Global deletion of Hdac8. (A) Mice with a global deletion of Hdac8 are viable but show a runted phenotype with hypoplastic, dysmorphic skulls. Mice on a mixed genetic background are shown at the age of 2 wk. (B) Global deletion of Hdac8 is lethal on a pure background. Mice were backcrossed for several generations in a C57/BL6 background. No hemizygous null animals (male, Y/−) were found after the first generation of backcrosssing. Mice were genotyped between 14 and 21 d of age. Heterozygous females (X/−) were recovered at sub-Mendelian ratios. (C) Strategy for transgenic rescue experiment. The CAG promoter was used to drive ubiquitous expression of a bicistronic mRNA encoding TAP-tagged Hdac8 and an IRES-linked eGFP. The transgene was crossed into Hdac8 heterozygous females (X/− tg+). These were then crossed with wild-type C57/BL6 males to obtain hemizygous null males, which were either transgene-negative (null) or transgene-positive (rescue). (D) Genotypes obtained in the transgenic rescue experiment. No Hdac8-null animals (Y/− tg−) were recovered, but mice with transgenic re-expression of Hdac8 on the null background (Y/−, tg+) were recovered at expected frequencies. (E,F) Phenotype and weight of mutant animals at E18. Mutant animals were recovered at Mendelian frequencies before birth, although they were smaller and weighed less.

Analysis of Hdac8-null NCCs. (A) Lineage trace of Hdac8-null NCCs using a double-transgenic ROSA26(lacZ), Wnt1-Cre system. Wnt-1 Cre marks the delaminating NCCs by activating lacZ expression. All the descendant cells are marked blue. (B) Lineage trace of Hdac8 using the ROSA26(GFP), Wnt-Cre system. In this system, NCCs are marked by GFP expression. (C) Strategy for microarray analysis of FACS sorted cranial NCCs. The anterior part of the skull was dissected from E15.5 embryos that expressed GFP in NCCs as indicated. Cells were dissociated and used for FACS analysis. (D) Cranial NCCs in culture. (Top panel) DIC. (Bottom panel) GFP (E,F) Gene ontology analysis of microarray analysis showing molecular process and function of up-regulated genes in Hdac8 mutant cranial NCCs. (G) Fold changes of up-regulated homeobox transcription factors.

Generation of a conditional Hdac8 allele. (A) Strategy to generate a conditional Hdac8 allele. Protein domains, genomic structure, targeting vector, and targeted allele are shown. Exon 4 was flanked by loxP sites (red triangles), and the neomycin resistance cassette (Neo), flanked by FRT sites (gray ovals) was removed by crossing to FLPe transgenic mice. NcoI (N) and SacI (S) sites used for Southern blotting are indicated. Primers used for genotyping are depicted with arrows. (B,C) Representative Southern blots of genomic DNA showing correct targeting of the Hdac8 locus in ES cells. Genomic DNA was digested with NcoI and SacI and probed with 5′ and 3′ probes shown in A. (D) Genotyping of wild-type, heterozygous and homozygous null alleles from tail DNA by genomic PCR shows germline transmission and functionality of the conditional allele. (E) Deletion of Hdac8 in mouse embryonic fibroblasts using a tamoxifen-inducible Cre. Hdac8 mRNA levels were detected using primers spanning the deleted exon 4 at the indicated days after adding 4-hydroxy-tamoxifen (4OHT).

Skull dysmorphism in Hdac8 mutants. (A) H&E-stained sagittal sections of a P1 skull. The skin was removed for better fixation. Bar, 2 mm. Note the herniation of brain tissue (yellow square and black arrows) and hemorrhage (white arrows). (B) Skeletal prep of P3 mouse skull showing massive ossification defects. The dotted lines demarcate the boundaries of uncalcified soft tissue. Arrows point to the anterior and posterior fontanelle. (C) Tissue-specific deletion of Hdac8 with the indicated Cre deleter strains to determine the causative tissue for the observed phenotype of the global deletion. (D) Three-dimensional reconstruction of micro-CT scans of newborn mouse skulls. Note the severe ossification defects in Hdac8 mutant mice. (E) Neural crest deletion of Hdac8 leads to ossification defects in frontal (arrow) and interparietal bones (arrowheads) in E18 embryos.

Dysregulated expression of homeobox transcription factors causes skull dysmorphism. (A) Semiquantitative RT–PCR showing loss of Hdac8 and up-regulation of Otx2 and Lhx1 in NCCs. (B) Strategy for conditional, transgenic overexpression in NCCs. A ubiquitous promoter is separated by a STOP cassette from a bicistronic cDNA-IRES-GFP expression cassette. Wnt1-Cre deletes the STOP cassette, leading to transgenic expression in NCCs. (C) Phenotype of transgenic overexpression of Lhx1 in NCCs. Skeletal prep of a P1 skull is shown. Note the severe skeletal dysmorphism in the transgenic animal (arrows). (D) Model of Hdac8 function in cranial NCCs. Hdac8 represses the expression of several homeobox transcription factors in cranial NCCs, which thus adopt a mesodermal fate. Loss of Hdac8 leads to up-regulation of these factors with cranial NCCs consequently adopting a neuroectodermal fate.