Figure 6. The adrenergic receptor Adrb2 is expressed in cells ventral to the central canal in which norepinephrine can be detected.

(A) Double immunodetection of GFP (left) and endogenous norepinephrine (middle) in the spinal cord of scospondin^ut24Tg embryos at 30 hpf imaged laterally. Merged signals and close up of boxed regions are represented on the right. 5 representative examples (#1 to #5) out of 26 embryos are shown (no signal was detected in 6 embryos out of 26 in total, n = 2 independent experiments). In the central canal, norepinephrine positive signals can be detected as colocalized with the Reissner fiber itself (black arrowheads; embryos #1, 2, 3), with extrafibrillar material in the central canal (black arrowheads; embryos #4 and #5), and closely apposed to the massa caudalis located after the caudal limit of the central canal (black arrowhead; embryo #6). Scale bars: 10 µm. r: rostral; c: caudal; d: dorsal; v: ventral; cc: central canal; fp: floor plate. (B) Immunohistochemistry for the adrenergic receptor Adrb2 in a 30 hpf control sibling (left, representative example out of 8 embryos) and a scospondin^icm15/icm15 embryo (right, representative example out of 9 embryos). Adrb2 is distributed along the midline of the ventral most region of the neural tube (corresponding to the floor plate, fp) at the interface with the central canal (cc). Embryos are oriented rostral to the left and dorsal to the top. Scale bars: 10 µm. (C) Sagittal views of double immunostainings against GFP and Adrb2 in the spinal cord of Tg(pkd2l1:GCaMP5G) embryos at 30 hpf. Maximal z-projections are shown for a control sibling (left; one embryo out of 12) and a scospondin^icm15/icm15 mutant (right; one embryo out of 8). Merged signals show that GFP-positive and Adrb2 positive signals cover different domains in the ventral most region of the neural tube. 3D reconstructions of the same field of views (bottom panel: GFP: green; Adrb2: purple) further illustrate that Adrb2 is enriched in a distinct cell population that is medial to ventral CSF-contacting neurons. Scale bars: 10 µm. L: left; R: right; See also Figure 6—video 1.

Figure 6—video 1. Adrb2 is expressed in the midline of the ventral most region of the neural tube that is different from that of CSF-contacting neurons.

Download video file ^{(1.9MB, mp4)}

Z-stack of a Tg(pkd2l1:GCaMP5G) control sibling (top) and a Tg(pkd2l1:GCaMP5G); scospondin^icm15/icm15 mutant (bottom) at 30 hpf immunostained against GFP (green) and Adrb2 (magenta). In both cases, Adrb2-positive domains are inserted between two rows of GFP-positive ventral CSF-contacting neurons (both domains are not colocalized). The video represents a 20.5 µm deep slice (0.5 µm z-step) for both embryos. Scale bar: 10 µm.