Gfi1 and Gfi1b act equivalently in haematopoiesis, but have distinct, non-overlapping functions in inner ear development

Abstract

Gfi1 is a transcriptional repressor essential for haematopoiesis and inner ear development. It shares with its paralogue Gfi1b an amino-terminal SNAG repressor domain and six carboxy-terminal zinc-finger motifs, but differs from Gfi1b in sequences separating these domains. Here, we describe two knock-in mouse models, in which the N-terminal SNAG repressor domain was mutated or in which the Gfi1 coding region was replaced by Gfi1b. Mouse mutants without an intact SNAG domain show the full phenotype of Gfi1 null mice. However, Gfi1:Gfi1b knock-in mice show almost normal pre-T-cell and neutrophil development, but lack properly formed inner ear hair cells. Hence, our findings show that an intact SNAG domain is essential for all functions of Gfi1 and that Gfi1b can replace Gfi1 functionally in haematopoiesis but, surprisingly, not in inner ear hair cell development, demonstrating that Gfi1 and Gfi1b have equivalent and domain-dependent, cell type-specific functions.

Figure 1

Generation of Gfi1:Gfi1b and Gfi1:P2A knock-in mice. (A) Schematic structure of Gfi1 and Gfi1b proteins. (B) Schematic representation of the targeting vector (top), the murine Gfi1 locus (middle) and the targeted allele (bottom). Exons are shown as open boxes and the Gfi1b coding region as a grey box. The removal of the neomycin resistance gene was achieved by mating to CMV-Cre transgenic mice. The probes used for genotyping by Southern blotting are indicated. B, BamHI; B^*, BamHI deleted; E, EcoRI; H, HindIII; S, SalI; Sc, SacI; Sp, SpeI; St, StyI; TK, thymidine kinase; wt, wild type; X, XbaI. (C) Schematic representation of the knock-in strategy for introducing the P2A mutation into the Gfi1 locus. Top: targeting vector; middle: Gfi1 locus; bottom: targeted allele. Exons are depicted as open boxes. EV, EcoRV. Loss of the neomycin resistance gene was carried out by mating with Flp recombinase-expressing mice. Localization of the 5′ probe for Southern blotting is shown.

Figure 2

Normal thymocyte and granulocyte development requires Gfi1 with an intact SNAG domain. (A) Total thymocyte numbers of 4- to 7-week-old animals (n=3–8). (B) Fluorescence-activated cell sorting (FACS) analysis of CD4 and CD8 expression on electronically gated c-Kit⁺ thymocytes (n=3). (C) FACS analysis of bone marrow and blood cells stained with labelled anti-Mac-1 and anti-Gr-1 antibodies (n=4).

Figure 3

Early stages in haematopoietic development are influenced by the repressor function of Gfi1. (A,B) Representative pictures of May–Grünwald–Giemsa-stained cytospins of bone marrow cells (after erythrocyte lysis; A) and blood smears (B) from the indicated mouse mutants (n=3–8). Note atypical features of some monocytes in Gfi1^−/− and Gfi1^P2A/P2A mutants (n=4–8). (C) Orderly granulocytic differentiation in Gfi1^+/+ bone marrow and accumulation of atypical monocytoid cells beyond the myelocyte stage in Gfi1^−/− and Gfi1^P2A/P2A mutants. The pictures were taken with a × 100 objective and a × 10 ocular (n=3–8).

Figure 4

Evaluation of inner ears of Gfi1^−/−, Gfi1^1b/1b and Gfi1^P2A/P2A mice. (A) Auditory brainstem response (ABR) thresholds of both Gfi1^−/− and Gfi1^1b/1b mice at age 3–3.5 months were determined to be over 100 dB at 8 kHz and over 90 dB at 16 and 32 kHz, whereas ABR thresholds of age-matched wild-type mice were within the normal range of hearing. N=2 for wild type; n=4 for all mutants. Error bars represent 1 standard error measured. (B–E) Cochlear epithelia from the mid-basal turn of P0 (B) wild type, (C) Gfi1^−/−, (D) Gfi1^1b/1b and (E) Gfi1^P2A/P2A mice. Cochlear epithelia were stained with an antibody for myosin VI (red) that labels the hair cells' cuticular plate and cytoplasm, and with phalloidin (green) to label filamentous actin at cell boundaries and in the stereocilia. (F–I) Cochlear epithelia from the mid-basal turn of P0 (F) wild type, (G) Gfi1^−/−, (H) Gfi1^1b/1b and (I) Gfi1^P2A/P2A mice. Cochlear epithelia were stained with an antibody for acetylated tubulin (green) that labels the hair cell kinocilia, pillar cells and various other tubulin-based structures in the cochlear sensory epithelium, and with phalloidin (red) to label filamentous actin at cell boundaries and in the stereocilia. IHC, inner hair cell; OHC, outer hair cell, Scale bar, 20 μM.