A Novel Mutant Allele of Pw1/Peg3 Does Not Affect Maternal Behavior or Nursing Behavior.

Parental imprinting is a mammalian-specific form of epigenetic regulation in which one allele of a gene is silenced depending on its parental origin. Parentally imprinted genes have been shown to play a role in growth, metabolism, cancer, and behavior. Although the molecular mechanisms underlying parental imprinting have been largely elucidated, the selective advantage of silencing one allele remains unclear. The mutant phenotype of the imprinted gene, Pw1/Peg3, provides a key example to illustrate the hypothesis on a coadaptation between mother and offspring, in which Pw1/Peg3 is required for a set of essential maternal behaviors, such as nursing, nest building, and postnatal care. We have generated a novel Pw1/Peg3 mutant allele that targets the last exon for the PW1 protein that contains >90% of the coding sequence resulting in a loss of Pw1/Peg3 expression. In contrast to previous reports that have targeted upstream exons, we observe that maternal behavior and lactation are not disrupted upon loss of Pw1/Peg3. Both paternal and homozygous Pw1/Peg3 mutant females nurse and feed their pups properly and no differences are detected in either oxytocin neuron number or oxytocin plasma levels. In addition, suckling capacities are normal in mutant pups. Consistent with previous reports, we observe a reduction of postnatal growth. These results support a general role for Pw1/Peg3 in the regulation of body growth but not maternal care and lactation.

The apical/basal-polarity determinant Scribble cooperates with the PCP core factor Stbm/Vang and functions as one of its effectors.

Most tissues display several features of cellular polarization. Besides the ubiquitous epithelial polarization in the Apical-Basal (A/B) axis, many epithelia (and associated organs) display a Planar Cell Polarization (PCP). Recently, a crosstalk between the PCP and A/B polarity determinants has been suggested, i.e. the activity or stability of the PCP factor Frizzled is regulated by the A/B determinants aPKC and Bazooka in the Drosophila eye. We have systematically investigated genetic and physical interactions between the Drosophila A/B factors and the core PCP component Strabismus (Stbm)/Van Gogh (Vang). The A/B determinant Scribble was found to interact both genetically and physically with Stbm/Vang. We demonstrate that Scribble binds Stbm/Vang through its PDZ domain 3 and that it cooperates with Stbm/Vang in PCP establishment. Our data indicate that Scribble, in addition to its role in A/B polarity, has a distinct requirement in PCP establishment in the Drosophila eye and wing. We define a scribble allele that is largely PCP specific. Our data show that Scribble is part of the Stbm/Vang PCP complex and further suggest that it might act as an effector of Stbm/Vang during PCP establishment.

Scribble participates in Hippo signaling and is required for normal zebrafish pronephros development.

Spatial organization of cells and their appendages is controlled by the planar cell polarity pathway, a signaling cascade initiated by the protocadherin Fat in Drosophila. Vertebrates express 4 Fat molecules, Fat1-4. We found that depletion of Fat1 caused cyst formation in the zebrafish pronephros. Knockdown of the PDZ domain containing the adaptor protein Scribble intensified the cyst-promoting phenotype of Fat1 depletion, suggesting that Fat1 and Scribble act in overlapping signaling cascades during zebrafish pronephros development. Supporting the genetic interaction with Fat1, Scribble recognized the PDZ-binding site of Fat1. Depletion of Yes-associated protein 1 (YAP1), a transcriptional co-activator inhibited by Hippo signaling, ameliorated the cyst formation in Fat1-deficient zebrafish, whereas Scribble inhibited the YAP1-induced cyst formation. Thus, reduced Hippo signaling and subsequent YAP1 disinhibition seem to play a role in the development of pronephric cysts after depletion of Fat1 or Scribble. We hypothesize that Hippo signaling is required for normal pronephros development in zebrafish and that Scribble is a candidate link between Fat and the Hippo signaling cascade in vertebrates.

Abnormal mammary gland development in MMTV-CBLC transgenic mouse.

The CBL family of E3 ubiquitin ligases regulates cell signaling in a number of tissues by promoting degradation of tyrosine kinase receptors such as epidermal growth factor receptor. CBLC, the third member of the CBL family, is expressed in epithelial tissues, including the mammary gland. A transgenic mouse strain expressing a tetracyclin-inducible CBLC in the mammary gland was derived. It was found that CBLC transgene expression reduces the number and length of ducts during the development of the gland. In vivo results support the concept of CBLs as negative regulators of cell proliferation. Alternatively, the phenotype may be due to increased apoptosis. This mouse model may be used to further study regulatory components of the CBL pathway and may be crossed with mice susceptible to develop mammary tumors.

Interaction between two ubiquitin-protein isopeptide ligases of different classes, CBLC and AIP4/ITCH.

In metazoans, CBL proteins are RING finger type ubiquitin-protein isopeptide (E3) ligases involved in the down-regulation of epidermal growth factor tyrosine kinase receptors (EGFR). Among the three CBL proteins described in humans, CBLC (CBL3) remains poorly studied. By screening in parallel a human and a Caenorhabditis elegans library using the two-hybrid procedure in yeast, we found a novel interaction between Hsa-CBLC and Hsa-AIP4 or its C. elegans counterpart Cel-WWP1. Hsa-AIP4 and Cel-WWP1 are also ubiquitin E3 ligases. They contain a HECT (homologous to E6-AP C terminus) catalytic domain and four WW domains known to bind proline-rich regions. We confirmed the interaction between Hsa-CBLC and Hsa-AIP4 by a combination of glutathione S-transferase pull-down, co-immunoprecipitation, and colocalization experiments. We show that these two E3 ligases are involved in EGFR signaling because both become phosphorylated on tyrosine following epidermal growth factor stimulation. In addition, we observed that CBLC increases the ubiquitination of EGFR, and that coexpressing the WW domains of AIP4 exerts a dominant negative effect on EGFR ubiquitination. Finally, coexpressing CBLC and AIP4 induces a down-regulation of EGFR signaling. In conclusion, our data demonstrate that two E3 ligases of different classes can interact and cooperate to down-regulate EGFR signaling.