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Figure 1 Comparison of p75NTRa and p75NTRb with other members of the neurotrophin receptor/
tumor necrosis factor receptor family. (A) Alignment of partial amino acid sequences of p75NTRa
(Genbank accession number AF246462) and p75NTRb (Genbank accession number AF246463), and
the corresponding amino acid sequences from chicken (a.a. 107â257), human (a.a. 115â268), and rat
(a.a. 116â269) p75NTR; mouse TNFR (a.a. 133â272); and the Xenopus and zebrafish neurotrophin
receptor homologues, xNRH1A (a.a. 108â233), xNRH1B (a.a. 105â234), and zNRH1A (a.a. 1â72).
The sequences designated zNRH1, xNRH1a, and xNRH1b represent Genbank Accession numbers
AI437140, AF131890, and AI031422, respectively. The fragments encompass a portion of the
ligand-binding domain, the highly divergent subset of the extracellular domain, and most of the
highly conserved transmembrane domain. Red indicates similarity over at least seven of the
sequences, while blue indicates similarity over at least two sequences. Xenopus p75NTRa and
p75NTRb contain all of the conserved cysteines present in the ligand-binding domain, and they show
a high degree of conservation with other published p75NTR genes in the putative transmembrane
domain. Key to consensus sequence: !: I or V; $: L or M; %: F or Y; #: N, D, Q, or E. (B)
Phylogenetic relationships between p75NTR-related genes. xp75NTRa and xp75NTRb are more
closely related to each other than to p75NTR from other species, suggesting they represent two allelic
variants. While Xenopus NRH1A appears to be more closely related to zebrafish NRH1A than to
Xenopus NRH1B in this region, this is an artifact of the fact that the zebrafish NRH1A sequence is
incomplete. Pairwise sequence comparisons clearly show that xNRH1B is the closest relative of
xNRH1A, suggesting that, like xp75NTRa and xp75NTRb, the two Xenopus NRH genes are allelic
variants.
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Figure 2 Multiple sequence alignment between conceptually translated Xenopus p75NTRa and
p75NTRs from chicken, human, and rat. Red indicates residues similar in all four species. Blue
indicates residues similar in at least two species. The location in the figure of each conserved domain
is as follows: cysteine repeat domain: a.a. 30â195; highly conserved trans/juxtamembrane domain:
a.a. 252â318; âdeath domainâ: a.a. 360â415. Key to consensus sequence: !: I or V; $: L or M; %:
F or Y; #: N, D, Q, or E. Genbank accession numbers: p75NTRa-1, AF172399; p75NTRa-2,
AF172400.
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Figure 3 RNase protection assays showing developmental expression of p75NTRa and p75NTRb.
(A) p75NTRa and (B) p75NTRb mRNA in 20 mg whole embryo total RNA. Undigested probes are
approximately 500 bases. Protected fragments of p75NTRa and p75NTRb are 431 and 428 bases,
respectively. Undigested probe for EF-1a, used as an internal control, is approximately 290 bases,
and the protected fragment is approximately 200 bases. mRNA for EF-1a increases gradually during
development as previously reported (Krieg et al., 1989). (C) Quantification of p75NTRa and p75NTRb
mRNAs normalized to EF-1a mRNA and subsequently normalized to p75NTRa expression level at
stage 28. Abcissa labels refer to embryonic stage.
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Figure 4 Whole mount in situ hybridization in stage 26
tadpoles. (A) p75NTRa. Note expression in cranial ganglia
(white arrow) and segmental expression in dorsal neural
tube (black arrows). Expression in dorsal neural tube is
believed to correspond to spinal senory neurons, possibly
Rohon-Beard cells. (B) p75NTRb. Expression of p75NTRb is
also observed in cranial ganglia and dorsal spinal sensory
neurons, although at lower levels. (C) Control in situ hybridization
containing combined sense strand probes for
both p75NTRa and p75NTRb. Anterior is to the right in all
panels.
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Figure 5 p75NTRa in situ hybridization in sections. I. CNS structures. (A) Transverse section
through stage 39 ventral telencephalon. Brackets indicate expression in ventral telencephalon in both
neuroepithelium and mantle zone. The neural tube is demarcated by a white dotted line. We did not
detect expression in olfactory epithelium above background. OE, olfactory epithelium. (B) Sense
strand control in same plane as (A). (C) Sagittal section through stage 45/46 retina showing
expression in the ganglion cell layer (innermost ring of cells, adjacent to lens), in the inner nuclear
layer (inner portion of outer ring of cells), and in the outer nuclear layer (outer portion of outer ring
of cells). No mRNA hybridization is observed in the inner plexiform layer, which contains the axons
of bipolar cells and dendrites of retinal ganglion cells. ipl, inner plexiform layer; le, lens; PE,
pigment epithelium. (D) Sense strand control. Anterior is to the left in (C) and (D). The epidermis
and pigment epithelium appear white in dark-field microscopy due to the presence of reflective
melanocytes, and not as a result of probe hybridization. Scale bar: 50 mm.
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Figure 6 p75NTRa in situ hybridization in sections. II. Neural crest- and placode-derived structures.
(A) Transverse section through stage 39 anterior otic vesicle showing expression in presumptive
sensory epithelium of the otic vesicle (bracket) and in seventh and eighth cranial ganglia. (B)
Sense strand control. (C) Parasagittal section through stage 45/46 medial otic vesicle showing
expression in otic epithelium (brackets) and in cranial ganglia. V, fifth cranial ganglion; VII, seventh
cranial ganglion; VIII, eighth cranial ganglion; IX/X, ninth and tenth cranial ganglia; ov, otic
vesicle; ph, pharynx. (D) Sense strand control. (E) Sagittal section through stage 45/46 intestines
demonstrating expression in enteric ganglia (arrows). int, intestine. (F) Sense strand control.
Anterior is to the left in (C) through (F). Scale bar: 50 mm.
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Figure 7 p75NTRa in situ hybridization in sections. III. Mesoderm-derived structures. (A) Sagittal
section through stage 45/46 esophagus and pharyngeal mesenchyme. es, esophagus; ph, pharynx.
(B) Sense strand control. (C) Sagittal section showing expression in cloaca at stage 45/46. clo,
cloaca. (D) Sense strand control. (E) Parasagittal section showing expression in stage 45/46 axial
skeletal muscle. p75NTR mRNA appears to be concentrated in the center of the muscle fibers. Somite
boundaries are indicated by dotted lines. (F) Sense strand control. (G) Hematoxylin stained section
through somites at the same stage as tadpole shown in (E). Row of nuclei is delimited with arrows.
Anterior is to the left in all panels. Scale bar: 50 mm.
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Figure 8 Cell death in developing retina. (A) and (B) Epifluorescence images of TUNEL-labeled
dying cells in transverse sections through retina. Lateral is to the left and medial is to the right, and
eyes are outlined in red stipple. Some labeled nuclei may appear to be out of focus due to thickness
of sections and intensity of labeling. (A) Transverse section through anterior retina at stage 37/38.
Most of the dying cells observed are in the ventronasal quadrant of the retina. (B) Transverse section
through central retina at stage 47. At this stage, most of the dying cells are observed in the retinal
ganglion cell layer (delimited by arrows) and the proliferating ciliary marginal zones (brackets).
Scale bar: 50 mm. (C) Quantification of cell death using TUNEL as described in Methods. n 5 2–7.
Two relative peaks in cell death are observed at stage 37/38 and stage 47. Mitotic index redrawn
from Harris and Hartenstein (1991).
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Figure 9 Schematic diagram of constructs used for transfection. p75.FL contains the entire coding
region of Xenopus p75NTRa. DDD lacks the death domain and the C-terminal serine-rich region.
CDD contains only the first five amino acids of the cytoplasmic domain.
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Figure 10 In vivo lipofections and cell death. Transverse sections through ventral stage 37/38
retina transfected with GFP/p75.FL or GFP/CDD. Lateral is to the left in all images. All transfections
were followed with GFP. A neutral construct containing an array of tandem myc epitopes was
cotransfected with GFP as a control for nonspecific toxicity (see Methods). (A) Schematic diagram
of the eye. RPE, retinal pigment epithelium; PRL, photoreceptor layer; OPL, outer plexiform layer;
INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (B) Cotransfection
of GFP and p75.FL. GFP (green) indicates cells in which p75.FL is expressed (see Methods). Red
indicates nuclei of dying cells labeled by TUNEL. None of the transfected cells are positive for
TUNEL. (C) Cotransfection of GFP and CDD. (D) High magnification of area outlined by box in
(C). Red arrows indicate GFP-positive cells with TUNEL-positive nuclei. White arrows indicate
GFP-positive cells with TUNEL-negative nuclei. A fifth GFP-positive cell can be seen above the
lower of the two TUNEL-positive cells, but its nucleus is not visible in this optical section. Scale
bar: (A)–(C), 50 mm. Scale bar: (D), 10 mm.
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Figure 11 Quantitative effects of p75NTR on cell death.
Fractional cell death was quantified at each stage by dividing
the total number of GFP-expressing cells with TUNELpositive
nuclei by the total number of GFP-expressing cells
with observable nuclei (see Methods). Relative cell death
was determined by dividing the fraction of dying cells for
each sample by the fraction of dying cells in controls. Thus,
relative cell death in controls equals 100%. At stage 37/38,
during the first phase of normal cell death, p75.FL caused a
61% reduction in cell death, CDD caused an 84% increase
in cell death, and DDD caused a slight (23%) increase in
cell death. At stage 41, when the amount of cell death is
normally low, CDD caused a 222% increase in cell death,
and DDD caused a 52% increase in cell death. p75.FL did
not have a significant effect at this stage.
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