XB-ART-7951J Comp Neurol December 17, 2001; 441 (3): 266-75.
Embryonic expression of pituitary adenylyl cyclase-activating polypeptide and its selective type I receptor gene in the frog Xenopus laevis neural tube.
The genes encoding pituitary adenylyl cyclase-activating peptide (PACAP) and its selective type I receptor (PAC1) are expressed in the embryonic mouse neural tube, where they may be involved in neurogenesis and neural tube development. We examined here the early expression and potential actions of PACAP and PAC1 in the vertebrate developmental model Xenopus laevis. PACAP and PAC1 mRNAs were first detected by RT-PCR in stage 16-18 embryos (18 hours after fertilization). Two distinct PACAP precursor mRNAs were identified. One encoded both growth hormone-releasing hormone and PACAP, whereas the other encoded only full-length PACAP. Unlike that in the adult, the latter represented the predominant embryonic PACAP mRNA species. In situ hybridization revealed that PACAP and PAC1 mRNAs were restricted to neural cells. PAC1 gene expression was observed mainly in the ventricular zone in the ventral parts of the prosencephalon, mensencephalon, rhombencephalon, and anterior spinal cord. In contrast, PACAP mRNA was localized exclusively in postmitotic cells in the dorsolateral parts of the rhombencephalon and entire spinal cord. Most PACAP mRNA-containing cells were characterized as Rohon-Beard neurons. Exposure of early embryos to UV irradiation, which ventralizes embryos and inhibits neural induction, reduced the expression of PACAP and PAC1 genes. These results suggest that PACAP may be involved in the early development of the embryonic Xenopus neural tube.
PubMed ID: 11745649
Article link: J Comp Neurol
Species referenced: Xenopus
Genes referenced: adcyap1 adcyap1r1 ghrh klf6 tubb2b
Article Images: [+] show captions
|Fig. 1. PACAP (A,B) and PAC1 receptor (C,D) gene expression in developing Xenopus embryos and PACAP gene expression in several adult brain regions (E) assayed by RT-PCR. RT-PCR products in shown A and C were subjected to Southern transfer and hybridized with oligonuleotides probes corresponding to sequences between the PCR primers (B and D, respectively). Two PACAP cDNA products were amplified from embryos and from dissected adult brain areas (A,B,E), whereas a signal band is amplified with the PAC1 receptor primers (C,D). Upper arrows correspond to the mRNA encoding both full-length GHRH and PACAP. Lower arrows correspond to the mRNA encoding a GHRH/PACAP precursor, which lacks the first 32 amino acids of GHRH. Developmental stages in A–D are indicated. Con, PCR of embryonic RNA minus reverse transcriptase. Adult brain regions analyzed in E are olfactory bulb (OB), forebrain (FB), hindbrain (HB), and spinal cord (SC).|
|Fig. 2. Whole-mount in situ hybridization illustrating the expression of PACAP (A–I) and PAC1 (J–O) genes in embryos at different developing stages. A–I show PACAP mRNA distribution in embryos at stage 18, 21, 23, 25, 26, 27, 29, 31, and 33, respectively. PACAP mRNA signals were first localized at stage 21 in the embryonic spinal cord (small arrow in B). At the later stages, PACAP mRNA signals were continuously detected in the dorsal part of the entire spinal cord and middle/posterior rhombencephalon (small arrows in B–I). Hybridization signals were not present in the embryonic forebrain over the course of study (open arrows in D–H). Starting from stage 23 (C), however, PACAP gene transcripts were observed in the trigeminal ganglia (large arrows in C–H). J–O show PAC1 mRNA distribution in embryos at stage 18, 21, 26, 28, 31, and 33, respectively. PAC1 mRNA signals were first detected in the forebrain area at stage 21 (solid arrow in K). At the later stages, PAC1 mRNA signals were continuously detected in deep portions of the forebrain, including the tuberculum posterior, prosencephalon, mesencephalon, rhombencephalon, and anterior spinal cord (solid arrows in L–O). PAC1 gene transcripts were not discernible in the forebrain of embryos at stages 18 (open arrow in J) or in the middle/posterior spinal cord at any stage (open arrows in K–O). SC, spinal cord; F, forebrain; TG, trigeminal ganglion. Scale bar 5 2 mm.|
|Fig. 3. Representative sections showing PACAP mRNA distribution in stage 26–28 embryos. Embryos were sectioned after wholemount in situ hybridization. PACAP mRNA was not detected in the forebrain or anterior rhombencephalon (open arrows in A–C) but was found to be present from the middle portion of the rhombencephalon (arrows in D) and throughout the spinal cord (arrows in E–H). Outside the neural tube, PACAP gene expression was also noted in the trigeminal ganglia (arrows in B,C). Dashed lines in C–H outline the boundary of the neural tube. N, notochord; M, mesencephalon; P, prosencephalon; R, rhombencephalon; T, tuberculum posterior; SC, spinal cord. Scale bar 5 500 mm in A (applies to A,B), 100 mm in H (applies to C–H).|
|Fig. 4. Comparison of the distribution of PACAP mRNA with that of neuronal-specific class II b-tubulin (N-tubulin) in the neural tube of Xenopus embryos. A,B and C,D illustrate the localization of N-tubulin and PACAP mRNA in the neural tube of stage 28 embryos, respectively. Whole-mount in situ hybridization revealed a distribution of N-tubulin mRNA (arrow in A) similar to that of PACAP mRNA (arrow in C) in the spinal cord (SC). Inside the spinal cord, N-tubulin mRNA hybridization signals (arrows in B) were present mainly in the Rohon- Beard neurons (RB) and motoneurons (MN). N-tubulin mRNA was also detected in the interneurons between Rohon-Beard and motoneurons (arrowhead in B). Conversely, throughout the spinal cord, PACAP mRNA signals were found only in the dorsolateral part of the spinal cord, corresponding to the positions of Rohon-Beard neurons (arrow in D). Dashed lines in B,D outline the approximate boundary of the neural tube. N, notochord. Scale bar 5 300 mm in C (applies to A,C), 100 mm in D (applies to B,D).|
|Fig. 5. Representative sections showing PAC1 receptor mRNA distribution in stage 28–30 embryos. Embryos were subjected to whole-mount in situ hybridization before sectioning. In the anterior forebrain, PAC1 mRNA signals were moderately abundant in the ventral prosencephalon and mesencephalon (arrows in A,B). High levels of PAC1 gene expression were also noted in the ventral parts of the anterior, middle, and posterior rhombencephalon (arrows in C–E, respectively). In the anterior spinal cord, PAC1 mRNA signals were observed only in the ventral area (arrows in F,G). No obvious PAC1 gene transcripts were present in the dorsal region or floor plate of the neural tube. PAC1 gene expression was undetectable from the middle (open arrows in H) to caudal spinal cord. Dashed lines in E–H outline the approximate boundary of the neural tube. M, mesencephalon; P, prosencephalon; T, tuberculum posterior; R, rhombencephalon; SC, spinal cord; N, notochord. Scale bar 5 200 mm in A (applies to A,B), 50 mm in H (applies to C–H).|
|Fig. 6. Detailed distribution of PAC1 mRNA in representative sections of the prosencephalon (A,B) and rhombencephalon (C,D) of the embryos at stage 30. B and D are higher magnifications of the boxed areas in A and C, respectively. Note the presence of PAC1 hybridization signals in the ventricular zone (large solid arrows in B,D) and in areas of the neural tube containing presumed postmitotic cells (small arrows in B,D). PAC1 mRNAs were not detected in the floor plate (open arrow in D) or alar region. N, notochord. Scale bar 5 0.5 mm in C (applies to A,C), 200 mm in D (applies to B,D).|
|Fig. 7. Changes of PACAP and PAC1 gene expression in the Xenopus embryos treated with UV irradiation and lithium. UV treatment resulted in an obvious down-regulation of PACAP and PAC1 gene expression as analyzed after 22 and 35 PCR cycles. Both G-PACAP and T-PACAP gene expressions (upper and lower bands in top gel, respectively) were inhibited after UV treatment. Lithium treatment did not induce an obvious change in the gene expression of PACAP or PAC1. Globin and H-4 mRNA levels (controls) were unchanged after UV irradiation or lithium treatment (25 cycles). Con, control embryos without UV or LiCl treatment; NC, negative control (minus reverse transcription control sample).|
|adcyap1 (adenylate cyclase activating polypeptide 1 (pituitary) ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 31, lateral view, anterior right, dorsal up.|
|adcyap1r1 (adenylate cyclase activating polypeptide 1 (pituitary) receptor type I) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 31, lateral view, anterior right, dorsal up.|