In Vivo and In Situ Detection of the Draxin/Ntn1a Interaction in Zebrafish Embryos

(A) Schematic illustration of the assay design. mRNAs encoding the indicated fluorophore tagged open reading frames (ORFs) were injected into one-cell stage zebrafish embryos and imaged at blastula stage (4 hr post fertilization). The imaging plane corresponds to a region approximately 15 µm beneath the enveloping layer of the embryos.

(B) Single optical section confocal images of the sphere stage embryos. (Ba) Embryos injected with 100 pg Draxin-sfGFP mRNA displayed uniform distribution of Draxin protein in the extracellular space. (Bb) Injection of 100 pg Ntn1a-sfGFP mRNA resulted in dense membrane associated speckles positive for Ntn1a-sfGFP protein. In (Ba2) and (Bb2), memRFP was used to label the cell surface. Upon co-injection of 200 pg Draxin-sfGFP mRNA and 200 pg of Ntn1a-mCherry mRNA (Bc), Draxin-sfGFP, and Ntn1a-mCherry proteins co-localized into membrane associated spots (arrowheads). Scale bars, 10 µm; n = 7.

(C) Schematic representation of in situ detection of the Draxin/Ntn1a interaction in zebrafish using an affinity probe. A Draxin_aa209–284-hFc fusion protein was generated in HEK293-6E cells as a probe to detect endogenous Netrins (Draxin_aa209–284 fragment is indicated in blue rectangle; human IgG Fc fragment is indicated in black ellipsoids). Mildly fixed 24- to 48-hpf embryos were incubated with the affinity probe (Draxin_aa209–284-hFc).

(D) Representative microscope images showing lateral views of whole-mount zebrafish embryos stained with Draxin_aa209–284-Fc fusion protein probe and VasnA antibody in floor-plate region (anterior left, 33 hpf). Anti-VasnA antibody shows outline of the membrane of floor-plate cells. The signal from the Draxin_aa209–284-hFc fusion probe is detectable in the floor-plate region in wild-type fish (Db, arrowheads) but strongly reduced or even absent in same-stage netrin1b knockdown embryos (Dc). forebrain; MB, midbrain; HB, hindbrain; FP, NC, notochord. A star labels the most anterior region of the notochord, which was use as the reference location. The dashed box in Da indicates the regions is scaled up in Db and Dc. Single plane confocal images were taken using 10× (Da) and 25× objective lens of Zeiss LSM 780 NLO confocal (Db and Dc), Scale bar, 100 µm in Da, 20 µm in Db and Dc, n > 10.

Co-expression of draxin and netrin mRNA in Proximity of Main Axonal Tracts in 24hpf Zebrafish Embryos

Whole mount double in situ hybridization of draxin mRNA (red) and netrin1b (green) in wild-type 24hpf zebrafish embryos in combination with anti acetylated tubulin antibody (Acte-Tubulin, white), dorsal view (a-a′′, anterior left) and lateral view (b-b′′, anterior left and dorsal up). Arrow heads in figure a show co-expression of draxin and netrin1b mRNA in the region of postopic commissure (POC) in dorsal view. In figure b, arrow heads show the coexpression in the region of supraoptic tract (SOT) in lateral view. tel: telencephalon; di: diencephalon; AC: Anterior Commissure, POC: Postoptic Commissure, TPOC: Tract of the Postoptic Commissure, SOT: Supraoptic Tract. Single plane confocal images were taken using 25X objective lens (Zeiss LSM 780 NLO confocal); scale bar: 20µm.