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. 2011 Feb;46(2-4):469–482. doi: 10.1016/j.mcn.2010.11.012

Fig. 3.

Fig. 3

PTPγ loss-of-function effects. Panels A–G demonstrate permanent loss of Lim1/2-expressing neurons. Embryos were electroporated at HH10–11 with either a GFP vector (A and B) or shRNA vectors (C–F) and developed to HH26–27 (E5). Brachial sections were immunostained for Isl1/2 (A, C, and E) and Lim1/2 (B, D, and F). Square brackets indicate the LMCl population of Lim1-expressing motor neurons (B). Treatment with either Si1 (E and F) or Si6 (C and D) results in relatively normal Isl1/2 expression, but reduced Lim1/2-expression in LMCl (arrows in D and F); “e” indicates electroporated side. Isl1/2- and Lim1/2-expressing cells were counted on both sides of each spinal cord and ratios generated (G). Means and standard deviations of these ratios are shown for embryos treated with control shRNA (dark bars), Si3 (grey bars for Isl1/2), or a combination of Si1, Si3 or Si6-treated embryos (grey bars, Lim1/2). The Lim1/2(MN) bars represent motor neuron counts in ventral horns only; Lim1/2(IN) bars represent dorsal interneuron counts (dorsal to white arrowheads in B and D). The numbers within columns show sample sizes. Asterisks indicate statistical significance (P < 0.01; Student T-test). Panels H–P demonstrate mitotic reduction and increased apoptosis after PTPγ shRNA treatment. Embryos electroporated with Si3 (I, J, L, and M) or negative control shRNA vector (H and K) (electroporated side marked e) were fixed at HH20 and HH22 and immunostained for phosphohistone-3 (H–J) or activated caspase-3 (K–M). Phosphohistone-positive cells were counted and graphed (N). Negative controls, black columns; PTPγ shRNA Si3, grey columns. Numbers in columns indicate sample size. Asterisks represent statistical significance (** P < 10− 5; * P < 0.001). O and P show counts of activated caspase-immunostained cells (HH20, O; HH22, P); each spot represents one embryo, with open circles representing Si3 electroporated embryos and closed circles representing control shRNA-treated embryos. Arrows in L and M indicate caspase-expressing cells. Scale bars = 100 μm (A–F), 50 μm (H–M).