Zhao H et al. (2001), Isolation and characterization of a Xenopus gen...

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Int J Dev Biol 2001 Oct 01;457:817-26.

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Isolation and characterization of a Xenopus gene (XMLP) encoding a MARCKS-like protein.

Zhao H , Cao Y , Grunz H .

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We have identified a cDNA coding for a Xenopus MARCKS-like protein (XMLP) from a cDNA library prepared from activin-treated ectoderm. Using whole-mount in situ hybridization and RT-PCR, we found XMLP maternal transcripts during the cleavage stages. After MBT, the signals were restricted to the neural plate. Subsequently XMLP was expressed predominantly in the brain, somites and pronephros. Ectopic expression of XMLP resulted in eye and axis defects and in a change of the expression pattern of Krox 20, a neural marker for rhombomeres 3 and 5. Injected XMLP caused apoptosis. It was characterized by loss of intercellular adhesion contacts, transient plasma membrane ruffling at gastrula, and epithelial disruption attailbud stage. Overexpression of mutant XMLPs showed that this phenotype was correlated with its putative PSD domain and glycine at position 2. The embryos injected with a morpholino oligo complementaryto XMLPmRNA showed malformations of the anterior axis and eye defects. Extirpation experiments indicated that the phenotypes might be correlated with disturbed morphorgenetic movements rather than an inhibition of induction process. Overexpression of XCYP26 resulted in a shift of the expression pattern of XMLP. In the early tailbud stage (stage 20) the signal stripe in the XCYP26 injected half of the embryo got diffuse or even disappeared. This observation suggests that retinoic acid plays an important role in the regulation of XMLP. Our results suggest that XMLP might participate in pattern formation of the embryonic axis and the central nervous system.

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Species referenced: Xenopus laevis
Genes referenced: cyp26a1 egr2 gal.2 got2 grap2 marcks marcksl1 psd uqcc6
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	Fig. 1. XMLP nucleotide and the deduced amino acid sequence. The nucleotide sequence in bold in the 3�UTR is the putative polyadenylation signal. The numbers on the left indicate the positions of the nucleotides and on the right the positions of the amino acids. The stop codon is marked by an asterisk. A purine at position -3 is in agreement with the Kozak sequence (Kozak, 1986). Glycine is found at position 2. The conserved region at 22 to 27 surrounding the site of intron splicing is also found in other members of the MARCKS familiy. The domain from K78 till K99 is the putative PSD domain. The three conserved sites mentioned above are underlined.
	Fig. 2. Whole-mount in situ hybridization using XMLP antisense RNA. Maternally expressed XMLP was present in cleave stages (A,B). Afterwards signals were found in the ectoderm and mesoderm of gastrula stages in (C,D). A sagittal section of stage11.5 is shown in (D). However, at neurula stage, signals were no longer evenly distributed and were restricted to neural ectoderm only (E,F,G). Subsequently, XMLP transcripts could be found in the neural folds at early tailbud stage and presumptive brain area only (H,I,M). The sagital section of (I) is shown in (J) and (K), while (L) shows a transversal section of (I). In later stages XMLP was expressed in ectoderm derivatives and pronephros during stage 27 to 34, (N,O,Q). (P) shows a transversal section of stage 34 (ac, archenteron; bt, blastocoel; bv, brain ventricle; ed, ectoderm; en, endoderm; hm, head mesenchyme; mc, mesencephalon; md, mesoderm; nc, notochord; ne, neural ectoderm; nt, neural tube; pn, pronephros; rc, rhombencephalon; sc, spinal chord).
	Fig. 3. Temporal expression pattern of XMLP at different stages analyzed by RT-PCR. Transcripts could be detected at all stages (stage 1-40, Nieuwkoop and Faber, 1975) especially at a high level in stage 8, when zygotic gene expression just starts. The analysis also revealed a decrease of the transcription level from uncleaved egg to stage 23, while it increased slightly in later stages.
	Fig. 4. RT-PCR of adult tissues with XMLP specific primers. XMLP transcripts were seen in all tested tissues. However, lower abundance was detected in kidney and intestine. (br, brain; ey, eye; he, heart; in, intestine; ki, kidney; li, liver; mu, muscle; ov, ovary; sk, skin; sp, spleen; st, stomach; te, testis).
	Fig. 5. The effect of overexpression with XMLP capped RNA. Ectopic XMLP results in protrusions of bulge cells distributed around the injection site (A). (B) Section of an XMLP-injected embryo at stage 10 as shown in (A). Note that there were some giant nuclei around the injection area (asterisk); a normal nucleus is indicted by an arrow. In addition, ectopic XMLP caused eye defects (C) where injected sides of the larvae were identified by LacZ as lineage marker. The larvae in the left column of panel (C) show smaller eyes at the injected side while the right column shows the unjected side of embryos of the same series (compare with larva shown from the dorsal side in (E) ). (D) Larva (injected with XMLP) with bended axis and one eye. (E) Larva with missing right eye injected with both XMLP and LacZ into one dorsal blastomere of the 4-cell stage. (F) The injected larvae of stage 40 with one eye only. A comparison of the histology of notochord in the head area between normal larvae (G) and larvae with a small eye (H). Note that less mesenchyme in (H) has been found in injected embryos compared to normal ones. (I) Transversal section of the tadpole with one eye shown in (F). (J,K) The differentiation of dorsal blastopore lip with adjacent dorsal ectoderm isolated from normal embryos (J) and injected embryos (K), respectively. Note that XMLP-treated embryos differentiated into notochord and brain structures with a rudimentary eye as the controls. No significant differences have been found in dorsal blastopore lips either isolated from injected or uninjected embryos (abbreviations: br, brain; bv, brain ventricle; gu, gut; le, lens; me, mesenchyme; nc, notochord; pe, pigmented epithelium; re, retina)
	Fig. 6. Effect of Morpholino (MO) XMLP and MO b-catenin injected into embryos. (A) 24 ng MO XMLP was injected into two-cell stage embryos resulting in embryos with a shorter anterior axis and eye defects. (B) The phenotype of apoptosis induced by XMLP mRNA. (C) The phenotype of apoptosis can be rescued by MO XMLP (16 ng MO XMLP and 0.8 ng XMLP mRNA were injected simultaneously). (D) The injected tadpole with MO XMLP and XMLP mRNA showed a relatively normal morphological phenotype or a mildly bent axis (the upper one). (E) shows larvae with reduced anterior axis after microinjection of 4 ng of MO b-catenin into two dorsal animal blastomeres of 8-cell stage embryos.
	Fig. 7. Schematic drawing of XMLP and its mutant constructs used in this study. Functional ED is indicated as a dark-slashed box, the site of intron splicing is indicated by vertical dashed box, and the glycine at position 2 is indicated by black box and the substituted alanine is shown by box with grid (A in the G2A mutant). The white-slashed box in the XMLP S83A mutant represents the mutant ED domain changing serine to alanine (A); the alanine is also shown by a box with grid.
	Fig. 8. Comparison of the phenotype induced by XMLP and its four mutants. The phenotypes at neurula stage induced by 0.4 ng XMLP G2A, SD, S83A, ED are shown in (A), (B), (C), (E) and (F) respectively. Note that apoptosis could be induced by 0.4 ng mRNA wild type of XMLP (A) and SD (C), but not by G2A (B), S83A (E) and ED (F). However the embryos showed apoptosis (D) when 0.8 ng S83A mRNA was injected to dorsal blastomeres of 4-cell stage embryos. The arrows in (A), (C), and (D) show the apoptosis area. The phenotypes in tadpole stages induced by G2A, SD, S83A, and ED with the dose of 0.4 ng mRNA are shown in (H) to (K) and (M). Larvae after injection of 0.4 ng XMLP are shown in (G). (H), (J), (K), and (M) indicate the phenotypes induced by G2A, S83A, ED, and SD, respectively. The injected embryos showed eye defects and a reduced anterior region. (M) indicates the extreme case of injected embryos with SD mutant. Note that the phenotype shown in (L), induced by 1.6 ng ED mRNA, is quite similar to the phenotype of exencephaly. The neural tube was not closed completely, and also the epidermis did not cover the internal yolk-rich yolk tissue.
	Fig. 9 Effect of ectopic XMLP on the expression of Krox20 and the expression alternation of XMLP induced by XCPY26. 0.8 ng XMLP were injected into one dorsal blastomere of 4-cell stage embryos. b-gal encoding mRNA was co-injected as a lineage tracer (indicated by light blue). Signals were detected by whole-mount in situ hybridization with XMLP antisense RNA. (A) shows the expression of Krox 20 in the control embryos. The signal stripes of Krox20 at the injected side were disturbed (B). 2 ng XCYP26 capped mRNA was injected into one blastomere of 2-cell stage embryo which was visualized by LacZ staining (light blue stain). Signals were detected by whole-mount in situ hybridization with XMLP antisense RNA. The signal stripes that could be clearly seen on the normal embryos (C) vanished or got smeared (D) at the injected side indicated by LacZ.
	marcksl1 (MARCKS-like 1) gene expression in bisected Xenopus laevis embryos, NF stage 11.5, as assayed by in situ hybridization. Horizontal view
	marcksl1 (MARCKS-like 1) gene expression in Xenopus laevis embryos, NF stage 20, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.