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Reticulon1, also known as neuroendocrine-specific protein, belongs to the reticulon (RTN) family, whose members possess a conserved reticulon domain and are associated with the endoplasmic reticulum (ER) membrane. Here, we report cloning and expression of Xenopus homologues of Reticulon1-A (XRTN1-A) and -C (XRTN1-C). XRTN1-A and -C contain an open reading frame of 752 and 207 amino acids, respectively, each containing a conserved reticulon domain. Sequence analysis shows that XRTN1 proteins have an ER membrane retention signal and four putative membrane-spanning domains. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization showed that XRTN1-A is expressed in early neural precursors and differentiating neuronal populations, including the trigeminal placode, olfactory placode, lateral line placode, and otic vesicle. XRTN1-C is expressed in the developing brain and spinal cord. We found that XRTN1-C protein is localized to the ER of Xenopus and mammalian cells and the granules in neurites of primary neurons of the Xenopus spinal cord and rat hippocampus. We also showed that XRTN1-C protein is detected in the heavy membrane fraction, which contains lysosomal and ER-resident proteins, as well as in the nucleus and polysomal fractions of the Xenopus embryo. Finally, we showed that thyroid hormone specifically down-regulates XRTN1-A mRNA in the head of premetamorphic Xenopus tadpoles. Our work characterizes the intracellular roles of XRTN1 during Xenopus neural development.
Figure 3. The spatial expression patterns of XRTN1s. A: XRTN1-A. (a) Lateral side view of stage (St.) 8. (b) Animal view of St. 8. (c) Lateral view of St. 10.5. The arrowhead indicates the blastopore lip. (d) Animal view of St. 10.5. The XRTN1-A transcript is detected in the animal hemisphere, potential neural ectoderm at the blastula and gastrula stages. (e) Anterior view of St. 19. (f) Dorsal view of St. 19, anterior is to the left. (g) Transverse section view of St. 19. (h) Magnified view of g. The expression of XRTN1-A is localized to the anterior neural border (arrow) and panplacodal primordium (arrowheads) at the late neurula stage. Additionally, XRTN1-A is also expressed in the dorsolateral side of archenteron roof (asterisks). (i) Lateral view of St. 23. (j) Lateral view of St. 27. (k) Magnified view of j. (l) Lateral view of St. 35. (m) Magnified view of l. XRTN1-A is specifically expressed in the cranial placodes, such as the trigeminal placode, lateral line placode, olfactory placode, and otic vesicle. (n-q) XRTN1-A sense in situ. (n) Anterior view of St. 19. (o) Dorsal view of St. 19, anterior to the left. (p) Transverse section view of St. 19. (q) Lateral view of St. 35. No positive signals were detected. B: XRTN1-C. (a) Lateral side view of St. 8. (b) Animal view of St. 8. (c) Animal view of St. 10.5. (d) Vegetal view of St. 10.5. The arrow indicates the blastopore lip. The XRTN1-C mRNA is localized in the animal hemisphere at the blastula and gastrula stages. (e) Anterior view of St. 19. (f) Dorsal view of St. 19, anterior to the left. (g) Transversal section view of St. 19. (h) Magnified view of g. XRTN1-C is expressed in the anterior neural border (arrow) and panplacodal primordium (arrowheads) at the late neurula stage. In addition, XRTN1-C is also detected in the dorsolateral side of archenteron roof (asterisks). (i) Lateral view of St. 27. (j) Magnified view of i. (k) Lateral view of St. 35. (l) Magnified view of k. (m) Magnified dorsal view of k. The expression of XRTN1-C is detected in the spinal cord, prosencephalon, mesencephalon, and rhombencephalon, as well as in the lateral line placode, otic vesicle, and pronephros. (n-q) XRTN1-C sense in situ. (n) Anterior view of St. 19. (o) Dorsal view of St. 19, anterior to the left. (p) Transversal section view of St. 19. (q) Lateral view of St. 35. No positive signals were detected. fb, forebrain; hb, hindbrain; ll, lateral line placode; mb, midbrain; mc, mesencephalon; of, olfactory palcode; ot, otic vesicle; pc, prosencephalon; pn, pronephros; sc, spinal cord; tg, trigeminal placode.
Figure 6. The effect of thyroid hormone (TH) on XRTN1 gene expression. A: The schematic illustration of TH and methimazole (Meth.) treatment and reverse transcriptase-polymerase chain reaction (RT-PCR). B: The phenotype of Xenopus tadpole embryos treated with T3 or methimazole. The T3-treated embryo shows the re-structured brain and tail regression. The control and methimazole-treated embryos undergo normal development. C: RT-PCR analysis from the head of T3- or methimazole-treated embryos. Upon the treatment of active TH T3, the expression of XRTN1-A was repressed, but not that of XRTN1-C. N-tubulin and L8 (L8-ribosomal protein) were used as loading controls.
