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Xenopus laevis is a valuable model system for the study of vertebrate neuroembryogenesis. However, very few well-characterized nervous system-specific molecular markers are available for studies in this organism. We screened a X. laevis adult brain cDNA library using a cDNA probe for mouse low molecular weight neurofilament protein (NF-L) in order to identify neuron-specific intermediate filament proteins. Clones for two distinct neuron-specific intermediate filament proteins were isolated and sequenced. One of these encoded for a Xenopus NF-L (XNF-L) and the other for a novel neuron-specific Xenopus intermediate filament protein (XNIF) that was present earlier and more abundantly than XNF-L during development. XNIF contained a central rod domain with multiple sequence features characteristic of IF proteins. The XNF-L was very similar to mouse NF-L, with a 77% sequence identity in the rod domain and the presence of a polyglutamic acid region in the tail domain, characteristic of type IV neurofilament proteins. In contrast, XNIF showed only 60% identity to mouse NF-L in the rod domain and lacked the glutamic acid-rich sequence in the tail domain. XNIF also had a very low (approximately 38%) sequence identity in the head and tail domains as compared to NF-L and other neurofilament proteins (45% identity to the head domain of alpha-internexin). In the adult frog, XNIF mRNA is detected by Northern blots only within the nervous system and by in situ hybridization histochemistry exclusively in neurons, particularly in the medullary reticular system and spinal cord. Antisera raised against the unique tail region of XNIF detected a single distinct 60 kDa band in Western blots of nervous system cytoskeletal preparations, and this XNIF immunoreactivity was concentrated in axons in the PNS and in small perikarya in the dorsal root ganglion. In contrast, NF-L immunoreactivity was principally in the large perikarya in the dorsal root ganglion. In development, XNIF mRNA appears more abundant than XNF-L mRNA in all premetamorphic stages examined. XNIF mRNA is first detectable at stage 24 (26 hr), whereas stable expression of XNF-L is at stage 35/36 (50 hr). XNIF immunoreactivity is detectable within the cement gland, within many neuronal cell bodies and axon tracts within the developing nervous system, and within all cellular layers of the developing retina. The availability of these two distinct neuron-specific intermediate filament proteins, with different temporal and spatial expression patterns, should provide new markers as well as targets for functional perturbation in the developing X. laevis nervous system.
Figure 1. A, Nucleotide and predicted amino acid sequences of the Xenopus neuronal intermediate filament protein XNIF derived from cDNA
clone 1 Oa 1. Translation was begun at the first in-phase methionine of the longest open reading frame. Nucleotide numbers are indicated above the
DNA sequence, and amino acid residue numbers are indicated at the ends of the translated sequence. The junctions between head, rod, and tail
domains are indicated with brackets. The amino acid sequence from the tail domain used to make the synthetic peptide to raise antisera is doubly
underlined. Two potential polyadenylation sequences in the 3’ untranslated portion are indicated with dotted underlines. Not all adenosine residues
present in the 3’ end of the clone are included in this figure. The GenBank accession number for XNIF is M86653. B, Schematic drawing indicating
relative sizes and overlap of the sequence information of clones lOa and XNK3. Both clones lOa and XNK3 encoded a single open reading
frame that corresponded to an intermediate filament protein (bottom line). Predicted 5’ and 3’ untranslated regions of the cDNAs are indicated on
the bottom line as dotted lines. The a-helical segments (IA, IB, and 2) of the rod domain of the predicted XNIF protein are shown as crosshatched
bars, and the head, tail, and rod domain linker segments are depicted as solid lines. The polyadenylation sequence present in clone lOal, but not
XNK3, is indicated by AAAA.
Figure 2. A, Assembled partial cDNA sequence and deduced amino acid sequence of XNF-L. The junctions between head, rod, and tail domains
are indicated with brackets. The glutamic acid-rich portion of the tail domain is double underlined. The GenBank accession number for XNF-L
is M86654. B, Schematic drawing indicating relative sizes and overlap of sequence information of clones H4, HIO, and XNF21. The relative
position of the assembled, predicted sequence of the XNF-L protein is shown on the bottom line. Other conventions are as in Figure 1B
Figure 3. Alignments of the predicted amino acid sequences of the head and tail domains of XNF-L to mouse NF-L (A, head; B, tail) and XNIF to mouse NF-L (C, head; D, tail)
and rat a-intemexin (E. head, F, tail). Sequences were aligned using the GAP program of the Wisconsin Genetics Computer Group with a gap weight of 3.0 and a length weight of
0.10. Dotted positions in the amino acid sequence show locations where gaps were inserted to obtain the highest degree of similarity between two sequences. Bars connecting upper
and lower sequences indicate amino acid identity; two vertical dots indicate a conservative substitution; one dot indicates a nonconservative, but similar substitution (i.e., pairings of
two polar/charged, or two nonpolar residues); no dot indicates dissimilar amino acid substitutions. Regions of amino acid sequences beyond the overlaps are not shown. MNF-L,
mouse NF-L, ZNEX, rat a-intemexin.
Figure 4. Northern blot analysis of the tissue specific distribution of XNIF mRNA. Aliquots of 1 rcgo f polyA+ RNA from XTC (aX enopusk idney
celll ine)c ells,a dultX . luevis brain,l iver, muscles, kin,a ndo vary, andw holes tage4 7 (5-d-olds wimmingta dpolesX) enopuse mbryosw eres eparated
on 1.2%d enaturinga garoseg els,b lottedt o nylon membranea,n dh ybridized with a cDNA probet o XNIF (XNK3) asd escribedin Materialsa nd
Methods.P ositionso f the 28s and 18s ribosomaRl NAs are asi ndicated.A prominent2 .4 kb bandw asd etectedin adult brain and weaklyi n
wholes tage4 7 embryos.T he blot wase xposedfo r 6 d.
Figure 5. Distribution of XNIF mRNA in adult .Y. laevis nervous system as shown by in situ hybridization with cRNA probes: bright-field (A,
C, E) and corresponding dark-field (II, D, F, respectively) photomicrographs of transverse sections of medulla (A, B), lumbar spinal cord (C, O),
and ventral horn (E, F). The photomicrograph of the ventral horn (E, F) is magnified from the region indicated by the arrowhead at vh in C and
D. Cresyl violet-stained nuclei of unlabeled cells are seen in the bright-field photomicrographs. Abbreviations: e, ependymal cells; c, spinal cordcentral canal; w, white matter containing nuclei of glial cells. The 400 pm scale bar is for A-D. The 100 pm scale bar is for E and F.
Figure 7. The distribution of selected intermediate filament protein immunoreactivities in the adult PNS. Transverse sections from sciatic nerve
(A-C) and dorsal root ganglion (D-F) were stained by antibodies to XNF-M (A, D), XNIF (B, E), GFAP (C), and XNF-L (0. Anti-XNIF
immunoreactivity in the sciatic nerve (B) was clearly axonal as seen by comparing B to sections stained by anti-XNF-M (A) and anti-GFAP (c)
antibodiesI.n the dorsalr oot ganglion,a nti-XNIF immunoreactivityQ was preferentiallyf ound in smallerp erikaryaa nd finer processeass,
comparedto anti-NF-L immunoreactivityQ found in largerp erikaryaa ndt hickerp rocesseasn, da nti-XNF-M immunoreactivity( D), whichw as
homogeneousdlyis tributed.T he scalefo r A-C is showni n C, and for D-F in F.
Figure 8. Northernb lot analysiso f XNIF (A) and XNF-L (B) mRNA expressiond uringd evelopmentI.d enticala liquotso f 10p go f total RNA
from indicateds tageso r juvenile brain weres eparatedan d transferredto nitrocellulosea sd escribedB. lotsw ere then hybridized with identical
amounts(2 0 ml at 5 x lo5c pm/ml)o f labeledc DNA probesfo r XNIF and XNF-L, washeda, nd exposedto x-ray film over the sameti mep eriod
(7 d). The lowerp anels howsth e ethidiumb romide-staineda garoseg elsp rior to transfer.X NIF mRNA wasf irst detectablea t stage2 4, whereas
XNF-L mRNA wasd etectedt ransientlya t stage1 8, and thenn ot againu ntil stage3 5/36.R NA in the stage1 8l anesm igrateds lightlyf astert han
the others.
Figure 9. Distribution of anti-XNIF antibodyi mmunoreactivity in a
stage 35136 embryo A. , Whole-mounteedm bryos howingth e heads tained
by the anti-XNIF antibody. Arrowheads at V, VIZ, and VIII indicate
positionso f theser espectivec ranialn erves.T he curveda rrow pointst o
ventral longitudinal tracts in the brain. The urrow at cfpoints to the
cephalicfl exure;c h andc gi ndicatet he optic chiasma ndc emengt lands,
respectivelyB. , View from the samee mbryos hownin A, at the site of
the futurec ervicomedullarjyu nction. The large arrow pointst o ventral
longitudinal tracts of axons; the small arrow shows ventral neuronal
perikarya. C, Same as in B, but with a different plane of focus. The
large arrow points to the same position as the large arrow in B. The
smaller arrows point to perikarya and processeosf dorsaRl ohon-Beard
neurons.
Figure 10. Anti-XNIF antibody immunoreactivity in the developing CNS and PNS. A, Horizontal section through the head of a stage 41 (3-dold)
swimming tadpole; anterior is up. Anti-XNIF antibody immunoreactivity is Seen in the optic tract (ot), and Vth cranial nerve (v). op. olfactory
placode. B, Horizontal section through the rhombencephalon of the embryo in A; anterior is up. Arrowheads at VII, VIZZ, and IX point to anti-
XNIF immunoreactivity in respective cranial nerves. XNIF immunoreactivity is also seen in longitudinal tracts of axons (It) and in the axons of
the Mauthner neurons at the point of decussation (arrowheads at M). C, Horizontal section (anterior is to the right) showing anti-XNIF-immunoreactive
neuronal cell bodies (arrowheads) and axons (k, longitudinal tracts) of the tailspinal cord of a stage 45 @-d-old) swimming tadpole. D,
An anti-XNIF antibodv-immunoreactive. rhombencenhalic neuronal nerikarya from the tadpole shown in C. The 100 pm scale bar is for A and
B, the 25 pm scale bar-is for C and D.
