Requirement for the c-Maf transcription factor in crystallin gene regulation and lens development

Abstract

The vertebrate lens is a tissue composed of terminally differentiated fiber cells and anterior lens epithelial cells. The abundant, preferential expression of the soluble proteins called crystallins creates a transparent, refractive index gradient in the lens. Several transcription factors such as Pax6, Sox1, and L-Maf have been shown to regulate lens development. Here we show that mice lacking the transcription factor c-Maf are microphthalmic secondary to defective lens formation, specifically from the failure of posterior lens fiber elongation. The marked impairment of crystallin gene expression observed is likely explained by the ability of c-Maf to transactivate the crystallin gene promoter. Thus, c-Maf is required for the differentiation of the vertebrate lens.

Figure 1

Targeted disruption of the murine c-maf gene. (A) Intron–exon structure of genomic DNA including the c-maf gene isolated from a 129/sv genomic library (Top). The thick black line represents the transcribed part of the c-maf locus, which is a single exon. The open box represents the ORF. Insertion of neo at the NotI site (amino acid codon 170) disrupts the gene 150 bp 3′ of the acidic transactivation of the c-Maf protein (Middle). The resultant mutant allele with the position of the probes used in the diagnostic Southern blot analysis is shown (Bottom). B, BamHI; H, HindIII; N, NcoI; and X, XhoI. (B) Southern blot analysis of embryonic stem cell DNA. As predicted from the restriction map of the wild-type locus, digestion of DNA with NcoI generates a 3-kb fragment, which is replaced by a 4-kb fragment when hybridized with the 3′ probe. (C) Southern blot analysis of tail DNA from mice resulting from the matings of c-maf^+/− mice. (D) Western blot of kidney nuclear extracts from +/+ or −/− mice using a polyclonal anti-c-Maf antiserum. Control lane is a truncated recombinant c-Maf protein indicated by an asterisk.

Figure 2

Histological analysis of heterozygote (+/−) and c-maf mutant (−/−) adult, neonatal, and embryonic lens. Richardson staining of c-maf^−/− lens and heterozygote^+/− lens at E12, E14, E16, postnatal day zero (P0), and 5-week-old adult. Loss of c-maf results in failure of posterior fiber elongation, apparent by E12, with subsequent absence of lens formation. Sections (1 μm) of Epon plastic embedded material were stained with Richardson stain.

Figure 3

c-Maf controls the expression of multiple crystallin genes. (A) Semiquantitative RT-PCR analysis of αA-, αB-, γA-, B-, C-, E/F-crystallins in the adult wild-type (+/+) and c-maf mutant (−/−) lens. For each gene, amplification of hypoxanthine phosphoribosyltransferase (hprt) cDNA was used as an internal control. Lane c indicates PCR performed in absence of reverse transcriptase. (B) c-Maf-dependent transactivation of the γF-crystallin promoter. The Psi2 fibroblast cell line was cotransfected with a c-Maf expression plasmid, pMex/maf, or control pMex vector alone, and a γF-crystallin promoter-luciferase reporter plasmid containing 1.4 kb of upstream sequence. One representative experiment of four is shown. The fold transactivation of the γF-crystallin promoter by c-Maf ranged from 100- to 1000-fold.