Int J Dev Biol
January 1, 2009;
Loss of REEP4 causes paralysis of the Xenopus embryo.
Members of the REEP (Receptor expression enhancing protein) family contain a TB2/DP1
, HVA22 domain that is involved in intracellular trafficking and secretion. Consistent with the presence of this domain, REEP1
enhance the expression of odorant and taste receptors in mammals, while mutation of these genes causes defects in neural development. REEP4
was identified in the course of a functional antisense morpholino oligonucleotide screen searching for genes involved in the early development of Xenopus tropicalis: although over-expression of the gene causes no phenotype, embryos lacking REEP4
develop a slightly kinked body axis and are paralysed. At tailbud
stages of development, REEP4
is expressed in the somites
and neural tube
. The paralysis observed in embryos lacking REEP4
might therefore be caused by defects in the nervous system
or in muscle
. To address this point, we examined the expression of various neural and muscle
markers and found that although all are expressed normally at early stages of development, many are down regulated by the tailbud stage
. This suggests that REEP4
plays a role in the maintenance of both the nervous system
and the musculature.
Int J Dev Biol
[+] show captions
Fig. 2. Expression pattern of Xenopus REEP4. (A) RT-PCR based expression profile of X. laevis REEP4 from stage 1 to tailbud stage 35. Values have been normalized to those of ornithine decarboxylase (ODC). (B-F) Whole-mount in situ hybridisation analysis of X. laevis REEP4 expression. (G-H) Transverse sections of X. laevis embryos at stage 30. Note expression of REEP4 in neural tube, otic vesicles and somites. (I,J) Expression of X. tropicalis REEP4 at tailbud stage 30 and tadpole stage 42. Note strong expression in somites in (I) and in the nervous system in (J). (K,L) REEP4 localises to plasma and nuclear membranes. Embryos at the one-cell stage were left uninjected (J) or received injections of 2 ng RNA encoding C-terminally GFP-tagged REEP4 (I). They were examined at stage 15. Note presence of tagged REEP4 in nuclear and plasma membranes.
Fig. 3. Inhibition of translation of X. laevis and X. tropicalis REEP4 by antisense morpholino oligonucle- otides. (A) Positions of X. tropicalis MO1 and MO2 and X. laevis MO are indicated in red. The start codon of each open reading frame is shown in blue. (B) Left panel: X. tropicalis REEP4 MO1 and MO2 inhibit translation of REEP4 mRNA in an in vitro transcription-translation reaction. MOs were used at concentrations of 5, 2.5 and 1 μM. Right panel: X. laevis REEP4 MO (90 ng) inhibits translation of REEP4-HA mRNA. The indicated amounts of REEP4 mRNA were injected into embryos of X. laevis at the one-cell stage in the presence or absence of MO. They were cultured to gastrula stage 11 and then processed for Western blotting. (C-I) Rescue of the effects of REEP4 antisense morpholino oligonucleotides. (C-F) Experiments in X. laevis. Embryos were injected with 90 ng control MO (C), 90 ng REEP4 MO (D) or 90 ng REEP4 MO together with 2 ng mRNA encoding X. tropicalis REEP4-HA. There are five base mis-matches between the X. laevis MO and X. tropicalis REEP4. Note the morphological rescue of the REEP4 MO phenotype in (E). (F) Measurements of the lengths of embryos cultured to stage 32 after injection with 90 ng control MO, REEP4 MO, or REEP4 MO together with mRNA encoding X. tropicalis REEP4-HA confirm the ability of REEP4 mRNA to rescue the REEP4 MO phenotype. *Indicates a significant difference in axis length between control MO and REEP4 MO, and between Rescue and REEP4 MO (Student t, P < 0.0001). (G-I) Rescue experi- ments performed in X. tropicalis. Embryos were injected with 30 ng control MO (G) or MO2 (H) or with 30 ng MO2 together with 1.2 ng RNA encoding REEP4-GFP (I).
Fig. 4. Expression of neural markers in embryos lacking REEP4 function. X. laevis or X. tropicalis embryos were injected, respectively, with 90ng MO or 30ng MO2. Control embryos received the same amounts of control MO. They were cultured to neurula (stage 15-18) or tailbud (stage 29/30) stages and analysed by in situ hybridisation for expression of N-tubulin, Sox3, Pax6 and Pax3. Expression of the neural markers N-tubulin, Sox3 and Pax6 is normal in embryos of X. laevis at neurula stages (A,B; E,F; I,J) and has declined little by tailbud stages (C,D; G,H; K,L). Early expression of the neural crest marker Pax3 is little affected by loss of REEP4 function (M,N), but expression in somites and pronephros becomes disrupted at tailbud stages (O,P). More prolonged culture to stage 38 (Q-T) reveals that Islet1 expression becomes disrupted in embryos lacking REEP4. Thus, in embryos of X. laevis, some embryos injected with a REEP4 MO appear normal (R') but in others, Islet1 expression is reduced. This phenotype is observed more frequently in embryos of X. tropicalis (T')
Fig. 5. Activation of muscle markers in embryos lacking REEP4 is normal during neurula stages but their expression becomes disorganised or reduced thereafter. X. laevis (A-BB) or X. tropicalis (CC-FF) embryos were injected, respectively, with 90 ng MO or 30 ng MO2. Control embryos received the same amounts of control MO. They were cultured to neurula or tailbud stages and analysed by in situ hybridisation for expression of Myf5 MyoD, Mrf4, the 12/101 epitope, Dystrophin, Cardiac actin and Myosin Heavy Chain. Note in X. laevis that expression of markers at the neurula stage is normal (A,B; E,F; U,V; Y,Z) but that they become disorganised (C,D; G,H; K,L; O,P; S,T) or reduced (W,X; AA,BB) thereafter. The decrease in Cardiac actin expression in the heart in (X) is not a consistent observation. Expression of the 12/101 epitope in X. tropicalis is more sensitive to loss of REEP4 function than is expression in X. laevis. Note the decreased levels of 12/101 staining in (DD) compared with (P).
reep4 (receptor accessory protein 4) gene expression in Xenopus laevis embryos, NF stage 29 & 30, as assayed by in situ hybridization, dorsal and lateral views, anterior left.