Evx1 and Evx2 are required for lmx1b expression. Lateral view of zebrafish spinal cord at 30 h (a-d), anterior, left dorsal top. in situ hybridization for lmx1ba (a, b) and lmx1bb (c, d). Brown coloration in (a-d) is pigment from melanocytes. e Mean number of cells (y-axis) that express lmx1ba and lmx1bb in WT (white) and evx1;evx2 double mutants (grey) (x-axis). Error bars indicate standard error of the mean. Statistically significant (p < 0.05) comparisons are indicated with square brackets and stars. One experiment was conducted for (a and b). Two independent experiments were conducted for (c and d) and cell count results were similar in each replicate. Data presented here (e) are average values for 5-12 embryos. Precise number of embryos counted and p values are provided in Table 8. f. Proposed mechanism for how the excitatory (glutamatergic) neurotransmitter phenotype of at least a subset of V0v neurons is specified and/or maintained. We previously demonstrated that Evx1 & Evx2 specifies the excitatory (glutamatergic) neurotransmitter phenotype and represses inhibitory (glycinergic) phenotypes in V0v cells [11]. The current study demonstrates that Evx1 & Evx2 are also required for lmx1ba and lmx1bb expression. Furthermore, we show that Lmx1bb is required at later developmental stages either to maintain the excitatory (glutamatergic) neurotransmitter phenotype for at least a subset of V0v neurons or to specify the glutamatergic phenotype of a later-forming subset of V0v cells. Scale bar = 50 µm
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