Developmental Characterization of Schizophrenia-Associated Gene Zswim6 in Mouse Forebrain

Abstract

Schizophrenia is a devastating neuropsychiatric disease with a globally 1% life-long prevalence. Clinical studies have linked Zswim6 mutations to developmental and neurological diseases, including schizophrenia. Zswim6's function remains largely unknown. Given the involvement of Zswim6 in schizophrenia and schizophrenia as a neurodevelopmental disease, it is important to understand the spatiotemporal expression pattern of Zswim6 in the developing brain. Here, we performed a comprehensive analysis of the spatiotemporal expression pattern of Zswim6 in the mouse forebrain by in situ hybridization with radioactive and non-radioactive-labeled riboprobes. Zswim6 mRNA was detected as early as E11.5 in the ventral forebrain. At E11.5-E13.5, Zswim6 was highly expressed in the lateral ganglionic eminence (LGE). The LGE consisted of two progenitor populations. Dlx⁺;Er81⁺ cells in dorsal LGE comprised progenitors of olfactory bulb interneurons, whereas Dlx⁺;Isl1⁺ progenitors in ventral LGE gave rise to striatal projection neurons. Zswim6 was not colocalized with Er81 in the dorsal LGE. In the ventral LGE, Zswim6 was colocalized with striatal progenitor marker Nolz-1. Zswim6 was highly expressed in the subventricular zone (SVZ) of LGE in which progenitors undergo the transition from proliferation to differentiation. Double labeling showed that Zswim6 was not colocalized with proliferation marker Ki67 but was colocalized with differentiation marker Tuj1 in the SVZ, suggesting Zswim6 expression in early differentiating neurons. Zswim6 was also expressed in the adjacent structures of medial and caudal ganglionic eminences (MGE, CGE) that contained progenitors of cortical interneurons. At E15.5 and E17.5, Zswim6 was expressed in several key brain regions that were involved in the pathogenesis of schizophrenia, including the striatum, cerebral cortex, hippocampus, and medial habenular nucleus. Zswim6 was persistently expressed in the postnatal brain. Cell type analysis indicated that Zswim6 mRNA was colocalized with D1R-expressing striatonigral and D2R-expressing striatopallidal neurons of the adult striatum with a higher colocalization in striatopallidal neurons. These findings are of particular interest as striatal dopamine D2 receptors are known to be involved in the pathophysiology of schizophrenia. In summary, the comprehensive analysis provides an anatomical framework for the study of Zswim6 function and Zswim6-associated neurological disorders.

Keywords: Zswim; basal ganglia; dopamine; schizophrenia; striatum.

Figure 1

Zswim6 mRNA expression pattern in E11.5 mouse forebrain. Expression pattern of Zswim6 mRNA analyzed by in situ hybridization with digoxigenin-labeled probes (A1–A6) and ³⁵S-labeled probes (B1–B10) from rostral to caudal levels. (C) Zswim6 is strongly expressed in the SVZ of LGE (C,C1), but is weaker in the SVZ of MGE (C,C2). A, amygdala; CGE, caudal ganglionic eminence; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; MZ, mantel zone; Sep, septum; SVZ, subventricular zone; VZ, ventricular zone.

Figure 2

Zswim6 mRNA expression pattern in E12.5 mouse forebrain. Expression pattern of Zswim6 mRNA analyzed by in situ hybridization with digoxigenin-labeled probes (A1–A6) and ³⁵S-labeled probes (B1–B10) from rostral to caudal levels. (C) Zswim6 is strongly expressed in the SVZ of LGE (C,C1), but is weaker in the SVZ of MGE (C,C2). (D) Zswim6 is highly expressed in the SVZ of LGE. There is a boundary at the dorsal-most part of the SVZ of LGE (arrowheads). A, amygdala; CGE, caudal ganglionic eminence; CP, cortical plate; H, hypothalamus; Hip, hippocampus; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; MZ, mantel zone; SVZ, subventricular zone; VZ, ventricular zone.

Figure 3

Zswim6 mRNA expression pattern in E13.5 mouse forebrain. Expression pattern of Zswim6 mRNA detected by in situ hybridization with digoxigenin-labeled probes (A1–A6) and ³⁵S-labeled probes (B1–B9) from rostral to caudal levels. (C) Zswim6 is strongly expressed in the SVZ of LGE (C,C1). Weak expression of Zswim6 is present in the SVZ of MGE (C,C2). A, amygdala; CGE, caudal ganglionic eminence; CP, cortical plate; FF, Fields of Forel; H, hypothalamus; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; MZ, mantel zone; SVZ, subventricular zone; VZ, ventricular zone.

Figure 4

Zswim6 mRNA expression pattern in E15.5 mouse forebrain. Expression pattern of Zswim6 mRNA detected by in situ hybridization with digoxigenin-labeled probes (A1–A7) and ³⁵S-labeled probes (B1–B9) from rostral to caudal levels. Zswim6 is mainly expressed in the SVZ of developing striatum with a border at the dorsal-most part (arrowheads in C1). Zswim6 is expressed in the developing cortex (C2). A, amygdala; CGE, caudal ganglionic eminence; CP, cortical plate; Hip, hippocampus; IZ, intermediate zone; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; Sep, septum; SVZ, subventricular zone; VZ, ventricular zone.

Figure 5

Zswim6 mRNA expression pattern in E17.5 mouse forebrain. Expression pattern of Zswim6 mRNA detected by in situ hybridization with digoxigenin-labeled probes (A1–A6) and ³⁵S-labeled probes (B1–B9) from rostral to caudal levels. A, amygdala; AcbC, nucleus accumbens, core; AcbSh, nucleus accumbens, shell; CP, cortical plate; GP, globus pallidus; H, hypothalamus; HIP, hippocampus; IZ, intermediate zone; LOT, nucleus of the lateral olfactory tract; MGE, medial ganglionic eminence; MHb, medial habenular nucleus; Pd, pallidum primordium; Pir, piriform cortex; POA, preoptic area; Sep, septum; SVZ, subventricular zone; T, thalamus; Tu, olfactory tubercle.

Figure 6

Zswim6 mRNA expression pattern in P0 mouse forebrain. Zswim6 mRNA signals were detected by in situ hybridization with digoxigenin-labeled probes (A1–A7) and ³⁵S-labeled probes (B1–B10) from rostral to caudal levels at P0. Moderate to strong Zswim6 signals were expressed in the olfactory bulb, dorsal and ventral striatum, cortex, hippocampus, thalamus, amygdala and medial habenular nucleus. A, amygdala; AcbC, nucleus accumbens, core; AcbSh, nucleus accumbens, shell; AO, anterior olfactory nucleus; Cere, cerebellum; Cg, cingulate cortex; CP, cortical plate; CTX, cortex; DG, dentate gyrus; GP, globus pallidus; GrO, granular cell layer of the olfactory bulb; H, hypothalamus; Hip, hippocampus; MHb, medial habenular nucleus; Mi, mitral cell layer of the olfactory bulb; ne, neuroepithelium; OB, olfactory bulb; Pir, piriform cortex; T, thalamus; Tu, olfactory tubercle; STh, subthalamic nucleus; STR, striatum; ZI, zona incerta.

Figure 7

Zswim6 mRNA expression pattern in P7 mouse forebrain. Zswim6 mRNA signals were detected from rostral to caudal levels at P7 (A–C). Moderate to strong Zswim6 signals were expressed in the olfactory bulb (D,E), dorsal and ventral striatum (A–C, I), cortex (F–I), hippocampus (J–L), thalamus (M,N), cerebellum (O), and medial habenular nucleus (J). AcbC, nucleus accumbens, core; AcbSh, nucleus accumbens, shell; AO, anterior olfactory nucleus; Cere, cerebellum; Cg, cingulate cortex; CP, cortical plate; CTX, cortex; DG, dentate gyrus; GP, globus pallidus; GrO, granular cell layer of the olfactory bulb; H, hypothalamus; Hip, hippocampus; MHb, medial habenular nucleus; Mi, mitral cell layer of the olfactory bulb; OB, olfactory bulb; Pir, piriform cortex; T, thalamus; Tu, olfactory tubercle; STh, subthalamic nucleus; STR, striatum; ZI, zona incerta.

Figure 8

Zswim6 mRNA expression pattern in P14 mouse forebrain. Zswim6 mRNA signals were detected from rostral to caudal levels at P14 (A1–A15). AcbC, nucleus accumbens, core; AcbSh, nucleus accumbens, shell; BLA, basolateral amygdala; Cbll, cerebellum; CTX, cortex; GP, globus pallidus; H, hypothalamus; Hip, hippocampus; La, lateral amygdaloid nucleus; LOT, nucleus of the lateral olfactory tract; MHb, medial habenular nucleus; OB, olfactory bulb; Pir, piriform cortex; SNC, substantia nigra; STR, striatum; T, thalamus; Tu, olfactory tubercle; ZI, zona incerta.

Figure 9

Zswim6 mRNA expression pattern in adult mouse brain. Zswim6 mRNA signals were detected from lateral to medial levels in parasagittal sections of the adult brain (A1–A6). Cbll, cerebellum; FrA, frontal association cortex; GrO, granular cell layer of the olfactory bulb; Hip, hippocampus; MO, medial orbital cortex; OB, olfactory bulb; Pir, piriform cortex; PrL, prelimbic cortex; RSD, retrosplenial dysgranular cortex; RSG, retrosplenial granular cortex; Str, striatum; Tu, olfactory tubercle.

Figure 10

Zswim6 are expressed in Nolz1-positive neurons in the LGE at E13.5 and striatonigral and striatopallidal neurons in adult brains. Zswim6 is mainly expressed in the SVZ of the LGE at E13.5 (A1,B1), whereas Ki67-positive (A2) and Tuj1-positive (B2) cells are located in the VZ and the MZ of the LGE, respectively. In the overlapping zone (A3), Zswim6-positive cells express none or at most low levels of Ki67 (arrows, A4). Cell expressing high levels of Ki67 contained none or low levels of Zswim6 (arrowheads, A4). Colocalization of Tuj1 protein and Zswim6 mRNA (B3) is detected in cells of SVZ (B4) and MZ (B5). (C,D) Both Zswim6 (C1) and Nolz-1 (C2) are expressed in the SVZ of LGE, and their expression domains are mostly overlapped in the ventral part of SVZ (C3). In the dorsal non-overlapping zone, cells expressing Zswim6 without Nolz-1 are present (C3, the area between arrowheads; C4, insets show high magnification of the cells indicated by arrows). In the Zswim6 and Nolz-1 overlapped regions, Zswim6 is colocalized with Nolz-1 (C5, insets show high magnification of cells indicated by arrowheads). At E15.5, Zswim6 mRNA (D1) is expressed in the SVZ of the striatal primordium, while Er81 marks the dorsal-most part of the striatal primordium (D2, between arrowheads). Although Zswim6 expression is overlapped with Er81 at the dorsal-most striatum (D3), at the single-cell level, Zswim6 mRNA signals (D4, arrows) are not co-localized with Er81-positive cells. (E,F) Confocal images of double in situ hybridization show that some Zswim6-positive cells (E1,E3,E4; arrowheads) were co-localized with D1R (E2–E4) in the mature striatum, whereas others express Zswim6 only (E1,E3,E4; arrows). In the double in situ hybridization of Zswim6 and D2R, some Zswim6-positive cells (F1,F3,F4; arrowheads) were co-localized with D2R (F2–F4) in the mature striatum, while others expressed Zswim6 only (F1,F3,F4; arrows). (G) Quantification of the co-localization ratios of Zswim6 and D1R or D2R in the mature striatum from the rostral to caudal levels. *p < 0.05, **p < 0.001, n = 3, student’s t-test. LGE, lateral ganglionic eminence; MZ, mantle zone; STR, striatum; SVZ, subventricular zone.