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Teneurin-1 Is Expressed in Interconnected Regions of the Developing Brain and Is Processed in Vivo

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Teneurin-1 Is Expressed in Interconnected Regions of the Developing Brain and Is Processed in Vivo

Daniela Kenzelmann et al. BMC Dev Biol.

Abstract

Background: Teneurins are a unique family of transmembrane proteins conserved from C. elegans and D. melanogaster to mammals. In vertebrates there are four paralogs (teneurin-1 to -4), all of which are expressed prominently in the developing central nervous system.

Results: Analysis of teneurin-1 expression in the developing chick brain by in situ hybridization and immunohistochemistry defined a unique, distinct expression pattern in interconnected regions of the brain. Moreover we found complementary patterns of teneurin-1 and-2 expression in many parts of the brain, including the retina, optic tectum, olfactory bulb, and cerebellum as well as in brain nuclei involved in processing of sensory information. Based on these expression patterns, we suspect a role for teneurins in neuronal connectivity. In contrast to the cell-surface staining of the antibody against the extracellular domain, an antibody recognizing the intracellular domain revealed nuclear staining in subpopulations of neurons and in undifferentiated mesenchyme. Western blot analysis of brain lysates showed the presence of N-terminal fragments of teneurin-1 containing the intracellular domain indicating that proteolytic processing occurs. Finally, the teneurin-1 intracellular domain was found to contain a nuclear localization signal, which is required for nuclear localization in transfected cells.

Conclusion: Teneurin-1 and -2 are expressed by distinct interconnected populations of neurons in the developing central nervous system. Our data support the hypothesis that teneurins can be proteolytically processed leading to the release of the intracellular domain and its translocation to the nucleus.

Figures

Figure 1
Figure 1
Teneurin-1 is expressed in many parts of the developing CNS. In situ hybridization at E17 with a teneurin-1 antisense probe (sense controls were negative). In the olfactory bulb (A), there is a strong hybridization signal in the mitral cell layer (mcl). The ganglion cell layer (gcl) is also positive, but the external plexiform layer (epl) is not. In the retina (B), the ganglion cell layer (gcl) is labelled intensely, and there is a faint signal in neurons of the inner nuclear layer (inl) adjacent to the inner plexiform layer (ipl). The pigment retina (pr) has dark melanosomes. The nucleus rotundus (C) contains large, scattered neurons that are positive for teneurin-1 mRNA. In the optic tectum (D) teneurin-1 mRNA is widespread, but is seen most clearly in the large neurons of the stratum griseum centrale (sgc). In the hindbrain (E) the nucleus laminaris (La) and nucleus magnocellularis (MCC) are labelled, as are Purkinje cells (Pcl) and other neurons in the cerebellum (F).
Figure 2
Figure 2
Teneurin-1 is expressed in interconnected regions of the brain and is complementary to teneurin-2 expression. Immunostaining of teneurin-1 and teneurin-2 in E17 brain. Green, teneurin-1; red, teneurin-2, blue (Hoechst), nuclei. In the olfactory bulb (A, B) the teneurin-1 antibody labels the mitral cell layer (mcl) and the glomerular layer. The external plexiform layer (epl) is positive for teneurin-2. In the temporal retina (C, D) subsets of neurons within the ganglion cell layer (gcl) and inner portion of the internal nuclear layer (inl) are positive for teneurin-1, as are laminae 2 and 5 within the inner plexiform layer (ipl). Teneurin-2 immunoreactivity is concentrated in laminae 1 and 3 of the ipl. The nucleus rotundus (E, ROT) is intensely labelled with teneurin-1 antibody, but not with anti-teneurin-2 (F). In the optic tectum (G, H) large neurons in the stratum griseum centrale (SGC) are positive for teneurin-1, but not teneurin-2. In the hindbrain (I, J), the nucleus laminaris (La) and nucleus magnocellularis (MCC) are labelled with the teneurin-1 antibody. Teneurin-1 and teneurin-2 immunoreactivity are found in distinctive dendritic fields within the La. In the cerebellum (K, L) teneurin-1 is found in Purkinje cells (Pcl) and in the molecular layer (ml) adjacent to the external granular layer (egl). In contrast, the anti-teneurin-2 labels the internal portion of the ml.
Figure 3
Figure 3
Nuclear teneurin-1 intracellular domain is observed in subsets of neurons and in specific tissues. Schematic diagram of teneurin domain architecture and antibodies used in this study (A). Comparison of immunostaining with antibodies to the extracellular and intracellular domains in sections and in vitro (B to I). Neurons of the piriform cortex at E17 (B, C) and neurons found in the retinal ganglion cell layer at E12 (D, E) stained with anti-ICD. The anti-ICD labels nuclei in the piriform cortex and labels a subset of retinal ganglion cells (arrow), but other TuJ1-positive neurons (label not shown) in the retinal ganglion cell layer are labeled with anti-ICD teneurin-1 but do not exhibit nuclear staining (arrowhead). There are also TuJ1-positive neurons in the retinal ganglion cell layer that are negative for teneurin-1 (asterisk). Head mesenchyme at E7 stained with anti-EGF (F, G) and anti-ICD (H, I). In the head mesenchyme, the anti-ICD labels the nucleus of most cells, whereas the anti-EGF exhibits cell-surface staining. Green, teneurin-1; blue (Hoechst), nuclei.
Figure 4
Figure 4
Teneurin-1 appears to be processed in the developing chicken brain. Western blot analysis of embryonic chicken brain lysates. Detection of endogenous teneurin-1 using anti-ICD (A) and anti-ECD antibodies (B). The numbers to the left indicate the positions of molecular weight standards. The anti-ICD detects a prominent N-terminal fragment at 65 kDa (arrowhead), which is much smaller than expected for the full-length protein (arrow), suggesting proteolytic processing. In contrast, using the anti-ECD antibody results in detection of the full-length protein (arrow) and a large C-terminal fragment (arrowhead). Asterisks label additional processing products (see text for details). Postulated cleavage sites and calculated molecular weights of processing products (C). Postulated cleavage sites: 1 = ectodomain shedding, 2 = intramembrane cleavage releasing the ICD. The sizes indicated are predicted from the amino acid sequence; known apparent molecular weights are shown in parentheses (see text for details). The calculated molecular weights are approximate, as the exact cleavage sites are not known.
Figure 5
Figure 5
The teneurin-1 intracellular domain contains a nuclear localization signal. The amino acid sequence representing a putative nuclear localization signal (NLS) which was mutagenized to alanines (A). Western blot analysis of total lysates, cytoplasmic and nuclear fractions of COS-7 cells mock transfected, transfected with wild type ICD (ICD-WT) or transfected with the mutated NLS (NLS-Mut) constructs (B). Blots were probed with the anti-ICD, an antibody to the cytoplasmic protein vinculin, or an antibody to the nuclear protein Oct-1. Western blot quantification and calculation of the nuclear-to-cytoplasmic ICD ratio (C). Immunofluorescence staining of transfected COS-7 cells (D-G) and quantification of nuclear localization by cell counting (H). Both western blot analysis and immunofluorescence show that the overexpressed wild-type ICD is localized in the nucleus, whereas the NLS-Mut construct is predominantly found in the cytoplasm.
Figure 6
Figure 6
Model of teneurin signalling. This model describes how an intracellular domain (ICD) could be generated from a transmembrane protein and transported to the nucleus. This process takes place only in specific cells and tissues where the anti-ICD labeled nuclei. 1) A first protease sheds the extracellular domain (ECD) of teneurins. 2) The remainder of teneurin-1 can now become a substrate for an intramembrane protease. 3) The ICD is released. 4) The soluble ICD translocates to the nucleus via nuclear localization signal (NLS). EGF, EGF-like repeats; YD, YD-repeats. Red arrows indicate postulated cleavage sites and the yellow spheres proteins interacting with teneurin-1 ICD in the nucleus.

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