Hutson LD and Bothwell M (2001), Expression and function of Xenopus laevis p75(N...

XB-ART-8265

J Neurobiol 2001 Nov 05;492:79-98. doi: 10.1002/neu.1067.

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Expression and function of Xenopus laevis p75(NTR) suggest evolution of developmental regulatory mechanisms.

Hutson LD , Bothwell M .

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Neurotrophins signal through two different classes of receptors, members of the trk family of receptor tyrosine kinases, and p75 neurotrophin receptor (p75(NTR)), a member of the tumor necrosis factor receptor family. While neurotrophin binding to trks results in, among other things, increased cell survival, p75(NTR) has enigmatically been implicated in promoting both survival and cell death. Which of these two signals p75(NTR) imparts depends on the specific cellular context. Xenopus laevis is an excellent system in which to study p75(NTR) function in vivo because of its amenability to experimental manipulation. We therefore cloned partial cDNAs of two p75(NTR) genes from Xenopus, which we have termed p75(NTR)a and p75(NTR)b. We then cloned two different cDNAs, both of which encompass the full coding region of p75(NTR)a. Early in development both p75(NTR)a and p75(NTR)b are expressed in developing cranial ganglia and presumptive spinal sensory neurons, similar to what is observed in other species. Later, p75(NTR)a expression largely continues to parallel p75(NTR) expression in other species. However, Xenopus p75(NTR)a is additionally expressed in the neuroepithelium of the anterior telencephalon, all layers of the retina including the photoreceptor layer, and functioning axial skeletal muscle. Finally, misexpression of full length p75(NTR) and each of two truncated mutants in developing retina reveal that p75(NTR) probably signals for cell survival in this system. This result contrasts with the reported role of p75(NTR) in developing retinae of other species, and the possible implications of this difference are discussed.

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Species referenced: Xenopus laevis
Genes referenced: cda myc ngfr ntsr1 prl.2 rpe zic1

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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. 107–257), human (a.a. 115–268), and rat (a.a. 116–269) p75NTR; mouse TNFR (a.a. 133–272); and the Xenopus and zebrafish neurotrophin receptor homologues, xNRH1A (a.a. 108–233), xNRH1B (a.a. 105–234), and zNRH1A (a.a. 1–72). 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.
	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. 30–195; highly conserved trans/juxtamembrane domain: a.a. 252–318; “death domain”: a.a. 360–415. 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.
	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.
	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.
	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.
	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.
	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.
	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).
	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.
	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.
	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.