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Fig. 1. Representation of the domain structure and alternative splicing sites of neurexins. Modified from Missler and Sudhof (1998). (A) Diagram
shows proteins schematically, with the extracellular portion on the left-hand side. Abbreviations: SP, signal peptide; LNS(A), LNS(B), two types of
laminin/neurexin/sex hormone-binding globulin domain; EGF, epidermal growth factor-like domain; C, carbohydrate attachment site; TMR,
transmembrane domain. Arrows indicate positions of variable regions generated by the alternative splicing of transcripts. (B,C) Protein sequence
alignments of Xenopus α-neurexins I, II and III. (B) Sequences of the N-terminal regions. Note the inserts generated by alternative splicing in nrxn
III, splice site 1 and nrxn I and II, site 2. (C) Alignment of the highly conserved transmembrane and cytoplasmic regions.
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Fig. 2. Developmental series RT-PCR showing the temporal pattern
of α-nrxn expression. Primers specific for each nrxn alpha cDNA were
used to identify transcripts in Xenopus oocytes and across early development.
Transcripts from each nrxn are found in the oocyte. However
zygotic expression of nrxn I is first apparent in the early tailbud embryo
(stage 24), which correlates with the onset of synaptogenesis. Transcripts
for nrxn II and III are present in the early embryo, but whereas the
level of nrxn II increases during tailbud stages, those of nrxn III show no
marked increase by the tailbud stage. Consequently, each gene shows
a specific temporal pattern of expression. ODC is used as a control for
equivalent loading. Oo, oocytes; numbers correspond to stages of
Xenopus development (Nieuwkoop and Faber, 1994) where 1 is the
fertilised egg; 10, the onset of gastrulation; 18, the late neurula stage;
28, the tailbud stage and 36 is a swimming tadpole.
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Fig. 3. Transcripts of α-neurexins are expressed in Xenopus oocytes.
Ovarian tissue consisting of connective tissue, follicle cells and oocytes
at different stages of development was subject to wholemount in situ
hybridisation using probes specific for each of the neurexin transcripts.
(A) An nrxn I specific probe identifies transcripts equally distributed
through small, early stage oocytes (yellow arrows). At later stages, the
nrxn Iα transcript is localised to a crescent within the large oocyte (red
arrows). Transcripts were not detected in cells other than oocytes. (B,C)
Nrxn II (B) and nrxn III (C) transcripts were found in all stages of oocyte.
At the later stages, in the large oocyte, the signal is less intense probably
due to the dilution of the mRNA in the larger cytoplasmic volume. There
was no evidence for localisation of these transcripts and neither were
they detected in cells other than oocytes. Right-hand-side panels: negative
controls, hybridised with corresponding sense neurexin cRNA probes
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Fig.4. Expression of α-neurexins is predominantly in neural tissue in
the tailbud embryo. (A,B) In situ hybridisation with a nrxn Iα probe
showing whole embryo (A) and an anterior neural section (B). Nrxn I is
found in cells along the length of the CNS but with a distinct gap at the
midbrain-hindbrain boundary. Sections show expression in the dorsolateral
neural tube and within the eye. (C,D) Embryos hybridised with a
nrxn IIα-specific probe show a similar pattern of staining to that seen with
nrxn I. (E,F) Embryos hybridised with a nrxn IIIα-specific probe show
much more restricted expression in the CNS and in section show ventrolateral
and eye expression in the anterior CNS (arrows).
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Fig. 5 (Left). Nrxn III α is expressed in a subset of neural cells. (A,B) In
situ hybridisation with an α-nrxn III-specific probe identifies a row of
primary neurons at stage 24 (A), that lies either side of the midline towards
the anterior end of the embryo. (C) By the tailbud stage, expression
resolves into two stripes either side of the midline (red and green arrows).
Those arrowed in red are located in the position expected for primary
interneurons. The second band is located more ventrally. Within the head there are discrete areas of expression (yellow arrows). (D,E) Transverse
sections at the level of the hindbrain (D) and anterior spinal cord (E). nrxnIIIα expression (red arrows) is restricted to the lateral neural tube.
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Fig. 6 (Right). The alternative splicing of the α-neurexins is under developmental regulation. Nrxn I was found to have isoforms based on
alternative splicing at site 1 (ssp1), which generated two forms in the oocyte but only one in the tailbud embryo. The larger form included the equivalent
of mouse exon 4 but lacked the other exons at ssp1. The smaller transcript lacked all the variable exons at ssp1. Nrxn II was also found in two forms
based on alternative splicing at splice site 2. The larger form included exon 6 and was detected only in the tailbud and later stages. Nrxn III existed
in three isoforms varying at splice site 1. Again the forms showed a developmental profile with the longer form being detectable only in the tailbud
and later stages. Left-hand side: representative images of PCR products. Right-hand side: diagrammatic representations of splice variants. Open
boxes: non-variant exons present in Xenopus, filled boxes, variant exons present in Xenopus transcripts. Predicted protein sequences resulting from
alternative splicing shown underneath. Numbering identifies equivalent exon in the mouse (Tabuchi and Sudhof, 2002) and bold indicates range of
splice site. Exons labelled with an asterisk can occur in more than one form in the mouse.
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nrxn1 (neurexin 1) gene expression in Xenopus laevis embryo as assayed by in situ hybridization, NF stage 32. Dorsal view: anterior left.
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nrxn2 (neurexin 2) gene expression in Xenopus laevis embryo as assayed by in situ hybridization, NF stage 32. Dorsal view: anterior left.
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nrxn3 (neurexin 3) gene expression in Xenopus laevis embryo as assayed by in situ hybridization, NF stage 32. Lateral view: Dorsal up, anterior left.
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