XB-ART-1009Int J Dev Biol January 1, 2006; 50 (1): 39-46.
The expression and alternative splicing of alpha-neurexins during Xenopus development.
The neurexins are involved in the formation and function of synapses. Each of three genes encodes alpha- and beta-neurexins. Additional diversity (particularly of alpha-neurexins) arises from alternative splicing, resulting in a large number of protein isoforms, the significance of which is currently unclear. We have analysed alpha neurexin expression and alternative splicing during development of the frog Xenopus laevis. Surprisingly, each alpha-neurexin gene is expressed in immature oocytes. During embryonic development, each Xenopus neurexin (nrxn) gene has a distinct temporal expression pattern, with expression being almost exclusively within the neuroepithelium. The spatial expression of nrxnIalpha and nrxnIIalpha is similar in the developing CNS, with staining being observed in the optic cup and in dorsolateral regions of anterior neural tube, but not adjacent to the ventral midline. The pattern of nrxnIIIalpha expression is more restricted, in several domains of the anterior neural tube. In the forebrain, expression was confined to an area in the ventrolateral neural tube; nrxnIIIalpha was also expressed in the hindbrain and spinal cord. By stage 32, a period when synaptogenesis occurs, nrxnIIIalpha is expressed midway along the neural tube''s dorso-ventral axis in the hindbrain and anterior spinal cord, at the site of the primary interneuron column. Because of the striking diversity of neurexin isoforms, we analysed alternative splicing of Xenopus transcripts during development and found examples of alternative splice variants of each neurexin. The data demonstrate differential regulation of the alpha neurexins with respect to the gene temporal and spatial expression and alternative splicing.
PubMed ID: 16323076
Article link: Int J Dev Biol
Species referenced: Xenopus laevis
Genes referenced: egf frzb2 nrxn1 nrxn2 nrxn3 odc1
Article Images: [+] show captions
|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.|
|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.|
|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|
|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).|
|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.|
|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.|
|nrxn1 (neurexin 1) gene expression in Xenopus laevis embryo as assayed by in situ hybridization, NF stage 32. Dorsal view: anterior left.|
|nrxn2 (neurexin 2) gene expression in Xenopus laevis embryo as assayed by in situ hybridization, NF stage 32. Dorsal view: anterior left.|
|nrxn3 (neurexin 3) gene expression in Xenopus laevis embryo as assayed by in situ hybridization, NF stage 32. Lateral view: Dorsal up, anterior left.|