Prdm14 promotes proliferation and the expansion of neural progenitors 44

Prdm14 overexpression at neural plate stage inhibited the expression of the postmitotic neuron marker tubb2b (Fig. 3.5B, F) and at tailbud stages resulted in an enlarged neural tube and ectopic neuronal differentiation (Fig.

3.5H’). To analyze if these effects were caused by the expansion of progenitor cells, the effect of prdm14-GR overexpression on the neural plate marker sox2 was evaluated, as it is a definitive marker of neural progenitors (Graham et al., 2003; Ellis et al., 2004) (Fig. 3.6A). Microinjection of prdm14-GR results in the expansion of the sox2 expression domain on the injected side of the embryo (n= 20; 75% increased) (Fig. 3.6B).

To determine if the expansion of sox2 expression by prdm14-GR is the result of increased proliferation, the number of mitotically active cells in the dorsal ectoderm was quantified by staining for phosphorylated histone 3 (pH3) (Dent et al., 1989) (Fig. 3.6C). To quantify the number of mitotically active cells, three embryos were sectioned and the pH3 positive cells were counted in the dorsal halves of 15 consecutive sections. The number of mitotically active cells

in the neuroectoderm was increased almost two-fold on the prdm14-GR injected side compared to the uninjected side (Fig. 3.6C’-D). These results suggest that prdm14 promotes the proliferation of progenitor cells, resulting in the inhibition of neuronal differentiation at neural plate stage and in an enlarged neural tube in later stages.

Fig. 3.6 Prdm14-GR overexpression promotes proliferation. (A) Prdm14-GR (500 pg) mRNA together with β-Gal (75 pg) mRNA (light blue staining) were injected into one blastomere of a two-cell stage embryo. Embryos were treated from four-cell stage on with dex. (B-C) Gene expression was analyzed at stage 14 by whole mount in situ hybridization using markers indicated on the bottom left side. The injected side is on the right, dorsal view, anterior up. (B) The white brackets indicate width of expression domain. (C’) Transverse section at indicated levels of embryo shown in C. (D) The graph shows the statistical evaluation of 15 consecutive sections of three embryos based on the number of pH3 positive cells in the dorsal half on the injected and uninjected site as indicated in the cross-section C’. Error bars represent the standard error of the mean of two independent experiments (+/-SEM).

3.4 Prdm14 promotes ectopic sensory neuron formation

Prdm14-GR induces ectopic neurons in the non-neural ectoderm of tailbud stage embryos (Fig. 3.5G-H’) raising the question as to which neuronal subtype they belong to. As the dorsal neural tube, which arises from the neural plate border (NPB), was strongly expanded, the expression of tlx3 was analyzed, which is expressed in the NPB and the dorsally located glutamatergic Rohon-Beard sensory neurons (Patterson and Krieg, 1999; Rossi et al., 2009).

Whole mount in situ hybridizations of embryos overexpressing prdm14-GR showed ectopic expression of tlx3 in the non-neural ectoderm (n= 22; 76%

ectopic staining) (Fig. 3.7C, red arrows) and an increased expression in the dorsal neural tube (Fig. 3.7C’, red arrows). The glutamatergic neurotransmitter identity of these neurons was further supported by the ectopic expression of the vesicular glutamate transporter 1 slc17a7 (vglut1) (n= 25; 80% ectopic staining) (Fig 3.7D-E’). To determine if other ectopic neuronal subtypes are induced by Prdm14-GR, the expression of mnx1 (also known as hb9) was analyzed, which is expressed in cholinergic motor neurons in the most ventral aspect of the neural tube (Saha et al., 1997). Ectopic mnx1 expression was never observed in the non-neural ectoderm. Nonetheless, an expansion of the endogenous expression domain was observed, which may be the result of an overall enlargement of the neural tube caused by prdm14-GR overexpression (Fig.

3.7F-G’).

Fig. 3.7 Prdm14-GR overexpression promotes sensory neurons in tailbud stage embryos.

(A) Prdm14-GR (500 pg) mRNA together with β-Gal (75 pg) mRNA (light blue staining) were injected into one blastomere of a two-cell stage embryo. Embryos were treated from four-cell stage on with dex. (B-G’) Gene expression was analyzed at stage 27 by whole mount in situ hybridization using markers indicated on the left. The injected side is on the right, lateral view.

is, injected side (C’, E’, G’) Transverse sections at indicated levels of embryo shown in C, E, G respectively. Black brackets indicate width of mesenchymal tissue at the level of the notochord.

Dashed line indicates midline of the neural tube. nt, neural tube; nc, notochord

To determine if prdm14 overexpression is sufficient to induce glutamatergic sensory neurons in pluripotent cells, the animal cap assay was used. Control and prdm14-GR mRNA-injected embryos were treated with dex at stage 3 and animal caps were isolated from blastula stage embryos (Fig. 3.8A).

At the equivalent of neural plate stage (stage 14) and tailbud stage (stage 27) total RNA was isolated and analyzed by RT-PCR. Similar to the phenotype in embryos, at the equivalent of tailbud stages, tubb2b as well as tlx3 were induced. These markers were not observed in prdm14-GR mRNA-injected animal caps not treated with dex, or control uninjected caps treated with dex. To elucidate if prdm14 directly promotes neural differentiation or requires the activity of proneural genes, the expression of neurog2 was analyzed. At the equivalent of stage 14, neurog2 is induced in caps expressing Prdm14-GR. The

absence of tubb2b at the equivalent of neural plate stage is similar to the transient inhibition of neurogenesis observed in the embryos. The regulation of neurog2 by Prdm14-GR is unexpected as neurog2 strongly promotes prdm14 (Fig. 3.4B), suggesting a positive feedback loop between these two factors.

Hence, active Prdm14-GR is able to promote neuronal differentiation in whole embryos as well as animal caps.

Fig. 3.8 Prdm14-GR overexpression activates sensory neuron marker tlx3 in animal caps.

(A-B) Prdm14-GR (500 pg) mRNA was injected into both blastomeres of a two-cell stage embryo. Control and prdm14-GR mRNA-injected embryos were treated with dex from four-cell stage on and animal caps were isolated from blastula stage embryos. At the equivalent of neural plate stage (stage 14) and tailbud stage (stage 27) total RNA was isolated and analyzed by RT-PCR using markers indicated on the right. Expression levels were shown by odc expression.