MOESM1 of Update on oral-facial-digital syndromes (OFDS)

Additional file 1: Table S1. Summary of mutations identified in OFDI patients

MOESM2 of Update on oral-facial-digital syndromes (OFDS)

Additional file 1: Table S2. Mutations identified in OFD genes other than OFD1

Markers of the ventral telencephalon are ectopically expressed in the dorsal telencephalon of Ofd1 mutant embryos.

<p>ISH analysis showed that <i>Gsh2</i>, <i>Mash1</i> and <i>Dlx2</i> genes are all expanded dorsally into the neocortex of <i>Ofd1^Δ</i>^4–5/+ heterozygous females with a severe phenotype (red arrows in C, F, I) compared to <i>Ofd1</i>^+/+ wild-type embryos (A, D, G). Immunohistochemical analysis for the ventral marker Nkx2.1 reveals an ectopic expression in the dorsal part of the brain with a severe phenotype (L) when compared to <i>Ofd1</i>^+/+ wild-type embryos (J). We do not observe any difference in the expression pattern of ventral markers on brain sections of Ofd1 mutants displaying a mild phenotype (B, E, H) with the exception of Nkx2.1, which is slightly mis-expressed in the LGE (red arrows in K) and it is ectopically induced at lower levels in the dorsal telencephalon (red arrows in L). The analysis of the expression pattern of <i>Sfrp2</i> and <i>Dbx1</i> genes at the boundary between pallial and subpallial zones (PSBP) demonstrated that these transcripts show a normal expression domain in <i>Ofd1^Δ</i>^4–5/+ heterozygous females (black arrows in N, O, Q, R) when compared to wild-type animals (black arrows in M, P). Dorsal is upwards, ventral is downwards. Scale bars: 150 µm. NCX: neocortex. GE: ganglionic eminences.</p

Neuronal primary cilia are severely affected in <i>Ofd1^Δ</i>

<p>^{<b>4–5/+</b>}<b> forebrains.</b> SEM analysis of neocortex from embryos at E12.5 (A, C, F, H). Numerous primary cilia (white asterisks) are present in <i>Ofd1</i>^+/+ wild-type neocortex (A). A higher magnification of a primary cilium where the pit is more evident (dashed white circle) is reported in inset <i>A</i>. The distribution of primary cilia (white asterisks) is reduced in <i>Ofd1^Δ</i>^4–5/+ heterozygous females displaying a mild phenotype (C). In severely affected <i>Ofd1^Δ</i>^4–5/+ heterozygous females, we observe regions with short protrusions of membrane indicated by white arrows (F) and regions completely devoid of cilia (H). TEM analysis of neocortex from embryos at E12.5 (B, D, E, G, I, J, K, L). Primary cilium in <i>Ofd1</i>^+/+ wild-type neocortex (B). Primary cilium in <i>Ofd1</i>^+/+ wild-type neocortex where the ciliary pocket is evident, as indicated by the black arrow (D). Transverse section of a normal basal body, where the nine triplets of microtubules are easily recognized (E). No ciliary axonemes can be detected in severely affected <i>Ofd1^Δ</i>^4–5/+ heterozygous females (G). The basal body in <i>Ofd1^Δ</i>^4–5/+ heterozygous females appear mature as indicated by the presence of appendages (black asterisk) on which microtubules are correctly anchored (dashed black lines demarcate the area where microtubules can be observed) (G). The basal body in severely affected <i>Ofd1^Δ</i>^4–5/+ heterozygous female appear to be correctly docked as indicated by the presence of the sheath (black arrow) and, as described above, they appear to be mature, given the presence of appendages (black asterisks) (I). Similar to SEM analyses, TEM analyses reveal that severely affected <i>Ofd1^Δ</i>^4–5/+ heterozygous female show short protrusions of membrane indicated by white arrow (J). One of the few ciliary axonemes present in a severely affected <i>Ofd1^Δ</i>^4–5/+ heterozygous female is showed (K). Transverse section of the basal body in a severely affected <i>Ofd1^Δ</i>^4–5/+ heterozygous female showing a normal ultrastructure with nine triplets of microtubules (L).</p

Cytoskeletal organization and cell polarity are altered in <i>Ofd1^Δ</i>

<p>^{<b>4–5/+</b>}<b> embryos.</b> Staining for F-actin with fluorescent phalloidin reveals a reduced signal and actin disorganization at the cell apex in severely affected <i>Ofd1^Δ</i>^4–5/+ mutants (B) with respect to wild-type <i>Ofd1⁺</i>^/+ embryos (A). Scale bars: 200 µm. A higher magnification of the actin boundary indicates a reduced thickness in mutant embryos (inset B) compared to wild-type <i>Ofd1⁺</i>^/+ embryos (inset A). Immunofluorescence with β-catenin reveals a continuous band in wild-type embryos (C) but a punctuate and discontinuous band in <i>Ofd1^Δ</i>^4–5/+ severe mutants (D). Scale bars: 100 µm. Immunofluorescence analysis of ZO1, a marker of tight junctions, shows a discontinuous pattern along the margin of the ventricular surface (F) compared to wild-type embryos (E). Scale bars: 50 µm. Immunostaining of Par3 with a discontinuous pattern along the cells lining the ventricular zone in <i>Ofd1^Δ</i>^4–5/+ severe mutant embryos (H) compared to wild-type embryos (G). Scale bars: 50 µm. White arrows in panels D, F, H indicate expression discontinuity along the apical membrane. Immunostaining of Ki67 (in red) and Par3 (in green) indicate reduced Ki67 expression and presence of abnormally located cortical progenitors, which tend to protrude outside the ventricular zone in <i>Ofd1^Δ</i>^4–5/+ severe mutant embryos (J) compared to wild-type embryos (I). Scale bars: 200 µm. vz: ventricular zone. Immunofluorescence analysis of Caspase 3 revealed no difference in the number of apoptotic cells in wild-type embryos (K) and in <i>Ofd1^Δ</i>^4–5/+ severe mutant embryos (L). Dashed rectangles designate enlarged areas indicated in panels K′ and L′ showing apoptotic cells in the ganglionic eminence (GE) region. Scale bars: 500 µm.</p

Markers of the dorsal telencephalon are preserved in the absence of Ofd1.

<p><i>Pax6</i> and <i>Ngn2</i> expression in the forebrain of wild-type (A, D) and <i>Ofd1</i> mutants (B, C, E, F) analyzed by ISH on coronal sections at E12.5. These transcripts are normally expressed in <i>Ofd1</i>^+/+ wild-type and <i>Ofd1^Δ</i>^4–5/+ heterozygous females in the dorsal part of telencephalon. In <i>Ofd1^Δ</i>^4–5/+ heterozygous females displaying a severe phenotype expression was detected exclusively in the presumptive neocortex (red arrows in B, D), but was absent in the dorsomedial regions. mRNA expression of <i>Lhx2</i> was detected in the dorsal telencephalon in a high dorsomedial to low lateral gradient (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052937#pone-0052937-g002" target="_blank">Figure 2E</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052937#pone.0052937-Mangale1" target="_blank">[32]</a>. Similarly to <i>Pax6</i> and <i>Ngn2</i>, <i>Lhx2</i> expression lost its expression gradient, but was still maintained in the malformed cortex (red arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052937#pone-0052937-g002" target="_blank">Figure 2F</a>), and not ectopically expressed in the protruded dorsomedial structure in severely affected O<i>fd1^Δ</i>^4–5/+ mutant embryos. mRNA expression of <i>Wnt8b</i> was detected in dorsomedial cortical structures, but not in the cortical primordium (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052937#pone-0052937-g002" target="_blank">Figure 2G</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052937#pone.0052937-Richardson1" target="_blank">[33]</a>. Severely affected O<i>fd1^Δ</i>^4–5/+ mutant embryos showed only a slight expansion of <i>Wnt8b</i> expression dorsomedially (red arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052937#pone-0052937-g002" target="_blank">Figure 2H</a>), indicating that the abnormally protruded dorsomedial structure in mutant embryos has not a cortical origin. Immunohistochemical analysis for Tbr2 reveals that its expression is still maintained in severely affected <i>Ofd1^Δ</i>^4–5/+ heterozygous females (I, J) although some areas lack Tbr2 expression (red arrows in J). Similarly, immunohistochemical analysis for Tbr1 reveals that its expression is mainly preserved in <i>Ofd1^Δ</i>^4–5/+ heterozygous females with a severe phenotype (K, L) although Tbr1-negative patches can be detected (red arrows in L). Dorsal is upwards, ventral is downwards. Scale bars: 100 µm. NCX: neocortex. GE: ganglionic eminences.</p

Shh signalling is altered in the developing forebrain of <i>Ofd1^Δ</i>

<p>^{<b>4–5/+</b>}<b> embryos.</b> ISH showed a normal expression pattern of <i>Shh</i> in <i>Ofd1^Δ</i>^4–5/+ heterozygous females (black arrows in B, C) when compared to <i>Ofd1</i>^+/+ wild-type embryos (black arrow in A). <i>Gli1</i> displays an ectopic expression in the neocortex in mutants with a severe phenotype (red arrows in F) while it is confined to the ventral part in <i>Ofd1</i>^+/+ wild-type embryos (black arrow in D) and <i>Ofd1^Δ</i>^4–5/+ heterozygous females with a mild phenotype (black arrow in E). <i>Ptch1</i> expression is upregulated in the neocortex in both mutants with a mild (red arrow in H) and severe phenotype (red arrows in I). Dorsal is upwards, ventral is downwards. Scale bars: 200 µm. NCX: neocortex. GE: ganglionic eminences. Western blot analysis of Gli3 protein on E12.5 <i>Ofd1</i>^+/+ wild-type and <i>Ofd1^Δ</i>^4–5/+ heterozygous brains with a severe phenotype. An increase level of the larger isoform 190 kDa Gli3^FL isoform is observed in Ofd1 mutant animals when compared to controls, thus indicating an impairment during Gli3 processing (J). Quantification of the ratio of Gli3^FL versus Gli3^R indicates a 3.4-fold increase in the mutant animals (J). Asterisk (*) denotes statistically significant changes with <i>p</i><0.05. Quantitative RT-PCR is performed upon mRNA extraction from E12.5 total brain (K; ***<i>p</i><0.01). Error bars indicate standard error of the mean. ISH analysis showed that no difference in Gli3 mRNA expression pattern was observed in <i>Ofd1^Δ</i>^4–5/+ heterozygous females with a severe phenotype (M) when compared to <i>Ofd1</i>^+/+ wild-type embryos (L). Dorsal is upwards, ventral is downwards. Scale bars: 100 µm. NCX: neocortex. GE: ganglionic eminences.</p

Identification of regulatory deletions telomeric to <i>PAX6</i>.

<p>Regulatory deletions telomeric to <i>PAX6</i> were identified in individual 1514 (chr11:30,874,642–31,654,833), individual 753 (chr11:30,967,000–31,704,000), individual 555 (chr11:31,108,579–31,649–842), individual 2014 (chr11:31,234,395–31,751,815) and individual 659 (chr11:31,379,000–31,708,000). The schematic diagram shows how the ‘critical region’ (delimited by grey dotted lines) required for <i>PAX6</i> transcriptional activation was delineated by combining our data with published deletions with known coordinates [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153757#pone.0153757.ref055" target="_blank">55</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153757#pone.0153757.ref067" target="_blank">67</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153757#pone.0153757.ref068" target="_blank">68</a>]. <i>PAX6</i> regulatory deletions from the present study are shown by red blocks. Genes transcribed on the forward strand are in blue and those transcribed on the reverse strand are in green, also indicated by arrows. Genomic coordinates are based on the Human Genome Assembly hg18.</p

Mutation analysis of the <i>FOXC1</i> locus.

<p><b>(A)</b> Genome-wide array CGH identified two deletions encompassing the <i>FOXC1</i> gene in individuals 1449 (chr6:1,543,591–1,675,085) and 1246 (chr6:1,543,591–1,675,085). <b>(B)</b> Direct sequencing of the <i>FOXC1</i> coding region identified a heterozygous substitution in individual 1839 (c.235C>A, p.(Pro79Thr)) and another in individual 1634 (c.302T>C, p.(Leu101Pro)). <i>FOXC1</i> mutation screening in unaffected parents of both patients showed that the mutations had occurred <i>de novo</i>. The locations of both mutations within the fork-head domain of the FOXC1 protein are indicated by vertical arrows. Genes transcribed on the forward strand are in blue and those transcribed on the reverse strand are in green, also indicated by arrows. Genomic coordinates are based on the Human Genome Assembly hg18. The genomic sequence identifier for <i>FOXC1</i> is NG_009368.</p

Identification of a potential <i>PITX2</i> regulatory deletion.

<p>Genome-wide array CGH identified a deletion of approximately 3.5 Mb in individual 1194 (chr4:111,994,000–115,504,000) (red bar). The deletion is located telomeric to the <i>PITX2</i> gene on chromosome 4. The positions of conserved elements (CE) in the deleted region, as identified by Volkmann et al., 2011 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153757#pone.0153757.ref047" target="_blank">47</a>] are marked by orange ellipses. Genes transcribed on the forward strand are in blue and those transcribed on the reverse strand are in green, also indicated by arrows. Genomic coordinates are shown on the x-axis and are based on the Human Genome Assembly hg18.</p