Transcript Analysis of Zebrafish GLUT3 Genes, slc2a3a and slc2a3b, Define Overlapping as Well as Distinct Expression Domains in the Zebrafish (Danio rerio) Central Nervous System

Abstract

The transport of glucose across the cell plasma membrane is vital to most mammalian cells. The glucose transporter (GLUT; also called SLC2A) family of transmembrane solute carriers is responsible for this function in vivo. GLUT proteins encompass 14 different isoforms in humans with different cell type-specific expression patterns and activities. Central to glucose utilization and delivery in the brain is the neuronally expressed GLUT3. Recent research has shown an involvement of GLUT3 genetic variation or altered expression in several different brain disorders, including Huntington's and Alzheimer's diseases. Furthermore, GLUT3 was identified as a potential risk gene for multiple psychiatric disorders. To study the role of GLUT3 in brain function and disease a more detailed knowledge of its expression in model organisms is needed. Zebrafish (Danio rerio) has in recent years gained popularity as a model organism for brain research and is now well-established for modeling psychiatric disorders. Here, we have analyzed the sequence of GLUT3 orthologs and identified two paralogous genes in the zebrafish, slc2a3a and slc2a3b. Interestingly, the Glut3b protein sequence contains a unique stretch of amino acids, which may be important for functional regulation. The slc2a3a transcript is detectable in the central nervous system including distinct cellular populations in telencephalon, diencephalon, mesencephalon and rhombencephalon at embryonic and larval stages. Conversely, the slc2a3b transcript shows a rather diffuse expression pattern at different embryonic stages and brain regions. Expression of slc2a3a is maintained in the adult brain and is found in the telencephalon, diencephalon, mesencephalon, cerebellum and medulla oblongata. The slc2a3b transcripts are present in overlapping as well as distinct regions compared to slc2a3a. Double in situ hybridizations were used to demonstrate that slc2a3a is expressed by some GABAergic neurons at embryonic stages. This detailed description of zebrafish slc2a3a and slc2a3b expression at developmental and adult stages paves the way for further investigations of normal GLUT3 function and its role in brain disorders.

Keywords: GABA; GAD1; brain development; brain disorders; glucose transporter; nervous system; psychiatric disorders.

Figure 1

Multiple sequence alignment of glucose transporter3 (GLUT3) orthologs from selected vertebrate species. Alignment was performed with CLUSTAL Omega. Hydrophobic transmembrane (TM) regions as predicted by the TMMHM algorithm are underlined and boxed. Note the additional sequence stretch between transmembrane domain 9 and 10 in zebrafish Glut3b sequence. Accession numbers of protein sequences are given in the “Materials and Methods” section.

Figure 2

Semi-quantitative analysis of PCR products generated by reverse transcription and subsequent PCR showing temporal expression levels of slc2a3a and slc2a3b in zebrafish. Total RNA was collected at the different developmental stages as indicated. Beta actin (actb1) served as a loading control and cDNA from pooled RNA from a mixture of developmental stages as a positive control.

Figure 3

Whole mount RNA in situ hybridization of slc2a3a at early embryonic stages [18–36 hours post fertilization (hpf)]. Pictures in the left column depict alternating lateral (A,C,E,G) and dorsal (B,D,F,H) views. Anterior is to the left. Higher magnifications (a₁–h₃) corresponding to boxes in (A–H). Arrowheads indicate examples of labeled cells or cell populations. Note staining in spinal cord, hindbrain, ventral midbrain, ventral thalamus, ventral telencephalon and epiphysis. Two bilateral rows, one medially (black arrowheads) and one laterally (white arrowheads) of positive cells are situated in the ventral hindbrain along the floor plate (f₂ and h₂). For abbreviations of anatomical terms see Table 1. Scale bars in (A), 100 μm and pertains to (A–H); scale bar in a₁, 50 μm and pertains to a₁–h₃.

Figure 4

Whole mount RNA in situ hybridization of slc2a3a at late embryonic and early post-hatching stages (48–120 hpf). Pictures in the left column depict alternating lateral (A,C,E) and dorsal (B,D,F) views. Anterior is to the left. Higher magnifications (a₁–f₃) corresponding to boxes in (A–F). Note staining in hindbrain, ventral midbrain, optic tectum, ventral thalamus, ventral telencephalon and retina. Detailed descriptions can be found in the text, for abbreviations see Table 1. Scale bar in (A), 100 μm and pertains to (A–F); scale bar in a₁, 50 μm and pertains to a₁–f₂, d₃, f₃ and scale bar in a₃, 50 μm and pertains to a₃, b₃.

Figure 5

Cryosections (20 μm) of 120 hpf embryo processed for RNA in situ hybridization for slc2a3a. The sections (A–P) are cross sections arranged from anterior to posterior at the levels indicated in Figure 4E. Detailed descriptions can be found in the text, for abbreviations see Table 1. Scale bar, 100 μm.

Figure 6

Whole mount RNA in situ hybridization for slc2a3b at blastula 128-cell, 24 hpf and 72 hpf stages. For comparison, a sense probe was included (B,E). Comparison of embryos incubated with antisense (A,A’,C,D) and sense (B,B’,E) show diffuse staining only in embryos incubated with the antisense probe. Pictures (F; lateral) and (G; dorsal) show 72 hpf larvae stained with antisense probe. Boxes in (F) and (G) depict locations of high magnification pictures in (F’,G’). For abbreviations see Table 1. Scale bars in (A,C,D), 100 μm and pertains to (A–G), respectively, scale bar in (A’,F’), 50 μm and pertains to (A’,B’,F’,G’), respectively.

Figure 7

RNA in situ hybridization of slc2a3a in the adult brain. Pictures (A–R) show cross sections of an adult zebrafish brain (80 μm) from anterior to posterior as indicated in the scheme. (B’–R’) are high magnifications of boxed areas in (B–R). Detailed descriptions can be found in the text, for abbreviations see Table 1. Scale bar in (A), 100 μm and pertains to (A–R); scale bars in (B’–R’), 20 μm.

Figure 8

RNA in situ hybridization of adult expression of slc2a3b. Pictures show cross sections of an adult zebrafish brain (80 μm) from anterior to posterior as indicated in the scheme. Panels (A’–J’) are high magnifications of boxed areas in (A–J). Black arrowheads in (A’,B’,J’) indicate example of single-positive cells. Detailed descriptions can be found in the text, for abbreviations see Table 1. Scale bar in (A), 100 μm and pertains to (A–J); scale bars in (A’–J’), 20 μm.

Figure 9

RNA in situ hybridization of gad1b and double RNA in situ hybridization for slc2a3a and gad1b, a marker for GABAergic neurons. For comparison, a single in situ hybridization for gad1b was performed at 24 hpf (A,B,B’) and 36 hpf (C,D,D’) old zebrafish embryos. Boxes in (A,B,B’,C,D,D’) depict location of high magnification pictures a₁,a₂,b₁,b₂ and c₁, c₂, d₁, d₂, respectively. In addition, a 24 hpf old zebrafish embryo was stained with slc2a3a DIG in situ probe (blue) and gad1b FLUO probe (red). Upper rows show lateral views (A,C,E) and lower rows ventral views (B,B’,D,D’,F). Anterior is to the left. Boxes in (E,F) depict location of high magnification pictures e₁–e₄ and f₁–f₄, respectively. Boxes in e₁ and e₂ and f₁, f₂, and f₄) represent higher magnification pictures shown in e₁’, e₂’ and f₁’, f₂’, and f₄’. Box in e₁’ represents location of even higher magnification shown in e₁”. White arrows mark double-positive cells or cell populations. Red arrow marks an example of cells only positive for gad1b and blue arrows cells only positive for slc2a3a. Scale bars in (A,E), 100 μm and pertains to (A–F); scale bar in a₁, 50 μm and valid for a₁–d₂; scale bar in e₁, 50 μm and pertains to e₁–e₂ and f_1–2; scale bar in e₃, 50 μm and valid for e₃-e₄, and f_3–4, scale bars in e₁’–f₄’, e₁’, 10 μm.