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doi: 10.1242/10.1242/dev.00190

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The zinc finger transcription factor Gfi1, implicated in lymphomagenesis, is required for inner ear hair cell differentiation and survival

¹ Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
² Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA
³ Division of Hematology/Oncology, Howard Hughes Medical Institute, Children's Hospital and the Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA
⁴ Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA
⁵ Institute for Cellular Therapeutics and Department of Surgery, University of Louisville, Louisville, KY 40202, USA
⁶ Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA

View larger version (131K): [in a new window]   Fig. 1. Gfi1 wild-type expression pattern. In situ hybridization using a specific antisense probe derived from the 3' UTR of Gfi1 and immunohistochemistry using a Gfi1-specific antibody. For in situ, positive cells are purple, and for immunohistochemistry positive cells are brown and counterstained with Hematoxylin (purple). (A) Sagittal section of E12.5 embryo. Areas of high expression of Gfi1 mRNA are denoted and include the developing brain (FB, forebrain; HB, hindbrain), optic epithelia (Op), dorsal root ganglia (DRG) and gut epithelia (Gut). In addition, Gfi1 mRNA is expressed in the developing otic vesicle (OV) and several ganglia including the trigeminal (V) and vestibulo-cochlear ganglia (VIII) (see Fig. 2A for enlarged view of ear and associated ganglia). (B) Sagittal section of E18.5 retina showing Gfi1 expression primarily in the retinal ganglion cell layer. (C) Sagittal section of E16.5 retina showing immunohistochemistry of Gfi1 expression in specific cells denoted with arrows that are likely to be retinal ganglion cells. (D) Gfi1 expression in E16.5 section through the upper lip and mouth area where the whiskers develop. Positive cells around the shaft of the whiskers are thought to be the Merkel cells. (E) Gfi1 expression in E15.5 section through the skin. Positive cells are located under the touch domes and correspond in size and position to the Merkel cells. (F) E18.5 section through the upper lip and mouth area where the whiskers develop. Immunohistochemically positive cells (brown cells) around the shaft of the whiskers denoted by arrows are thought to be the Merkel cells. (G) Gfi1 expression in E16.5 section of the olfactory epithelia. (H) Gfi1 expression in E18.5 section of the tongue and its dorsal epithelium where taste papilla develop. (I) Gfi1 expression in E15.5 section of the developing lung. Clusters of cells as well as individual cells express Gfi1. (J) Gfi1 protein expression in E16.5 section of the developing lung. Clusters of cells denoted by arrows express Gfi1. (K) Gfi1 mRNA expression in E15.5 section of the developing thymus.

View larger version (123K): [in a new window]   Fig. 2. Gfi1 wild-type expression in the sensory epithelia of the inner ear. mRNA in situ hybridization using a specific antisense probe derived from the 3' UTR of Gfi1 and immunohistochemistry using a Gfi1-specific antibody. For in situ hybridization, positive cells are purple, and for immunohistochemistry, positive cells are brown and counterstained with Hematoxylin (purple). (A) Sagittal section of E12.5 embryo. Gfi1 is expressed in the otic vesicle (OV) and several ganglia including the vestibulo-cochlear ganglia (VIII). (B) Sagittal section of E14.5 ear. Gfi1 is expressed in the saccule (S) and utricle (U), but high levels of expression are not yet visible in the cochlea (Co). (C) Sagittal section of E16.5 ear. Gfi1 is expressed in the saccule (S), utricle (U) and cochlea (Co). An arrow indicates the hair cells in the organ of Corti. (D) Sagittal section of E18.5 cochlea. High expression levels of Gfi1 mRNA can be seen in the hair cells. The inner hair cell is indicated by a red arrow and the outer hair cells by green arrows. Lower levels of expression can be seen in the cochlear ganglia (asterisk). (E) Sagittal section of E14.5 ear. Gfi1 protein is expressed in the saccule (S) and utricle (U). (F) Sagittal section of E18.5 cochlea. High expression levels of Gfi1 protein can be seen in the hair cells. The inner hair cell is indicated by a red arrow and the outer hair cells by green arrows.

View larger version (126K): [in a new window]   Fig. 3. Expression profile of markers in Gfi1 mutant ears. (A,C,E,G,I,K) Gfi1 wild type (+/+); (B,D,F,H,J,L) Gfi1 null (-/-) mice. The inner hair cell is denoted by a red arrow and the outer hair cells by green arrows. (A-F) Anti-myosin VI/VIIa (Myo) staining of inner ear hair cells showing abnormal cell shape (arrow) and organization (arrowhead) at E13.5 (A,B) and E14.5 (C,D) in the developing vestibular organs. Note that some mutant hair cells are maintained in the support cell layer (arrowhead). (E,F) Hair cells in the mutant organ of Corti are disorganized. (G,H) Math1 in situ showing that mRNA expression in the organ of Corti at E17.5 is unaffected in the mutants, but hair cells are disorganized. (I,J) Brn3c in situ showing Brn3c mRNA expression in the organ of Corti at E18.5 is unaffected in the mutant, but the hair cells are disorganized. (K,L) Anti-TUJ1 staining marking the neurons at E17.5 showing abnormal TUJ1 staining in the outer hair cells of the mutant. Note that in wild type, innervation stops at the base of the outer hair cells, but the TUJ1 staining of the mutant outer hair cells appears to be all over the cell body. The inner hair cell is indicated by a red arrow and the outer hair cells by green arrows.

View larger version (93K): [in a new window]   Fig. 4. Gfi1 and Math1 expression in hair cells of Math1 and Gfi1 mutants. (A-F) Gfi1 expression in wild-type and Math1 mutant mice. (G-J) Math1/ß-Gal expression in Gfi1 mice. (A,C,E,G,I) Wild-type littermates (+/+). (B,D,F) Math1 null mutants (-/-). (H,J) Gfi1-null mutants (-/-). (A,B) Gfi1 mRNA expression in both wild-type and Math1 null otic vesicle (OV) at E12.5. (C,D) Gfi1 protein expression in wild-type but not Math1 null otic vesicle (OV) at E12.5. Arrow indicates position of Gfi1-positive cells (E,F) Loss of Gfi1 mRNA expression at E18.5 in the Math1 null mutant utricle (U), saccule (S) and cochlea (Co), presumably due to loss of hair cells. (G-J) Math1/ß-Gal expression (blue) and hair cell placement are visualized by the ß-galactosidase cassette driven by the Math1 promoter in P0 coronal sections of the ear. Sections are counterstained with Nuclear Fast Red (pink). Some of the stereocilli are visible (small arrows). (G) Gfi1 wild-type saccule showing a single layer of blue stained hair cells and a single layer of pink stained support cells. (H) Gfi1-null saccule showing the disorganization of the hair cell `layer'. Note that some hair cells are placed completely beneath others in the layer (large arrows) (see Fig. 3A-D for comparison). (I) Gfi1 wild-type cristae showing normal development of hair cells and expression of Math1. (J) Gfi1-null cristae showing normal development of hair cells and expression of Math1.

View larger version (111K): [in a new window]   Fig. 5. Cochlear hair cells degenerate rapidly in Gfi1 mutants. Math1/ß-Gal expression (blue) and hair cell placement are visualized by the ß-galactosidase cassette driven by the Math1 promoter in whole mounts of the cochlea. (A,C,E,I) Gfi1 wild type (+/+). (B,D,F-H,J-L) are Gfi1 null (-/-). (A,B) Math1/ß-galactosidase at E15.5. (A) Gfi1 wild-type (+/+) region of the basal cochlea, showing the development of the orderly arrangement of three outer and one inner row of hair cells. (B) Gfi1 null (-/-) basal cochlea showing Math1 expression and hence specification of hair cells is unaffected in the null cochlea. However, the characteristic rows of inner and outer hair cells are not as clearly defined as in wild type. (C,D) Math1/ß-Gal expression at E17.5. (C) Gfi1 wild-type region of the basal cochlea showing the orderly arrangement of three outer and one inner row of hair cells. In the wild-type mouse, these orderly rows are continuous along the entire length of the cochlea. (D) Gfi1-null basal cochlea showing persistent disorganization of hair cells and decreased hair cell numbers. (E-H) Math1/ß-galactosidase at P0. (E) Gfi1 wild-type region of the basal cochlea showing the orderly arrangement of three outer and one inner row of hair cells. (F) Gfi1 null basal cochlea, (G) Gfi1 null medial cochlea, (H) Gfi1 null apical cochlea. Note the degeneration of the cochlea in a basal to apical gradient. (I-L) Math1/ß-galactosidase at P3. (I) Gfi1 wild-type region of the basal cochlea showing the orderly arrangement of three outer and one inner row of hair cells. (J) Gfi1 null basal cochlea; (K) Gfi1 null medial cochlea; (L) Gfi1 null apical cochlea. Note the degeneration of the cochlea in a basal to apical gradient.

View larger version (88K): [in a new window]   Fig. 6. The inner ear phenotype in Gfi1 mutant mice. (A,C,E,G,I) are Gfi1 wild-type (+/+) mice and (B,D,F,H,J) are Gfi1 null (-/-) mice. (A,B) Hematoxylin and Eosin (H&E) morphological stains of the organ of Corti at P14. Note the development of the organ of Corti with one inner (red arrow) and three outer (green arrows) hair cells in the wild type, but its complete degeneration in the mutant section. (C,D) Hematoxylin and Eosin morphological stains of the saccule at 5 months of age. Note the presence of hair cells with stereocilli in both the wild-type and mutant. However, the mutant saccule has disorganized layering of the hair and support cells. (E-J) The progressive degeneration of the cochlear ganglion neurons in the mutant mice. (E-H) Sections stained with anti-activated-caspase-3 (C3) as a marker of apoptosis (brown cells) and counterstained with Hematoxylin. Arrows in E, F and H indicate activated-caspase-3-positive cells. (E,F) P7 mice. Note similar cell densities and levels of apoptosis in both samples. (G,H) P21 mice. Note that by P21 we see a slightly lower cell density in the mutant as well as a high level of apoptosis in the mutant that is not present in the wild-type mouse. (I,J) 5-month-old mice. Sections stained with Hematoxylin and Eosin.

View larger version (174K): [in a new window]   Fig. 7. Ultrastructural analysis of the organ of Corti at E 18.5 using transmission electron microscopy (TEM). (A,C) Gfi1 wild-type mice. (B,D) Gfi1-null mice. (A,B) An overview of the outer hair cell. (C,D) The same outer hair cells as in A,B at a higher magnification. Double arrowheads indicate stereocilli of the wild-type outer hair cell, while single arrowheads indicate the blebbing seen in the mutant hair cell. Note the shrinkage of the cell body in the mutant outer hair cell. The mutant hair cell also contains vacuoles (asterisk). There is no detectable difference between wild-type and mutant hair cell mitochondria.

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