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- Fig. 1.
Expression of Suz12 in mouse development. (A) Detection of Suz12 expression through mouse embryo development by northern blotting. Stages E4.5–E6.5 include extra-embryonic tissues and maternal uterus; stages E7.5–E9.5 include embryo and extra-embryonic tissues. There are two peaks of Suz12 expression, including one at the very early stages of development. (B) The Suz12 transcript remains expressed in mouse brain up to older ages, as shown by northern blotting. (C–K) Expression of Suz12 in the mouse embryo. β-galactosidase (β-gal) staining on whole-mount Suz12+/− mouse embryos at E10.5 (F) and E11.5 (G) shows strong expression in the developing first branchial arch (br) and liver (li). In situ hybridization (ISH) of Suz12 on coronal (C) and sagittal (D,E,H,J,P) sections at E12.5 (C,D,H,J) and at E14.5 (E,P) correlates with the β-gal staining on whole-mount Suz12+/− mouse embryos at E12.5 (I) and E18.5 (K), and with β-gal ISH of Suz12+/−mouse embryos at E14.5 (O). The Suz12 transcript shows a posterior (p)-to-anterior (a) gradient of expression in the primordium of the cerebral cortex (D,E,I,O,P), with no expression in the cortical hem (hem) (C–E), and expression in the tectal neuroepithelium (tc) (J,K), eye primordium (H,I), nasal epithelium (ne), kidney (ki), lung (lu) and pancreas (pa) (O,P). (L–N) β-gal ISH (L) and X-gal reaction (M,N) of Suz12+/− mouse embryos at P9 reveals Suz12 expression in the external granular layer (egl) of the cerebellum, as well as in the Purkinje cell layer (pcl) (L), which represents the only expression in the adult cerebellum (M). Suz12 is also expressed in the hippocampus, with enrichment in the adult CA2 (N). (Q,R) Transcripts of Ezh2 (Q) and Eed (R), two other PRC2 partners, show an expression pattern similar to Suz12. Bars, 250 μm (C,D,I–N); 100 μm (E,H); 500 μm (F,G); 1 mm (O–R).
- Fig. 2.
Suz12+/− mice show a reduced occipital cortex and enlarged tectum. (A,B) Inactivation of Suz12. (A) Schematic representation of wild-type Suz12+/+ (top) and mutated Suz12−/− (bottom) alleles. Exons are represented by black boxes. The sizes of the restriction fragments that were expected to be recognized by the Southern probe (indicated) are shown. (B) Southern blot. (C–D″) Suz12+/− mice show different degrees of brain reduction. A comparison between the brains of 46-day-old wild-type (C,D) and Suz12+/− (C′,C″,D′,D″) mice shows different degrees of reduction in the size of the P-CTX of the heterozygote (arrowheads). D–D″ show Nissl-stained sections of C–C″ brains. (E–G) Histological measurements show a specific reduction of the P-CTX and enlargement of the tectum in the newborn Suz12+/− brain (discordant asymmetry). (E,F) We subdivided the cortex into two parts, anterior (A-CTX) and posterior (P-CTX), and measured them, as well as the inferior and superior colliculus (IC and SC, respectively), hippocampus (HIPP) and caudate-putamen (CP). (G) Statistical analysis showed that Suz12+/− brains had a reduced P-CTX, as well as an enlarged IC and SC (tectum), with no differences in the other regions. (H–K) Expression of the posterior cortical developmental markers, Id2 (H,I) and COUP-TFI (J,K), showed shortened domains in the Suz12+/− cortex (I,K) compared with the wild-type cortex (H,J). Arrowheads indicate the anterior boundary of the expression domain; OB, olfactory bulb (for reference). The values in G represent the mean±s.d. of measurements from seven wild-type and 13 mutant brains. Bars, 1 mm (C–E); 250 μm (H–K).
- Fig. 3.
Beaked tectum, cerebellar herniation and hydrocephalus, as well as altered Zac1 expression, in Suz12+/− mice. (A,B) An enlarged beaked tectum (tc) in Suz12+/− mice (B) results in herniation of the cerebellum when compared with wild-type littermates (A). In the most dramatic herniations, hydrocephalus (HYD) was observed (C,D), including severe cerebellar (CB) abnormalities (D). (E–H) MRI studies of 42-day-old mice. The comparison between sagittal views of T2-weighted MRIs of wild-type (E,F) and Suz12+/− (G,H) mice shows how the ventral paraflocculus (the structure that corresponds to the cerebellar tonsils in humans) goes into the foramen magnum in Suz12+/− mice as a consequence of cerebellar herniation (yellow arrowhead) (F,G). At the same time, the Suz12+/− mice have a crowded posterior fossa, with the volumes of both the cisterna cerebellomedularis and pontis reduced (white arrowheads) (F,G). (I) BrdU labeling showed strongly reduced proliferation, specifically in the P-CTX (but not the A-CTX), as well as increased proliferation in the tectum (TC). (J–P) Zac1 is specifically upregulated in areas of altered proliferation in the Suz12+/− brain. Zac1 shows an expression pattern that is very similar to Suz12, as observed by ISH on Suz12+/− mice at E14.5 (J). Zac1 (red in K) and Suz12 (green in K) colocalize in the cortical neuroepithelium (ctx) but not in the hem (K). Zac1 is upregulated in the Suz12+/− cortex (CTX) (L–N) and tectal central gray neuroepithelium (tcn) (O,P), as revealed by ISH (J,L,M,O,P) and real-time PCR (N). In addition, real-time PCR revealed increasing expression of Zac1 in Suz12+/− E13.5 embryonic fibroblasts (MEF) (N). (Q) SUZ12 knockdown in human osteosarcoma U2Os cells resulted in upregulation of ZAC. Transfection with specific siRNA dramatically reduced SUZ12 expression, as confirmed by western blotting and real-time PCR (Q), resulting in upregulation of ZAC at both the protein and mRNA levels (Q). (R,S) Detection of P-Creb by immunohistochemistry at E14.5 revealed an increase in the Suz12+/− P-CTX (arrowheads). The values in I represent the mean±s.d. of measurements on three brains; the values in N,Q represent the mean±s.d. of three experiments. Bars, 500 μm (A–D,F,G); 1 mm (E,H,J); 100 μm (K,R,S); 250 μm (L,M,O,P).
- Fig. 4.
Spina bifida, diastematomyelia and callosal alterations in Suz12+/−mice. (A) In the most impaired cases, Suz12+/− mice showed a protuberated back (white arrowhead) and paraplegia of the posterior legs (yellow arrowhead). (B–D) Suz12 expression in the neural tube (NT) at E8.5 (B), E9.5 (C) and E10.5 (D). (E–H) Malformations observed in the vertebral column of Suz12+/− mice. Diverse degrees of spina bifida (black arrows) were observed in heterozygous mice (F,G) in contrast to wild-type mice (E). In addition, diastematomyelia (H) was also found, caused by bony septa in lumbar L3–L4 vertebrae (white arrowheads). (I–L) Other alterations observed in the brain were deformations of the hippocampal cortex (I,J), partial agenesis of the corpus callosum (black arrows) (K,L), and loss of the gray matter in a gradient (K,L; see also Fig. 3C) that follows the posterior-to-anterior Suz12 expression gradient (Fig. 1D,E), with disorganization of cortical layers in the P-CTX of 24-day-old Suz12+/− mice compared with wild-type brains (K,L). Bars, 500 μm (B–F,I–L); 1 mm (G); 250 μm (H).
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