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Fig. 1. Xenopus expressed tau isoforms during development. Total RNAs
from stage (st.) 20, 29/30, 37/38, juvenile frog brain, spinal cord or st. 56
tadpole tail were reverse transcribed and amplified by PCR. PCR products
were then separated by agarose gel electrophoresis and stained with ethidium
bromide. The contrast has been reversed to improve visibility of the bands.
Arrowheads point to the four major bands that mark the earliest forms of tau
expressed at the beginning of axon development (st. 20; 1.2, 1.4, 1.9 and
2 kb). These bands were excised and cloned for sequencing. During development,
large isoforms (1.9â2 kb) became the most prominent ones in the tail
of metamorphosing tadpoles, which is enriched with peripheral neurons. This
expression and close resemblance to human peripheral nervous system tau
(see text) are consistent with their identification as Xenopus big taus. The
smaller isoform, at 1.2 kb, became most prominent in adult CNS.
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Fig. 2. Comparison of Xenopus laevis and human mapt genes. (A) To identify the exons, the nucleotide and the amino acid sequences of the four tau PCR
products from Fig. 1, as well as those of two previously characterized X. laevis mapt cDNAs (XTP-1 and XTP-2) cloned from late-stage tadpole tail (Olesen
et al., 2002), were aligned against nucleotides of the X. tropicalis genomic sequence and against the amino acid sequences of nine human tau isoforms (see
Supporting Information), respectively. The six Xenopus isoforms were generated by alternative splicing of four exons (exons 4, 4a, 6 and 12a) as indicated.
Exons that were constitutively present in all four isoforms are indicated in black. As a result of the insertion of exon 12a (124 nt), which has no human parallel,
into XTP-2, a frameshift is introduced and a premature stop codon (*) is encountered after the first amino acid of exon 13. (B) The degree of amino acid identity
[Identities (%)] as well as the number of amino acids (aa#) encoded for each exon of X. laevis and human tau.
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Fig. 3. Tau protein expression began at early stages of axon outgrowth and increased with neuronal development through adulthood. (AâC) Parasagittal views
of st. 22, 29/30 and 37/38 embryos processed for tau immunofluorescence in whole-mount and imaged by confocal laser-scanning microscopy. Images were
taken with the 109 (NA 0.5) objective and represent maximal intensity projections created from stacks of 26, 17 and 20 optical sections, respectively, with
voxel sizes of 1.8 9 1.8 9 8.0 lm (A and B) and 2.5 9 2.5 9 7.8 lm (C). At st. 22 (A), representing early axonal outgrowth, tau immunoreactivity was most
intense in axon tracts within the hindbrain (arrow) and ventral spinal cord (S.C.), and was also visible in peripheral motor axons projecting to anterior somites.
At st. 29/30 (B), representing a stage of maximal axonal outgrowth, tau immunoreactivity became more intense throughout the brain and spinal cord, and could
be visualized in axons of the trigeminal nerve (Vmd, mandibular branch of the trigeminal nerve). By st. 37/38 (hatchling tadpole; C), tau immunoreactivity was
abundant in axon tracts throughout the brain and spinal cord, in the retina (eye), and cranial nerves (X, vagus; IX, glossopharyngeal; VII, facial; Vmd). The
image in (C) was cropped when rotated. (D) Tau immunoreactivity in neuronal perikarya and axons within a transverse section through the ventral diencephalon
of a juvenile frog. The bracket indicates the optic tract. The image is a single optical confocal laser-scanning microscopy section taken with the 209 objective
(NA 0.75), with a pixel size of 0.45 9 0.45 lm. (E and F) Tau immunoreactivity within transverse sections through the retina of a juvenile frog at low (E) and
high (F) magnifications. Images are single confocal laser-scanning microscopy optical sections taken with the 209 objective (NA 0.75) with the scan zoom at
0.7 and 2.0, and pixel sizes of 0.60 9 0.60 lm and 0.45 9 0.45 lm for (E) and (F), respectively. Abbreviations: GCL, ganglion cell layer; INL, inner nuclear
layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; PL, photoreceptor layer. Scale bars: 100 lm (AâE); 20 lm (F).
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Fig. 4. In intact embryos, tau knockdown reduced peripheral nerve development. Embryos were injected at the two-cell stage with tau antisense morpholino
oligonucleotide (MO) and immunostained at st. 37/38 in whole-mount for tau, to visualize effects of the MO on tau expression (A), and for peripherin (B1â2,
C1â2), to visualize effects on peripheral nerve development. (A) View in the horizontal plane illustrating normal tau immunostaining within the brain (arrowhead
1), cranial peripheral nerves (e.g. arrowhead 2) and spinal cord (arrowhead 3) on the uninjected side of the embryo (left of the dotted line) and reduced
tau immunostaining on the side descended from the blastomere injected with MO (right of the dotted line). Rostral is toward the top. The image represents a
maximal intensity projection stack of 33 confocal laser-scanning microscopy sections (objective: 109; NA 0.5) with a voxel size of 2.5 9 2.5 9 6.4 lm. (B1
and 2) Parasagittal views through trunk level somites illustrating diminished robustness of peripheral motor nerve development (arrowheads) on the injected
(B2) vs. uninjected (B1) side. Images represent a single optical section (109; NA 0.5) taken at comparable depths through the somites; voxel size is
1.8 9 1.8 9 5.1 lm. (C1 and 2) Parasagittal views through the head illustrating diminished robustness of cranial nerve development on the injected (C2) vs.
uninjected (C1) side. Rostral is toward the right. Images represent a maximal intensity projection stack of 15 confocal laser-scanning microscopy sections (109;
NA 0.5) with a voxel size of 1.6 9 1.6 9 3.1 lm. Scale bars: 100 lm for all panels; (B1 and 2) are at the same magnification, as are (C1 and 2). (D) Average
lengths ( SEM) of cranial and spinal peripheral nerves of tau and control MO-injected animals immunostained for peripherin at st. 37/38 (N = 3). **Significantly
different from controls, as determined by t-test, P < 0.01. Abbreviations: II, optic nerve; IX, glossopharyngeal nerve; Vmd, mandibular branch of the
trigeminal nerve; Vop, ophthalmic branch of the trigeminal nerve; VII, facial nerve; SN, spinal motor nerves; X, vagus nerve.
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Fig. 5. Tau knockdown selectively disrupted the structural integrity of microtubules containing a neuronal class II b-tubulin. Newly differentiating, embryonic
cultured spinal cord neurons containing tau antisense morpholino oligonucleotide (tau MO; B1â3, D2, E2, F2, G2) or control MO (A1â2, D1, E1, F1, G1) were
stained for the neuron-specific b-tubulin II isotype (Nb-tubulin; Xenopus gene symbol, tubb2b), ubiquitous a- and b-tubulins, tyrosinated (Tyr-), and acetylated
(Ac-) a-tubulins, respectively. (A1âB3) In contrast to the generally filamentous structures seen in the axons and growth cones (A2) of control MO neurons
stained for Nb-tubulin, significantly more short, discontinuous fragments (arrowheads in B1 and 2) were seen in the axons and growth cones (B3) of tau MO
neurons. (C) Quantitation of the number ( SEM) of discontinuous, fragmented microtubules (puncta) stained by the Nb-tubulin antibody along the neuritic
shaft (left; /100 lm) and within growth cones (right; /100 lm2) of control and tau MO cultured neurons (three cultures each). **Significantly different from
control, as determined by t-test; P < 0.01. (D1âG2) As determined by immunostaining, neither the levels of expression nor distributions of ubiquitous b-tubulins
(D1 and 2), ubiquitous a-tubulins, Tyr-tubulin (F1 and 2) and Ac-tubulin (G1 and 2) differed between tau MO and control MO cultured neurons. Scale bars
(20 lm in all panels) in (A2), (B1), (E1), (F1) and (G1) apply to (B3), (B2), (E2), (F2) and (G2), respectively. Neurons were imaged by conventional epifluorescence
with a 1009 objective (NA 1.4). (H) Quantitation of the relative fluorescence intensity (RFI; see Statistical analyses in Materials and methods for
details) within neurites for each of the tubulin antibodies in (D1âG2) revealed no significant differences in tau MO neurons relative to that of control MO neurons
(RFI = 1.0; t-test, three cultures each).
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