Impaired megakaryopoiesis and behavioral defects in mafG-null mutant mice

Abstract

The small Maf proteins (MafG, MafK, and MafF), which serve as heterodimeric partner molecules of CNC family proteins for binding in vitro to MARE sites, have been implicated in the regulation of both transcription and chromatin structure, but there is no current evidence that the proteins fulfill these functions in vivo. To elucidate possible contributions of the small Maf proteins to gene regulation, we have ablated the mafG and mafK genes in mice by replacing their entire coding sequences with the Escherichia coli lacZ gene. mafG homozygous mutant animals exhibit impaired platelet formation accompanied by megakaryocyte proliferation, as well as behavioral abnormalities, whereas mafK-null mutant mice are phenotypically normal. Characterization of the mafG and mafK embryonic expression patterns show that their developmental programs are distinct and intersecting, but not entirely overlapping. These results provide direct evidence that the small Maf transcription factors are vital participants in embryonic development and cellular differentiation.

Figure 1

Targeted disruption of the mafG and mafK genes. (a) Structure of the mafG genomic locus, targeting vector, and targeted locus. (b) PCR screening of R1 ES cell clones using the primers whose positions are indicated by single headed arrows in a. (c) Southern blot confirmation of PCR positive and negative clones, using probe 1G, a 1.6-kb BamHI–SacI fragment. Clone 3E8 shows an additional band that was not seen after further expansion. (d) Southern blot of tail DNAs from litters of mafG^+/− intercrosses, using probe 1G. (e) Structure of the mafK genomic locus, targeting vector, and targeted locus. (f) Southern blot screening of R1 ES cell clones, using probe 1K, a 6.6-kb XbaI fragment. (g) Southern blot of tail DNAs from litters of mafK^+/− backcrosses, using probe 2K, a 1.3-kb SacI–XbaI fragment. (M) Mutant allele; (WT) wild-type allele.

Figure 2

Stimulation of megakaryopoiesis in mafG mutant mice. (a) A transverse section through a mafG^+/− adult spleen shows few β-gal staining megakaryocytes (indicated by arrowheads). (b) A similar section through the spleen of a mafG^−/− littermate shows expansion of the megakaryocyte population. In addition, these show a dosage dependent increase in staining, attributable to the presence of one additional expressed copy of lacZ. mafG^−/− megakaryocytes from adult bone marrow are positive for anti-glycoprotein IIb antibody reactivity (c) and for acetylcholinesterase activity (d) (Jackson 1973; Beckstead et al. 1986).

Figure 3

Behavioral and weight deficits in mafG mutant animals. (a) Six-month-old mafG^+/− (left) and mafG^−/− (right) male littermates display wild-type and abnormal behaviors, respectively. Homozygous mutant animals spontaneously clench their hindlegs together when suspended in an inverted position. (b) A photograph of the mice in a shows that the mafG^−/− mutant (right) is smaller than its heterozygous littermate (left). (c,d) Developmental profile of mafG postnatal weights in B6/129 background animals. Heterozygous () and homozygous (♦) mutant littermates were weighed and averaged from the first week after birth up to 11 weeks of age.

Figure 4

Embryonic expression patterns of mafG and mafK. mafG^−/− (a,c,e,g,i) and mafK^−/− (b,d,f,h,j) embryos were dissected, fixed, and stained for β-gal activity as described in Materials and methods. (a,b) Whole-mounts of E6.5 embryos show that mafG expression is detected strongly in embryonic tissues, whereas mafK is predominantly extraembryonic. Both genes are expressed in a diffuse pattern in the complementary region of the embryos. (c,d) E8.5 embryos show that mafG and mafK continue to be embryonic and extraembryonic, respectively, with expansion of both into the ectoplacental cone, and mafK initiating expression in the yolk sac endoderm. (e,f) Transverse sections through E9.5 embryos indicate expression of mafG in the embryo, placenta, and yolk sac, with mafK limited to the placenta and yolk sac endoderm. (g,h) At E12.5, transverse sections of mafG and mafK stained embryos exhibit strong expression in the hematopoietic compartment of the fetal liver. (i) A transverse section of an E14.5 embryo shows strong mafG staining in the developing lens and retina. (j) At E14.5, the strongest site of mafK expression is seen specifically in the intestinal epithelium. (Arrowheads in a and b) Embryonic/extraembryonic boundary; (e) embryonic pole of the egg cylinder; (ex) extraembryonic pole of the egg cylinder; (ec) ectoplacental cone; (ys) yolk sac; (emb) embryo; (pl) placenta; (fl) fetal liver; (ret) retina; (len) lens epithelium; (int) intestine.

Figure 5

Small maf expression in megakaryocytes. E12.5 fetal liver (a,b) and adult bone marrow (c,d) were isolated, fixed, and stained for lacZ expression as described (Materials and methods). (a) mafG^−/− and mafK^−/− (b) E12.5 embryos display similar intensities of β-gal activity in fetal liver megakaryocytes. (c) mafG exhibits intense expression in adult mutant megakaryocytes, whereas mafK expression is much weaker (d).

Figure 6

Neuronal mafG and mafK expression. Newborn and adult mice were sacrificed, their organs dissected, fixed, and stained for β-gal activity as described (Materials and methods). (a) The inner ear of newborn pups shows mafG expression in the vestibulocochlear ganglion (arrowhead) and saccule sensory epithelium (arrow). (b) Adult cerebellar Purkinje and granular cells exhibit high levels of mafG, but no detectable mafK (c). (co) Cochlea.