XB-ART-51903Development April 1, 2016; 143 (7): 1134-48.
Tumor protein Tctp regulates axon development in the embryonic visual system.
The transcript encoding translationally controlled tumor protein (Tctp), a molecule associated with aggressive breast cancers, was identified among the most abundant in genome-wide screens of axons, suggesting that Tctp is important in neurons. Here, we tested the role of Tctp in retinal axon development in Xenopus laevis We report that Tctp deficiency results in stunted and splayed retinotectal projections that fail to innervate the optic tectum at the normal developmental time owing to impaired axon extension. Tctp-deficient axons exhibit defects associated with mitochondrial dysfunction and we show that Tctp interacts in the axonal compartment with myeloid cell leukemia 1 (Mcl1), a pro-survival member of the Bcl2 family. Mcl1 knockdown gives rise to similar axon misprojection phenotypes, and we provide evidence that the anti-apoptotic activity of Tctp is necessary for the normal development of the retinotectal projection. These findings suggest that Tctp supports the development of the retinotectal projection via its regulation of pro-survival signalling and axonal mitochondrial homeostasis, and establish a novel and fundamental role for Tctp in vertebrate neural circuitry assembly.
PubMed ID: 26903505
PMC ID: PMC4852495
Article link: Development
Genes referenced: actb b2m bcl2 cox5a cycs gcg hist1h4a hprt1 idh3a map2 mcl1 pax6 rho rhot1 rps13 tbp tpt1
Morpholinos: mcl1 MO1 tpt1 MO2
Article Images: [+] show captions
|Fig. 1. Expression of tctp in the Xenopus neural retina. (A) Coronal section of stage 43 retina probed with an anti-Tctp antibody and counterstained with DAPI. Arrowheads indicate the optic fibre layer (OFL). The boxed area is enlarged beneath. The dashed contour delineates the outer plexiform layer. (B) Stage 32 eye explants grown in vitro for 24 h were stained with anti-Tctp antibody (left, phase contrast image; right, Tctp antibody staining). Tctp is detected in the axon shaft, central domain and filopodia. (C) In situ hybridisation (ISH) detection of tctp mRNA expression on coronal sections of stage 43 retinas. Arrowheads indicate the OFL. The boxed area is enlarged in the middle panel. (D,E) Quantitative ISH detection of tctp mRNA expression in the RGC axonal and growth cone compartments was performed using stage 32 eye explants grown in vitro for 24 h. Mean±s.e.m.; ***P<0.0001, one-way ANOVA with Bonferroni correction. (F) RACE amplifications of tctp mRNAs using retinal RNA extracts. FP, forward primer; NUP, nested universal primer; RP, reverse primer; UP, universal primer. (G) Organisation of the tctp gene in X. laevis. cds, coding region; poly(A) signal, polyadenylation signal. (H) Schematic of the laser-capture microdissection procedure used to collect RGC axonal extracts. (I) RACE amplifications of tctp mRNAs using laser-captured axonal extracts. (J) Purity assessment of laser-captured material by RT-PCR. –RT, RNA samples not reverse transcribed. (K) RT-qPCR experimental design. (L,M) Axonal and whole-eye content of tctp mRNAs were analysed by RT-qPCR and normalised to actb expression. In L, data are plotted as ‘tctp-S+tctp-L’ to ‘tctp-L’ expression ratios (*P=0.0175, one-way ANOVA), whereas in M the quantification cycle (Cq) difference relative to actb is shown. Scale bars: 50 μm in A,C; 5 μm in B,D. CMZ, ciliary marginal zone; GCL, ganglion cell layer; IPL/OPL, inner/outer plexiform layer; ONH, optic nerve head; PR, photoreceptor layer.|
|Fig. 5. The retinotectal projection develops unerringly in Tctp-deficient brains. (A) Experimental outline illustrating the two experimental scenarios created to investigate the contribution of extracellular Tctp to the optic tract pathway substrate. (B) Dorsal view of embryos microinjected unilaterally with fluorescein-tagged con-MO or tctp-MO. (C) Unilateral tctp-MO injection leads to a targeted knockdown in Tctp expression in half of the CNS, as shown by immunoblot analysis of eye or brain lysates. The ‘ipsilateral’ label refers to the MO-injected half of the embryo; the uninjected half is designated ‘contralateral’. (D-G) DiI-filled stage 40 retinotectal projections. Dashed lines approximate the boundary of the optic tectum. (H) Relative projection lengths. Mean±s.e.m.; n, number of brains analysed; **P=0.0002, Kruskal–Wallis and Dunn's multiple comparison test (for details of statistics see Fig. S2F). (I) Number of brains displaying axon extension defects. Eye-MO:Brain-wt backgrounds, *P=0.0352; tctp-MO backgrounds, *P=0.0364; Fisher's exact test; analyses performed on frequencies but plotted as percentage. Scale bars: 100 μm.|
|Fig. 8. Axonal Tctp interacts with pro-survival Mcl1. (A) Coronal section of stage 43 retina probed with an anti-Mcl1 antibody and counterstained with DAPI. (B,C) PLA signal for Tctp and Mcl1 in cultured rat cortical neurons (E18.5+3 DIV) counterstained with DAPI and phalloidin. The boxed areas are enlarged beneath. In C, anti-Mcl1 serum and blocking peptide were co-incubated before proceeding with the assay. (D) Representative control and Tctp morphant RGC growth cones stained for P53. Mean±s.e.m.; n, number of growth cones analysed; ***P=0.0002, unpaired t-test. (E) Representative control and Tctp morphant RGC growth cones stained with an antibody that specifically recognises the cleaved (activated) form of Caspase-3. Mean±s.e.m.; n, number of growth cones analysed; ***P=0.0002, unpaired t-test. Scale bars: 50 μm in A; 10 μm in B,C; 5 μm in D,E.|
|Figure S5. Pro-survival Mcl1 is expressed in the axonal compartment. (A) Complements Fig. 8A: Optic nerve head (ONH) region of a stage 43 wild-type retina (coronal section) probed for Mcl1 and counterstained with DAPI. Mcl1 was detected in the optic fiber layer and the optic nerve head, indicating that, like Tctp, this protein localizes to retinal ganglion cell axons in vivo. (B) Complements Fig. 8A: Outer nuclear layer of a stage 43 wild-type retina (coronal section) probed for Mcl1 and Rhodopsin, and counterstained with DAPI. Similar to Tctp, Mcl1 was detected in the inner segment of photoreceptors. (C and D) Cultured rat cortical neurons (E18.5 + 3DIV) stained for Mcl1 or Tctp, and counterstained with Phalloidin. Scale bars: 25 μm in (A), (C) and (D), 10 μm in (B).|
|Figure S6. Axonal Tctp interacts with pro-survival Mcl1. (A) PLA signal (green) obtained using anti-Tctp and anti-Mcl1 sera in control- or tctp-shRNA-infected HCT116 cells, counterstained with DAPI (blue) and phalloidin (red). (B) PLA signal obtained using anti-Tctp and anti-Mcl1 sera in cultured rat cortical neurons (E18.5 + 14 DIV), counterstained with DAPI and phalloidin. (C) PLA signal obtained using anti-Tctp and anti-Mcl1 sera in cultured rat cortical neurons (E18.5 + 3 DIV), counterstained with MitoTracker. Arrowheads denote PLA positive puncta co-localizing with mitochondria. The bottom three panels are centred on a distal region of the neurite. The data indicates that approximately 5-10% of Tctp-Mcl1 PLA puncta co-localize with mitochondria in neurites. This is in agreement with observations by Yang and colleagues who found by immunocytochemistry means that Tctp and Bcl-XL partially co-localize not only in mitochondria but also in the cytosol (Yang et al., 2005). It is noteworthy that while anti-apoptotic Bcl-2 family proteins are generally integrated within the outer mitochondrial membrane, they can also be found in the cytosol or in the endoplasmic reticulum membrane (Chipuk et al., 2010). Furthermore, pro-apoptotic Bax, whose homodimerization in the outer mitochondrial membrane is prevented by Tctp (Susini et al., 2008), is usually cytosolic (Chipuk et al., 2010). Likewise, the cellular localization of Tctp is mainly cytoplasmic in healthy cells (Bommer and Thiele, 2004; Gachet et al., 1999; Yang et al., 2005). One possibility is that Tctp shuttles between the cytosol and the outer mitochondrial membrane in non-apoptotic conditions. (D) Stage 43 Mcl1-depleted retina probed with anti-Opsin antibody, counterstained with phalloidin and DAPI. Signs of morphological disruption were not detected on examination of the retina in Mcl1 morphants, suggesting that the mcl1-MO does not elicit widespread toxicity. Scale bars: 5 μm in (A), 10 μm in (B) and (C), 50 μm in (D).|
|Figure S8. Axonal Tctp interacts with Mcl1-related Bcl-XL. (A) Cultured rat cortical neurons (E18.5 + 3DIV) stained for Bcl-XL, and counterstained with Phalloidin. (B) PLA signal obtained using anti-Tctp and anti- Bcl-XL sera in cultured rat cortical neurons (E18.5 + 14 DIV), counterstained with DAPI and phalloidin. (C) Co-delivery of tctp-MO and tctp40-172 mRNA, encoding a truncated Tctp protein devoid of anti-apoptotic activity, fails to rescue the effects of Tctp depletion on the development of the retinotectal projection. Number of embryos displaying axon extension defects (‘tctp-MO’ versus ‘tctp-MO + tctp40-172 mRNA’: n.s., P = 1.00; Fisher’s exact test, performed on number of observations but plotted as percentages). Scale bars: 25 μm in (A), 10 μm in (B).|