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. 2017 Mar 1;525(4):818-849.
doi: 10.1002/cne.24100. Epub 2016 Sep 16.

Cellular distribution of the fragile X mental retardation protein in the mouse brain

Diego A R Zorio  1 , Christine M Jackson  1 , Yong Liu  1 , Edwin W Rubel  2 , Yuan Wang  1   3
Affiliations

Cellular distribution of the fragile X mental retardation protein in the mouse brain

Diego A R Zorio et al. J Comp Neurol. .

Abstract

The fragile X mental retardation protein (FMRP) plays an important role in normal brain development. Absence of FMRP results in abnormal neuronal morphologies in a selected manner throughout the brain, leading to intellectual deficits and sensory dysfunction in the fragile X syndrome (FXS). Despite FMRP importance for proper brain function, its overall expression pattern in the mammalian brain at the resolution of individual neuronal cell groups is not known. In this study we used FMR1 knockout and isogenic wildtype mice to systematically map the distribution of FMRP expression in the entire mouse brain. Using immunocytochemistry and cellular quantification analyses, we identified a large number of prominent cell groups expressing high levels of FMRP at the subcortical levels, in particular sensory and motor neurons in the brainstem and thalamus. In contrast, many cell groups in the midbrain and hypothalamus exhibit low FMRP levels. More important, we describe differential patterns of FMRP distribution in both cortical and subcortical brain regions. Almost all major brain areas contain high and low levels of FMRP cell groups adjacent to each other or between layers of the same cortical areas. These differential patterns indicate that FMRP expression appears to be specific to individual neuronal cell groups instead of being associated with all neurons in distinct brain regions, as previously considered. Taken together, these findings support the notion of FMRP differential neuronal regulation and strongly implicate the contribution of fundamental sensory and motor processing at subcortical levels to FXS pathology. J. Comp. Neurol. 525:818-849, 2017. © 2016 Wiley Periodicals, Inc.

Keywords: RRID:AB_10805421; RRID:AB_1157880; RRID:AB_476743; cortical laminar heterogeneity; fragile X syndrome; sensory information processing; subcortical sensory systems; whole brain analyses.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors have no identified conflict of interest.

Figures

Figure 1
Figure 1
Antibody characterization for FMRP in the mouse brain. A, Western blot of 2F5 and 7G1 on brain samples from wild-type (WT) and FMR1 knockout (KO) mice. 50 μg of protein was loaded to each lane with β-actin as the loading control. B–D, 7G1 immunostaining in the WT (C) and FMR1 KO (B, D) mouse brain. Note the nonspecific staining in the lateral ventricle (VL; D). E, 2F5 immunostaining in the WT mouse brain. Scale bar: 2 mm (A, B, C, E); 500 μm (D).
Figure 2
Figure 2
FMRP (7G1) immunostaining in the sagittal plane at the medial level of the wild-type mouse brain. This level is comparable to Lateral 0.60 mm according to the Mouse Brain Atlas (Paxinos and Franklin, 2013). The 7G1 immunostaining (A) and the Nissl stain (B) were taken from two sections at the comparable level. Note differential FMRP levels across brain regions, high in the olfactory bulb, cerebral cortex, hippocampus, thalamus, and cerebellum, and generally low in the hypothalamus and midbrain. The brainstem contains many distinct cell groups that are rich in FMRP. Scale bar: 2 mm.
Figure 3
Figure 3
FMRP (7G1) immunostaining in the sagittal plane at the middle level of the wild-type mouse brain. This level is comparable to Lateral 1.80 mm according to the Mouse Brain Atlas (Paxinos and Franklin, 2013). The 7G1 immunostaining (A) and the Nissl stain (B) were taken from two sections at the comparable level. The two inserts were taken from a section at the level of Lateral 2.04 mm, corresponding to the location marked with * and +, respectively. Scale bar: 2 mm.
Figure 4
Figure 4
FMRP (7G1) immunostaining in the sagittal plane at the lateral level of the wild-type mouse brain. This level is comparable to Lateral 2.28 mm according to the Mouse Brain Atlas (Paxinos and Franklin, 2013). The 7G1 immunostaining (A) and the Nissl stain (B) were taken from two sections at the comparable level. Scale bar: 2 mm.
Figure 5
Figure 5
FMRP (7G1) immunostaining in the coronal plane through the spinal cord and caudal brainstem. A: Schematic drawing showing the levels of the coronal sections illustrated in the Figures 5–14 (red vertical lines). The black lines outline the mouse brain in the sagittal plane. The numerical rostral-caudal levels of each section is indicated on the top of the drawing, determined by comparing to the Mouse Brain Atlas (Paxinos and Franklin, 2013). B–C: An example of the original image (B) and generated threshold image (C) at a high magnification. D–E: The photomicrograph (D) and the threshold image (E) of 7G1 immunostaining in the spinal cord. The threshold image was generated as described in the Material and Methods. The pixels with an optical intensity at or above the threshold are in red. The border of the brain section is outlined with black lines. F–G: The photomicrograph (F) and the threshold image (G) of FMRP immunostaining in the caudal brainstem at the level of the inferior olive complex (IO). Scale bar: 100 μm (B); 1 mm (G; applies to D–G).
Figure 6
Figure 6
FMRP (7G1) immunostaining in the coronal plane at the level of the cerebellum and cochlear nuclei. A, The photomicrograph. B, The threshold image. Grey shades indicate the ventricle and areas without brain tissue. Scale bar: 1 mm.
Figure 7
Figure 7
FMRP (7G1) immunostaining in the coronal plane at the level of the inferior colliculus. A, The photomicrograph. B, The threshold image. Grey shades indicate the ventricle and areas without brain tissue. Scale bar: 1 mm.
Figure 8
Figure 8
FMRP (7G1) immunostaining in the coronal plane at the level of the superior colliculus. A, The photomicrograph. B, The threshold image. Grey shade indicates the ventricle. Scale bar: 1 mm.
Figure 9
Figure 9
FMRP (7G1) immunostaining in the coronal plane at the level of the isocortex and caudal thalamus. A, The photomicrograph. B, The threshold image. Grey shades indicate the ventricle and areas without brain tissue. Scale bar: 1 mm.
Figure 10
Figure 10
FMRP (7G1) immunostaining in the coronal plane at the level of the isocortex and middle thalamus. A, The photomicrograph. B, The threshold image. Grey shades indicate the ventricle and areas without brain tissue. Scale bar: 1 mm.
Figure 11
Figure 11
FMRP (7G1) immunostaining in the coronal plane at the level of the isocortex and rostral thalamus. A, The photomicrograph. B, The threshold image. Grey shades indicate the ventricle and areas without brain tissue. Scale bar: 1 mm.
Figure 12
Figure 12
FMRP (7G1) immunostaining in the coronal plane at the level of the caudal striatum. A, The photomicrograph. B, The threshold image. Grey shades indicate the ventricle and areas without brain tissue. Scale bar: 1 mm.
Figure 13
Figure 13
FMRP (7G1) immunostaining in the coronal plane at the level of the rostral striatum. A, The photomicrograph. B, The threshold image. Grey shades indicate the ventricle and areas without brain tissue. Scale bar: 1 mm.
Figure 14
Figure 14
FMRP (7G1) immunostaining in the coronal plane at the level of the rostral cortex (A–B) and olfactory bulb (C–D). A and C, The photomicrographs. B and D, The threshold images. Scale bar: 1 mm.
Figure 15
Figure 15
Cellular quantification of FMRP intensity in selected neuronal cell groups. A, The spinal cord and cell groups in the caudal brainstem and vestibular and auditory nuclei. B, Cell groups in the rostral brainstem, midbrain, cerebellum, as well as the cortical subplate and nuclei. C, Layers of isocortical areas. D, Cell groups in the olfactory bulb, hippocampus, thalamus, and hypothalamus. FMRP intensities were normalized as described in the Materials and Methods. Higher intensities indicate higher levels of FMRP staining at the individual cell level. For each cell group, 30 neurons were measured and averaged. Measurements for each cortical area were taken at levels comparable to Figure 24. Error bars are S.D. * indicates p < 0.01. ** indicates p < 0.001. *** indicates p < 0.0001. “ns” indicates not significant.
Figure 16
Figure 16
High-magnification images of FMRP (7G1) immunostaining in the spinal cord. A, The spinal cord at the approximate level of C-2 segment. Closer looks of the boxes are displayed in B and C. Dashed lines indicate the borders between the white and grey matters. B, The grey matter contains darkly (arrows) and relatively lightly (arrowheads) stained cells. C, The white matter contains small immunoreactive cell bodies (arrowheads), presumably glial cells. Scale bar: 500 μm (A), 100 μm (C, applies to B–C).
Figure 17
Figure 17
High-magnification images of FMRP (7G1) immunostaining in the caudal brainstem. A–B, High FMRP levels in the inferior olive complex (IO; A) and the magnocellular part of the lateral reticular nucleus (LRNm; B). C: The dorsal motor nucleus of the vagus nerve (DMX) exhibits stronger FMRP staining than the adjacent hypoglossal nucleus (XII). These two nuclei are also illustrated in J and K. D–E, The giganticellular reticular nucleus (GRN) contains darkly labeled neurons. E is at a higher magnification than D. F–G, The external cuneate nucleus (ECU) is rich in FMRP at both the rostral (F) and caudal (G) levels. H–I, FMRP immunoreactive neurons in the facial motor nucleus (VII; H) and the abducens nucleus (VI; I). J–K, 7G1 immunostaining (J) and the Nissl stain (K) on the adjacent sections containing DMX and XII. Note the relatively lower level of FMRP in XII as compared to the DMX. A–I are coronal sections while J–K are sagittal sections. Scale bar: 200 μm (A; applies to A, B, D, F–H, J–K); 200 μm (C, I); 100 μm (E).
Figure 18
Figure 18
High-magnification images of FMRP (7G1) immunostaining in the vestibular and auditory brainstem nuclei. A, FMRP staining in the dorsal and ventral cochlear nuclei (DCO and VCO). B, FMRP staining in the three subnuclei of the nucleus of the lateral lemniscus (NLLd, NLLh, and NLLV). C–D, FMRP staining in the medial, spinal, lateral, and superior portions of the vestibular nucleus (MV, SPIV, LAV, and SUV). D was taken from a section more rostral than C. E, FMRP staining in the superior olive complex and the nucleus of the trapezoid body (NTB). Scale bar: 500 μm.
Figure 19
Figure 19
High-magnification images of FMRP (7G1) immunostaining in the rostral brainstem. A, Intense FMRP staining in the tegmental reticular nucleus (TRN) and the pontine gray (PG). Dashed line indicates the midline. B, FMRP staining in the principal sensory nucleus of the trigeminal (PSV) and the motor nucleus of the trigeminal (V). Note particularly high FMRP levels in neurons embedded in the motor root of the trigeminal nerve (moV). C, FMRP immunoreactive neurons in the compact pact of the substantia nigra (SNc). Scale bar: 200 μm (A, B); 100 μm (C).
Figure 20
Figure 20
High-magnification images of FMRP (7G1) immunostaining in midbrain cell groups. A–C, FMRP immunoreactivity in the external and central nuclei of the inferior colliculus (ICe and ICc). B and C are at a higher magnification than A. D, FMRP staining is weak in the adjacent periaqueductal gray (PAG). Note darkly labeled neurons in the midbrain trigeminal nucleus (MEV). E–F, Intense FMRP staining in the intermediate gray layer (ig), but not other layers, of the superior colliculus (SC). F is a closer look of the box in E. G, Neurons in the parabigeminal nucleus are darkly labeled for FMRP immunocytochemistry. Scale bar: 250 μm (A, D, E); 50 μm (B; applies to B, C); 100 μm (F, G).
Figure 21
Figure 21
High-magnification images of FMRP (7G1) immunostaining in the thalamus. A, Strong FMRP staining in most thalamic cell groups but not the ventral portion of the lateral geniculate complex (LGv) and the intergeniculate leaflet (IGL). B–C, Closer looks of the neurons in the dorsal portion of the LG (LGd; B) and LGv (C). D, FMRP staining appears stronger in the medial habenula (MH) than the lateral habenula (LH). E–F, Closer looks of the neurons in the LH (E) and MH (F). Note FMRP intensity at the individual cell level is comparable between these two cell groups, although MH has a higher cell density. Dashed lines outline the approximate location of different cell groups. Scale bar: 500 μm (A; applies to A, D); 50 μm (F; applies to B, C, E, F).
Figure 22
Figure 22
High-magnification images of FMRP (7G1) immunostaining in the hypothalamus. FMRP staining is strong in the lateral mammillary nucleus (LM; A), the ventral part of the tuberomammillary nucleus (TMv; B), the dorsomedial nucleus of the hypothalamus (DMH; C), the medial part of the medial mammillary nucleus (MMm; D), the tuberal nucleus (TU; E), the parasubthalamic nucleus (PSTN; G), and the subthalamic nucleus (STN; H). In contrast, FMRP staining is weak in the ventral premammillary nucleus (PMv; F). Note that FMRP staining is weaker in the lateral part of the medial mammillary nucleus (MMl) than in the MMm (D). Dashed lines outline the approximate location of different cell groups. Scale bar: 100 μm.
Figure 23
Figure 23
High-magnification images of FMRP (7G1) immunostaining in the cerebellum. A, Strong FMRP staining in purkinje neurons (arrows) and granular layer but not in the molecular layer. B, FMRP staining in the three cerebellar nuclei. Dashed lines outline the approximate location of these nuclei. Scale bar: 100 μm (A); 200 μm (B).
Figure 24
Figure 24
High-magnification images of FMRP (7G1) immunostaining in the isocortex. A–B, The primary somatosensory area taken from the level comparable to Figure 12. B are closer looks of individual layers in A. C–D, The ventral part of retrosplenial area taken from the level comparable to Figure 9. D are closer looks of individual layers in C. E–F, The primary visual area taken from the level comparable to Figure 9. F are closer looks of individual layers in E. G–H, The anterior cingulate area taken from the level comparable to Figure 12. H are closer looks of individual layers in G. Note that neurons in the layer 4 are more lightly stained than other layers in the somatosensory (A–B) and retrosplenial (C–D), but not in the visual (E–F), areas. Neurons in the layers 2–3 exhibit stronger staining than other layers in the somatosensory (A–B) and visual (E–F) areas. Arrowheads in H indicate the neurons with uniquely strong staining. Scale bar: 200 μm (A; Applies to A, C, E, G); 50 μm (B; applies to B, D, F, H).
Figure 25
Figure 25
High-magnification images of FMRP (7G1) immunostaining in the cortical subplate and striatum. A–G, Serials images throughout the cortical subplate from caudal (A) to rostral (G). Dashed lines outline the approximate boundaries of a number of nuclei. Scale bar: 500 μm.
Figure 26
Figure 26
High-magnification images of FMRP (7G1) immunostaining in the hippocampus and the main olfactory bulb (MOB). A, FMRP staining in the hippocampus CA1. Arrows indicate stained dendrites of the pyramidal neurons. B, FMRP staining in the dentate gyrus (DG). C, FMRP staining in the hippocampus CA3. D, FMRP staining in the MOB. Scale bar: 100 μm.
Figure 27
Figure 27
High-magnification images of FMRP (7G1) immunostaining in the striatum and pallidum. FMRP staining is stronger in the caudoputamen (CP; A), the islands of Calleja (isl; C), the diagonal band nucleus (NDB; D), and the globus pallidus (GP; E), than in the nucleus accumbens (ACB; B) and the central amygdalar nuclei (CEA; F). Arrows in B indicate two more darkly labeled neurons in ACB. Dashed line in C outlines the boundary of one island of Calleja. Scale bar: 100 μm.
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