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Christopher Wylie

Publications and source records attributed to Christopher Wylie.

12 recordsLinked to original sources
PubMed2007

Wnt11/beta-catenin signaling in both oocytes and early embryos acts through LRP6-mediated regulation of axin.

Current models of canonical Wnt signaling assume that a pathway is active if beta-catenin becomes nuclearly localized and Wnt target genes are transcribed. We show that, in Xenopus, maternal LRP6 is essential in such a pathway, playing a pivotal role in causing expression of the organizer genes siamois and Xnr3, and in establishing the dorsal axis. We provide evidence that LRP6 acts by degrading axin protein during the early cleavage stage of development. In the full-grown oocyte, before maturation, we find that axin levels are also regulated by Wnt11 and LRP6. In the oocyte, Wnt11 and/or LRP6 regulates axin to maintain beta-catenin at a low level, while in the embryo, asymmetrical Wnt11/LRP6 signaling stabilizes beta-catenin and enriches it on the dorsal side. This suggests that canonical Wnt signaling may not exist in simple off or on states, but may also include a third, steady-state, modality.

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PubMed2006

Steel factor controls midline cell death of primordial germ cells and is essential for their normal proliferation and migration.

During germ-cell migration in the mouse, the dynamics of embryo growth cause many germ cells to be left outside the range of chemoattractive signals from the gonad. At E10.5, movie analysis has shown that germ cells remaining in the midline no longer migrate directionally towards the genital ridges, but instead rapidly fragment and disappear. Extragonadal germ cell tumors of infancy, one of the most common neonatal tumors, are thought to arise from midline germ cells that failed to die. This paper addresses the mechanism of midline germ cell death in the mouse. We show that at E10.5, the rate of apoptosis is nearly four-times higher in midline germ cells than those more laterally. Gene expression profiling of purified germ cells suggests this is caused by activation of the intrinsic apoptotic pathway. We then show that germ cell apoptosis in the midline is activated by down-regulation of Steel factor (kit ligand) expression in the midline between E9.5 and E10.5. This is confirmed by the fact that removal of the intrinsic pro-apoptotic protein Bax rescues the germ-cell apoptosis seen in Steel null embryos. Two interesting things are revealed by this: first, germ-cell proliferation does not take place in these embryos after E9.0; second, migration of germ cells is highly abnormal. These data show first that changing expression of Steel factor is required for normal midline germ cell death, and second, that Steel factor is required for normal proliferation and migration of germ cells.

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PubMed2005

Maternal Xenopus Zic2 negatively regulates Nodal-related gene expression during anteroposterior patterning.

During the development of Xenopus laevis, maternal mRNAs and proteins stored in the egg direct early patterning events such as the specification of the dorsoventral axis and primary germ layers. In an expression screen to identify maternal factors important for early development, we isolated a truncated cDNA for maternal Zic2 (tZic2), encoding a zinc-finger transcription factor. The predicted tZic2 protein lacked the N-terminal region, but retained the zinc-finger domain. When expressed in embryos, tZic2 inhibited head and axial development, and blocked the ability of full-length Zic2 to induce neural crest genes. Depletion of maternal Zic2 from oocytes, using antisense oligonucleotides, caused exogastrulation, anterior truncations and axial defects. We show that loss of maternal Zic2 results in persistent and increased expression of Xenopus nodal-related (Xnr) genes, except for Xnr4, and overall increased Nodal signaling. Injection of a Nodal antagonist, Cerberus-short, reduced the severity of head and axial defects in Zic2-depleted embryos. Depletion of Zic2 could not restore Xnr expression to embryos additionally depleted of VegT, a T-domain transcription factor and an activator of Xnr gene transcription. Taken together, our results suggest a role for maternal Zic2 in the suppression of Xnr genes in early development. ZIC2 is mutated in human holoprosencephaly (HPE), a severe defect in brain hemisphere separation, and these results strengthen the suggestion that increased Nodal-related activity is a cause of HPE.

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PubMed2004

Primordial germ cell migration.

Mutational and antisense screens in Drosophila and zebrafish, and transcriptional profiling and time-lapse analysis in the mouse, have contributed greatly to our understanding of PGC development. In all three systems, the behavior of PGCs is controlled by growth factors which signal through G-protein coupled receptors and/or tyrosine kinase receptors. Additionally, regulated cell-cell and cell-substrate adhesion is important for PGC motility. Finally, localized growth factors may control PGC survival and consequently PGC position. Chemotaxis, regulated adhesion and cell survival are important for multiple migration processes which occur during development and disease. PGC migration shares these features.

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PubMed2003

The Xenopus LIM-homeodomain protein Xlim5 regulates the differential adhesion properties of early ectoderm cells.

One of the earliest lineage restriction events in embryogenesis is the specification of the primary germ layers: ectoderm, mesoderm and endoderm. In Xenopus, germ layer specification occurs prior to gastrulation and requires the transcription factor VegT both for the cell-autonomous specification of endoderm and the generation of mesoderm-inducing signals. In the absence of VegT, ectoderm is formed in all regions of the embryo. In this work, we show that VegT-depleted vegetal cells (prospective endoderm) behave like animal cells in sorting assays and ectopically express early markers of ectoderm. To gain insight into how ectoderm is specified, we looked for candidate ectoderm-specific genes that are ectopically expressed in VegT-depleted embryos, and examined the role of one of these, the LIM homeobox gene Xlim5, in ectoderm development. We show that overexpression of Xlim5 in prospective endoderm cells is sufficient to impair sorting of animal cells from vegetal cells but is not sufficient (at similar doses) to induce ectoderm-specific genes. In whole embryos, Xlim5 causes vegetal cells to segregate inappropriately to other germ layers and express late differentiation markers of that germ layer. Inhibition of Xlim5 function using an Engrailed repressor construct or a morpholino oligonucleotide causes loss of animal cell adhesion or delay in neural fold morphogenesis, respectively, without significantly affecting early ectoderm gene expression. Taken together, our results provide evidence that a primary role for Xlim5 is to specifically regulate differential cell adhesion behaviour of the ectoderm.

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PubMed2003

GP130, the shared receptor for the LIF/IL6 cytokine family in the mouse, is not required for early germ cell differentiation, but is required cell-autonomously in oocytes for ovulation.

GP130 is the shared receptor for members of the IL6 family of cytokines. Members of this family have been shown to enhance the survival of migratory (E10.5) or postmigratory (E12.5) murine primordial germ cells (PGCs) in culture; however, it is uncertain what role these cytokines play during PGC development in vivo. We have examined PGC numbers in E13.5 GP130-deficient mouse embryos and found that males exhibited a slight decrease in PGC numbers; females were normal. Also, we used the Cre-loxP system to inactive GP130 specifically in germ cells and found that this resulted in a fertility defect in females. These animals were found to have a slight reduction in the number of primary follicles and a major defect in ovulation. This data suggests that GP130 is required in female germ cells for their normal function, but is dispensable in male germ cells.

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PubMed2003

The pro-apoptotic gene Bax is required for the death of ectopic primordial germ cells during their migration in the mouse embryo.

In the mouse embryo, significant numbers of primordial germ cells (PGCs) fail to migrate correctly to the genital ridges early in organogenesis. These usually die in ectopic locations. In humans, 50% of pediatric germ line tumors arise outside the gonads, and these are thought to arise from PGCs that fail to die in ectopic locations. We show that the pro-apoptotic gene Bax, previously shown to be required for germ cell death during later stages of their differentiation in the gonads, is also expressed during germ cell migration, and is required for the normal death of germ cells left in ectopic locations during and after germ cell migration. In addition, we show that Bax is downstream of the known cell survival signaling interaction mediated by the Steel factor/Kit ligand/receptor interaction. Together, these observations identify the major mechanism that removes ectopic germ cells from the embryo at early stages.

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PubMed2002

Surface contraction waves (SCWs) in the Xenopus egg are required for the localization of the germ plasm and are dependent upon maternal stores of the kinesin-like protein Xklp1.

During the first four cell cycles in Xenopus, islands of germ plasm, initially distributed throughout the vegetal half of the egg cortex, move to the vegetal pole of the egg, fusing with each other as they do so, and form four large cytoplasmic masses. These are inherited by the vegetal cells that will enter the germ line. It has previously been shown that germ plasm islands are embedded in a cortical network of microtubules and that the microtubule motor protein Xklp1 is required for their localization to the vegetal pole [Robb, D., Heasman, J., Raats, J., and Wylie, C. (1996). Cell 87, 823-831]. Here, we show that germ plasm islands fail to localize and fuse in Xklp1-depleted eggs due to the abrogation of the global cytoplasmic movements known as surface contraction waves (SCWs). Thus, SCWs are shown to require a microtubule-based transport system for which Xklp1 is absolutely required, and the SCWs themselves represent a cortical transport system in the egg required for the correct distribution of at least one cytoplasmic determinant of future pattern.

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PubMed2002

Repression of organizer genes in dorsal and ventral Xenopus cells mediated by maternal XTcf3.

In the early Xenopus embryo, the dorsal axis is specified by a Wnt signal transduction pathway, involving the movement of beta-catenin into dorsal cell nuclei and its functional association with the LEF-type transcription factor XTcf3. The subsequent function of XTcf3 is uncertain. Overexpression data has suggested that it can be both an activator and repressor of downstream genes. XTcf3 mRNA is synthesized during oogenesis in Xenopus and is stored in the egg. To identify its role in dorsal axis specification, we depleted this maternal store in full-grown oocytes using antisense deoxyoligonucleotides, and fertilized them. The developmental effects of XTcf3 depletion, both on morphogenesis and the expression of marker genes, show that primarily, XTcf3 is an inhibitor, preventing both dorsal and ventral cells of the late blastula from expressing dorsal genes. We also show that simple relief from the repression is not the only factor required for dorsal gene expression. To demonstrate this, we fertilized eggs that had been depleted of both XTcf3 and the maternal transcription factor VegT. Dorsal genes normally repressed by XTcf3 are not activated in these embryos. These data show that normal dorsal gene expression in the embryo requires the transcriptional activator VegT, whilst XTcf3 prevents their inappropriate expression on the ventral side of the embryo.

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PubMed2002

The roles of three signaling pathways in the formation and function of the Spemann Organizer.

Since the three main pathways (the Wnt, VegT and BMP pathways) involved in organizer and axis formation in the Xenopus embryo are now characterized, the challenge is to understand their interactions. Here three comparisons were made. Firstly, we made a systematic comparison of the expression of zygotic genes in sibling wild-type, VegT-depleted (VegT(-)), beta-catenin-depleted (beta-catenin(-)) and double depleted (VegT(-)/beta-catenin(-)) embryos and placed early zygotic genes into specific groups. In the first group some organizer genes, including chordin, noggin and cerberus, required the activity of both the Wnt pathway and the VegT pathway to be expressed. A second group including Xnr1, 2, 4 and Xlim1 were initiated by the VegT pathway but their dorsoventral pattern and amount of their expression was regulated by the Wnt pathway. Secondly, we compared the roles of the Wnt and VegT pathways in producing dorsal signals. Explant co-culture experiments showed that the Wnt pathway did not cause the release of a dorsal signal from the vegetal mass independent from the VegT pathway. Finally we compared the extent to which inhibiting Smad 1 phosphorylation in one area of VegT(-), or beta-catenin(-) embryos would rescue organizer and axis formation. We found that BMP inhibition with cm-BMP7 mRNA had no rescuing effects on VegT(-) embryos, while cm-BMP7 and noggin mRNA caused a complete rescue of the trunk, but not of the anterior pattern in beta-catenin(-) embryos.

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