Laura W. Burrus

β-Catenin-dependent Wnt signalling controls the epithelial organisation of somites through the activation of paraxis

The regulation of cell adhesion in epithelia is a fundamental process governing morphogenesis in embryos and a key step in the progression of invasive cancers. Here, we have analysed the molecular pathways controlling the epithelial organisation of somites. Somites are mesodermal epithelial structures of vertebrate embryos that undergo several changes in cell adhesion during early embryonic life. We show that Wnt6 in the ectoderm overlaying the somites, but not Wnt1 in the neighbouring neural tube, is the most likely candidate molecule responsible for the maintenance of the epithelial structure of the dorsal compartment of the somite: the dermomyotome. We characterised the signalling pathway that mediates Wnt6 activity. Our experiments suggest that the Wnt receptor molecule Frizzled7 probably transduces the Wnt6 signal. Intracellularly, this leads to the activation of theβ -catenin/LEF1-dependent pathway. Finally, we demonstrate that the bHLH transcription factor paraxis, which was previously shown to be a major player in the epithelial organisation of somites, is a target of theβ -catenin signal. We conclude that β-catenin activity, initiated by Wnt6 and mediated by paraxis, is required for the maintenance of the epithelial structure of somites.

Differential Inhibition of Wnt-3a by Sfrp-1, Sfrp-2, and Sfrp-3

Secreted frizzled related proteins (Sfrps) are extracellular attenuators of Wnt signaling that play important roles in both embryogenesis and oncogenesis. Although Sfrps are generally thought to bind and sequester Wnts away from active receptor complexes, very little is known about the specificity of Sfrp family members for various Wnts. In the developing chick neural tube, sfrp‐1, 2, and 3 transcripts are expressed in and adjacent to the dorsal neural tube, where Wnt‐1 and Wnt‐3a are expressed. To better define the possible roles of Sfrp‐1, 2, and 3 in the neural tube, we first tested the ability of purified Sfrps to inhibit Wnt‐3a‐induced accumulation of β‐catenin in L cells. We find that both Sfrp‐1 and Sfrp‐2 can inhibit Wnt‐3a activity while Sfrp‐3 cannot. To determine where Sfrp‐1 and Sfrp‐2 impinge on the Wnt signaling pathway, we tested the ability of these Sfrps to inhibit Wnt signaling induced by the addition of LiCl, an inhibitor of GSK‐3. Sfrp‐1 and Sfrp‐2 are unable to inhibit the accumulation of β‐catenin in LiCl‐treated cells, suggesting that the ability of Sfrps to inhibit the accumulation of β‐catenin is GSK‐3 dependent. We have further shown that Sfrp‐2 inhibits the ability of ectopic Wnt‐3a to stimulate proliferation in the developing chick neural tube. These results provide the framework for understanding how Sfrps function to regulate Wnt‐3a activity in developing embryos and in cancer. Developmental Dynamics 235:681–690, 2006.

Porcupine-mediated lipid-modification regulates the activity and distribution of Wnt proteins in the chick neural tube

A long-term goal of developmental biology is to understand how morphogens establish gradients that promote proper tissue patterning. A number of reports describe the formation of the Wg (Wnt1) gradient in Drosophila and have shown that Porcupine, a predicted membrane-bound O-acyl transferase, is required for the correct distribution of Wg protein. The discovery that Wnts are palmitoylated on a conserved cysteine residue suggests that porcupine activity and Wnt palmitoylation are important for the generation of Wnt gradients. To establish the role of porcupine in Wnt gradient formation in vertebrates, we tested the role of porcupine/Wnt palmitoylation in human embryonic kidney 293T cells and in the chick neural tube. Our results lead us to conclude that: (1) vertebrate Wnt1 and Wnt3a possess at least one additional site for porcupine-mediated lipid-modification; (2) porcupine-mediated lipid-modification of Wnt proteins promotes their activity in 293T cells and in the chick neural tube; and (3) porcupine-mediated lipid-modification reduces the range of activity of Wnt1 and Wnt3a in the chick neural tube. These findings highlight the importance of porcupine-mediated lipid modifications in the formation of vertebrate Wnt activity gradients.

Sfrp-1 and sfrp-2 are expressed in overlapping and distinct domains during chick development.

Secreted frizzled related proteins (Sfrps) are thought to bind and regulate Wnt activity through a cysteine rich domain that is highly similar to that of Frizzled receptors. To investigate possible roles for Sfrps in chick development, we have isolated partial cDNAs encoding Sfrp-1 and Sfrp-2 and have thoroughly characterized the expression patterns of both genes. Both sfrp-1 and sfrp-2 are expressed at all stages of development analyzed, ranging from Hamburger and Hamilton stage 4 through stage 32. Expression of both sfrp-1 and sfrp-2 is observed in mesodermal and ectodermal derivatives, while sfrp-1 is also found in endodermal lineages.

Comparative analysis of the expression patterns of Wnts and Frizzleds during early myogenesis in chick embryos

During embryogenesis, the transduction of Wnt signals through Frizzled receptors is thought to play integral roles in myogenesis and somite patterning. However, little is known about which Wnt-Frizzled interactions are required for skeletal myogenesis. Thus, we sought to determine which Wnts and Frizzled exhibit expression patterns that are spatiotemporally consistent with the expression of two myogenic determination factors: Myf-5 and MyoD. To accomplish this, we first isolated partial cDNAs for six chick Frizzled orthologues and then compared the expression patterns of chick Frizzleds and Wnts to myogenic and somite patterning factors, such as Myf-5, MyoD, Sonic Hedgehog (Shh), Pax-1 and Pax-3 in Hamburger and Hamilton stage 10 chick. We used these data to generate a schematic composite of expression patterns at the level of the segmental plate and developing somites (stage V) that shows multiple Frizzled and Wnt transcripts expressed in tissues that are overlapping and adjacent to Myf-5 and MyoD expressing tissues.

The expression patterns of Wnts and their antagonists during avian eye development.

To determine the possible involvement of Wnt signaling in eye development, we analyzed the expression patterns of Wnts and Wnt inhibitors in the chicken eye at stage 25, when the first wave of neural crest migration into the cornea begins, and stage 27, just prior to the second wave of neural crest migration. Wnt expression is developmentally regulated in the chicken eye, and antagonists of Wnt signaling are generally expressed in patterns that are temporally distinct from the Wnts.

Differential Palmit(e)oylation of Wnt1 on C93 and S224 Residues Has Overlapping and Distinct Consequences

Though the mechanisms by which cytosolic/intracellular proteins are regulated by the post-translational addition of palmitate adducts is well understood, little is known about how this lipid modification affects secreted ligands, such as Wnts. Here we use mutational analysis to show that differential modification of the two known palmit(e)oylated residues of Wnt1, C93 and S224, has both overlapping and distinct consequences. Though the relative roles of each residue are similar with respect to stability and secretion, two distinct biological assays in L cells show that modification of C93 primarily modulates signaling via a ß-catenin independent pathway while S224 is crucial for ß-catenin dependent signaling. In addition, pharmacological inhibition of Porcupine (Porcn), an upstream regulator of Wnt, by IWP1, specifically inhibited ß-catenin dependent signaling. Consistent with these observations, mapping of amino acids in peptide domains containing C93 and S224 demonstrate that acylation of C93 is likely to be Porcn-independent while that of S224 is Porcn-dependent. Cumulatively, our data strongly suggest that C93 and S224 are modified by distinct enzymes and that the differential modification of these sites has the potential to influence Wnt signaling pathway choice.

Identification and characterization of subpopulations of Pax3 and Pax7 expressing cells in developing chick somites and limb buds

Pax3 and Pax7 are closely related paired‐boxed family transcription factors that are known to play important roles in embryonic and adult myogenesis. Previous reports describing the expression of Pax3 and Pax7 transcripts reveal expression in many overlapping domains. In this manuscript, we extend these studies by examining the protein expression profiles for Pax3 and Pax7 in developing chick somites and limbs with cellular resolution. Our studies show the existence of distinct subpopulations of cells in the somite and developing limb that are defined by the relative expression levels of Pax3 and Pax7. We also show that Pax3 and Pax7 negatively regulate each others expression in the dermomyotome, thus providing a possible mechanism for the maintenance of observed expression patterns in the dermomyotome. Further characterization of Pax3‐ and/or Pax7‐positive cells in the dermomyotome and myotome with respect to proliferation and differentiation reveals subpopulations of cells with distinct properties. Developmental Dynamics 237:1862–1874, 2008.

Identification of the WNT1 residues required for palmitoylation by Porcupine

The post‐translational palmitoylation of WNT morphogens is critical for proper signaling during embryogenesis and adult homeostasis. The addition of palmitoyl groups to WNT proteins is catalyzed by Porcupine (PORCN). However, the Wnt amino acid residues required for recognition and palmitoylation by PORCN have not been fully characterized. We show that WNT1 residues 214–234 are sufficient for PORCN‐dependent palmitoylation of Ser224. Substitution of Ser224 with Thr, but not Cys, is tolerated in palmitoylation and biological assays. Our data highlight the importance of palmitoylation for WNT1 activity and establish PORCN as an O‐acyl transferase for WNT1.

Beyond the Biology: A Systematic Investigation of Noncontent Instructor Talk in an Introductory Biology Course

We define a new construct termed Instructor Talk that may be a key mechanism for effectively implementing active learning and that may contribute to minimizing student resistance and stereotype threat, while building instructor immediacy. We used a mixed-methods approach and analyzed more than 600 instructor quotes. Presented here is a novel framework revealing the emergent categories and subcategories of the framework as well as examples of Instructor Talk.