Temporal-specific roles of fragile X mental retardation protein in the development of the hindbrain auditory circuit

Abstract

Fragile X mental retardation protein (FMRP) is an RNA-binding protein abundant in the nervous system. Functional loss of FMRP leads to sensory dysfunction and severe intellectual disabilities. In the auditory system, FMRP deficiency alters neuronal function and synaptic connectivity and results in perturbed processing of sound information. Nevertheless, roles of FMRP in embryonic development of the auditory hindbrain have not been identified. Here, we developed high-specificity approaches to genetically track and manipulate throughout development of the Atoh1⁺ neuronal cell type, which is highly conserved in vertebrates, in the cochlear nucleus of chicken embryos. We identified distinct FMRP-containing granules in the growing axons of Atoh1⁺ neurons and post-migrating NM cells. FMRP downregulation induced by CRISPR/Cas9 and shRNA techniques resulted in perturbed axonal pathfinding, delay in midline crossing, excess branching of neurites, and axonal targeting errors during the period of circuit development. Together, these results provide the first in vivo identification of FMRP localization and actions in developing axons of auditory neurons, and demonstrate the importance of investigating early embryonic alterations toward understanding the pathogenesis of neurodevelopmental disorders.

Keywords: Auditory circuit; Autism spectrum disorder; Axon development; Axon fasciculation; Axon targeting; CRISPR-Cas9; Fragile X syndrome; RNA-binding proteins.

Fig. 1.

High-specificity genetic labeling of NM precursors and neurons. (A) Schematic of the NM-NL circuit. (B) Plasmid design for Atoh1-mGFP. Electroporation was performed following plasmid injection into rhombomeres 5-6 (r5-6; blue). (C) E15 brainstem sections showing a restricted localization of mGFP⁺ cell bodies in the NM on the transfection (ipsi) side. Yellow asterisks indicate bilateral NM axons to NL. (E) Proportion of transfected neurons in the NM. The mean value±s.d. are indicated for this and all plots in subsequent figures. ANF, auditory nerve fiber; contra, contralateral; ipsi, ipsilateral. Scale bar: 200 µm.

Fig. 2.

Axon development of NM precursors and neurons. Images were taken from embryos electroporated with Atoh1-mGFP at E2-2.5. (A) Flat-mount view at E4.5 showing two contralateral projection bundles (LF and DF) of Atoh1/dA1 cells. (B) Top view at E5 showing that axons of Atoh1/dA1 cells at r5-6 join the DF bundle. Dashed lines indicate the midline in A and B. (C) Transverse section at E4.5 at the level of r5-6. mGFP⁺ axons have crossed the midline and arrived in their contralateral target area (yellow arrow). White and yellow arrows indicate the axon bundle at the ipsilateral and contralateral sides, respectively. (D) Illustration describing the measurements used to quantify axonal growth patterns of NM precursors. Axon bundle width was calculated as the ratio of B (GFP⁺ fascicule width) divided by A (mantle-ventricular width). Axonal midline crossing rate was calculated as D (area of GFP⁺ contralateral axons) divided by C (area of GFP⁺ ipsilateral axons). A° is the angle between the most medial GFP⁺ projecting axon and the mantle plate. (E,F) Box plot analysis of the ratio of axonal midline crossing (E) and bundle width (F) of Atoh1:cre-tagged axons at E4.5. Each data point represents one embryo (n=7). (G) Plasmid design for SV2-GFP with Atoh1-enhancer and PiggyBac (PB) transposase. (H-I′) SV2-GFP (green) distribution in transverse sections counterstained with NeuroTrace (magenta) on the ipsilateral (H,H′) and contralateral (I,I′) sides. H′ and I′ are enlarged views of the boxes in H and I, respectively. NM is outlined by dashed circles. The cell body layer (c) as well as the dorsal (d) and ventral (v) dendrite domains of the NL are indicated. LF, lateral funiculus; DF, dorsal funiculus. Scale bars: 1 mm in A (applies to A,B) and C; 100 µm in H,I; 20 µm in H′,I′.

Fig. 3.

Morphological maturation of presynaptic terminals of NM neurons. Images were taken from embryos electroporated with Atoh1-mGFP at E2. (A,B) NM axon terminals in the dorsal neuropil of the ipsilateral NL (A) and in the ventral neuropil of the contralateral NL (B) at E12 and E15. NM axons show a growth cone structure with filopodia (white arrows) at E12 and bouton-like terminals (yellow arrows) at E15. (C,D) Frequency distribution (C) and population analysis (D) of the number of filopodia per terminal at E11-13 (n=51 terminals) and E15 (n=42 terminals). Additional images and data analyses are shown in Figs S3 and S4. Scale bar: 2 μm.

Fig. 4.

Endogenous FMRP is localized in distal axons of NM precursors. (A) FMRP immunostaining of an E4 embryo. (B-B″) FMRP immunostaining (B′) of an E5 embryo transfected with Atoh1-mGFP (B). B″ is the merged image. Two photomicrographs from the left and right halves of the section were manually tiled for the whole view in B-B″. Note the FMRP immunostaining in the region where transfected cell bodies are located (green arrows). The terminal region on the contralateral side (yellow arrows) is low in FMRP immunoreactivity. (C-C″) High-magnification images of the box in B″ from the transfection (ipsi) side. Transfected cells (green) contain FMRP immunoreactivity (red) in the cytoplasm (c in insets) and a weaker staining in the nuclear (n in insets). (D-D″) High-magnification images of the box in B″ from the contralateral side (contra). A subset of FMRP puncta (arrows) are localized in mGFP⁺ axon processes (insets). FMRP puncta that are localized beyond mGFP⁺ axon processes are presumably in untransfected axons because this region contains no cell bodies as indicated with the lack of DAPI-labeled nuclei. (E) Colocalization analysis of a representative FMRP punctum with Atoh1-mGFP⁺-labeled axon, confirming the axonal location of FMRP. Dashed line indicates the region of interest for colocalization analysis. Scale bars: 100 µm in A; 200 µm in B″ (applies to B-B″); 5 µm in C,D (applies to C-D″); 2 µm in insets; 1 µm in E.

Fig. 5.

Axon localization of FMRP in NM neurons. (A) Plasmid designs for constitutive expression of chicken and human FMRP (chFMRP and hFMRP). (B) Schematic of the co-transfection protocol for chFMRP-mCherry and Atoh1-mGFP. NM cells are transfected either with chFMRP-mCherry only (red circles) or with both plasmids (half red and half green circles). This co-transfection protocol yields very few NM cells transfected with Atoh1-mGFP only. Grey circles indicate nontransfected neurons in NM and NL. Green lines indicate axons of Atoh1-mGFP-labeled NM cells on the dorsal NL ipsilaterally and ventral NL contralaterally (green). chFMRP-mCherry-labeled puncta are indicated as small red points in these two NL neuropil regions. mCherry-labeled puncta are also located in the fiber regions adjacent to NL neuropil regions. (C) Transverse sections at E15 showing transfected cell bodies in the NM (left column) and their contralateral projection in the NL (right column), following the co-transfection shown in B. White arrows indicate a co-transfected NM neuron. On the contralateral side, chFMRP-mCherry puncta are detected within the ventral neuropil domain of NL as well as the fiber region containing incoming NM axons. (D) High-magnification images of the ventral neuropil of the contralateral NL at E11 and E15. A subset of FMRP-mCherry puncta are located in Atoh1-mGFP⁺ NM axons (arrows). (E) Images of the contralateral NL at E15 following transfection with hFMRP-EGFP and MAP2 counterstaining (red), a somatodendritic marker. hFMRP puncta are distributed in the ventral fiber region and the ventral NL neuropil. Inset shows a high magnification of the boxed area. MAP2, microtubule-associated protein 2. Scale bars: 100 µm in C, left column; 20 µm in C, right column; 5 µm in D; 50 µm in E; 7.5 µm in inset.

Fig. 6.

FMRP knockout with CRISPR/Cas9 strategy. (A) CRISPR design of FMRP sequence in exon 8. (B) Gel electrophoresis of PCR products from hindbrains electroporated with gRNA_control and gRNA₃₊₄ plasmids. Red arrow points to a ∼260 bp fragment obtained by Cas9 deletion. (C-D″) Sagittal-section views of E6.5 brainstems expressing gRNA_control (C-C″) or gRNA₃₊₄ (D-D″) plasmids (green) and stained for FMRP antibody (red). High-magnification views of the boxed areas in C,D and C′,D′ appear to the right of each image. Arrows and arrowheads point to FMRP⁺ and FMRP⁻ cells, respectively. (E) Box plot quantification of FMRP-immunoreactive cells out of total GFP⁺ cells. Each data point represents one embryo (n=7 embryos for each group). (F,G) Cross-section views of E4.5 hindbrains obtained from embryos that were electroporated with gRNA_control or gRNA₃₊₄ (green) and stained with Lhx2/9 antibody (red). Higher-magnification views of the boxed areas in F and G are represented in the right of each panel in different channels. Arrows indicate the same cells in all channels. (H) Box plot quantification of Lhx2/9-immunoreactive cells out of total GFP⁺ cells. Each data point represents one section (n=3 embryos for each group). Scale bars: 100 μm in C (applies to C,D); 100 µm in F,G (main panels); 50 μm in C′ (applies to C′,D′) and F,G (right panels); 20 μm in C″ (applies to C″,D″).

Fig. 7.

CRISPR-mediated FMRP knockout induces disoriented axonal growth. (A-B′) E4.5 flat-mounted hindbrains from embryos electroporated at E2.5 with gRNA_control (A) or gRNA₃₊₄ (B). Higher-magnification views of the boxed areas in A,B are represented in A′,B′. Arrows point to organized axons in A,A′. Dashed arrows in B′ indicate disoriented axons. (C-G′) Transverse sections of r5-6 level at E4.5 (C-E′) and E6.5 (F-G′) from embryos electroporated with gRNA_control (C,C′,F,F′) or gRNA₃₊₄ (D-E′,G,G′) plasmids. Higher-magnification views of the boxed areas in C-G are represented in C′-G′. Arrows in the left panels indicate axons that crossed the midline. Arrows and arrowheads in the right panels point to organized and disorganized axons, respectively. (H,I) Box plot analysis of the width of the GFP⁺ axonal bundle at E4.5 (H) and E6.5 (I). (J,K) Box plot analysis of the axonal midline crossing rate at E4.5 (J) and E6.5 (K). Each data point represents one embryo. FP, floor plate. Dashed lines outline the border of embryos and sections. Scale bars: 100 µm in A (applies to A,B) and C (applies to C-G); 50 µm in A′ (applies to A′,B′) and C′ (applies to C′-G′).

Fig. 8.

shRNA-mediated FMRP knockdown induces axonal disorganization. (A-B′) E4.5 flat-mounted hindbrains from embryos that were electroporated at E2.5 with scrambled-shRNA-EGFP (sc-shRNA; A) or Fmr1-shRNA-EGFP (B). Higher-magnification views of the boxed areas in A,B are represented in A′,B′. Plasmid design for Fmr1-shRNA is illustrated on the top. Arrows and dashed arrows represent organized and disoriented axons, respectively. (C-G′) Transverse sections of r5-r6 level at E4.5 (C-E′) and E6.5 (F-G′) from embryos electroporated with sc-shRNA (C,C′,F,F′) or Fmr1-shRNA (D-E′,G,G′) plasmids. Higher-magnification views of the boxed areas in C-G are represented in C′-G′. Arrows in left panels indicate axons that crossed the midline. Arrows and arrowheads in the right panels point to organized and disorganized axons, respectively. (H,I) Box plot analysis of the width of the GFP⁺ axonal bundle at E4.5 (H) and E6.5 (I). (J,K) Box plot analysis of the axonal midline crossing rate at E4.5 (J) and E6.5 (K). Each data point presents one embryo. FP, floor plate. Dashed lines outline the border of embryos and sections. Scale bars: 100 µm in A (applies to A,B) and C (applies to C-G); 50 µm in A′ (applies to A′,B′) and C′ (applies to C′-G′).

Fig. 9.

CRISPR-mediated FMRP knockout induces neurite overgrowth and overbranching in hindbrain culture. (A-H′) Time-lapse analysis of cell cultures obtained from E3.5 hindbrains that were electroporated at E2.5 with gRNA_control (A,C,E,G) and gRNA₃₊₄ (B,D,F,H) plasmids. Cells were documented every 6 h for 5 days. Representative phase (A-H) and green fluorescence (A′-H′) images in different time points are shown. GFP⁺ neurites are evident in all images. (I-L) Higher-magnification views of the boxed areas in E′-H′. Arrows in J,L show overbranching along the neurite up to its terminal. (M,N) Quantification of neurite branch point (M) and neurite length (N) along 5 days using NeuroTrack analysis. Each data point represents six different wells of a 48-well plate. Scale bars: 200 µm in H′ (applies to A-H′); 50 µm in L (applies to I-L).

Fig. 10.

FMRP knockdown leads to axon projection errors in NL. (A) Transfection protocol for late-onset shRNA expression. Blue arrows indicate the days for Dox treatment. (B,C) Schematics of normal (B) and aberrant (C) axon targeting of NM neurons in the contralateral NL. (D,E) Photomicrographs of NM axons in the contralateral NL at E15 following scrambled-shRNA (D) and Fmr1-shRNA (E) expression. Arrows point to abnormally projected NM axons through the cell body layer into the dorsal neuropil. (F) NM axons in the contralateral NL at E19 following Fmr1-shRNA expression. The axons are predominantly distributed in the ventral neuropil, similar to the control. Dashed lines indicate the cell body layer. (G) Quantification of the dorsal/ventral ratio of axon area. This ratio is significantly increased in Fmr1-shRNA transfected embryos at E15 (red squares) but not E19 (blue triangles), compared with control embryos (black circles). d, dorsal; v, ventral. Scale bar: 50 μm.

Fig. 11.

Aberrantly projected NM axons form synapses on NL dendrites. NM precursors were unilaterally transfected with Fmr1-shRNA-EGFP. Images were taken from the side contralateral to the transfection. (A-A″) Images of a dye-filled NL neuron (red) the dorsal and ventral dendrites of which are in close contact with EGFP⁺ NM axons (white arrows).Inset in A′ shows higher magnification of the boxed area. (B-B″) Double labeling of Syt2 immunoreactivity with EGFP⁺ NM axons. Asterisks indicate NL cell bodies in B″. Higher-magnification views of the boxed area in B″ are represented to the right. EGFP⁺ axonal terminals (white arrows) contain Syt2 immunoreactivity. Scale bars: 10 μm in A; 2 μm in inset in A′ and in right-hand panels in B; 20 μm in B. Syt 2, synaptotagmin 2.

Fig. 12.

FMRP knockdown does not affect the morphological maturation of NM axonal terminals. (A,B) Frequency distribution (A) and population analysis (B) of the number of filopodia per terminal following transfection with Atoh1:cre-mGFP (black bars; n=21 terminals) or Fmr1-shRNA (green bars; n=14 terminals). All terminals were measured from the ventral neuropil of the contralateral NL.