Remy P et al. (1996), Overexpression of the Xenopus Xl-fli gene durin...

XB-ART-18108

Int J Dev Biol 1996 Jun 01;403:577-89.

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Overexpression of the Xenopus Xl-fli gene during early embryogenesis leads to anomalies in head and heart development and erythroid differentiation.

Remy P , Sénan F , Meyer D , Mager AM , Hindelang C .

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The product of the Xl-fli gene, a Xenopus laevis transcription factor of the ets family, specifically expressed in several lineage of migratory cells during Xenopus development (Meyer et al., Int. J. Dev. Biol. 39: 909-919, 1995) was overproduced during Xenopus embryogenesis, upon microinjection of a synthetic transcript in the fertilized egg or in the early embryo. This results in anomalies of the antero-posterior and dorso-ventral polarities, and in tissue differentiation, particularly in the eye- and head cartilage development, as well as erythroid differentiation (absence of erythrocyte differentiation in the circulating blood, often accompanied by ectopic localization of mature erythrocytes, leading to important hemangiomas). Cytological examination reveals at gastrulation the existence of abnormal cells separating the different embryonic layers, suggesting modifications of the cellular adhesion properties. The possible involvement of the fli gene in controlling the dissemination of migratory cells is discussed.

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Species referenced: Xenopus laevis
Genes referenced: abcc6 aktip fli1 tbx2 tbxt.2

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	Fig. 1. Analysis of XI.f1i overexpression in injected embryos. IA) Polyacrylamide gel elecrrophoresis of the 35$-labeled translation products encoded by the XI.fli synthetic transcript, in a rabbit reticulocytesystem (fanes 1)and in a Xenopus oocytesystem (lane 2. 3 and4).Lane 1shows the migrationprofile of the polypeptides recovered from the rabbit reticulocyte translation mixture. Lane 2 shows the polypeptidesrecoveredfrom the oocyte extract upon immunoprecipitation with antl.FL/ antibodies. In lane 3. the anti-FLIantibody was preincubated with the antigen prior to the immunoprecipitationreacrion. Lane 4 shows the complete oocyte translation mixture, in the absence of immunoprecipitation. (8) Bandshifting activity of the overproduced FLI protein in extracts of Xenopus embryos at neurula stage towards the fTS-target sequence present In MSV-LTR. The embryos have been injected at the one-celf stage, either with the sense- (lanes 3 and 4) or antisense fIi-synthetic transcript (lanes 1 and 2). The specificity of the campfeJl: formed with extracts of embryos injected with the sense transcript is assessed by the disappearance of the retarded band upon preincubation of the protein eJl:tract with an anti-FLI antibody (lane 3). No bandshifting activity is observed in control embryos, probably because endogenous FLI protein is present at a too low concentration. mRNA: S. injection with the sense transcript; AS. injection with the antisense transcript. antibody: (+), incubation in the presence of the anti-FLI antibody; (-), incubation in the absence of the anvbody. (C) Immunocharacterization of the FLI protein overproduced in Xenopus embryos. Fertilized eggs were injected with increasing amounts of synthetic fll mRNA and allowed to develop for 2. 16 and 24 h. Embryos were then homogenized and the proteins were separated by 50S-PAGE and blotted to a nitrocellulose membrane. Proteins of control embryos of the same developmental stages were also analyzed. The Western blot was then probed with a rabbit polyclonal antl-FLI antibod". L. MW ladder; T1. T2. T3, control embryos analyzed at 2. 16 and 24 h. respectively. 1.2,3: proteins recovered 2 h after injection of the fertilized eggs with 0.55, 1.1 and 2.2 ng of mRNA. respectively. 4,5 and 6. proteins recovered after a 16 h incubation. 7,8 and 9. proteins recovered after a 24 h Incubation. The bands correspondmg to the XI-fli protem present in the mjected samples and absent from the control samples are indicated by an arrowhead. With the eJl:ception of a minor polypeptide of MW ~30 kOa (probably corresponding to a degradation product), all other bands are non specific since they are observed both in the control and injected samples.
	Fig. 2. Perturbation of gastrulation accompanying XI.fJjoverexpres. sion. (AI Control Xenopus laevis embryos at the gastrula stage (st. 11.5/12). No superficial feature can be observed, except the blastoporal rmg m the vegetal hemisphere. IBI Fertilized eggs injecred with the sense fli svnthetlc transcript exhibir at the gastrula stage (Sf. 10.5/171 a distinctive cellular outgrowth (arrows) in the ventTO-anterior region.
	Fig. 3. Injected embryos exhibit hyperswollen neural folds. IAI ControlXenopus laevis embryos at neurula stage (Sf. 18). (B) Fertilized eggs injected with the sense ffi synthetic transcnpt exhibit at neurula stage (st. 17/18) a swelling of the border of the neural fold.
	Fig. 4. Injected embryos show at neurula stage 1st. 18/191 distinct streaks of cells extending from the dorsal to the ventral region, evocarive of an amplified and/or precocious streaming of neural crest cells towardsbranchial arches (curved arrows). Note the hyperpigmented region at the tip of the most anterior cell streak (arrowhead).
	Fig. 5. Injected embryos exhibit blisters and cellular outgrowths at the level of ectoderm. (AI Embryosinjected with high amounts of the fli sense synthetic transcripr (1 to 1.4 ngleggJ present head rruncation phenotypes. similar to the one shown in this picture. Note also the ectodermal outgrolNths in the most anterior region (arrows} and the important blisters in the ventral region (hand pointers). (8) A stage 33 injected embryo, showing a unique malformed eye (full arrow), ventro-anteflor blisters (hand pOinters), and ectodermal outgrowths (open arrows) which may look digitated like the most posterior one.
	Fig. 6. Developmental anomalies affecting the eyes in embryos injected with the (Ii sense synthetic mRNA 1350 pg/egg). (A) A single-eyed embryo. IBIAn embryo exhibiting a deeply buried vestigial eye. ICI An embryo presenting a third eye in rhe median plane. probablv arising from a duplication of the A-P axis in the anterior-most region. IDI Embryos showmg heavily pigmenred optic sralks
	Fig. 7. XI-fli overexpression leads to a blocking of erythropoiesis in circulating blood. (Top) Stage 45 embryo injecred with the sense fli synthetic transcript at the fertilized egg stage (700 pg/egg). Note the malformations affecting the eyes and guts. and the complete absence of red blood at the level of rhe heart. although the latter could clearly be seen beating under the microscope. (BottOm) Conrrol embryo. Arrowhead, heart; large full arrow, reddish internal mass suggestingan ectopic accumulation of erythrocytes.
	Fig. 8. Formation of a periocular hemangioma in a stage 45 X. fae. vis embryo injected with a ffi sense synthetic mRNA (350 pg/eggJ.
	Fig. 9. XI-fli overexpression results in a lack of adhesion between ectodermal layers. (A) Section of an ara/dire-embedded Xenopus laevis embryo (st. 10.5/11), injected with the sense fh synthetic mRNA at the fertiftzed egg stage. Note the .scar. of the injection in the vegetal hemisphere, containing round-shaped. isolated cells. These abnormal cells can also be observed on the blastocoel floor. Simultaneously, the eel/layers in the non involuting dorsal and ventral marginal zones appear to separate from each other. 8ar, 200 pm_ 81, blastocoel; o8L, dorsal blastoporal lip; DMZ. VMZ, dorsal and ventral marginal zones; M, mesoderm. (BI Transverse section of an araldlte-embedded gastrula embryo (st. 17/12) injected with the sense fli synthetic transcript at the fertilized egg stage, exhibiting a hyperswollen blastocoel. Note that the blastocoel appears limired by a single cell layer (the outer layer of epidermal ectoderm), the inner layer of the epidermal ectoderm looking broken at the level of the blastocoel floor. AS. archentera! slit; BI, blastocoel; Ec, ectoderm; M. mesoderm: y. yolk. Bar. 200 pm.
	Fig. 10. XI-fli overexpression leads to the accumulation of nonadherent cells at the borders of the different embryonic layers. (AI Sagittal section of an araldite-embedded X. laevis embryo at neurula stage (control). The embryo exhibits a large archenteral cavity and in the dorsal region the three embryonic layers (neuroectoderm. mesoderm and endoderm) are in perfect contiguity. Anterior is to the right. dorsal to the top. Bar. 200 f,Jm. (B) Section equivalent to (A' in a neurula embryo microinjected with a sense fJj synthetic transcript (700 pg) at the fertilized egg stage. The archenteron is reduced to a mere slit. Although the mesoderm has actively migrated under the ecroderm. the borders between the three embryonic layers are now full of round-shaped isolated cells (thick arrows). probably present In an excess of ECM. Notice the .scar' of the iniection site. exhibiting rhe same anomalous cells. Also remark the separation between the epithelial and sensorial layers of the ectoderm in the ventro-anterior region (arrowheads). Anterior is to the left. dorsal to the tOp. Bar. 200 j.Jm. (CI Two-fold enlargement of the dorsal region of the section of (B) showing in more details the separation of the embryonic layers by anomalous cells (thick arrows). Bar. 100 11m. Ar: archenteron; Ar.d.. archenteron (dorsa/); Ar.R.. archenteron roof; Ar.v.. archenteron (ventra/); Bcd, blastoporal coffar (dorsa!}; Bcv. blastoporal collar (ventral); EA. eye anlage; Ee. ectoderm; M. mesoderm; Nee. neuroectoderm; SM. somitic mesoderm,- Y. yolk.
	Fig. 11. Two para sagittal sections of a paraffin-embedded stage 37/38 embryo injected with a sense fIi synthetic mRNA (700 pg/egg) at the fertilized egg stage. Note the curvature of the A-P axis. the presence of blisters and the eye anomalies. On the one side. the eye is almost normal (A. long arrow), whereas on the other side the optic cup did not invaginate properly, the retinal layer does not appear differentiated as on the opposite side, and the crystalline lens is stiff attached to the ectoderm (B) (see x2 enlargemenr, C,. Simultaneously, the anterior region of the eye appears to contain an excess of ECM (long arrow in the inserrJ containing elongated mesenchymal cells. Bar. 200 pm.
	Fig. 12. Anomalies in heart development accompanying XI.fIi overexpression. (A) Sagittal section of an araldite-embedded stage 45 embryo (control). Notice the layer of endothelial cells (EC) lining the cardiac jelly (CJ) and the red blood cells (BC) filling the cardiac cavity. (B) Equivalent section In a stage 45 embryo injected with the fli sense synthetic mRNA (700 pg/egg) at the fertilized egg stage. Notice the thickening of the cardiac jelly (CJ) reducing the volume of the cardiac cavity and suggesting an increase in the secretion of a component of the ECM. The endothelial cells (EC) instead of forming a monolayer are now piling up as if they were proliferating abnormally or had modified adhesion properties. Bar. 30um.
	Fig. 13. Transverse section of a paraffin-embedded stage 45 embryo exhibiting a hemangioma in the periocular region (see Fig. 8). demonstrating the accumulation of erythrocytes (ellipsoidal nucle. ated cells) in avascular structures (thin arrows). Also note the fibrous remnants of a fiffing marerial evocarive of an excessive production of ECM(thickarrows).C. cartilage:E. eye: Er. erythrocytes: M, muscle. Bar. 20 /)m.