Journal: The Journal of Neuroscience

Article Title: Expression of Neuregulins and their Putative Receptors, ErbB2 and ErbB3, Is Induced during Wallerian Degeneration

doi: 10.1523/JNEUROSCI.17-05-01642.1997

Figure Lengend Snippet: Identification of neuregulin splice variants expressed in axotomized sciatic nerve and the corresponding neuronal populations. A, Poly(A+) RNA was isolated from a pool of surgically transected sciatic nerve distal to the site of transection collected 16 hr, 3 d, and 7 d postinjury and a pool of L4, L5, and L6 dorsal root ganglia and lumbar spinal cord collected 7 and 10 d postaxotomy. Each RNA pool was reverse-transcribed to cDNA with random hexamer primers and used as templates for PCR with primers, the positions of which are indicated byarrows (modified from Ben-Baruch and Yarden, 1994). Distinctly different subsets of neuregulin splice variants were identified in axotomized sciatic nerve as opposed to the regions of the nervous system projecting axons into this same structure. Regions encompassed by these cDNAs include the EGF-like common domain (EGF), EGF-like variable domains (α or β), the juxtamembrane domains (numbers 1–4), and the transmembrane domain (TM). Note that splice variants with a “3” juxtamembrane domain terminate within the juxtamembrane domain (indicated by a dark bar at the C terminus). Ratios beneath each juxtamembrane domain indicate the number of times a clone with each α/β-juxtamembrane combination was isolated relative to the number of clones generated with primers “A” and “B.” Ratios in parenthesesindicate the same values for clones generated using the “A” primer in combination with “C” (4 juxtamembrane-specific) or “D” (3 juxtamembrane-specific) reverse primers. B, Schematic representation of currently known members of the GGF and SMDF neuregulin subfamilies. Although the encoded proteins contain domains in common [e.g., the EGF-like common domain (EGF)], each NRG subfamily is distinguished from one another by unique N termini [represented by SP (signal peptide) andKringle for GGF and the SMDF N terminus (Apolar) above]. Only truncated and presumably secreted (β3) variants have been described previously for GGF (Marchionni et al., 1993) and SMDF (Ho et al., 1995). In contrast, the clones described in this work represent transmembrane precursors (β1a variants) of SMDF and GGF; the presence of all domains illustrated has been confirmed by sequence analysis. These transmembrane precursors may either be cleaved to release soluble factor (Burgess et al., 1995) or potentially remain embedded in the membrane to mediate juxtacrine interactions (Bosenberg and Massague, 1993). Domains not defined above are as follows:Ig-like, Immunoglobulin-like; Glyco, glycosylation region; β, EGF-like β variable; 1 and3, juxtamembrane domains; Cytoplasmic, cytoplasmic domain common to all neuregulin transmembrane precursors;a, one of three potential neuregulin C termini.

Article Snippet: To make an initial assessment of whether appreciable levels of neuregulin mRNAs are expressed in normal and axotomized sciatic nerve and the neuronal populations projecting axons into this nerve, RNA samples were probed by Northern blotting using a cDNA fragment encoding the extracellular domain of a GGF (kringle-β1a variant) cDNA (Fig. A ).

Techniques: Isolation, Random Hexamer Labeling, Modification, Clone Assay, Generated, Sequencing

Journal: The Journal of Neuroscience

Article Title: Expression of Neuregulins and their Putative Receptors, ErbB2 and ErbB3, Is Induced during Wallerian Degeneration

doi: 10.1523/JNEUROSCI.17-05-01642.1997

Figure Lengend Snippet: Neuregulin mRNA expression in sciatic nerve is induced by axotomy. A, A cDNA fragment (Probe) spanning the neuregulin transmembrane (TM), EGF-like (EGFβ1), immunoglobulin-like (Ig-like), and Kringle domains of a GGF (kringle-β1a) neuregulin splice variant was used to probe a Northern blot (15 μg of total cellular RNA/lane). Up to three mRNA species (2.0, 3.5, and 7.5 kb) are seen in this exposure. This blot has been purposefully overexposed to demonstrate the presence of the 7.5 kb transcript. B, A total of 10 μg per lane of cellular RNA from noninjured (Control) sciatic nerve and the nerve segment distal to a site of surgical transection taken 1, 3, 5, 7, 18, or 30 d postaxotomy was probed with the same probe used in A. Repeat experiments performed with a mixture of GGF, SMDF, and NDF N-terminal probes confirmed these observations and also demonstrated levels of neuregulin mRNA expression at 10 d postaxotomy similar to those at 7 and 18 d. Aphotograph of the ethidium bromide-stained gel before transfer is presented beneath the Northern blot to demonstrate the uniform loading of these samples; equivalent transfer was confirmed by examining the membrane under ultraviolet illumination.C, D, Northern blots of total cellular RNA (10 μg/lane) isolated from the lumbar enlargement of the spinal cord (SpCord), the nerve segment distal to a site of surgical transection 7 d postaxotomy (7d Nerve), JS1 rat schwannoma cells (JS1), gastrocnemius/soleus muscle (Muscle), Lung, small intestine (Sm Int), and large intestine (Lg Int) were probed with the indicated probes, which are specific for the N termini of GGF (C) or SMDF (D) neuregulin splice variants. Except for the 7 d postaxotomy nerve, all tissues were collected from animals that had not undergone any surgical manipulation. Expected positions of the 2.0, 3.5, and 7.5 kb mRNAs are indicated. Note that the GGF probe hybridizes to the 2.0 kb transcript, whereas the SMDF probe recognizes both the 3.5 and 7.5 kb mRNAs. Although SMDF expression was most prominent in JS1 cells, longer exposures of this blot also demonstrated mRNAs of the same size in spinal cord (data not shown). Again, photographs of the ethidium bromide-stained gels before transfer are presentedbelow each autoradiograph.

Article Snippet: To make an initial assessment of whether appreciable levels of neuregulin mRNAs are expressed in normal and axotomized sciatic nerve and the neuronal populations projecting axons into this nerve, RNA samples were probed by Northern blotting using a cDNA fragment encoding the extracellular domain of a GGF (kringle-β1a variant) cDNA (Fig. A ).

Techniques: Expressing, Variant Assay, Northern Blot, Staining, Isolation, Autoradiography