QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online May 5, 2004
doi: 10.1242/10.1242/dev.01132

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kofron, M. Articles by Heasman, J. Search for Related Content PubMed PubMed Citation Articles by Kofron, M. Articles by Heasman, J. Social Bookmarking                         What's this?

The role of Mixer in patterning the early Xenopus embryo

Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA

View larger version (49K): [in a new window]   Fig. 4. Mixer depletion causes ectopic expression of VegT target genes. (A) Embryos injected with Mixer MO-1 were cultured until the blastula stage, and dissected into vegetal and equatorial explants. These explants were cultured until the mid-gastrula stage, frozen and analyzed for expression of mesodermal and endodermal markers by real-time RTPCR. Expression levels were normalized to ODC. we, wild-type embryo; eq, equatorial explant; bs, vegetal explant. Expression levels were normalized to ODC. (B) Half-mount in situ for Cerberus, eomesodermin, Bix3, Fgf8 and Xbra in Mixer MO-1-injected embryos. Arrows indicate dorsal. Embryos were injected with 25 ng of Mixer MO-1 into both vegetal cells of a two-cell embryo. These embryos were cultured until stage 10 and 11, fixed in MEMFA, bisected, re-fixed in MEMFA and assayed for gene expression by whole-mount in situ hybridization for the indicated gene. (C) Whole-mount in situ for eomesodermin in Mixer MO-1-injected embryos. Embryos were injected with 6 ng of Mixer MO-1 into one vegetal cell of an eight-cell embryo. These embryos were cultured until stage 10.5, fixed in MEMFA and analyzed for eomesodermin expression by whole-mount in situ hybridization. The arrow indicates the expanded region of eomesodermin mRNA. The pinkish color is due to ß-gal staining from co-injection of ß-galactosidase mRNA as a lineage tracer.

View larger version (69K): [in a new window]   Fig. 6. Vegetal cells from Mixer-depleted embryos can induce ectopic somite tissue. Schematic of vegetal-cell transplantation experiment shown at top of figure. (A) Progeny from transplanted vegetal cells from control late blastula embryos become endoderm in tailbud stage host embryos (TRITC labeled cells in red, arrows). (B) The progeny of transplanted vegetal cells from Mixer-depleted embryos are found in endoderm and lateral plate mesoderm (arrows). (C) Control host embryo with progeny of wild-type vegetal cells (red) and somite staining (green, 12/101 MAB). (D,E) Ectopic patches of 12/101 positive cells are found near to the progeny of transplanted Mixer-depleted vegetal cells (arrows).

View larger version (40K): [in a new window]   Fig. 1. Alignment of Mixer morpholinos against Mixer and related Xenopus sequences (A) and morpholino inhibition of in vitro translation (B) of wild-type and MO-R Mixer mRNA. (A) Xenopus Mixer aligned against the reverse complement of the three morpholinos used in this study using Clustal alignment algorithem in Macvector. These sequences were also compared to a potential pseudo-allele identified by the Sanger EST sequencing project and the most closely related homeodomain proteins Mix.1 and Mix.2. (B) Mixer MO-1, MO-2 and MO-3 block translation of wild type but not MO-R Mixer mRNA in an in vitro translation assay as described in the Materials and methods.

View larger version (98K): [in a new window]   Fig. 2. Non-complementary Mixer mRNA rescues the effects of Mixer depletion. Gastrulation is delayed in embryos injected with MO-1 (A) and MO-3 (B) as marked by formation of the dorsal blastopore lip. The gastrulation delay is partially rescued by the injection of Mixer MO-R mRNA into the four vegetal cells of eight-cell MO-1-injected embryos (C). Anterior and gut abnormalities in Mixer-depleted embryos is partially rescued by injection of Mixer MO-R mRNA into the four vegetal cells of the eight-cell embryo (D).

View larger version (45K): [in a new window]   Fig. 3. Analysis of gene expression in Mixer-depleted gastrulae (A) and in mRNA rescued gastrulae (B). (A) Embryos were injected at the two-cell stage with 20 ng or 40 ng of MO-1 and cultured until the early or mid-gastrula stage. The resulting Mixer-depleted embryos were collected at indicated stages and analyzed by real time RT-PCR for the expression of various mesodermal markers Eomesodermin, Fgf8, Xnot1, Gata2, Fgf3, eFGF, antipodean, mesendodermal markers Xnr1, Xnr5, Antivin, Bix1, Bix4, endodermal markers Xsox17, Gata5, Endodermin, Cerberus, Xlim-1 and Ectodermal markers Xlim-5 and epidermal keratin. Expression levels are normalized to ODC. (B) The rescue experiment was carried out by injecting MO-1 into the vegetal region of both cells of the two-cell embryos. Embryos were cultured until the eight-cell stage and injected into the four vegetal blastomeres with either 50, 125 or 500 pg of Mixer MO-R mRNA. Embryos were cultured until the mid-gastrula stage (stage 10.5), frozen and assayed by real-time RT-PCR. Expression levels were normalized to ODC.

View larger version (18K): [in a new window]   Fig. 5. Mixer-depleted vegetal masses have increased mesoderm inducing activity in Nieuwkoop recombinants. (Top) Schematic of real-time RT-PCR of Nieuwkoop recombinant experiment in which the capacity of wild-type and Mixer-depleted vegetal masses to induce mesoderm in animal caps is compared. eomesodermin, Fgf8, Xbra and Xnr1 are induced more efficiently in animal caps by Mixer-depleted vegetal masses than control vegetal masses. we, wild-type embryos; WT base-treated caps, 10 animal caps co-cultured with wild-type vegetal masses; MO-1 base-treated caps, 10 animal caps co-cultured with Mixer-depleted vegetal masses. Expression levels were normalized to ODC.

View larger version (13K): [in a new window]   Fig. 7. Model for Mixer function in the early Xenopus embryo. This and previous work suggest the following model for formation of boundaries of expression of several VegT target genes. After MBT, VegT activates the expression of Xnr genes, Xsox17, Gata4, Gata5 and Gata6, and Fgf genes in vegetal cells. The secreted mesoderm inducing molecules induce adjacent marginal cells, which do not express VegT, to express more of themselves as well as the presumptive mesodermal genes including eomesodermin and Xbra. At the early gastrula stage, Mixer is cell autonomously induced by VegT-expressing cells. It reduces, directly or indirectly, the expression of Xnr1 and Xnr5, Fgf3 and Fgf8 and eomesodermin by vegetal cells and therefore prevents further mesoderm inducing signals being released by the vegetal mass. Mixer also activates a second round of Xsox17 and Gata5 expression, consolidating endodermal fates for vegetal cells. Together with maternal XTcf3/ß-catenin, Mixer initiates cerberus expression and cerberus protein blocks further Xnr, Wnt and BMP signaling in the anterior endoderm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter