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Brain Res Mol Brain Res
2000 Oct 20;821-2:35-51. doi: 10.1016/s0169-328x(00)00180-7.
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Structure, biological activity of the upstream regulatory sequence, and conserved domains of a middle molecular mass neurofilament gene of Xenopus laevis.
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During development, the molecular compositions of neurofilaments (NFs) undergo progressive modifications that correlate with successive stages of axonal outgrowth. Because NFs are the most abundant component of the axonal cytoskeleton, understanding how these modifications are regulated is essential for knowing how axons control their structural properties during growth. In vertebrates ranging from lamprey to mammal, orthologs of the middle molecular mass NF protein (NF-M) share similar patterns of expression during axonal outgrowth, which suggests that these NF-M genes may share conserved regulatory elements. These elements might be identified by comparing the sequences and activities of regulatory domains among the vertebrate NF-M genes. The frog, Xenopus laevis, is a good choice for such studies, because its early neural development can be observed readily and because transgenic embryos can be made easily. To begin such studies, we isolated genomic clones of Xenopus NF-M(2), tested the activity of its upstream regulatory sequence (URS) in transgenic embryos, and then compared sequences of regulatory regions among vertebrate NF-M genes to search for conserved elements. Studies with reporter genes in transgenic embryos found that the 1. 5 kb URS lacked the elements sufficient for neuron-specific gene expression but identified conserved regions with basal regulatory activity. These studies further demonstrated that the NF-M 1.5 kb URS was highly susceptible to positional effects, a property that may be relevant to the highly variant, tissue-specific expression that is seen among members of the intermediate filament gene family. Non-coding regions of vertebrate NF-M genes contained several conserved elements. The region of highest conservation fell within the 3' untranslated region, a region that has been shown to regulate expression of another NF gene, NF-L. Transgenic Xenopus may thus prove useful for testing further the activity of conserved elements during axonal development and regeneration.
Fig. 1. Sequence of the upstream regulatory region of the Xenopus laevis NF-M(2) gene. The nucleotide marking the start of transcription is designated +1, whereas the one immediate upstream is −1. All others are numbered relative to this. Nucleotides of various features discussed in the text are underlined and labeled underneath. The beginning and end of the various deletion constructs used in the plasmid embryonic injection experiments are also designated below the appropriate nucleotide sequences. The initial codons of the predicted sequence of the NF-M(2) protein are labeled below the sequence with the single amino acid code. The full sequence of the NF-M(2) gene is available from GenBank under accession number AF237379.
Fig. 2. Verification of the transcription initiation site. Molecular weight standards (left lane; Hinc II ΦX174) and the product of a 5′ RACE PCR (right lane) were separated on a 1% TBE/agarose gel and stained with ethidium bromide. The RT-PCR was performed with three, successive, 3′-directed NF-M(2) gene-specific primers, beginning with total RNA isolated from brain. The final PCR was performed with an NF-M(2) gene-specific primer directed against sequences 250 nucleotides downstream from the initiation codon, which is approximately 310 and 690 nucleotides away from each of the two putative TATA boxes. A single strong band of PCR product occurs at approximately 300 bp.
Fig. 3. β-Galactosidase reporter gene expression in plasmid-injected albino tadpoles (stages 35/36–39). Embryos were injected at the 2-cell stage with 100 pg of a given β-galactosidase reporter construct in pBluescript plus 100 pg of pCSKA-GFP. Each β-galactosidase construct contained a different URS, either that of CSKA or different lengths of that of NF-M(2). Two typical specimens from each series are shown. (A) tadpoles injected with β-galactosidase driven by the CSKA URS. (B)–(G) tadpoles injected with β-galactosidase driven by −1468/+33 URS (B), −857/+33 URS (C), −570/+33 URS (D), −330/+33 URS (E), −53/+33 URS (F), and 0 URS (G). (H) uninjected tadpoles. Dorsal is up in all cases. Most tadpoles are shown anterior to the left, but the upper tadpoles in (B) and the lower tadpoles in (D) and (E) are shown anterior to the right.
Fig. 4. GFP-reporter gene expression [pNF-M(2)URS-GFP] in living transgenic pigmented embryos and tadpoles. Animals were photographed with a Dage CCD 300 video camera on an Olympus SZX12 fluorescence-dissecting microscope. Arrows indicate the orientation of the posterior (left) to anterior (right) axis. (A) GFP expression in scattered epidermal cells, dorsal axial structures (nt, neural tube), and undifferentiated ventro-lateral mesoderm (vlm) of JR27-1 at stage 22, several hours before endogenous NF-M(2) expression. (B) GFP expression in the ear (ov), gill (g) arches (slightly out of focus) and scattered epidermal cells (examples at unlabeled arrowheads) of JR27-2 at stage 33/34. (C) GFP expression in midbrain (mb), descending spinal tracts (sct) and trigeminal nerve (tn) of JR27-10 at stage 37/38. (D) GFP expression in notochord (n) of JR27-15 at stage 41. (E) GFP expression in tail bud (tb) and scattered epidermal (examples at unlabeled arrowheads) cells of JR27-2 at stage 26. (F) GFP expression in tail (t) of JR27-11 at stage 37/38. Autofluorescence within the ventral yolk mass is labeled “af” in panels (C)–(F).
Fig. 5. Confocal microscopic image of GFP-reporter gene expression in fixed preparations of stage 48 pigmented transgenic tadpoles. (A) Expression of GFP-reporter gene in tail somites of specimen JR27-5. GFP expression is resolvable in individual muscle cells within the somites. The image was composed from a stack of 168 image planes, 2 μm apart. Anterior is up. z, example of z-line at the inter-somitic junction; m, example of a melanophore. (B) Expression of GFP-reporter gene in the head of JR27-20. The image was composed from a stack of 159 image planes, 10 μm apart. Anterior is down. GFP expression occurs in a variety of head structures including lens (le), iris (ir), extra ocular muscles (eom), olfactory epithelia (oe), gills (g), midbrain (mb), and scattered epidermal cells.
Fig. 6. Conserved sequence motifs found in non-coding regions of the NF-M(2) gene. Sequences were initially identified by the “Meme” routine of version 10 of GCG. Groups of sequences from each of the species listed were run separately for each untranslated region of the NF-M gene (upstream regulatory, intron I, intron II and 3′ untranslated). Numbers in the location column designate the position of the beginning of each sequence alignment relative to the beginning of the first exon (upstream sequence), the start of the intron (intron II) or the termination codon (3′ untranslated), respectively, for each group of sequences. Nucleotides that are shared among at least three of the four species are shaded in black; and those that are shared among two of the four are shaded in gray. See the text for a full description of the parameters.
