A family of rat CRMP genes is differentially expressed in the nervous system

Abstract

Members of the collapsin/semaphorin family play an important role in creating the complex pattern of neuronal connectivity. Inhibition of growth cone motility by chick collapsin is mediated by the intraneuronal protein CRMP-62. We have now isolated four rat sequences that are highly related to chick CRMP-62. All four genes are expressed exclusively in the nervous system and primarily during development. Rat CRMP-2/TOAD-64 is most closely related to chick CRMP-62 and is the most widely expressed CRMP within the nervous system. Rat CRMP-1 and CRMP-4/rUlip are expressed during discrete periods of neuronal development and are not found in the adult nervous system. Rat CRMP-3 has a distinct distribution, being expressed transiently in developing spinal cord and selectively in the postnatal cerebellum. The differential expression of these genes suggests that CRMPs may transduce signals from different semaphorins and that semaphorins may regulate the plasticity of the adult nervous system.

Fig. 1.

Amino acid alignments of CRMP-related proteins. The deduced amino acid sequence for four rat CRMP-related genes (R-1, R-2, R-3, and R-4) and three human CRMP-related genes (H-1, H-2, and H-3) is compared with CRMP-62 (CH). Those residues that differ from the consensus are shown in bold type. GenBank accession numbers for R-1, R-2, and R-3 areU52102, U52103, and U52104, respectively.

Fig. 2.

Northern analysis of CRMP-related RNAs in various rat tissues. Fifteen micrograms of total RNA from the indicated P1 rat tissues were separated on an agarose–formaldehyde gel, transferred onto nylon membranes, and hybridized to ³²P-labeled CRMP cDNA probes. The probes were theEagI–NsiI 2.0 kb fragment of rCRMP-1 cDNA, the SspI–PstI 1.7 kb fragment of rCRMP-2 cDNA, the entire 2.5 kb cDNA fragment of CRMP-3, and the 1.1 kb PCR product corresponding to amino acids 25–390 of rCRMP-4/rUlip, respectively. RNAs were visualized with ethidium bromide for examining the equivalent amount of each sample. The position of 28S and 18S ribosomal RNA is shown. Note that the expression of all four CRMPs is neural-specific.

Fig. 3.

Northern analysis of rCRMP expression in the developing nervous system. Total RNA from the indicated tissues was prepared. RNA blots were performed as described in Figure 2. The position of 28S and 18S ribosomal RNA is shown. Note that the expression of rCRMP-3 is specific for cerebellum of postnatal rats. Rat CRMP-2/TOAD-64 has the most persistent expression during development.

Fig. 4.

In situ hybridization of rCRMP mRNAs in developing rat forebrain. Coronal sections (14 μm) ofE15 or P1 rat forebrain were hybridized separately with digoxigenin-labeled antisense riboprobes derived from rCRMP-1, rCRMP-2/TOAD-64, rCRMP-3, or rCRMP-4/rUlip as described in Materials and Methods. The probes were theBglII–NsiI 1.0 kb fragment of CRMP-1, the BamHI–EcoRI 1.2 kb fragment of CRMP-2/TOAD-64, thePstI–EcoRI 1.3 kb fragment of CRMP-3, and the 1.1 kb cDNA fragment corresponding to amino acid residue 25–390 of CRMP-4/rUlip, respectively. At E15, only CRMP-2/TOAD-64 mRNA is expressed in the ventricular zone (arrowhead), whereas the signals of CRMP-1 and CRMP-4/rUlip are detected only in the differentiated neurons (arrow). At P1, the mRNAs of CRMP-1, CRMP-2/TOAD-64, and CRMP-4/rUlip are enriched in the cortex (C), thalamus (T), and hippocampus (H). The pyramidal cells display higher intensity than that of dentate granule cells. The location of basal ganglia is marked by an asterisk. Scale bar, 400 μm.

Fig. 5.

In situ hybridization of rat CRMP mRNAs in cerebellum. Horizontal sections (14 μm) ofP15 or adult rat (AD) cerebellum were hybridized with digoxigenin-labeled CRMP cRNA probes as described in Figure 4. Note the staining of the external germinal layer (g), the Purkinje cell layer (p), and the granule cell layer (gc). The molecular layer (m) is not stained. Rat CRMP-1 staining is widely distributed at P15 and is markedly decreased in adult. The expression of rCRMP-2/TOAD-64 in Purkinje cells persists through adulthood. rCRMP-3 mRNA is localized primarily to granule cells and is reduced only slightly from P15 to the adult. rCRMP-4/rUlip is expressed primarily in the external germinal layer at P15 and is absent in adult. Scale bar, 100 μm.

Fig. 6.

In situ hybridization of rat CRMP mRNA in E13 developing spinal cord. Cross sections (14 μm) of E13 rat embryo were hybridized with digoxigenin-labeled CRMP cRNA probes as described in Figure 4. The staining of rCRMP-2/TOAD-64 and rCRMP-3 mRNA was observed at both dorsal and ventral sides of the spinal cord as well as the DRG. The signals of rCRMP-1 and rCRMP-4/rUlip were less abundant and were limited to the ventral side and DRG. Scale bar, 250 μm.

Fig. 7.

In situ hybridization of rat CRMP mRNAs in developing spinal cord. Cross sections (14 μm) ofE15 or P1 rat spinal cord were hybridized with digoxigenin-labeled CRMP antisense riboprobes as described in Figure 4. At E15, the strongest signal of CRMP-3 is detected in the dorsal germinal matrix layer, whereas the staining of CRMP-1 and CRMP-4/rUlip is reciprocal to that of CRMP-3. ByP1, CRMP-3 signal is undetectable in the spinal cord. Scale bar, 200 μm.

Fig. 8.

Localization of rat CRMP mRNAs in DRG or sympathetic ganglion neurons by in situ hybridization. Cross sections of the P1 or adult rat (AD) thorax were hybridized with digoxigenin-labeled CRMP cRNA probes as described in Figure 4. DRG (P1 and adult) or sympathetic ganglia (P1) are shown. Note that the rCRMP-3 signal is absent in the P1 sympathetic ganglia (s). The expression of rCRMP-2/TOAD-64 and rCRMP-4/rUlip is prominent in P1 DRG neurons (d) and persists through adulthood. The mRNA of rCRMP-1 and rCRMP-3 is widely expressed in P1 DRG neurons and is differentially detected in adult DRG neurons. Scale bar, 200 μm.