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First published online August 25, 2008
doi: 10.1242/10.1242/dev.022236

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A crucial role for hnRNP K in axon development in Xenopus laevis

The Department of Biological Sciences and the Center for Neuroscience Research, University at Albany, State University of New York, Albany, NY 12222, USA.

View larger version (97K): [in this window] [in a new window]   Fig. 1. Expression of hnRNP K in whole-mount embryos. (A) Stage 10 gastrula; hnRNP K was found in all germ layers, but was most intense in mesectoderm (M). yp, yolk plug. (B) Stage 15 neural plate; hnRNP K staining intensified in mesectoderm. NF, neural plate and adjacent neural folds; NC, notochord; SM, somitic mesoderm. (C-F) Stage 22 tailbud (C), stage 37/38 tadpole (D), stage 42 tadpole (E,F). hnRNP K was abundant in brain and spinal cord (SC), in aligned nuclei of somitic muscle (unlabeled arrowheads) and all retinal layers by stage 42 (E). OV, optic vesicle; RGC, retinal ganglion cell layer; IN, interneuron layer; PR, photoreceptor layer; CF, choroidal fissure. NC, notochord. Scale bars: in A, 100 µm for A-D; 50 µm in E,F.

View larger version (94K): [in this window] [in a new window]   Fig. 2. Transition of hnRNP K from predominantly nuclear to mixed nuclear and cytoplasmic localization during neuronal development. (A-C) hnRNP K immunofluorescence (A), DAPI-fluorescence (B) and phase-contrast (C) images of embryonic muscle cell and neurons in dissociated cell culture. hnRNP K immunostaining colocalized with DAPI-stained nuclei (arrowheads, A,B); neurites and growth cones (gc) had no immunostaining (arrows in A,C). (D,E) hnRNP K immunoperoxidase staining of sections of juvenile frog retina (D) and spinal cord ventral horn (E). Retinal ganglion cells (RGC) exhibited more intense cytoplasmic than nuclear staining; motoneuron staining was both in the nucleus and cytoplasm (arrowhead, E). Arrow in E indicates interneuron with predominantly nuclear staining.

View larger version (53K): [in this window] [in a new window]   Fig. 3. Suppression of hnRNP K expression by antisense MO. Embryos were unilaterally injected with two separate non-overlapping hnRNP K MOs (MO1 in A-D; MO2 in E). (A1,A2) Stage 10 gastrula; persistent maternal hnRNP K expression. (B1,B2) Stage 15 neural plate stage; suppression of hnRNP K expression on the injected side (left of broken line). Upper arrowhead, neural fold; lower arrowhead, somitic mesoderm, on the uninjected side. (C1,C2) Stage 22 tailbud stage; hnRNP K expression in myotome, spinal cord and brain (arrowheads, left to right, respectively) was suppressed on the injected side (C2) when compared with the uninjected side (C1). Because the animals are bent, the optical section in C2 also contained some cells from the uninjected side of the embryo (rectangle). (D,E) Three-day tadpole (stages 39 and 40); the uninjected side exhibited normal staining and morphology, but the injected side exhibited only background immunostaining and some defects in somites. (A1-C2,E) Confocal optical sections; (D) fluorescence dissecting microscopic view of whole animal. hnRNP K immunostaining, red; co-injected FITC-dextran, green.

View larger version (113K): [in this window] [in a new window]   Fig. 4. Suppression of axonal outgrowth by hnRNP K MO in intact animals. Embryos unilaterally injected with control MO (A), hnRNP K MO1 (B,D-H) or hnRNP K MO2 (C) processed at stage 37/38 by whole-mount immunofluorescence. (A-C) Embryos injected with control MO (A), hnRNP K MO1 (B) or MO2 (C), and immunostained for NF-M. Broken lines separate injected (lower) from uninjected (upper) sides. (B,C) Arrowheads on the injected side indicate peripheral motor axons; arrowheads on the uninjected side indicate residual wispy peripheral axons. (A) View under the fluorescence dissecting microscope; (B,C) stacked confocal optical sections. (D1) Neuronal β-tubulin staining in neuronal perikarya and peripheral axons of spinal cord, uninjected side. (D2) Tubulin staining in perikarya and a few scattered fibers, injected side. (E1-G2) Peripherin immunostaining of head (E1,E2) and spinal cord (F1,F2,G1,G2). (E1,F1,G1) Uninjected side; (E2,F2,G2) injected side. (E1,E2) Trigeminal ganglion neuronal perikarya (upper arrowhead) and nerves (lower arrowhead). (F1,F2) Spinal cord with peripheral motor axons. (G1,G2) Spinal cord neuronal perikarya. (H) Spinal cord immunostained for HNK-1. Robust staining of neuronal perikarya and axons on the uninjected side (below the broken line); clusters of stained neuronal perikarya (arrowheads) and very few fibers on the injected side (above the broken line). Scale bars in D1,E2,F1 and G2 also apply to D2,E1,F2 and G1, respectively.

View larger version (112K): [in this window] [in a new window]   Fig. 5. Rescue from hnRNP K MO by co-injection of hnRNP K RNA. Two-cell embryos were unilaterally injected with hnRNP K MO1 plus 100 pg of hnRNP K RNA, then processed for whole-mount immunostaining for hnRNP K (A1,A2), neuronal β-tubulin (B1,B2) or NF-M (C1,C2). Images are stacks of five confocal microscopic optical sections taken from the uninjected (A1,B1,C1) and injected (A2,B2,C2) sides of the same animal, parasagitally through the spinal cord. SC, spinal cord; MA, motor axons; SN, cellular nuclei of somitic myotomes. Scale bars in A2,B2,C2 also apply to A1,B1,C1, respectively.

View larger version (92K): [in this window] [in a new window]   Fig. 6. Binding to hnRNP K and expression of NF-M mRNA in vivo. (A,B) Co-immunoprecipitation and RT-PCR of NF-M (A) and peripherin (B) mRNAs with hnRNP K from juvenile brain. (1) NF-M PCR from plasmid template with Xenopus NF-M cDNA insert, which served as a positive control for NF-M PCR amplification. (2) NF-M RT-PCR of sample prior to co-IP, demonstrating NF-M mRNA presence in the lysate. (3) NF-M RT-PCR of anti-hnRNP K co-IP. (4) NF-M RT-PCR of anti-β-galactosidase co-IP, a control for non-specific IP. (5) EF1- RT-PCR of hnRNP K co-IP, demonstrating absence of mRNAs not associating with hnRNP K. (6) EF1- RT-PCR of TIC, demonstrating its presence in lysate. (B) (1,2) peripherin PCR from plasmid template, which served as positive controls for peripherin PCR with each primer set. (3) peripherin RT-PCR of lysate prior to co-IP using the first pair of primers (30 cycles). (4,5) peripherin RT-PCR of anti-hnRNP K co-IP with the first (30 cycles) and second (15 additional cycles) pair of nested primers, respectively. Std, 1 kb DNA ladder (NE Biolabs). (C-E) Expression of NF-M and peripherin mRNAs in unilaterally injected hnRNP K MO animals. Dorsal views in whole mount of the entire animal (C), spinal cord (C1) and head (C2) of a stage 39/40 tadpole, processed for NF-M in situ hybridization (digoxigenin-alkaline phosphatase). Stained neurons are on both sides of the spinal cord (C; arrowheads in C1) as well as in brain, trigeminal ganglion (Vth) and retinal ganglion cells (Rgc, C2). (D) Horizontal confocal optical section of a unilaterally injected stage 39/40 hnRNP K MO tadpole processed for NF-M FISH. Rostral is towards the upper left. (E) Dorsal view of the head of a stage 39/40 hnRNP K MO tadpole stained for peripherin mRNA. R, rhombomeres.

View larger version (64K): [in this window] [in a new window]   Fig. 7. Effects of hnRNP K knockdown on neurite outgrowth in culture. (A,A') Neuron from control MO culture with neurite. (B,B') Cells from bilaterally injected hnRNP K MO cells lacking neurites. Arrowheads in A and B indicate cells identified as neurons because they were positive for NF-M mRNA (by FISH). (C) The fraction (±s.d., n=4 cultures) of NF-M mRNA-positive cells that had neurites. In parentheses are the total number of NF-M mRNA-positive cells scored. Asterisks indicate significant variation relative to control MO and uninjected cultures (P<0.01, one-way ANOVA). (D-E') Cells in control cultures immunostained for NF-M. Whereas most cells that expressed NF-M had neurites (D), a few did not (E). Scale bars in A and D apply to A-B' and D-E', respectively.

View larger version (35K): [in this window] [in a new window]   Fig. 8. FISH and immunohistochemistry for NF-M. (A,A') NF-M FISH of single confocal optical sections from opposite sides of spinal cord or unilaterally injected hnRNP K MO1 tadpole (stage 39/40). Broken outlines surround individual neurons. (B1-D4) Cells from dissociated cell culture. (B1-B3) Control MO neuron stained for NF-M protein (B1), RNA (B2) and RNA/DAPI merged (B3). (C1,C2) Adjacent cells from a unilaterally injected hnRNP K MO culture, viewed for NF-M protein (C1) and RNA (C2). Arrow indicates normal staining for both protein and RNA; arrowhead indicates a cell with no protein and FISH pattern typical of hnRNP K MO-injected spinal cord neurons from whole mount. (D1-D4) Neurons from a bilaterally injected hnRNP K MO culture stained for protein (D1), RNA (D2), DAPI (D3) and RNA/DAPI merged (D4). Scale bars in B1,C1 and D2 apply to B1-3,C1-2 and D1-4, respectively. Bar graph shows qRT-PCR of nuclear and cytoplasmic fractions for NF-M and peripherin RNAs from bilaterally injected hnRNP K MO and control animals assayed at stage 29/30. CT, mean (±s.d.) difference in the number of PCR cycles to reach threshold (see text). *CT was significantly less for NF-M RNA in hnRNP K MO embryos than for other groups (P<0.01, one-way ANOVA).

View larger version (46K): [in this window] [in a new window]   Fig. 9. Polysome profiling of NF-M and peripherin mRNAs. Black bars indicate the distribution of NF-M or peripherin mRNA across fractions, represented as a percentage of the total of each respective mRNA in the gradient (right ordinate). The black line depicts A260 values (left ordinate) across fractions. `M' indicates the position of the monosomal peak in the A260 trace. (Top four panels) During normal development, NF-M (left) and peripherin (right) profiles shift from right to left, indicating mRNA moving from poorly translated to efficiently translated fractions. (Bottom two panels) Bilaterally injected hnRNP K MO1 embryos processed at stage 29/30. hnRNP K knockdown strongly inhibited the shift into heavier polysomal fractions for NF-M but not peripherin mRNA.

View larger version (47K): [in this window] [in a new window]   Fig. 10. hnRNP K suppression disrupts cytoskeletal cytoarchitecture. (A,C,E) Bilaterally injected control MO cultures exhibited normal distributions for all three cytoskeletal elements (A, phalloidin-stained F-actin; C, peripherin-immunostained neurofilaments; E, neuron-specific β-tubulin). (B,D,F) The cytoarchitectures of these elements was significantly disrupted in bilaterally injected hnRNP K MO cultures. F-actin circumscribed the cell body and was concentrated towards one side (B). Peripherin formed twisted filaments and aggregates (D), and neuronal β-tubulin formed ring-shaped structures (F).

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