Frizzled 3 acts upstream of Alcam during embryonic eye development.

Formation of a functional eye during vertebrate embryogenesis requires different processes such as cell differentiation, cell migration, cell-cell interactions as well as intracellular signalling processes. It was previously shown that the non-canonical Wnt receptor Frizzled 3 (Fzd3) is required for proper eye formation, however, the underlying mechanism is poorly understood. Here we demonstrate that loss of Fzd3 induces severe malformations of the developing eye and that this defect is phenocopied by loss of the activated leukocyte cell adhesion molecule (Alcam). Promoter analysis revealed the presence of a Fzd3 responsive element within the alcam promoter, which is responsible for alcam expression during anterior neural development. In-depth analysis identified the jun N-terminal protein kinase 1 (JNK1) and the transcription factor paired box 2 (Pax2) to be important for the activation of alcam expression. Altogether our study reveals that alcam is activated through non-canonical Wnt signalling during embryonic eye development in Xenopus laevis and shows that this pathway plays a similar role in different tissues.

A phospho-dependent mechanism involving NCoR and KMT2D controls a permissive chromatin state at Notch target genes.

The transcriptional shift from repression to activation of target genes is crucial for the fidelity of Notch responses through incompletely understood mechanisms that likely involve chromatin-based control. To activate silenced genes, repressive chromatin marks are removed and active marks must be acquired. Histone H3 lysine-4 (H3K4) demethylases are key chromatin modifiers that establish the repressive chromatin state at Notch target genes. However, the counteracting histone methyltransferase required for the active chromatin state remained elusive. Here, we show that the RBP-J interacting factor SHARP is not only able to interact with the NCoR corepressor complex, but also with the H3K4 methyltransferase KMT2D coactivator complex. KMT2D and NCoR compete for the C-terminal SPOC-domain of SHARP. We reveal that the SPOC-domain exclusively binds to phosphorylated NCoR. The balance between NCoR and KMT2D binding is shifted upon mutating the phosphorylation sites of NCoR or upon inhibition of the NCoR kinase CK2ß. Furthermore, we show that the homologs of SHARP and KMT2D in Drosophila also physically interact and control Notch-mediated functions in vivo Together, our findings reveal how signaling can fine-tune a committed chromatin state by phosphorylation of a pivotal chromatin-modifier.

Site-specific methylation of Notch1 controls the amplitude and duration of the Notch1 response.

Physiologically, Notch signal transduction plays a pivotal role in differentiation; pathologically, Notch signaling contributes to the development of cancer. Transcriptional activation of Notch target genes involves cleavage of the Notch receptor in response to ligand binding, production of the Notch intracellular domain (NICD), and NICD migration into the nucleus and assembly of a coactivator complex. Posttranslational modifications of the NICD are important for its transcriptional activity and protein turnover. Deregulation of Notch signaling and stabilizing mutations of Notch1 have been linked to leukemia development. We found that the methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1; also known as PRMT4) methylated NICD at five conserved arginine residues within the C-terminal transactivation domain. CARM1 physically and functionally interacted with the NICD-coactivator complex and was found at gene enhancers in a Notch-dependent manner. Although a methylation-defective NICD mutant was biochemically more stable, this mutant was biologically less active as measured with Notch assays in embryos of Xenopus laevis and Danio rerio. Mathematical modeling indicated that full but short and transient Notch signaling required methylation of NICD.

The Wnt/JNK signaling target gene alcam is required for embryonic kidney development.

Proper development of nephrons is essential for kidney function. ß-Catenin-independent Wnt signaling through Fzd8, Inversin, Daam1, RhoA and Myosin is required for nephric tubule morphogenesis. Here, we provide a novel mechanism through which non-canonical Wnt signaling contributes to tubular development. Using Xenopus laevis as a model system, we found that the cell-adhesion molecule Alcam is required for proper nephrogenesis and functions downstream of Fzd3 during embryonic kidney development. We found alcam expression to be independent of Fzd8 or Inversin, but to be transcriptionally regulated by the ß-Catenin-independent Wnt/JNK pathway involving ATF2 and Pax2 in a direct manner. These novel findings indicate that several branches of Wnt signaling are independently required for proximal tubule development. Moreover, our data indicate that regulation of morphogenesis by non-canonical Wnt ligands also involves direct transcriptional responses in addition to the effects on a post-translational level.

Comparative analysis reveals distinct and overlapping functions of Mef2c and Mef2d during cardiogenesis in Xenopus laevis.

The family of vertebrate Mef2 transcription factors is comprised of four members named Mef2a, Mef2b, Mef2c, and Mef2d. These transcription factors are regulators of the myogenic programs with crucial roles in development of skeletal, cardiac and smooth muscle cells. Mef2a and Mef2c are essential for cardiac development in mice. In Xenopus, mef2c and mef2d but not mef2a were recently shown to be expressed during cardiogenesis. We here investigated the function of Mef2c and Mef2d during Xenopus laevis cardiogenesis. Knocking down either gene by corresponding antisense morpholino oligonucleotides led to profound heart defects including morphological abnormalities, pericardial edema, and brachycardia. Marker gene expression analyses and rescue experiments revealed that (i) both genes are required for proper cardiac gene expression, (ii) Mef2d can compensate for the loss of Mef2c but not vice versa, and (iii) the γ domain of Mef2c is required for early cardiac development. Taken together, our data provide novel insights into the function of Mef2 during cardiogenesis, highlight evolutionary differences between species and might have an impact on attempts of direct reprogramming.

Wnt/ß-catenin signaling defines organizing centers that orchestrate growth and differentiation of the regenerating zebrafish caudal fin.

Zebrafish regenerate their fins via the formation of a population of progenitor cells, the blastema. Wnt/ß-catenin signaling is essential for blastemal cell proliferation and patterning of the overlying epidermis. Yet, we find that ß-catenin signaling is neither active in the epidermis nor the majority of the proliferative blastemal cells. Rather, tissue-specific pathway interference indicates that Wnt signaling in the nonproliferative distal blastema is required for cell proliferation in the proximal blastema, and signaling in cells lining the osteoblasts directs osteoblast differentiation. Thus, Wnt signaling regulates epidermal patterning, blastemal cell proliferation, and osteoblast maturation indirectly via secondary signals. Gene expression profiling, chromatin immunoprecipitation, and functional rescue experiments suggest that Wnt/ß-catenin signaling acts through Fgf and Bmp signaling to control epidermal patterning, whereas retinoic acid and Hedgehog signals mediate its effects on blastemal cell proliferation. We propose that Wnt signaling orchestrates fin regeneration by defining organizing centers that instruct cellular behaviors of adjacent tissues.

sox4 and sox11 function during Xenopus laevis eye development.

SoxC genes are involved in many developmental processes such as cardiac, lymphoid, and bone development. The SoxC gene family is represented by Sox4, Sox11, and Sox12. Loss of either Sox4 or Sox11 function is lethal during mouse embryogenesis. Here, we demonstrate that sox4 and sox11 are strongly expressed in the developing eye, heart as well as brain in Xenopus laevis. Morpholino oligonucleotide mediated knock-down approaches in anterior neural tissue revealed that interference with either Sox4 or Sox11 function affects eye development. A detailed analysis demonstrated strong effects on eye size and retinal lamination. Neural induction was unaffected upon Sox4 or Sox11 MO injection and early eye field differentiation and cell proliferation were only mildly affected. Depletion of both genes, however, led independently to a significant increase in cell apoptosis in the eye. In summary, Sox4 and Sox11 are required for Xenopus visual system development.