Top PDF Patterning the early Xenopus embryo

Patterning the early Xenopus embryo

Patterning the early Xenopus embryo

By contrast, there is accumulating evidence that pre-MBT maternal Wnt signaling occurs. First, Wnt11-induced dorsalization occurs most effectively if the mRNA is introduced into the oocyte rather than the fertilized egg (Tao et al., 2005b), suggesting that it is required soon after fertilization. Second, nuclear localization of ␤ catenin on the dorsal side of the embryo happens before MBT (Larabell et al., 1997; Schneider et al., 1996). Third, depletion of maternal ␤ -catenin protein (Heasman et al., 2000) or activation of a dominant-negative Xtcf3 [the transcription factor activated by maternal ␤ catenin (Yang et al., 2002)] blocks dorsal axis formation at the two- or four-cell stage, but not later. This indicates that the signaling pathway cannot be inactivated by the late cleavage stage. Finally, Xtcf3 activity in pre-MBT embryos is sensitive to the transcription inhibitor actinomycin D, and two of its target genes, the TGF ␤ nodal-family members Xnr5 and Xnr6, are expressed from the 256-cell stage onwards (Yang et al., 2002). These experiments provide strong circumstantial evidence that the earliest phase of ␤ -catenin/Xtcf3 interaction happens during the cleavage to early blastula stages.
Show more

13 Read more

Xnr3 affects brain patterning via cell migration in the neural epidermal tissue boundary during early Xenopus embryogenesis

Xnr3 affects brain patterning via cell migration in the neural epidermal tissue boundary during early Xenopus embryogenesis

of Xnr3 (Xtnr3) that is functionally identical to Xnr3 (Haramoto et al., 2004). Injection of pXtnr3 (Fig. 3A), encoding the pro-region of Xtnr3, into the VAP region hardly altered the expression pat- terns of krox20/En2, slug, and xOtx2 (Fig. 3B-C, F-G, J-K). On the other hand, injection of the mature region of Xtnr3 (mXtnr3; Fig. 3A) induced a bent pattern of krox20 expression, similar to that in Xnr3-injected embryo (Fig. 3D, E). The expression regions of both slug and Xotx2 were also smaller in mXtnr3-injected embryos, as in Xnr3-injected embryo (Fig. 3H-I and Fig. 3L-M). These results indicated that the mature region of Xnr3 is essential for conferring the specific effects on neural patterning. However, the effects were only weak in mXtnr3-injected embryos, compared with Xnr3 injec- tions, prompting us to investigate the role of the Xnr3 pro-region in pattern specification. Co-injection of pXtnr3 and mXtnr3 increased the severity of the specific effects on krox20 and En2 expression patterns to a level equivalent to that observed with full-length Xnr3 (Fig. 3N). These results suggested that pXtnr3 could enhance the
Show more

9 Read more

Formation of a functional morphogen gradient by a passive process in tissue from the early Xenopus embryo

Formation of a functional morphogen gradient by a passive process in tissue from the early Xenopus embryo

However, since our experiments were performed using TGF- β 1, it is not possible to be certain that other potentially more physiologi- cal TGF- β superfamily members behave in the same fashion. Although unlikely, it is also possible that the mechanism of gradient formation in our model system is substantially different from that which occurs in the undisrupted embryo. For example, dissection of the animal caps into culture might induce alterations in the tissue structure, although we obtained no evidence for such a change. In particular, we see no histological evidence for any disruption of the cell:cell interactions in our conjugates compared with whole em- bryos. In addition, at present it is unclear whether mesoderm induction, in vivo, involves short-range induction rather than the release of long-range signals. However, during gastrulation, long- range inhibitors are thought to diffuse from a localized source, the Spemann-Mangold Organizer, to create an activity gradient of a locally produced agonist. Since this agonist is soluble and extracel- lular, and not a transmembrane receptor, then establishing the mechanism of passage of the antagonists may prove technically difficult. The use of bead sources to generate model morphogen gradients has already provided powerful clues as to the mecha- nisms of tissue patterning in the vertebrate embryo, and it is likely that the results presented here will be similarly useful. In particular, the work we describe, while for technical reasons necessitating a model system, establishes that passage through the solid tissue of the early Xenopus embryo is possible by a passive process, most likely by diffusion through the extracellular milieu.
Show more

9 Read more

Evidence that platelet derived growth factor (PDGF) action is required for mesoderm patterning in early amphibian (Xenopus laevis) embryogenesis

Evidence that platelet derived growth factor (PDGF) action is required for mesoderm patterning in early amphibian (Xenopus laevis) embryogenesis

be expressed at the early gastrula stage (st. 10-11), then they are actively expressed in the dorsal (somitic) mesoderm at neurula (st. 18). When XMyoD and XMyf5 mRNA are microinjected together into Xenopus animal caps full myogenesis fails, but when XMRF4 is microinjected with them it succeeds to occur (Gurdon et al., 1992). The gene is expressed in the somitic mesoderm at neural fold stage and the gene product functions in late mesodermal differentiation (Jennings, 1992). Since MyoD family genes were induced by PDGF, we suggest that PDGF plays a role in the pattern formation of dorsal mesoderm. This notion is supported by the fact that PDGF induced the expression of SL1 (Fig. 3). The RSRF gene, belonging to the MEF2 family, is expressed in the dorsal mesoderm of the early Xenopus embryo (Chambers et al., 1992). The MEF2 protein family activates XMyoD transcription and XMEF2 expression in myogenic cells and contributes to the activation and stabilization of XMyoD transcription during muscle cell differentiation (Wong et al., 1994). SL1 (MEF2D) lies downstream of the myogenic factors in the skeletal myogenic pathway and is a dorsal mesoderm marker. SL1 regulates the cardiac muscle-specific transcription of XMLC2 in Xenopus embryos. XMLC2 is a direct target for trans- activation by the SL1 protein and a marker for terminal differentiation of cardiac muscle in the Xenopus embryo. Its expression is detected first in stage 28/29 tadpoles in the presumptive heart region and is subsequently confined to the developing heart tube (Chambers et al., 1992, 1994). In our experiment, both SL1 and XMLC2 were expressed in animal cap cells treated with PDGF, activin and FGF. This indicates that PDGF is involved in the formation of cardiac muscle originated from lateral mesoderm. However, the possibility of the PDGF inducing ventral mesoderm needs more research.
Show more

6 Read more

Dorsal Ventral Patterning and Gene Regulation in the Early Embryo of Drosophila melanogaster

Dorsal Ventral Patterning and Gene Regulation in the Early Embryo of Drosophila melanogaster

Additional evidence also suggests that TGF-ß signaling may also regulate the ind expression domains, but whether or not this signaling pathway functions through the A- box element was not known. Decapentaplegic (Dpp) is a TGFß/BMP homolog that is limited in its expression to dorsal regions of the embryo and functions as a morphogen to support patterning of the amnioserosa, at higher levels in dorsal-most regions of the embryo, and the non-neurogenic ectoderm, at lower levels in dorsal-lateral regions of the embryo (Ferguson and Anderson, 1992). A previous study found that in mutants in which Dpp signaling is expanded into lateral regions of the embryo, ind expression is lost (Von Ohlen and Doe, 2000). Likewise, ectopic expression of dpp in lateralized embryos that exhibit expanded ind expression throughout the embryo was able to repress ind in the domain where Dpp signaling was presented (Mizutani et al., 2006). Also, the ind CRM contains a 15 bp DNA sequence implicated in TGF- β signaling-mediated repression (Stathopoulos and Levine, 2005). Similar sites have been shown to mediate repression by recruiting a Dpp-dependent Schnurri/Mad/Medea (SMM) protein complex, but SMM dependent repression of ind has never been shown and in fact this mechanism of repression has only been shown to act at later stages of development (Dai et al., 2000; Pyrowolakis et al., 2004).
Show more

120 Read more

Systems Biology Derived Mechanism Of Bmp Gradient Formation

Systems Biology Derived Mechanism Of Bmp Gradient Formation

The extracellular BMP antagonist Chordin and its homologs are essential to proper regulation of BMP signaling in mouse, fish, and frog development. Chordin is the central node of a network of regulators that modulate Chordin function in the extracellular space. Chordin inhibits BMP signaling by binding BMP ligand, rendering BMP ligand unable to bind its receptors (Fig. 1.2A, Table 1.1) (Stefano Piccolo Yoshiki Sasai, Bin Lu, Eddy M. De Robertis 1996, Troilo et al. 2014, Zhang J. L. et al. 2007a). Chordin is expressed in dorsal tissues, including the dorsal organizer, throughout early development (Abe et al. 2014, Abe et al. 2016, Bachiller 2003, Bachiller et al. 2000, Branam et al. 2010, Kuroda et al. 2004, Miller-Bertoglio et al. 1997, Ramel and Hill 2013, Schulte-Merker et al. 1997, Shimizu et al. 2000, Xue et al. 2014). In zebrafish, the loss of chordin causes a modest expansion of ventral mesodermal and ectodermal structures such as blood and tail and a concomitant reduction of dorsal structures such as the somites, eyes, and brain (Fisher et al. 1997, Matthias Hammerschmidt 1996, Schulte- Merker et al. 1997). A similar expansion of ventral mesodermal markers and ventral structures is seen in Xenopus embryos deficient for Chordin (Oelgeschlager et al. 2003). In mice, the loss of chordin alone does not induce as severe a phenotype, causing an expansion of the allantois at the expense of the embryonic mesoderm, along with mild pharyngeal and bone defects (Bachiller 2003).
Show more

114 Read more

Differential requirements for Smad4 in TGFβ dependent patterning of the early mouse embryo

Differential requirements for Smad4 in TGFβ dependent patterning of the early mouse embryo

By contrast, our data suggest that in the early embryo, Smad4 is required for certain TGFβ/Bmp signaling pathways, but not obligate for others. A growing body of data corroborates our observations. For example, in Smad4- deficient MEFs as well as several Smad4-deficient human tumor cell lines, TGFβ addition still results in classical growth inhibition (reviewed by Derynck and Zhang, 2003; Wakefield and Roberts, 2002). In Drosophila, oogenesis is more severely disrupted by the loss of the R-Smad Mad than by Medea. Interestingly, in wing imaginal discs, loss of Medea most severely affects regions receiving low Dpp signal (Wisotzkey et al., 1998). One possible explanation is that Medea normally potentiates weak Dpp signals and that in the absence of Medea, R-Smads regulate target genes only when the Dpp signal strength is high. R-Smads may substitute for or bypass the requirement for Smad4, resulting in unpotentiated levels of downstream signal (Yeo et al., 1999). Additionally, alternate TGFβ pathways have been described, including the Ras/Mapk, Pp2a/S6 kinase, Rhoa and PI3K-Akt pathways, some of which are entirely Smad independent and others that involve crosstalk with Smad signals. Finally, an independently-derived Smad4 conditional allele was recently employed to eliminate Smad4 function during CNS and mammary gland development and the resulting phenotypes were surprisingly mild (Li et al., 2003; Zhou et al., 2003). These findings in combination with our genetic results emphasize the versatility in the intracellular transduction of TGFβ-related signals, and encourage a more careful consideration of the current canonical model of TGFβ signaling that places Smad4 as a central effector molecule.
Show more

12 Read more

Where do we stand now?   mouse early embryo patterning meeting in Freiburg, Germany (2005)

Where do we stand now? mouse early embryo patterning meeting in Freiburg, Germany (2005)

About two dozen active mouse researchers (see list of "Meeting Participants" on p. 581) literally from all over the world gathered in Freiburg in September last year, to discuss the current controversy regarding the patterning mechanisms of mouse pre-implantation embryos. This one-day meeting was apparently too short to resolve many of the issues, as summarized above. We apologize that this paper largely describes rather specific and technical aspects of the studies, which may be unfamiliar to non-mammalian researchers and perhaps even to many mammalian researchers. However, it is inevitable to describe a certain level of detail, because the source of the contradictions appears to stem from the differences in such details, e.g., experimental conditions, defini- tions of terms and interpretation of data. If we were to ignore these details, we would never be able to truly understand the mecha- nisms of mammalian development.
Show more

7 Read more

Guidance of mesoderm cell migration in the Xenopus gastrula
requires PDGF signaling

Guidance of mesoderm cell migration in the Xenopus gastrula requires PDGF signaling

To identify the nature of the guidance cues that direct mesoderm migration has been a goal since their discovery two decades ago. Previously, we have shown that signals emanating from the vegetal part of the embryo, which employ fibroblast growth factor (FGF)- and activin-like signaling pathways for propagation, polarize the BCR layer. This polarization is a prerequisite for the deposition of a correctly oriented extracellular matrix (Nagel and Winklbauer, 1999). However, the molecular basis of the matrix-borne directional cues remained elusive. We address this long-standing unresolved issue, and show that platelet-derived growth factor (PDGF) signaling plays an essential role in mesoderm guidance during Xenopus gastrulation. PDGF signaling is involved in a variety of processes, including early embryonic development, formation of the nervous system, heart and lung development, and angiogenesis. It has also been implicated in several pathological conditions, such as oncogenesis, atherosclerosis, lung and kidney fibrosis, and wound healing. Major target processes of PDGF signaling are the regulation of proliferation, and the control of cell motility and migration (Ataliotis and Mercola, 1997; Heldin and Westermark, 1999; Betsholtz et al., 2001).
Show more

10 Read more

Fox (forkhead) genes are involved in the dorso ventral patterning of the Xenopus mesoderm

Fox (forkhead) genes are involved in the dorso ventral patterning of the Xenopus mesoderm

chick HFH-8 (Peterson et al., 1997; Mahlapuu et al., 1998; Funayama et al., 1999) and is likely to be the same gene as XFD-13’ by Koster et al. (1999). At the amino acid level, XFD-13’(FoxF1b) and FREAC1 are 80% identical and they share almost complete conservation in the fork head domain with mouse and chick HFH8 genes. XFD-13’(FoxF1b) is activated during gastrulation in the presumptive ventrolateral mesoderm. During neurulation, the strongest expression is in the lateral plate mesoderm. The most ventral mesoderm has either no or much less expression than the lateral plate mesoderm (Fig. 1C). From neural tube stages on, XFD-13’(FoxF1b) is increasingly expressed in the lateral plate mesoderm. The expression appears to progress from anterior to posterior and from dorsal to ventral (not shown). In tadpoles this gene is expressed in the lateral plate mesoderm, the ventral mesoderm and the migrating neural crest cells of the head (Fig. 4C). Most of the expression in the head is ventral to the eyes, in the branchial arches. Figure 4A shows expression of XFD-13’(FoxF1b) in the head of a stage 35 embryo. Figure 4B shows the expression of XFD-13’(FoxF1b) in the trunk of the same embryo. Expression is in the lateral plate mesoderm and ventral mesoderm. The circulatory system, including blood cells, blood and blood vessels, is derived from the lateral plate mesoderm and ventral mesoderm. However, XFD-13’(FoxF1b) is not expressed in the heart anlage, as is demonstrated by double in situ hybridization with troponin, a marker specific for heart development (Drysdale et al., 1994) and XFD-13’(FoxF1b). As shown in Fig. 4D, there is no overlap be- tween the two signals. This pattern is very similar to the expression of HFH-8 in mouse and confirms the expression analysis of XFD- 13 by Koster et al., (1999). BMP4 is the most potent activator of XFD-13’(FoxF1b) in animal caps, but activin and FGF also activate this gene to a lesser degree (not shown).
Show more

7 Read more

Exploration of the extracellular space by a large scale secretion screen in the early Xenopus embryo

Exploration of the extracellular space by a large scale secretion screen in the early Xenopus embryo

egg. Despite some progress in recent years, the signals and pathways involved are only partially understood (De Robertis et al., 2000). Given that more than twenty-five thousand cDNA clones of the 32-cell stage cDNA library were screened, surpris- ingly few extracellular proteins with known signaling function were isolated. Among the growth factor antagonists, the only protein we found was Sizzled (Salic et al., 1997; Table 1). Other proteins previously reported to be secreted and not yet known in Xenopus included the calcium-binding EF-hand protein Nucleobindin and the translationally controlled tumor protein (TCTP; Table 2). In the unamplified maternal library, we isolated TCTP independently as three different cDNA clones suggesting a high abundance of this gene product in the Xenopus egg. A function for Sizzled, Nucleobindin and TCTP at this early stage of development remains to be shown. In addition, we identified the novel secreted proteins LAMA-like, SOUL and Darmin-related (Table 3). Their function remains to be determined. The relatively low yield of secreted signaling proteins suggest that cell-cell communication may not play a major role before mid blastula transition (MBT). It has previously been shown that pre-MBT cell contacts are dis- pensable for the expression of endodermal and dorsal mesoder- mal markers (Wylie et al., 1996; Yasuo and Lemaire, 1999). Thus, our data support the notion that early development up to MBT relies more on cell-autonomous signals rather than communica- tions exchanged between cells.
Show more

16 Read more

Retinol dehydrogenase 10 is a feedback regulator of retinoic acid signalling during axis formation and patterning of the central nervous system

Retinol dehydrogenase 10 is a feedback regulator of retinoic acid signalling during axis formation and patterning of the central nervous system

ventralization and influences mesodermal gene expression. Antisense morpholino oligonucleotides (MOs) were injected marginally at the two-cell stage (5.2 pmol per blastomere), followed by injection of non-targeted mRNA constructs ( XRDH10* and mRALDH2 ) at the four-cell stage (1 ng per blastomere). (A,B) MOs target the translation initiation sites of two pseudoalleles of Xenopus laevis RDH10 and RALDH2 . (C,D) Protein synthesis of XRDH10 and XRALDH2 is specifically inhibited by XRDH10 -MO and XRALDH2 -MO, but not by control-MO of random sequence. (E-J) Microinjection of XRDH10 -MO and XRALDH2 - MO leads to microcephaly and enlarged ventroposterior structures in tailbud embryos (E-G), and to reduced eye structures and shortened tails in tadpoles (H-J). Normal development is restored by XRDH10 * and mRALDH2 mRNAs, respectively (insets). (K-S) Gastrula embryos in dorsal view. XRDH10 - and XRALDH2 -morphants have reduced Chordin expression (K-M) and expanded expression domains of Goosecoid (N-P) and ADMP (Q-S). (T-V) Neurula embryos in dorsal view (anterior to the top) after a single injection of MOs with the lineage tracer nlacZ mRNA (red nuclei). XRDH10 -MO and XRALDH2 -MO reduce Xlim1 expression in the pronephros (arrowhead). The indicated phenotypes were observed in: E, 101/117; F, 62/84 (inset, 73/98); G, 44/67 (inset, 80/84); H, 74/79; I, 39/50 (inset, 56/68); J, 18/48 (inset, 58/64); K, 33/33; L, 21/36 (inset, 19/27); M, 28/36 (inset, 20/28); N, 51/56; O, 28/38 (inset, 46/53); P, 37/49 (inset, 34/44); Q, 52/52; R, 14/26 (inset, 51/62); S, 51/62 (inset, 23/32); T, 11/15; U, 14/20 (inset, 28/32); V, 16/24 (inset, 50/51) embryos.
Show more

12 Read more

Herbert Steinbeisser: a life with the Xenopus embryo

Herbert Steinbeisser: a life with the Xenopus embryo

In 1995 Herbert became a Group Leader at the Max Planck Institute for Developmental Biology in the Department of Peter Hausen, a phage geneticist turned a strong leader of Xenopus research. His first paper there was a collaboration with Stephan Schneider, a graduate student of Hausen. They made the important discovery that endogenous b-catenin protein translocated inside nuclei in the dorsal side of the midblastula embryo, both in Xenopus and zebrafish (Schneider et al., 1996). In this study we can see Steinbeisser’s imprimatur for he used LiCl to show stabilization of b-catenin all around the embryo and UV irradiation to block nuclear translocation on the dorsal marginal cells. This paper was a landmark study in embryology. The dorsal localization of b-catenin is caused by a cortical rotation of the egg cytoplasm, a mechanism to which Herbert was to return (Medina et al., 1997; Ding et al., 1998). Steinbeisser’s interest turned to Frizzled seven-transmembrane proteins, which had been recently discovered by Roel Nusse and Jeremy Nathans as the long sought Wnt receptors. In a series of papers he explored the role and structure-functional aspects of Fz7 during Xenopus development (Swain et al., 2001; Medina and Steinbeisser, 2000; Medina et al., 2000). This culminated in the discovery, together with Rudi Winklbauer, that Fz7/PKC signaling regulates sorting out of early involuted anterior endomesoderm from ectoderm. This comes about by a switch in cell adhesion and is essential in Xenopus gastrulation (Winklbauer et. al., 2001). The paper featured an elegant explant assay, where embryonic cells are placed on naïve ectoderm and either sink-in or stay put, the type of experiment that makes Xenopus such a unique system. This study set the stage for a series of follow-up papers on tissue separation, notably involving the then poorly understood protocadherins. These transmembrane proteins play an important role in cell adhesion and morphogenesis. In Tübingen, and later in Heidelberg, Herbert showed that paraxial protocadherin (PAPC) regulates tissue separation and that it is a signaling molecule, which activates Wnt/Planar Cell Polarity (PCP) signaling (Medina et al., 2004; Wang et al., 2008; Kraft et al., 2012) and inhibits canonical Wnt signaling by sequestering casein kinase 2 beta (Kietzmann et al., 2012). Steinbeisser’s studies on tissue separation renewed interest in an extracellular matrix region of the Xenopus embryo that separates the endomesoderm from the ectoderm called Brachet’s cleft (Gorny and Steinbeisser, 2012). This narrow region is now thought to provide a signaling highway during patterning of the gastrula.
Show more

5 Read more

Ras dva, a member of novel family of small GTPases, is required for the
anterior ectoderm patterning in the Xenopus laevis embryo

Ras dva, a member of novel family of small GTPases, is required for the anterior ectoderm patterning in the Xenopus laevis embryo

transcriptional regulators, the homeobox genes Otx2 and Xanf1, in the gastrula and neurula stage Xenopus embryos as revealed by whole-mount in situ hybridization. All embryos are shown from the anterior, dorsal side upwards. Broken line indicates the neural plate border. (A) At midgastrula (stage 11) the Ras-dva is diffusely expressed within a broad territory that includes the presumptive anterior neural and non-neural ectoderm. (B) At late gastrula (stage 12.5) the Ras-dva expression weakens in the area corresponding to the anterior neural plate but it increases in the surrounding area. (C) At the midneurula (stage 14) Ras-dva is expressed in cells of the non-neural anterior ectoderm and in the lateral neural folds. (D-F) Normal expression patterns of the homeobox genes Otx2 and Xanf1. Although Ras-dva and Xanf1 are expressed in mutually excluding domains, the expression domains of Ras-dva and Otx2 are largely overlapping. (G,H) The results of in situ hybridization in halves of Xenopus embryos on the 12.5 and 14 stages. Left halves show the expression of Otx2 gene, right halves show Ras-dva expression. (I) The Xenopus embryo (stage 13) in which the left half is stained for Xanf1 expression and the right half is stained for Ras-dva expression. (J) The scheme of expression patterns of Ras- dva, Otx2 and Xanf1 at the midneurula stage (stage 14).
Show more

10 Read more

Lin28 proteins are required for germ layer specification in Xenopus

Lin28 proteins are required for germ layer specification in Xenopus

It has recently been shown that mammalian Lin28a and Lin28b localise to different subcellular compartments and that this differential localisation in part accounts for the dissimilar modes of action of the two proteins; Lin28a protein is found mainly in the cytoplasm whereas Lin28b is highly enriched in the nucleus of cells in culture (Piskounova et al., 2011). We investigated whether amphibian lin28 proteins are also differentially localised within the cells of the early embryo. Immunofluorescence shows that lin28a and lin28b are localised in cells of the circumblastoporal region of the Xenopus embryo at the start of gastrulation (Fig. 1Q-V). Moreover, control experiments indicate that the observed immunofluorescence is specific for each of the proteins; the immunoreactivity can be effectively competed out by the relevant peptide immunogens (supplementary material Fig. S2).
Show more

11 Read more

PRDM15 loss of function links NOTCH and WNT/PCP signaling to patterning defects in holoprosencephaly

PRDM15 loss of function links NOTCH and WNT/PCP signaling to patterning defects in holoprosencephaly

Holoprosencephaly (HPE) is a congenital forebrain defect often associated with embryonic lethality and lifelong disabilities. Currently, therapeutic and diagnostic options are limited by lack of knowledge of potential disease- causing mutations. We have identified a new mutation in the PRDM15 gene (C844Y) associated with a syndromic form of HPE in multiple families. We demonstrate that C844Y is a loss-of-function mutation impairing PRDM15 transcriptional activity. Genetic deletion of murine Prdm15 causes anterior/posterior (A/P) patterning defects and recapitulates the brain malformations observed in patients. Mechanistically, PRDM15 regulates the transcription of key effectors of the NOTCH and WNT/PCP pathways to preserve early midline structures in the developing embryo. Analysis of a large cohort of patients with HPE revealed potentially damaging mutations in several regulators of both pathways. Our findings uncover an unexpected link between NOTCH and WNT/PCP signaling and A/P patterning and set the stage for the identification of new HPE candidate genes.
Show more

11 Read more

Patterning the Xenopus embryo: The role of Xom, a novel homeobox gene

Patterning the Xenopus embryo: The role of Xom, a novel homeobox gene

As mentioned already, RNAase protection analysis on dissected regions o f the embryo suggest that at gastrulation stages Xom is found throughout the embryo, but is less abundant in the dorsal organiser region. Whole mount in situ hybridisation supports this idea (Figure 5.4B), although the levels on the dorsal side are much lower than those predicted by RNAase protection on dissected pieces of the embryo. The higher levels predicted by the dissection data may be due to the fact that the exact extent of the dorsal marginal zone is very hard to judge. Another discrepancy between the RNAase protection data and using whole mount in situ hybridisation is the apparent lack of Xom transcripts in the vegetal pole in the whole mount embryo. It is known that the whole mount in situ hybridisation technique detects vegetally localised transcripts poorly. This is thought to be due to poor probe penetration into the yolk-rich vegetal hemisphere (Hemmati- Brivanlou et at., 1990). However, in some cases these transcripts can be observed in sections of overstained embryos. The vegetally localised transcripts appear as a punctate pattern and these can be seen in figure 5.5 A .
Show more

194 Read more

New roles for FoxH1 in patterning the early embryo

New roles for FoxH1 in patterning the early embryo

To confirm that these effects were specifically caused by the reduction of maternal Foxh1 mRNA, we determined the appropriate dose of mRNA for rescue experiments. When Foxh1 mRNA was over-expressed in oocytes in doses greater than 100 pg, embryos developed with a headless phenotype (data not shown). We therefore tested the effect of Foxh1 mRNA over-expression on the activity of the ARE-luciferase reporter: 15-120 pg Foxh1 mRNA was injected into the vegetal area of two-cell stage embryos that had been injected with ARE-luciferase at the one-cell stage, and luciferase activity was examined at the early gastrula stage. Fig. 2B indicates that 15 pg Foxh1 mRNA activates the ARE-reporter to a higher level than the level caused by endogenous signals, while higher doses repress ARE-luciferase activity, compared to control levels. In a second experiment, 30 pg Foxh1 mRNA activated luciferase, while higher doses caused inhibition (data not shown). Thus, FoxH1 acts as an activator or repressor of the ARE-luciferase reporter in a concentration-dependent manner. For rescue experiments, oligo 10-injected and control oocytes were incubated for 48 hours (to allow oligo and mRNA degradation) and then 15 and 30 pg Foxh1 mRNA was injected in the vegetal area. Oocytes were matured and fertilized and allowed to develop to the tailbud stage. Siblings were frozen at the gastrula stage for the analysis of molecular markers of axis formation. Foxh1 mRNA significantly rescued the expression of Xnr3, and other organizer genes, in a dose- responsive fashion (Fig. 2C); 30 pg also significantly rescued head formation in 80% of embryos (8/10 cases) compared to sibling FoxH1-depleted embryos which had 100%-reduced heads or headless phenotype (15/15 cases; Fig. 2D). The experiment was repeated with a similar result. These results indicate that the embryo is extremely sensitive to the level of expression of Foxh1 mRNA, and confirms that FoxH1 regulates the expression of Xnr3, and head formation.
Show more

14 Read more

The roles of maternal Vangl2 and aPKC in Xenopus oocyte and embryo patterning

The roles of maternal Vangl2 and aPKC in Xenopus oocyte and embryo patterning

Fig. 7. Maternal Vangl2 and aPKC depletion disrupts the pattern of expression of specific zygotic genes. (A)  Relative expression levels of Siamois, Xnr3 and Foxi1e mRNAs in control (Uninj) and Vangl2-depleted (VG–) embryos at the late blastula (st9) and early gastrula stages (st10) determined by real-time RT-PCR (mean±s.d.). (B)  Phenotype of embryos derived from sibling control (Uninjected) and Vangl2-depleted oocytes (VG AS5, 4 and 6 ng oligonucleotide injected; VG AS4, 4, 6 and 8 ng oligonucleotide injected) at the early tailbud stage. (C)  Relative expression levels of Siamois, Xnr3 and Foxi1e in control (Uninj) and aPKC-depleted embryos (AS5, 5 ng; AS6, 10 ng) determined by real-time RT-PCR at the late blastula and early gastrula stages. The experiment was repeated with a similar result. (D)  Phenotype of embryos derived from sibling control (Uninjected) and aPKC-depleted oocytes (AS5, 3, 4 and 5 ng; AS6, 5 and 10 ng) at the early tailbud stage. (E)  TOPflash reporter activation after injection into two dorsal vegetal cells of 8-cell stage control embryos compared with sibling Vangl2-depleted (left-hand graph) and aPKC-depleted (right-hand graph) embryos frozen at the late blastula stage, showing the reduction in canonical Wnt signaling activity (mean±s.d.). (F)  Percentage of embryos with different phenotypes derived from Vangl2-depleted (AS4 and AS5), aPKC-depleted (AS5 and AS6) and sibling control at the tailbud stage. (G)  Histological section of whole-mount stained, in situ hybridization of sibling control (upper) and aPKC-depleted (lower) early gastrulae using a Foxi1e probe. Arrow indicates ectopic expression of Foxi1e in the vegetal mass. (G ⬘ )  Whole-mount in situ hybridization of sibling control (upper) and Vangl2-depleted (lower) early gastrulae using a Sox17a probe. (H)  Relative expression levels of Xbra, Wnt11, Sox17a and Foxi1e in equatorial zones of control (Uninj.), Vangl2-depleted (VG–) and aPKC-depleted (aPKC–) embryos dissected at the late blastula stage and assayed by real-time RT-PCR
Show more

12 Read more

MRAS GTPase is a novel stemness marker that impacts mouse embryonic stem cell plasticity and Xenopus embryonic cell fate

MRAS GTPase is a novel stemness marker that impacts mouse embryonic stem cell plasticity and Xenopus embryonic cell fate

Therefore, we took advantage of the complementary Xenopus model to address Mras functions in pluripotency and early development. We used both the in vivo approach and the model of pluripotent animal cap cells (Sasai et al., 2008). Mras is highly conserved among vertebrates from zebrafish to human at both the gene and protein levels, suggesting a selection pressure and conserved function. As in mESCs, there are clearly two phases of Mras expression in Xenopus, with a maternal expression that decreases between the blastula and early gastrula stages followed by a zygotic expression that increases in the early tadpole, a time when Mras mRNA is found in RB cells and trigeminal neurons. RB neurons are a transient population of primary sensory neurons that is observed in non-mammalian vertebrates, such as zebrafish and Xenopus, and is replaced in the late embryo by dorsal root ganglia (Holland, 2009; Olesnicky et al., 2010; Roberts, 2000). Strikingly, in the mouse embryo, Mras is also expressed in dorsal root ganglia (Keduka et al., 2009). We conclude that the amphibian and murine Mras genes share both strong sequence conservation and expression pattern (Kimmelman et al., 2000).
Show more

12 Read more

Show all 10000 documents...