Amaravadi LS et al. (1997), Autonomous neural axis formation by ectopic exp...

XB-ART-15568

Dev Biol 1997 Dec 15;1922:392-404. doi: 10.1006/dbio.1997.8780.

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Autonomous neural axis formation by ectopic expression of the protooncogene c-ski.

Amaravadi LS , Neff AW , Sleeman JP , Smith RC .

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The ski oncogene was originally isolated as an avian retroviral gene with the ability to induce quail embryonic cells to differentiate into muscle. Mice containing a chicken c-ski transgene exhibit postnatal hypertrophy of skeletal muscle. Xenopus ski (Xski) protein is maternal and present throughout early development. We show that overexpression of Xski RNA in Xenopus embryos results in the cell-autonomous induction of secondary neural axis formation. Injection of Xski RNA into prospective endodermal cells resulted in the formation of an ectopic neural tube-like structure and cells derived from the injected blastomeres populated the spinal cord. Injected Xski RNA was able to induce neural-specific gene expression directly in ectodermal explants in the absence of the expression of mesodermal markers. The widespread distribution of ski protein in the early gastrula embryo including the dorsal animal region supports a role for ski in neural axis formation in vivo.

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Species referenced: Xenopus laevis
Genes referenced: lsamp ski
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	FIG. 1. Analysis of endogenous Xski protein expression during early embryogenesis and oogenesis. (A) Top panel shows a Western blot performed using affinity-purified polyclonal anti-Xski antibodies. Numbers on top of each lane represent the stage of embryogenesis. E, unfertilized egg. Total lysate from one embryo or egg was loaded in each lane. Arrows point to 90-kDa Xski protein and an additional 55-kDa (possibly ski-related) protein. Bottom panel is a similarly loaded blot probed with anti-enolase antibodies (43 kDa) as a guide for equivalent loading. M, marker. (B) Lysates from head, trunk, and tail segments of tadpole-stage (stage 45) embryos were probed with anti-ski antibodies. He, head; Tr, trunk; Ta, tail; W, whole embryo. Each lane contained lysate equivalent to one segment. (C) Equivalent amount of total protein from pooled oocytes was loaded in each lane. M, marker; 1/2, stage I and II oocytes; 3/4, stage III and IV oocytes; 5/6, stage V and VI oocytes.
	FIG. 2. Injection of Xski2 RNA into the VMZ of four-cell embryos results in the formation of an ectopic neural axis or ectopic neural tissue on the injected side. (A) Embryos were fixed and analyzed at stage 35–36. The top two embryos were injected with 0.1 ng of Xski2 RNA. Arrows point to the partial secondary axes. The bottom embryo was uninjected. (B) Dorsal view of neurula stage embryos injected with 50, 100, or 150 pg of Xski2 RNA. A dose-dependent response was seen at this stage. Anterior is at the top. (C thru E) Cross sections through embryos injected with 0.1 ng of Xski2 RNA into the VMZ at the four-cell stage. Curved arrow, ectopic neural tube structure induced by Xski2 RNA; arrowheads, pigmented cement gland-like structure; straight arrows point to the roof plate and to the lightly stained disorganized fiber tracts. (C) Note that more posteriorly the yolky mass of tissue which is continuous with the neural tissue is also continuous with the endoderm. (D) Cross section through another embryo which displayed an external bulge. Note the pigmented cement gland-like structure (arrowheads); curved arrow, additional neural tube-like structure with a roof plate (arrow) that was contained in the bulge. (E) More posterior section of the same embryo as in D shows that the yolky ectopic tissue is continuous with the spinal cord. Arrowheads, cement gland-like structure; curved arrow, ectopic neural tissue. Uninjected side developed normally. NC, notochord; S, somites.
	FIG. 3. Xski-induced ectopic bulge contains differentiated neural tissue and does not contain any ectopic muscle tissue. (A–D) Cross sections through a stage 35–36 embryos injected with 100 pg of Xski2 RNA into the VMZ at the four-cell stage. These embryos displayed bulges in the head region. (A and B) Sections were treated with a differentiated neural tissue-specific antibody (2G9) and visualized using a fluorescein-conjugated secondary antibody. Arrow points to additional 2G9-positive tissue in the bulge region. (B) DAPI staining of the same section as in A. (C and D) Sections were treated with 12/101 (muscle-specific) antibody and detected by immunofluoresence. Only the somites in the primary axis stained with 12/101 antibody. No additional 12/101 staining was noted in the ectopic tissue. (D) DAPI staining of the same section as in C. OV, otic vesicle; NT, neural tube; NC, notochord; SM, somites.
	FIG. 4. Cell-autonomous induction of a neural axis by Xski2 RNA. (A) Cont, control uninjected embryos (stage 35–36) stained for b-gal activity; Inj, embryos injected with 0.1 ng of Xski2 RNA together with 0.1 ng of nb-gal RNA injected as lineage tracer into the VMZ of four-cell-stage embryo, fixed, and stained for b-gal activity at stage 35–36. The majority of the blue staining is in the partial secondary axis. (B) Cross section through an embryo injected with 0.1 ng of Xski2 RNA together with 0.1 ng of nb-gal RNA (cross section through the third embryo from the left in A). Histology of the embryo stained for b-gal activity reveals a very similar neural tube-like structure as seen with Xski2 RNA injection alone (compare with Fig. 2A). nc, nototchord; sc, spinal chord; s, somites; e, endoderm; nt, secondary neural tube.
	FIG. 5. Injection of Xski2 RNA into prospective endodermal cells. (A) Cont, embryos injected with 0.1 ng of nb-gal RNA only; Expt, embryos injected with 0.1 ng of Xski2 RNA together with 0.1 ng of nb-gal RNA. RNAs were injected into any one blastomere of the Dtier (vegetal-most blastomeres) at the 32-cell stage. Embryos were fixed at stage 35–36 and analyzed by whole-mount staining for b-gal activity. (B) Cross section through a control embryo. All of the b-gal-positive cells remained in the endoderm. (C) Cross section through an embryo that contained an ectopic tube (bottom right embryo in A). Cells that received Xski2 RNA formed a tube-like structure with a lumen. (D) Cross section through an embryo pictured on the top right of A. Cells that received Xski2 RNA have migrated more dorsally and populated the spinal cord (arrows). Sc, Spinal cord.
	FIG. 6. Xski induces neural tissue-specific gene expression in the absence of mesodermal markers. Animal caps from injected and uninjected embryos were explanted at stage 9 and cultured until the control embryos reached stage 18. mRNA was extracted from lysed animal caps and whole embryos at this stage and subjected to RT–PCR with gene-specific primers as indicated. Lane 1, no template control for each of the primer sets; Lane 2, uninjected control animal caps; Lane 3, ski-injected animal caps; Lane 4, intact whole embryos. A control without adding the reverse transcriptase (-RT) was run for each of these reactions and is shown for EF1-a.
	FIG. 7. Western blots of (A) early-gastrula embryos dissected into ventral and dorsal halves (B) late-blastula embryos dissected into animal and vegetal halves. Lysates equivalent to two halves or one whole embryo were run in each lane and probed with purified anti- ski antibodies. V, ventral half; D, dorsal half; W, whole embryo; A, animal half; Vg, vegetal half. Bottom panels contained similar blots probed with anti-enolase antibodies to check for equivalent load- ing. (C) Localization of Xski protein in oocyte cytoplasm. Western blot of stage 6 oocytes fractionated into germinal vesicle and cyto- plasm. Lysates from one oocyte (St. 6), cytoplasm from one oocyte (Cyt), and five Gvs (GV) were run separately in each lane. No Xski protein was detected even when 20 Gvs were run in one lane (data not shown). M, marker.