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Figure 2. Downregulation of 40LoVe/Samba causes several head and neuronal defects.
(A) 40LoVe/Samba MO1 morphants display generalized head defects with the most prominent being a reduced eye size, eye size, dorsal pigmentation, craniocephalic shape and overall cranial volume. (A�) MO2-injected embryo (24 ng) displayed a milder phenotype compared to MO1-injected embryos. (A��) Co-injection of 80 pg of R40LoVe with 24 ng MO1 partially rescues the phenotype. (A���) Uninjected control embryo. Eye size was measured from embryos injected with MO1, MO2, control morpholino (CoMO), rescued (R2-R40Love co-injected with MO1 and R5-RhnRNPAB-GRD40LoVe co-injected with MO1 as shown in table 1) and control embryos (n = 80�120 for each category). (B) Graph shows that in MO1-injected embryos the eyes were 40% smaller than controls and it was successfully rescued by injection of R40LoVe mRNA (Rescue 2 - eye size 90% of controls). hnRNPAB-GRD40LoVe (Rescue 5) is also able to partially rescue the phenotype. (C) Optical sections from representative whole mount immunostained embryo for acetylated tubulin (red) to reveal neurons. The presence of using GFP (green) indicates the MO-injected side of the embryo (n = 20, three independent experiments). Compared to the uninjected side, the trigeminal nerve (tn), the ophthalmic nerve (on) and the nasocilliary nerves (nc) on the injected side of the embryo (marked with a white circle) are thinner and disorganized. The arrows show the properly formed neurons on the uninjected side of the tadpole. (D) Depth color coded (depth key at bottom of D) 3D reconstruction of optical sections of the trunk of a representative tadpole injected in the animal pole of one out two animal blastomeres with MO1 and then stained using an anti-acetylated tubulin antibody to reveal the axonal projections (n = 30, three independent experiments) 3D reconstruction is shown on the injected side (left) and was then rotated 180 degrees and shown on the uninjected side (right). Motor neuron projections rising from the spinal cord on the injected side of the embryo are absent or short in contrast to the un-injected side. (E) Whole mount in situ hybridization using N-tubulin (Ntub) and Sox10 show that the neural tissues are defined normally in MO1-injected embryos. However, tissues such as the cranial sensory ganglia and the branchial arches are misshapen and did not migrate normally (arrowheads) and expression levels Ntub and Sox 10 appear reduced in the injected side. (F) RT-PCR experiments confirm a reduction of neural marker expression in MO1 injected embryos. Actin was used as a loading control. |
