Pax2-Islet1 Transgenic Mice Are Hyperactive and Have Altered Cerebellar Foliation

Abstract

The programming of cell fate by transcription factors requires precise regulation of their time and level of expression. The LIM-homeodomain transcription factor Islet1 (Isl1) is involved in cell-fate specification of motor neurons, and it may play a similar role in the inner ear. In order to study its role in the regulation of vestibulo-motor development, we investigated a transgenic mouse expressing Isl1 under the Pax2 promoter control (Tg ^+/- ). The transgenic mice show altered level, time, and place of expression of Isl1 but are viable. However, Tg ^+/- mice exhibit hyperactivity, including circling behavior, and progressive age-related decline in hearing, which has been reported previously. Here, we describe the molecular and morphological changes in the cerebellum and vestibular system that may cause the hyperactivity of Tg ^+/- mice. The transgene altered the formation of folia in the cerebellum, the distribution of calretinin labeled unipolar brush cells, and reduced the size of the cerebellum, inferior colliculus, and saccule. Age-related progressive reduction of calbindin expression was detected in Purkinje cells in the transgenic cerebella. The hyperactivity of Tg ^+/- mice is reduced upon the administration of picrotoxin, a non-competitive channel blocker for the γ-aminobutyric acid (GABA) receptor chloride channels. This suggests that the overexpression of Isl1 significantly affects the functions of GABAergic neurons. We demonstrate that the overexpression of Isl1 affects the development and function of the cerebello-vestibular system, resulting in hyperactivity.

Keywords: Age-related deterioration of Purkinje cells; Attention deficit hyperactivity disorder; Calcium homeostasis; Cerebellum; Foliation defects; GABA signaling; Hyperactivity; Islet1 transcription factor; Purkinje cells; Transgenic mouse; Vestibular system.

Fig. 1

Behavior tests in an open field. Tg ^+/− mice display GABA receptor-mediated increased locomotor activity. a Traces of locomotion in an open field show significant hyperactivity and circling of Tg ^+/− mice. b Motor coordination of WT (n = 6) and Tg ^+/− (n = 3) mice on the accelerating rotarod was analyzed in three trials/session (repeated measures ANOVA: genotype effect, ***P < 0.0001; session effect, *P < 0.0117). The values represent means of three trials/session ± SEM. c Quantification of mouse locomotion in an open field showing that picrotoxin significantly reduced hyperactivity of mutants (n = 6 Tg ^+/−, n = 5 WT) but did not correct circling. There was a significant alleviation of the locomotor activity of Tg ^+/− mice compared to WT after the application of haloperidol-decanoate at 48 h. Data represent mean ± SEM (**P < 0.01; ***P < 0.001)

Fig. 2

The pattern of innervation in the vestibular system at P3 (a, b). Similar dense innervation of WT and Tg ^+/− sensory epithelia is shown by anti-tubulin staining of the fibers in whole mount. A misguided nerve fiber with the same aberrant trajectory was repeatedly observed in Tg ^+/− (white arrow). Scale bar 500 μm. c, d Less fibers in the transgenic saccule at P1. The utricle and anterior and horizontal canals are typically labeled at the same intensity. Lipophilic dyes were injected into the cerebellum. e, f A reduction of sensory epithelium of the saccular maculae in Tg ^+/− (e) compared to WT (f) at P6. Hair cells are visualized using anti-Myo7a (red) in whole-mount immunohistochemistry. Quantification of area saccule (g) and counting of Myo7a⁺ cells per 100 μm² (h) is done by ImageJ. The values represent means ± SEM (N = 3 individuals/group and 6 × 100 μm²/3 individuals/group). **P < 0.01; ***P < 0.001. Scale bar 500 μm. U, Utricle; Ac, anterior canal crista; Hc, horizontal canal crista; S, saccule

Fig. 3

Total number of calretinin-labeled neurons in the vestibular ganglion of WT (a) and Tg ^+/− (b). c The number of calretinin⁺ neurons in WT and Tg ^+/− ganglia is similar at 6 months of age (6M) and it is declining with age at a similar rate in both WT and Tg ^+/− (11 months of age, 11M). Single immunostaining with anti-calretinin (red) and visualization of nuclei with Hoechst (blue). Scale bar 500 μm

Fig. 4

Changes in the cerebellum. a, b P3 sagittal sections using Hoechst nuclear staining show the different organizations in the control (WT) and mutant (Tg ^+/−) littermate cerebellar foliation (insert a, b) and disorganization of lobule I + II. Note absence of a recognizable anterior medullary velum (AMV) and the rostral expansion of a hemilobe only in the transgenic mouse (arrowhead). c, d Pax2 (red) and calbindin (green; Purkinje cells) staining of sagittal sections of the anterior lobe of the cerebellar vermis at P3 shows a comparable distribution of Purkinje cells and Pax2⁺ cells in WT (C) and Tg ^+/− lobules (d). The altered foliation of lobules I–III is obvious in the Tg ^+/− cerebellum. e, f Hematoxylin-eosin staining of the brain sections at the level of vermis at P15. The predominant phenotype of altered formation of vermis lobules leading to the fusion of I–III and a hemilobule on top of or as part of the anterior medullary velum (arrow) is detected in the Tg ^+/− cerebellum. The remnant of the inferior colliculus (IC) is denoted by a red asterisk in the Tg ^+/− midbrain. The superior colliculus (SC) and IC are outlined by blue- and red-dashed lines, respectively. g, h The adult Tg ^+/− cerebellum shows the defect in the foliation of the anterior lobe compared to WT littermates as shown by Hoechst staining of the granule cell layer nuclei. The fissure (*) between anterior folia I/II and III failed to form properly, leading to the fusion of the lobules. A hemilobule is on top of or as part of the anterior medullary velum (arrow). The lobules IV–V in Tg ^+/− differ from controls. Roman numerals depict cerebellum lobules. AMV, anterior medullary velum; Calb, calbindin; EGL, external granule layer; IGL, internal granule layer; IC, inferior colliculus; ML, molecular layer; SC, superior colliculus. Scale bar 100 μm (a–d) and 1000 μm (e–h)

Fig. 5

Changes in the inferior colliculus of transgenic mice. Representative confocal images shows the expression of NF200 (red) and calbindin (green) in cerebellar sections from P16 WT (a, c) and transgenic (b, d) mice. c, d The brachium of the inferior colliculus (arrow) is profoundly reduced in Tg ^+/− compared to WT mice. An arrowhead indicates an aberrant tract of white matter fibers in the transgenic cerebellum expanding along the hemilobe that is fused with the anterior medullary velum (AMV). e, f The auditory brainstem response (ABR) waveforms of 3-week-old mice to a click stimulus. Individual responses at 80 dB SPL click are represented. Major waves I–IV are indicated above the peaks. The results show that the amplitude of ABR wave IV is lower, and the latency of ABR waves is prolonged in Tg ^+/− compared to WT. Scale bar 1000 μm (a, b) and 500 μm (c, d)

Fig. 6

Morphological changes in the adult transgenic cerebellum. Hoechst staining of the granule cell layer nuclei of the cerebellum shows a differential penetrance leading to variable foliation defects in Tg ^+/−. Severe foliation defects (a) compared to less affected Tg ^+/− (b–f). The formation of the anterior lobe (lobules I–V) is altered in all Tg ^+/−. The area of the anterior lobe is outlined by white dashed line and shows defects in all transgenic mice, including the AMV aberration. AMV, anterior medullary velum. Scale bar 1000 μm

Fig. 7

Changes in Purkinje cells in the anterior lobe (detail of lobules I–II). a, b Purkinje cells (PCs) are oriented in a monolayer with dendrites projecting into the ML at P16, as visualized by calbindin staining (green; nuclear staining, blue). More calbindin-negative PCs are visible in the Tg ^+/− anterior lobe (b, arrows). The density of PC dendrites stained by calbindin is noticeably reduced in Tg ^+/− compared to WT (a) at P16. c, d A profound reduction of calbindin expression in PCs and PC dendrites in the ML progresses with increasing age in the Tg ^+/− anterior lobe (d), as visualized by lack of staining with anti-calbindin. Anti-NF200 staining (red) of basket interneuron fibers wrapped around Purkinje cell bodies (arrowheads) is still detected in 4-month-old Tg ^+/− mice. ML, molecular layer; PCL, PC layer; GCL, granule cell layer. Scale bar 200 μm (a–b), 100 μm (c, d)

Fig. 8

Reduction of Purkinje cell (PC) immunogenicity and apparent loss of PC dendrites in the molecular layer of the adult transgenic cerebella. PCs form a monolayer with dense network of dendrites in the ML throughout all the lobules in control cerebellum (a). At 6 months, a profound loss of calbindin expression in PCs and PC dendrites in the ML progresses in all lobules of the Tg ^+/− cerebella (b) as visualized by lack of staining with anti-calbindin (green). A near complete loss of calbindin expression in PCs and PC dendrites (arrows in d, f) is detected in the Tg ^+/− cerebella compared to WT, in detail shown in the lobules I–II and X (c, e). ML, molecular layer; PCL, PC layer; GCL, granule cell layer. Scale bar 1000 μm (a, b); 250 μm (c–f)

Fig. 9

Altered distribution of calretinin-labelled cells in lobules X and IX of the transgenic cerebellum. Calretinin⁺ cells are primarily found in lobules X and half of IX as shown by calretinin staining (red) in both WT (a) and Tg ^+/− (b) cerebella. Double staining with anti-Calbindin (Calb, green) and anti-Calretinin (red) and visualization of nuclei with Hoechst staining of 100 μm sections of P16 cerebella. Scale bar 500 μm. Quantification of calretinin staining in lobules IX and X of the cerebellum at P16 (c) and 8-month-old (d) using ImageJ. The values represent an average percentage of calretinin⁺ area/lobule area ± SEM (n = 6 Tg ^+/− and 6 WT/each age group), t test *, P < 0.05

Fig. 10

The expression of Isl1 in the transgenic cerebellum at P3. Confocal microscopy of 100 μm sections shows the expression of Isl1 in the transgenic cerebellum (lobule IX) indicated by white arrows. Double staining with anti-Pax2 (b, red) and anti-Isl1 (c, green) and visualization of nucleus with Hoechst staining (a) and overlay of fluorescent channels (d). Scale bar 500 μm (whole cerebellum), 25 μm (detail a–d)

Fig. 11

RT-qPCR analysis of gene expression changes induced by the transgenic expression of Isl1 in the cerebellum. a Representative 2 % agarose gel electrophoresis of RT-qPCR products shows the expression of Isl1 in Tg ^+/− cerebella of 1-, 7-, and 11-month-old mice (two samples/genotype/age). Hprt1 was used as the reference gene. Lane: PC, positive control (hindbrain); NC, negative control (H₂O). b The expression of genes was analyzed in WT and Tg ^+/− cerebella from 1-month-old mice; the relative expression levels were quantified using −ΔΔCq method. The data represent the expression of mRNA relative to the control cerebella, normalized by the reference gene Hprt1. *P < 0.05; **P < 0.01, t test. The values are means ± SEM (each experiment in duplicate; N = 8/group). Dlg4, discs large homolog 4; Cacng1, voltage-dependent calcium channel gamma subunit 1; Cr, calretinin