XB-ART-43454Development July 1, 2011; 138 (14): 3079-90.
hnRNP K post-transcriptionally co-regulates multiple cytoskeletal genes needed for axonogenesis.
The RNA-binding protein, hnRNP K, is essential for axonogenesis. Suppressing its expression in Xenopus embryos yields terminally specified neurons with severely disorganized microtubules, microfilaments and neurofilaments, raising the hypothesis that hnRNP K post-transcriptionally regulates multiple transcripts of proteins that organize the axonal cytoskeleton. To identify downstream candidates for this regulation, RNAs that co-immunoprecipitated from juvenile brain with hnRNP K were identified on microarrays. A substantial number of these transcripts were linked to the cytoskeleton and to intracellular localization, trafficking and transport. Injection into embryos of a non-coding RNA bearing multiple copies of an hnRNP K RNA-binding consensus sequence found within these transcripts largely phenocopied hnRNP K knockdown, further supporting the idea that it regulates axonogenesis through its binding to downstream target RNAs. For further study of regulation by hnRNP K of the cytoskeleton during axon outgrowth, we focused on three validated RNAs representing elements associated with all three polymers - Arp2, tau and an α-internexin-like neurofilament. All three were co-regulated post-transcriptionally by hnRNP K, as hnRNP K knockdown yielded comparable defects in their nuclear export and translation but not transcription. Directly knocking down expression of all three together, but not each one individually, substantially reproduced the axonless phenotype, providing further evidence that regulation of axonogenesis by hnRNP K occurs largely through pleiotropic effects on cytoskeletal-associated targets. These experiments provide evidence that hnRNP K is the nexus of a novel post-transcriptional regulatory module controlling the synthesis of proteins that integrate all three cytoskeletal polymers to form the axon.
PubMed ID: 21693523
Article link: Development
Species referenced: Xenopus
Genes referenced: actl6a actr2 aicda hnrnpc hnrnpk mapt mark3 nefm neurog1 neurog2 neurog3 nif pard3 pard6b pkm prph tubb2b
Antibodies: Hnrnpk Ab1 Mapt Ab1 Mark3 Ab1 Nefm Ab1 Nif Ab1 Pard3 Ab1 Tubb2b Ab3
Morpholinos: hnrnpk MO3 mapt MO1 nif MO1
GEO Series: GSE25207: NCBI
Article Images: [+] show captions
|Fig. 1. Axon outgrowth and cytoskeletal organization, but neither neuronal specification nor early stages of cellular polarization, were severely compromised with hnRNP K knockdown. (A) Immunostained optical section of a stage 40 Xenopus tadpole demonstrates suppression of hnRNP K expression on the injected side by hnRNP K MO (below dots). SC (inside dashed lines), spinal cord; arrowheads, hnRNP K staining of muscle nuclei on the uninjected side. (B-Db) Suppression of axon outgrowth on the injected side of stage 40 tadpoles [B(bottom),Cb,Db] revealed by immunostaining for the indicated axonal markers. A and B are horizontal and Ca-Db parasagittal sections. Arrowheads in Cb and in Da and Db indicate examples of atrophied axons and neuronal perikarya, respectively. Arrowheads in B indicate normally projecting motor axons on the uninjected side. (Ea-Gc) Organization of the indicated cytoskeletal polymers was severely disrupted in neurons of bilaterally injected hnRNP K MO (PK MO) cultures compared with those of control MO (Ea,Fa,Ga), which appeared normal. With hnRNP K MO, F-actin circumscribed perikarya, but was asymmetrically distributed (Eb,Ec); microtubules formed an anastomosing network (Fb,Fc); neuronal intermediate filaments were twisted and radiated outward in multiple directions (Gb,Gc). (Ha-Id) Double immunostaining for N-β tubulin and Par6 as indicated. With control MO (Ha,Hb), Par 6 staining was punctate and filled neuronal perikarya and axons (arrowheads, growth cone). With hnRNP K MO (Ia-Id), neuronal perikaryal microtubules (Ic,Id) were poorly organized and failed to correspond with an asymmetric hotspot of Par6 immunostaining (Ia,Ib). Scale bars: 100 μm for A-Db; 20 μm for Ea-Id.|
|Fig. 5. Axon outgrowth was suppressed by injection into embryos of a non-coding RNA bearing the triple tripartite hnRNP K RNA-binding motif. (Aa-Cb) Parasagittal optical sections of whole-mount immunostained stage 37/38 animals. (Aa-Ad) Animals receiving the triple tripartite binding motif. Aa shows the uninjected (Uninj.) side with arrowheads indicating motor axons. Animals were immunostained for the indicated axonal markers (NF-M; peripherin; neuronal (N) β-tubulin). Arrowheads in Ab-Ad point to severely atrophied fibers. (Ba-Cb) Two control RNAs yielded normal axon outgrowth and expression of NF-M on the injected side. Scale bars: 100 μm. (Da-Dd) In dissociated embryonic spinal cord cultures, the triple tripartite binding motif RNA yielded neurons (positively stained for N-β tubulin) with marked defects in microtubule organization and that either lacked neurites (Da) or had only short, immature neurites (Db). Neurons from indicated control RNA cultures (Dc,Dd) were normal. Scale bars: 20 μm. (E) Top: Mean (± s.d.) fraction of N-β tubulin-positive cells that had neurites. Bottom left: Mean (± s.d.) total length of neuritic arbor per cell. Bottom right: Mean (± s.d.) number of higher order branches (secondary and higher) per cell. 1, uninjected; 2, single tripartite binding motif-injected; 3, triple tripartite binding motif-injected; 4, triple mutated tripartite motif-injected; 5, triple scrambled tripartite motif-injected cultures. Because embryos were unilaterally injected, only the half derived from the uninjected side was expected to be normal (indicated by dashed line). **P<0.01, t-test. Numbers of neurons tabulated were: upper graph (four cultures each), bars 1-5: 610, 625, 631, 591, 632, respectively; lower graphs (three cultures each), bars 1-5: 169, 158, 155, 142, 184, respectively.|
|Fig. 6. hnRNP K MO inhibits protein but not mRNA expression of select targets in vivo. (A) qRT-PCR (SYBR Green) for identified hnRNP K cytoskeletal target (Arp2, tau, XNIF) and non-target (peripherin) RNAs. Ordinate, fold difference relative to loading control (GAPDH RNA). Differences were insignificant among the groups (one-way ANOVA, P>0.1). W.T., uninjected wild type; Con., control MO-injected; Uni. or Bi., unilaterally or bilaterally injected, respectively, with hnRNP K MO. (B) Chemiluminescent western blot and quantified band intensities for Arp2, tau and XNIF (after normalization to GAPDH) relative to uninjected controls. (1) uninjected controls; (2) unilaterally injected, control MO: 95±6%; 102±8%; 96±5%, respectively; (3) unilaterally injected, hnRNP K MO: 54±6%, 53±9%, 52±4%, respectively; (4) bilaterally injected, hnRNP K MO: 18±3%, 12±2%, 15±3%, respectively. (Ca-Ec) Parasagittal, confocal, optical sections of stage 29/30 embryos injected with MO and immunostained for Arp2 (Ca-Cc), tau (Da-Dc), and XNIF (Ea-Ec). Normal expression is seen with control MO (Ca,Da,Ea) but was effectively suppressed by hnRNP K MO (Cb,Db,Eb). Inhibition of Arp2, XNIF and tau expression was effectively restored by co-injection of hnRNP K RNA with the antisense MO (Cc,Dc,Ec). Note that axon outgrowth was also rescued, as seen with tau and XNIF (Dc,Ec, arrowheads). Optical sections for Arp2 are too close to the midline to view axons (Ca-Cc). SC, spinal cord. Ca-Cc contain notochord (NC), whereas Da-Ec contain motor axons and somites. Rostral is left in Da-Dc and right in Ca-Cc,Ea-Ec. Scale bars: 100 μm.|
|Fig. 8. Triple knockdown of Arp2, tau and XNIF substantially phenocopied hnRNP K knockdown. (Aa-Cb) Parasagittal optical sections of stage 37/38 heads immunostained for peripherin in whole mount. Whereas Arp2 MO (Aa,Ab) and tau MO (Ba,Bb) injected individually yielded only moderate defects in peripheral axon outgrowth on the injected side, triple knockdown of Arp2, tau and XNIF yielded severe defects (Ca,Cb). II, optic; Vop and Vmd, opthalmic and mandibular branches of the trigeminal; VII, facial; IX, glossopharyngeal; and X, vagus nerves. (Da-Fe) In dissociated culture, Arp2 and tau MO injected individually yielded marked defects in actin filaments and microtubules, respectively, compared with control MO (Da-Dd, phalloidin stained; Ea-Ed, N-β tubulin immunostained). Triple knockdown of Arp2, tau and XNIF synergistically compromised axon outgrowth severely (Fb,Fd) compared with control MO (Fa,Fc). Fa,Fb, phalloidin stained; Fc,Fd, N-β tubulin immunostained. Scale bars: 100 μm for Aa-Cb; 10 μm for Db,Dd,Eb,Ed; 20 μm for Da,Dc,Ea,Ec,Fa-Fd. Bar graphs show mean total length of neuritic arbor and number of higher order branches (secondary and higher) per cell (±s.d., three cultures). White bars, uninjected; black bars, control MO-injected; gray bars, bilaterally injected with Arp2 (De), tau (Ee) or all three (Fe) MOs. **P<0.01, t-test. Numbers of uninjected, control MO and experimental MO neurons were: De: 128, 107, 106; Ee: 117, 104, 109; Fe: 108, 115, 126, respectively.|
|Fig. S1. Par1, 3 and 6 are expressed in juvenile frog brain. Immunostaining as indicated for Par1 A; also known as microtubule affinity-regulating kinase 2 (MARK2), Par 3 (B) and Par6 (C) in neuronal perikarya scattered throughout the neuropil of the dorsal pallium of the telencephalon of juvenile frog brain. Arrowheads point to examples of immunoreactive neurons. Scale bar applies to all panels.|
|Fig. S3. Arp2, tau and XNIF MOs suppress their respective protein expression in vivo. (Aa-Cb) Immunofluorescence, confocal microscope optical parasagittal sections of embryos unilaterally injected with Arp2 (Aa-Bb) or tau (Ca,Cb) MO and immunostained for their respective proteins. Substantially reduced immunostaining is seen on each injected side compared with the uninjected side. Aa and Ab are through somites (SM); Ba and Bb are through spinal cord (SC) and notochord (NC). Unmarked arrowheads in Ca depict motor axons. (Da,Db) Head of an immunoperoxidase labeled stage 33/34 tadpole injected unilaterally with XNIF MO, viewed through the stereomicroscope. XNIF-positive peripheral axons of the trigeminal (arrowhead, Da) and other cranial nerves are visible on the injected but not the uninjected side. Scale bars in Ab, Cb and Db apply to Aa-Bb, Ca and Da, respect|
|Parasagittal optical sections of stage NF 37/38 Xenopus tadpole head regions, immunostained for peripherin in whole mount. Position of cranial nerves II, V, VII, IX and X are indicated.|
|Comparative in situ hybridization analysis of X. tropicalis Ngn1, Ngn2, Ngn3, and X. laevis Ngn1, Ngnr-1, and Ngn3 in stage 30 embryos. Probes are indicated on the left of each line of embryos. Fourth panel, transverse section at the level of the retina; fifth panel, transverse section at the level of the otic vesicle. The transversal sections were performed with embryos stained for both Ngn (dark blue) and N-tubulin (red). VII, facial epibranchial placode; IX, glossopharyngeal epibranchial placode; X1, first vagal epibranchial placode; X2, second vagal epibranchial placode; X3, third vagal epibranchial placode; hb, hindbrain; mb, midbrain; nc, notochord; op, olfactory placode; vOT, otic vesicle; tg, trigeminal placode; pg, pineal gland; pP, posterior lateral line placode; pM, middle lateral line placode; pAD, anterodorsal lateral line placode; r, retina.|