The Wnt Co-Receptor PTK7/Otk and Its Homolog Otk-2 in Neurogenesis and Patterning

Abstract

Wnt signaling is a highly conserved metazoan pathway that plays a crucial role in cell fate determination and morphogenesis during development. Wnt ligands can induce disparate cellular responses. The exact mechanism behind these different outcomes is not fully understood but may be due to interactions with different receptors on the cell membrane. PTK7/Otk is a transmembrane receptor that is implicated in various developmental and physiological processes including cell polarity, cell migration, and invasion. Here, we examine two roles of Otk-1 and Otk-2 in patterning and neurogenesis. We find that Otk-1 is a positive regulator of signaling and Otk-2 functions as its inhibitor. We propose that PTK7/Otk functions in signaling, cell migration, and polarity contributing to the diversity of cellular responses seen in Wnt-mediated processes.

Keywords: Drosophila; PTK7; Wnt.

Figure 1

Otk and Wnt-4 in embryonic patterning. (A) Loss of segment polarity patterning in a wg^IG22 embryo or denticle lawn phenotype. (B) Loss of segment polarity or naked phenotype due to wg overexpression, as compared to wild-type segment polarity patterning (C). (D) otk-1^RNAi results in denticle pattern disruption but not a clear segment polarity phenotype. (E) otk-2^RNAi showed a disruption of segment polarity, with normally naked cuticles covered in denticles. (F) Co-expression of both otk-1^RNAi and otk-2^RNAi led to a more severe denticle lawn phenotype. The combination of otk-1^RNAi and wnt-4 overexpression (G) led to a slight increase in denticles over otk-1^RNAi alone. The combination of otk-1^RNAi and wnt-4^RNAi showed a loss of cuticle (H), making the observation of patterning phenotypes difficult, but the combination of otk-2^RNAi and wnt-4^RNAi showed a near denticle lawn phenotype (I). For RNAi experiments, we examined embryos n > 100 per experiment.

Figure 2

Effect of Otk on Engrailed expression. (A) Wild-type embryos show a striped pattern of En protein expression usually in two to three cell nuclei per stripe. (B) Overexpression of wnt-4 showed a small effect on stripe width. (C) wnt-4^RNAi did not appear to affect En protein. (D) otk-1^RNAi showed a very mild effect on stripe width. (E) otk-2^RNAi showed lower levels of En expression and loss of En-positive cells. (F) Co-expression of both otk-1^RNAi and otk-2^RNAi led to a complete disruption in the En expression pattern. Overexpression of Otk-1 showed a mild to no effect on En expression (G). The combination of otk-1^RNAi and wnt-4^RNAi showed some extopic En-positive cells (H). The combination of otk-1^RNAi and wnt-4 overexpression led to a slight decrease in En levels (I). For each condition, we examined at least three individual embryos of a similar stage.

Figure 3

Gene expression changes in Drosophila embryos downstream of Wnt-4 and Otk-1. (A) Heatmap of gene expression in embryos expressing various levels of Wnt-4, Otk-1, and Otk-2. Clusters I and II are Wnt-4 dependent, while Cluster III is Wnt-4 independent. (B) Gene Ontology (GO) enrichment analysis highlighting key signaling and metabolic pathways associated with each cluster (FDR < 10%). Downregulation of Wnt-4 results in reduced EGF, and VEGF signaling (Cluster I) and increased canonical Wnt and Hedgehog signaling (Cluster II). Downregulation of Wnt-4 and Otk-1 results in decreased tight junction assembly (Cluster III). (C) KEGG pathway enrichment analysis highlighting key signaling and metabolic pathways associated with each cluster (FDR < 10%). Downregulation of Wnt-4 results in reduced MAPK, Hippo signaling (Cluster I), and increased Notch signaling (Cluster II).

Figure 4

Otk-1 TrojanGal4 in Drosophila embryos. (A,A’) Still images from live imaging showing expression of Otk-1 in ventral, epithelial stripes during mid stages of embryogenesis. (B,B’) Still images from Lightsheet imaging, showing later stage expression of Otk-1 in neural tissue. Images and videos are representative of three independent experiments.

Figure 5

Still images from Otk-1, with two fluorescent proteins inserted into the locus, lightsheet imaging in Drosophila embryos. A rapidly folding fluorescent protein (super folder GFP) and a slow folding fluorescent protein (RFP) were inserted directly into the otk-1 locus. (A,A’) RFP signal was only observable in the stomatogastric cells in mid embryonic stages along with a strong sfGFP signal (B,B’) which overlapped (C,C’). In late embryonic stages both sfGFP and RFP were observed in stomatogastric cells (D–F,D’–F’). (G) Quantification of otk1Timer intensity in the ring gland for early and late-stage embryos. Median intensity was rendered via IMARIS. Statistical p-value calculated using unpaired t-test in R. Images and videos are representative of three independent experiments.

Figure 6

Overexpression of Otk-1 in embryos. (A) Still image from confocal microscopy live imaging of HA-tagged otk-1 imaged using αHA frankenbody fused to GFP shows an irregular pattern of overexpression at the membrane of epithelial cells. (B) Membrane localization and irregular levels were confirmed by fixed, αHA antibody staining. (C) Still image from lightsheet movie showing embryonic neural development using neuronally driven GFP. (D) Addition of RNAi for both Otks causes loss of GFP signal and disorganized neurogenesis. Images and videos are representative of three independent experiments.

Figure 7

Model of Otk Signaling. Canonical Wnt signaling determines the naked cell fate. Otk-2 represses Otk-1 function to prevent denticle cell fate activation. This repression can be overcome by the presence of the Wnt-4 ligand.