1. Lhx2, a LIM homeobox gene, is required for eye, forebrain, and definitive erythrocyte development Introduction Lhx2 is a member of the LIM homeodomain superfamily of transcription factors. There are at least 5 members of the Lhx family expressed in mammals. Most of these genes are expressed in the differentiation of ectodermal-derived tissues such as skin, CNS, and eyes. The vertebrate Lhx genes are orthologues of the Drosophila apterous gene which is involved in determination of the dorsal-ventral polarity of the fly wing. Lhx2 was originally found as a gene expressed in B-cells, but is also expressed in the developing brain. The gene was targeted by removing exons 2 and 3 and replacing them with a neomycin resistance gene. The resulting phenotype was a loss of eyes, disruption of forebrain formation and reduction in liver development. Jon B Scales Genetics Fall 2015 Figure 1. Construction of gene targeting vector and genomic analysis of targeted alleles. A) Schematic of targeting vector construction. A PGKneo cassette replaced an ~5Kbp region of the Lhx2 gene surrounding exons 2 and 3 (numbered black boxes). A PGK-TK cassette flanks the genomic sequences on the right. The PGK cassettes drive ex- pression of neomycin resistance or HSV thymidine kinase genes using the phosphoglycerate kinase 1 promoter. A probe made from the Xba-BglII frag- ment at the 5’ side of exon 2 will detect a 6Kbp fragment from the wildtype allele or an 18Kbp fragment from the targeted allele lacking exons 2 and 3. B) Southern blot analysis shows two stem cell lines (F6 and F101) which contain the correctly targeted allele. J1 is the non- transgenic stem cell line. C) Southern blot analysis of DNA isolated from transgenic homozygotes (-/-), heterozygotes (+/-) and non-transgenic (+/+) embryos. Figure 2. Comparison of Lhx2 knockout phenotype to hetero- zygous and wildtype litter mates. Lhx2 -/- mice lack eyes (arrow), have reduced forebrain (arrowhead) and reduced liver (L) sizes. L L L Figure 3. Histological analysis of developing eyes. The entire developing eye is missing from Lhx2 -/- embryos. A) E13.5 stage wildtype eye showing lens (L), retinal epithelium (RE) and neural retina (NR). B) E13.5 stage Lhx2 -/- embryos are completely devoid of eye structures. C) &D) E9.5 stage embryos Lhx2 -/- embryos (D) have and optic vesicle (OV) but lack lens placodes (LP) and neural retina (NR) development seen in wildtype (C). E) & F) Pax6 gene expression is lacking from the ectoderm overlying the optic vesicle (OV) in Lhx2 -/- embryos (F) compared to wildtype (E). Figure 4. Histological analysis of developing brain. The Lhx2 -/- embryos (B, D, & F), show marked reduction in brain structures compared to wildtype littermates (A, C, & E). A) Coronal sections of E12.5 stage embryos show a reduction in the cortical plate (C), and hippocampus (H) (compare panels A-B and C-D), while the diencephalon (D) appears enlarged. The Lhx2 -/- mice have a reduce proliferation of neuronal cells as determined by labeling mitotic cells with BrdU (E & F).