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First published online 26 November 2003
doi: 10.1242/dev.00908

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Atrophin 2 recruits histone deacetylase and is required for the function of multiple signaling centers during mouse embryogenesis

Department of Neurology and the Ernest Gallo Clinic and Research Center, University of California at San Francisco, 5858 Horton Street, Emeryville, CA 94608, USA

View larger version (35K): [in a new window]   Fig. 5. Atr2 interacts with histone deacetylase 1 in vivo. (A) The domain structure of Atr2 and the fragments used in transfection experiments are illustrated. N-Atr2 encodes the N-terminal BAH, ELM2 and SANT domains. C-Atr2 encodes the rest of the protein, from the GATA domain through the C terminus. (B) Each indicated protein was flag-tagged at the N terminus, overexpressed in 293 cells, then immunoprecipitated using anti-flag beads. Western blots show immunoprecipitated proteins [IP] run next to total soluble extract [E]. Hdac1 is associated with full-length Atr2, N-Atr2 and Mta2, but not with C-Atr2. The NuRD core subunits RbAp48 and RbAp46 are only found in association with Mta2. Extract lanes show that the total protein concentrations in each extract were similar. (C) Extracts were made from wild-type E9.5 embryos, and endogenous Atr2 was immunoprecipitated using the 286-1 antibody linked to protein G sepharose. 286-1 beads immunoprecipitated the full-length Atr2 as well as Atr2S. Endogenous Atr2 associates with Atr1 and Hdac1 in vivo, but not with RbAp46. Protein G beads did not pull down any of these proteins. (D) Endogenous Atr2 was immunoprecipitated from wild-type and om MEFS with 286-1 beads. Full-length Atr2 is present in wild-type cells, but is missing in om cells; Atr2S was present in both. Atr1 was pulled down with Atr2 in both cell types.

View larger version (173K): [in a new window]   Fig. 1. openmind embryos exhibit diverse developmental defects by E9.25. A wild-type embryo at E9.25 (A) and an om homozygous littermate (B) of the same stage and size are shown in side views. The mandibular component of the first branchial arch is well developed in the wild type but is poorly developed in the om embryo (red arrowheads). The om heart is abnormally dilated and is near failure. (C) The anterior neural tube is fully closed by E9.25 in a normal embryo (shown in front view with tail removed), the heart is looped, and the optic vesicles are visible. (D) In the om mutant, the anterior neural tube fails to close and is abnormally thickened, the heart remains as an unlooped tube, and the optic vesicles are morphologically absent. (E) Wild-type somites are uniform, and the neural tube is smooth and straight at the midline in this dorsal view of the tail. (F) om somites are irregular in size (red arrowhead points to a small somite) and the neural tube is kinked.

View larger version (44K): [in a new window]   Fig. 2. The openmind phenotype is caused by mutations in the Atr2 gene. (A) Diagram of the domain structure of Atr2. Atr2 has N-terminal homologies to mouse atrophin 1 (Atr1), C. elegans EGL-27, Drosophila Atro and mouse metastasis associated protein 2 (Mta2). Black lines illustrate percent identity to Atr2; only the regions of highest homology are shown. The N terminus of Atr2 includes four domains also found in Mta2; these are the BAH (bromo-adjacent homology), ELM2 (EGL-27 and Mta1 homology 2), SANT (SWI3, ADA2, N-CoR and TFIIIB) and GATA (zinc finger) domains. The C terminus of Atr2 (red oval) is homologous to Atr1. The exon structure of the Atr2 gene is shown with respect to the domain structure of the encoded protein. Exons are numbered from the 5' end of the gene; coding sequences are orange, non-coding sequences white. Positions of the om mutation, insertion alleles and the Atr2S initiation site are indicated. (B) RT-PCR of1200 bases of the amino end of Atr2 from om mutant embryos, and heterozygous and wild-type tissues. The primers amplify two bands from wild-type brain (br.) and testis (tes.), one full-length (right upper arrow) and the other a minor alternatively spliced form lacking exon 5 (right lower arrow). By contrast, shorter fragments amplify from om mutants (left arrows). Brain and testis from heterozygous animals show all transcripts. Mutant and wild-type full length fragments are illustrated. The mutant cDNA has exon 3 spliced to exon 5, whereas wild-type cDNAs always include exon 4. The reading frames of exons 3 and 5 are different, creating a stop codon. (C) The genomic sequence of the 3' end of exon 4 (shaded) and the start of intron 4 is shown. om homozygotes have an ENU-induced single base change of T to A; this mutation destroys the splice donor causing exon 4 to be omitted from the mutant mRNA. (D) Whole-mount in situ hybridization for Atr2 with a 5' probe at E9.5 reveals a dramatic reduction in Atr2 mRNA levels in mutant embryos (right) compared with normal littermates (left). (E) At E9.5, a ßgal-stained compound heterozygote with the Atr2om/Atr2PT026 genotype clearly duplicates the om phenotype (see text).

View larger version (83K): [in a new window]   Fig. 3. Atr2 expression is elevated in the developing notochord, apical ectodermal ridge and neurons. A time course of Atr2 expression, from E8.0 through E11.5, is illustrated using the PT026 Atr2-ßgal fusion protein. (A) At E8.0, Atr2 is expressed throughout the embryo and is elevated in the anterior midline (region between arrows; front view, anterior up; this embryo has been flattened). (B) At E8.25, Atr2 shows a more pronounced elevation in the anterior midline (arrows). (b) A transverse section through the anterior of the embryo in B shows elevated expression in midline cells (red arrow). (b') A more posterior section from the same embryo shows no elevated expression in the posterior midline (red arrow). (C) At E8.5, Atr2 is elevated throughout the anteroposterior extent of the notochord and is downregulated in the heart. (c,c') Transverse sections confirm elevated expression in anterior and posterior regions of the notochord (red arrows). (D) At E8.75, expression is increased in the ventral brain. (d) A section through the hindbrain reveals continued expression in the notochord (red arrow), and the beginning of elevation in the ventral brain (black arrow). (d') A more posterior section shows uniform expression in the spinal cord, and upregulation in the notochord (red arrow). (E) At E9.5, additional sites of elevation besides the notochord (black arrow) appear, including the apical ectodermal ridge, the isthmus and the ventral diencephalon (red arrows). (F) At E10.5, sites of elevation include the notochord (black arrow), the AER, and spinal and brain neurons (red arrows). (G) A wild-type embryo at E9.75, showing that the pattern of Atr2 transcripts detected with a 5' RNA probe is similar to the fusion protein at E10.5. (H) A wild-type forelimb bud at E9.5 shows concentrated expression of Atr2 mRNA in the AER (left). A section through the forelimb bud of an E9.5 Atr2PT026/+ embryo shows ßgal expression in the AER (right). (I) Formation of the AER occurs normally in Atr2PT026/+ embryos at E9.0. (J) The AER is defective in Atr2PT026/Atr2om embryos at E9.0. (K) A transverse section through the caudal hindbrain at E9.5 shows elevated expression in ventrolateral neurons (arrow). (L) A horizontal section through the optic region at E11.5 shows expression in telencephalic and diencephalic neurons. (M) A transverse section at sacral level at E11.5 shows high levels of expression in spinal cord neurons.

View larger version (133K): [in a new window]   Fig. 4. Atr2 is required for patterning the ventral forebrain and for expression of patterning signals by the anterior notochord and the ANR during E8. (A) A normal E9.5 embryo expresses Nkx2.1 in the ventral forebrain. (B) An om mutant littermate shows reduced Nkx2.1 expression. (C) At E8.5, Pax6 is excluded from the anterior neural plate (red arrowheads). (D) In om mutants, Pax6 is abnormally expanded across the anterior midline (red arrowhead). (E) At E8.5, Emx2 is excluded from the anterior midline (arrowheads). (F) om mutants express Emx2 across the anterior neural midline (arrowhead). (G) At E8.75, Shh is diminished in the anterior region of om mutants (left) compared with in normal littermates (right), except for a small spot (arrowhead). (H) At E8.0, Shh is expressed in the full extent of the developing midline, nearly reaching the anteriormost edge of the neural plate (red arrow). (I) In E8.0 mutants, Shh expression is reduced anteriorly. The anterior edge of the neural plate is indicated (red arrow). (J) Gli1 is reduced in the anterior region of the om mutant (left) compared with wild type (right). (K) Gli3 is normally absent from the midline of the neural plate at E8.75 (arrowheads), as seen in this transverse slice through the hindbrain. (L) In om, Gli3 is expanded toward the midline (arrowheads). (M) At E9.5, transverse sections show Shh in the spinal cord floorplate and the notochord (nc) of a wild-type embryo. The floorplate is thinner than the lateral wall (compare red lines). (N) In om mutants at E9.5, Shh is expressed normally but the notochord is enlarged and the floorplate has not thinned (red lines). (O,P) At E9.5, Hnf3b is expressed in the ventral spinal cord in both normal (O) and mutant (P) embryos; mutants have enlarged notochords and an absence of floorplate. Ventral neurocoels are outlined in yellow. (Q) Hnf3b expression in a wild-type embryo at E8.5. (R) Hnf3b is expressed normally in om embryos. (S) Brachyury marks the developing notochord at E8.25. (T) om mutants express brachyury normally at E8.25. (U) At E8.5, goosecoid is expressed in the anterior midline. (V) om mutants express goosecoid in a wild-type pattern. (W-Y) Parasagittal sections of the anterior neural plate of E8.5 embryos stained with Fgf8. Dorsal is up, anterior is right. Epidermal ectoderm is outlined in green, neural tissue in yellow. (W) In wild type, Fgf8 transcripts are tightly localized to the anterior neural ridge (ANR; red circle). (X,Y) In om embryos, Fgf8 expression is decreased in the ANR (red circles), and is shifted into the adjacent epidermal ectoderm and neurectoderm. (Z) At E8.5, Hesx1 is expressed in the anterior neural plate up to the ANR (yellow line). (Z') In om embryos, Hesx1 expression is reduced laterally and is absent from the anteriormost neural plate (yellow arrowheads).