cranial sensory ganglia for a subset of CNs. (Right) CN development is altered in an E10.5 LgDel embryo (asterisks). CN V appears sparse and de-fasciculated, the normal bifurcation of CN VII is not evident, and the ganglia of CN IX and X are fused. Middle bottom right: specific CN phenotypes in LgDel embryos. These examples, from additional WT and LgDel embryos, are representative of the features scored for quantitative phenotypic analysis. First row: the ophthalmic (op, arrow), maxillary (mx, bracket) and mandibular (md, arrows) branches of CN V appear dysmorphic in LgDel E10.5 embryos. Second row: the bifurcation (arrows) of CN VII that prefigures its division into multiple dorsal and ventral branches, including the chorda tympani, is frequently not evident in LgDel embryos. Third row: the sensory ganglia of CN IX/X and their immediate distal branches are frequently fused in LgDel embryos (compare arrows in left and right panels).
	Fig. 6. Statistically significant changes in phenotypic frequency in developing CN V, IX and X in LgDel versus WT embryos. Top panels: histograms showing the proportion of WT (green) and LgDel (red) embryos with each of the three phenotypes scored (CN V, VII and IX/X). Statistical significance determined using Fisher’s exact analysis. The CN V and IX/X phenotypes occur at significantly greater frequency than in the WT; the CN VII phenotype does not. Lower panels: frequency of single or multiple CN phenotypes in WT and LgDel embryos. Overall phenotypic frequency is substantially increased in LgDel mice, as is the number of individual embryos showing multiple (two or three) CN phenotypes.
	Fig. 7. Heterozygous Tbx1 mutation yields a CN IX/X phenotype parallel to that in E10.5 LgDel embryos. Left: the ganglia of CN IX and CN X are more frequently connected by axon fascicles or fused (arrows) in E10.5 Tbx1+/− embryos than WT littermates. Middle left: quantitative analysis of CN phenotypes shows a statistically significant CN IX/X phenotype with the same frequency in Tbx1+/− embryos as in LgDel embryos. Middle right: qPCR measurement of gene expression in the CN V ganglion of E10.5 Tbx1+/− embryos, normalized to WT littermate control levels. Robo2 (black bar), which increases significantly in the LgDel (see Fig. 5), decreases significantly in the Tbx1+/− CN V ganglion (asterisk). Right: Cyp26b1, which, in WT, is expressed at high levels in r5/6, lower levels in r3/4 and barely detectable in r2, has a similar pattern of expression in r6 through r2 in Tbx1+/− embryos. There is no noticeable expansion of r5/6 (compare brackets left) or compression of r2 to r4 (compare brackets right).
	Fig. 8. Heterozygous inactivation of Raldh2 rescues CN V, but not CN IX/X, phenotypes in LgDel embryos. Top left: (first row) comparison of CN V differentiation in E10.5 Raldh2+/−, which resembles the WT (see Fig. 5), Raldh2+/−:LgDel, which also resembles the WT, and LgDel, which shows clear disruption of fasciculation and differentiation of all major branches of CN V. The ophthalmic (op, arrow), maxillary (mx, bracket) and mandibular (md, arrows) branches of CN V are shown. (Second row) Comparison of CN IX/X differentiation in E10.5 Raldh2+/−, Raldh2+/−:LgDel and LgDel embryos. CNs in Raldh2+/− embryos resemble the WT, whereas Raldh2+/−:LgDel and LgDel have similar ganglion fusions (arrows) and disrupted axon trajectories. Top right: frequency of phenotypes in 12 individual CN V and CN IX/X from six E10.5 Raldh2+/−:LgDel, or from six LgDel embryos. Statistical comparisons made using Fisher’s exact analysis. Middle: A-P rhombomere selective genes, many of which are RA regulated (see Fig. 4), return to WT levels in E9.5 LgDel:Raldh2+/− hindbrains, based upon qPCR analysis of microdissected hindbrain samples from n=8 Raldh2+/−; 7 LgDel:Raldh2+/−; 13 WT; and 12 LgDel embryos. Ø: genes for which LgDel:Raldh2+/− levels are statistically indistinguishable from WT and Raldh2+/− levels; *: genes for which LgDel levels are significantly increased over both Raldh2+/− and WT. Bottom left: scatterplots showing the ranges of individual hindbrain expression values for four ‘rescued’ genes in WT, LgDel (LD) and LgDel:Raldh2+/− (LD:Ra). The minimum and maximum values in the WT and LgDel:Raldh2+/− are similar, and the minimum and maximum values for the LgDel are consistently increased. Bottom right: Cyp26b1 patterns in the Raldh2+/− hindbrain are similar to WT (compare to WT panels in Figs 4 and 7); those in LgDel:Raldh2+/− hindbrain resemble the Raldh2+/− and WT as well; in the LgDel, intensity increases in r6, r5, r4 and r3, the barely detectable expression in r2 is more robust, and, in this case, apparently extends into r1. Right hand brackets show expansion of r5/6 in LgDel but not Raldh2+/− or LgDel:Raldh2+/− hindbrain; left brackets show that r2-r4 are apparently compressed in the LgDel but not Raldh2+/− or LgDel:Raldh2+/− hindbrain.

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