Distribution of ELOVL4 in the Developing and Adult Mouse Brain

Abstract

ELOngation of Very Long chain fatty acids (ELOVL)-4 is essential for the synthesis of very long chain-fatty acids (fatty acids with chain lengths ≥ 28 carbons). The functions of ELOVL4 and its very long-chain fatty acid products are poorly understood at present. However, mutations in ELOVL4 cause neurodevelopmental or neurodegenerative diseases that vary according to the mutation and inheritance pattern. Heterozygous inheritance of different ELOVL4 mutations causes Stargardt-like Macular Dystrophy or Spinocerebellar Ataxia type 34. Homozygous inheritance of ELOVL4 mutations causes more severe disease characterized by seizures, intellectual disability, ichthyosis, and premature death. To better understand ELOVL4 and very long chain fatty acid function in the brain, we examined ELOVL4 expression in the mouse brain between embryonic day 18 and postnatal day 60 by immunolabeling using ELOVL4 and other marker antibodies. ELOVL4 was widely expressed in a region- and cell type-specific manner, and was restricted to cell bodies, consistent with its known localization to endoplasmic reticulum. ELOVL4 labeling was most prominent in gray matter, although labeling also was present in some cells located in white matter. ELOVL4 was widely expressed in the developing brain by embryonic day 18 and was especially pronounced in regions underlying the lateral ventricles and other neurogenic regions. The basal ganglia in particular showed intense ELOVL4 labeling at this stage. In the postnatal brain, cerebral cortex, hippocampus, cerebellum, thalamus, hypothalamus, midbrain, pons, and medulla all showed prominent ELOVL4 labeling, although ELOVL4 distribution was not uniform across all cells or subnuclei within these regions. In contrast, the basal ganglia showed little ELOVL4 labeling in the postnatal brain. Double labeling studies showed that ELOVL4 was primarily expressed by neurons, although presumptive oligodendrocytes located in white matter tracts also showed labeling. Little or no ELOVL4 labeling was present in astrocytes or radial glial cells. These findings suggest that ELOVL4 and its very long chain fatty acid products are important in many parts of the brain and that they are particularly associated with neuronal function. Specific roles for ELOVL4 and its products in oligodendrocytes and myelin and in cellular proliferation, especially during development, are possible.

Keywords: cerebellum; cortex; hippocampus; seizure; spinocerebellar ataxia; stargardt-like macular dystrophy; very long chain fatty acids.

Figure 1

Distribution of ELOVL4 protein and mRNA in the mouse brain. (A) Immunohistochemical labeling (IHC) for ELOVL4 in the mouse brain at P60 is widespread, but shows region-specific distribution. (B) In situ hybridization (ISH) for Elovl4 mRNA in the mouse brain at P56 also is widespread and shows distribution similar to ELOVL4 protein in (A). (Image from the Allen Institute for Brain Science Allen Mouse Brain Atlas for the P56 mouse brain, image number 69059903_134. http://mouse.brain-map.org). CB, cerebellum; BG, basal ganglia; CTX, cerebral cortex; HPF, hippocampal formation; HY, hypothalamus; M, medulla; MB, midbrain; MOB, main olfactory bulb; P, pons; cc, corpus callosum; fo, fornix; VL, lateral ventricle. Scale bars = 1 mm.

Figure 2

Expression of ELOVL4 in development of cerebral cortex. (A) Developing cortex at E18 shows intense ELOVL4 labeling in numerous cells throughout the cortex (CTX). Labeling is especially prominent in the periventricular zone (PVZ), particularly in the region directly adjacent to the lateral ventricle (VL). (B) Developing cortex at P10 shows strong labeling of cells in all layers, but the most intensely labeled cells are present in layers II/III and V. Numerous ELOVL4-positive cells also are present in the developing corpus callosum (cc). (C) Widespread, layered expression of ELOVL4 is preserved in the cortex at P20. (D) The characteristic layered pattern of ELOVL4 expression in cortex is maintained at P60. Scale bars = 250 μm.

Figure 3

Expression of ELOVL4 in the olfactory bulb. (A) Labeling for ELOVL4 is present in the main olfactory bulb (MOB) and accessory olfactory bulb (AOB). Mitral cells in the mitral cell layer (mi) of the MOB and the AOB show intense labeling. Cells surrounding the olfactory glomeruli (gl) and granule cells (gr) also show ELOVL4 labeling. Little labeling is present in the outer plexiform layer (opl) or inner plexiform layer (ipl). (B) Labeling for the neuronal marker, NeuN. (C) Overlay of (A,B). Scale bar = 1 mm.

Figure 4

Expression of ELOVL4 in development of the hippocampal formation. (A) The developing hippocampal formation (HPF) at E18 shows strong labeling for ELOVL4 in cells of the developing Cornu Ammonis (CA), dentate gyrus (DG), and subiculum (Sub). Intense ELOVL4 labeling is seen near the margins of the lateral ventricle (VL). The periventricular zone (PVZ) of the overlying and developing cerebral cortex iare visible above the hippocampal formation. (B). Developing hippocampal formation at P10, with a well-developed CA region and dentate gyrus. Cells in the CA region show ELOVL4 labeling, with cells in CA3 showing stronger labeling than cells in CA1. Cells in the dentate gyrus and subiculum also show labeling. Labeled cells are also present in the developing corpus callosum (cc) and fornix (fo). Developing thalamus (TH) is visible below the hippocampal formation. (C) Hippocampal formation at P20. ELOVL4 labeling is present throughout the CA region, with cells in CA3 showing the strongest labeling. Labeling for ELOVL4 in the granule cell layer of the dentate gyrus is reduced compared to earlier stages although ELOVL4-positive cells persist along the inner margin of the dentate gyrus. Strong labeling is also present in interneurons in the polymorph layer of the dentate gyrus and in the subiculum. (D) The hippocampal formation at P60 shows distribution of ELOVL4 labeling similar to that at P20. Scale bars = 500 μm.

Figure 5

Expression of ELOVL4 in development of the basal ganglia. (A) The developing basal ganglia at E18 show very high levels of ELOVL4 expression, particularly in the periventricular zone (PVZ) adjacent to the lateral ventricle (VL). Caudoputamen (CP), nucleus accumbens (ACB), and olfactory tubercle (OT) are shown. (B). Labeling for ELOVL4 in the basal ganglia decreases substantially by P10. (C) The basal ganglia maintain low levels of ELOVL4 expression at P20. (D) At P60, the basal ganglia maintain relatively low levels of ELOVL4 with sparsely distributed ELOVL4-labeled cells. Labeling for ELOVL4 is also evident in the subventricular zone (SVZ). HPF, hippocampal formation; HY, hypothalamus; PIR, piriform cortex; cc, corpus callosum; fo, fornix; ic, internal capsule. Scale bars = 500 μm.

Figure 6

Expression of ELOVL4 in the amygdala. Labeling for ELOVL4 is present in the amygdala, but varies among subnuclei. Prominent labeling in the central amygdalar nucleus (CEA), basolateral amygdalar (BLA), basomedial amygdalar nucleus (BMA), posterior amygdalar nucleus (PA), and the cortical amygdalar area (COA) is shown. CA1, Cornu Ammonis, field 1; EP, endopiriform nucleus; GPe, globus pallidus external; GPi, globus pallidus internal; HPF, Hippocampal formation; Pir, piriform cortex; Sub, subiculum; VL, lateral ventricle. Scale bar = 500 μm.

Figure 7

Expression of ELOVL4 in the developing cerebellum. (A) ELOVL4 labeling is present in the cerebellum (CB) during early development at E18. Intense labeling is present in the nascent external granule cell layer (egl) of the developing cerebellar cortex. Labeling for ELOVL4 is also present in the developing inner granule cell layer (igl). A few labeled cells also are present in the forming molecular cell layer (mcl). (B) Intense ELOVL4 labeling is present in the egl and igl of the cerebellar cortex at P10. Many cells traversing the mcl show ELOVL4 labeling. Strong ELOVL4 labeling is present in the deep cerebellar nuclei by P10 (interpositus nucleus, IP, shown). (C) Mature layering of cerebellar cortex is achieved by P20, and is characterized by very intense ELOVL4 labeling in cells in the granule cell layer (gcl). Purkinje cells in the Purkinje cell layer (pcl) also show ELOVL4 labeling, but are difficult to discern at this magnification. The sparsely distributed cells in the mcl also show labeling. Labeling of cells in the deep cerebellar nuclei also persists. (D) Distribution of ELOVL4 in the P60 cerebellar cortex and deep cerebellar nuclei (fastigial nucleus, FN, shown) is similar to that in the P20 cerebellum. IC, inferior colliculus; M, medulla; MB, midbrain; P, pons; V4, 4th ventricle. Scale bars = 500 μm for (A); 1 mm for (B–D).

Figure 8

Expression of ELOVL4 in the thalamus. (A) Prominent labeling for ELOVL4 in the lateral thalamus is present in the lateral geniculate nucleus (LG), reticular nucleus (RT), and subgeniculate nucleus (SubG). The medial geniculate nucleus (MG) shows much weaker ELOVL4 labeling. (B) Labeling for the neuronal marker, NeuN, in lateral thalamus. (C) Overlay of (A,B). (D) Labeling for ELOVL4 also is present in more medial regions of the thalamus. Strong labeling is present in the anteroventral (AV), ventral posterolateral (VPL), and ventral anterior-lateral complex (VAL) nuclei. Moderate ELOVL4 labeling is present in the lateral dorsal (LD), ventral posteromedial (VPM), and ventromedial (VM) nuclei. Lateral posterior (LP), posterior complex (PO), and subparafascicular (SPF) nuclei of the thalamus show less ELOVL4 labeling. (E) Labeling for NeuN in the medial thalamus. (F) Overlay of (D,E). HPF, hippocampal formation; MB, midbrain; ZI, zona incerta. Scale bars = 500 μm.

Figure 9

Expression of ELOVL4 in the hypothalamus and midbrain. (A) Extensive ELOVL4 labeling is present in the hypothalamus (HY). Prominent labeling is seen in the lateral preoptic area (LPO), lateral mammilary nucleus (LM), zona incerta (ZI), and in parts of the lateral hypothalamic area (LHA). TH, Thalamus; opt, optic tract. (B) Labeling for ELOVL4 in the midbrain is widespread but varies among nuclei. Prominent labeling is present in the superior colliculus (SC), inferior colliculus (IC), midbrain reticular nucleus (MRN), and anterior pretectal nucleus (APN). Substantia nigra (SN) shows much less labeling for ELOVL4 than other portions of the midbrain. Cerebellum (CB), cortex (CTX), and zona incerta (ZI) also are visible. Scale bars = 500 μm.

Figure 10

Expression of ELOVL4 in the pons and medulla. Many labeled cells are present in the pons, including cells in the pontine reticular nucleus (PRN), pontine gray (PG), and superior olivary complex (SOC). In the medulla, ELOVL4-positive cells are prominent in the medial vestibular nucleus (MV), facial motor nucleus (VII), spinal nucleus of the trigeminal (SPV), parvicellular reticular nucleus (PARN), medullary reticular nucleus (MDRN), and later reticular nucleus (LRN). CB, cerebellum; IP, interpositus nucleus of the cerebellum; MB, midbrain; V4, fourth ventricle. Scale bar = 500 μm.

Figure 11

ELOVL4 expression is primarily neuronal. (A–C) Cerebral Cortex (CTX). (A) Labeling for ELOVL4 is present in all layers of the cerebral cortex (CTX). Cells in the pyramidal layers (II/III and V) are most prominently labeled, but ELOVL4-positive cells also are present in the molecular layer (I), layer 4 (IV), and layer 6 (VI). (B) Labeling for the neuronal marker, NeuN. (C) Overlay of panels (A,B) shows close correspondence of ELOVL4 (red) and NeuN labeling (green), indicating that ELOVL4 is primarily expressed by neurons in the cortex. (D–F) Hippocampal formation. (D) Labeling for ELOVL4 is present in the cellular layers of the hippocampal formation (HPF), including the Cornu Ammonis, with field 1 (CA1) showing less prominent labeling than field 3 (CA3). Prominent ELOVL4 labeling also is present in the subiculum (sub) and interneurons in the polymorph layer (arrow). Cells along the inner margin of the dentate gyrus (DG) show moderate ELOVL4 labeling, but most dentate granule cells show little ELOVL4 labeling. (E) Labeling for the neuronal marker, NeuN. (F) Overlay of (D,E) shows close correspondence of ELOVL4 (red) and NeuN (green) labeling, indicating that ELOVL4 is primarily expressed by neurons in the hippocampal formation. TH, Thalamus. (G–I) Cerebellar cortex. (G) Cross section through a cerebellar folium showing ELOVL4 expression in the cerebellar cortex. Neurons (arrows) in the molecular cell layer (mcl) show strong ELOVL4 labeling, but the monolayer of large Purkinje cells (arrowheads) that form the Purkinje cell layer (pcl) shows only moderate levels of ELOVL4 labeling. The densely packed cells of the granule cell layer (gcl) show very intense labeling. (H) Labeling for the neuronal marker, NeuN, strongly labels neurons in the gcl, but not Purkinje cells, as appropriate. (I) Overlay of panels (G,H) shows close correspondence of intense ELOVL4 (red) and NeuN (green) labeling resulting in an orange color in the gcl. These results indicate that ELOVL4 in the cerebellum is primarily expressed by neurons. wm, white matter of the arbor vitae. Scale bars = 200 μm for (A–C,G–I); 500 μm for (D–F).

Figure 12

Astrocytes show little ELOVL4 labeling. (A) Labeling for ELOVL4 is present in many cells in the cerebral cortex (^*). (B) Astrocytes (arrowheads), identified by labeling for glutamine synthetase (GS). (C) Overlay of (A,B) shows little labeling for ELOVL4 in astrocytes. Nuclei counterstained with DAPI (blue) in all panels. Scale bar = 50 μm.

Figure 13

Radial glial cells show little ELOVL4 labeling. (A) Many cells (^*) in the external and internal granule cell layers (egl and igl, respectively), as well as cells migrating through the molecular cell layer (mcl), of the developing cerebellum show ELOVL4 labeling at P10. (B) Radial glial cells and their processes (arrowheads) spanning the layers of the cerebellum can be identified by labeling for glutamine synthtase (GS). (C) Overlay of (A,B) shows no colocalization of labeling for ELOVL4 and GS. (D) Many cells in the developing cerebral cortex show ELOVL4 labeling (^*) at P10. (E) Radial glial cells and their processes (arrowheads), identified by labeling for glial fibrillary acidic protein (GFAP). Non-specfic labeling of a blood vessel (bv) also is visible. (F) Radial glial cells and their processes do not show labeling for ELOVL4. Nuclei counterstained with DAPI (blue) in all panels. Scale bars = 50 μm.

Figure 14

Glial cells in white matter express ELOVL4. (A) Immunohistochemical labeling (IHC) for ELOVL4 identifies presumptive oligodendrocytes (arrowheads) in white matter. Corpus callosum (cc) shown. CTX, cerebral cortex; BG, basal ganglia. (B) In situ hybridization (ISH) for Elovl4 mRNA also reveals small cells in corpus callosum (arrowheads). VL, lateral ventricle. (Image from the Allen Institute for Brain Science Allen Mouse Brain Atlas for the P56 mouse brain, image number 69059903_134. http://mouse.brain-map.org). Scale bars = 100 μm.