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. 2018 Jun 15;12:153. doi: 10.3389/fncel.2018.00153

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Copyright © 2018 Ter-Avetisyan, Dumoulin, Herrel, Schmidt, Strump, Afzal and Rathjen.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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In the absence of MTN axon bifurcation a decreased maximal bite force was measured in Npr2^flox/flox;Engr1^Cre in comparison to control mice. (A) Strong reduction of Npr2 was observed in sagittal sections of midbrain or rhombomere 1 in conditional Npr2^flox/flox;Engr1^Cre mutants at E12.5. Scale bar, 20 μm. (B) Body weight of 15 weeks old Npr2^flox/flox control and Npr2^flox/flox;Engr1^Cre mutant mice (scatter blots). Body mass differed between males and females [F_{(1, 22)} = 24.84; P < 0.001]. However, body mass did not differ between the control and experimental groups [F_{(1, 22)} = 2.55; P = 0.13]. The interaction effect was also not significant [F_{(1, 22)} = 0.60; P = 0.45]. (C) A bifurcating MTN axon in rhombomere 2 in the Npr2^flox/flox line (control). Arrowheads point to a bifurcating MTN afferent. (D,E) MTN afferents do not bifurcate in the hindbrain in rhombomere 2 in conditional Npr2 mutants (Npr2^flox/flox;Engr1^Cre or Npr2^flox/flox;Wnt1^Cre). The broken lines indicate the margin of the hindbrain and arrowheads point to MTN afferents. In homozygous mutants (Npr2^flox/flox;Engr1^Cre and Npr2^flox/flox;Wnt1^Cre) 41 non-bifurcating and 1 bifurcating MTN afferent were observed in 8 embryos (see also Table 1). Scale bar for (C–E), 50 μm. (F–I) DiI tracings of trigeminal afferents in Npr2^flox/flox;Engr1^Cre or Npr2^flox/flox;Wnt1^Cre show normal bifurcation in the hindbrain at E12.5. In 7 control embryos (Npr2^flox/flox) 29 bifurcating trigeminal afferents and in 7 mutant embryos (Npr2^flox/flox;Engr1^Cre and Npr2^flox/flox;Wnt1^Cre) 37 bifurcating and 3 non-bifurcating trigeminal afferents were observed (see also Table 2). Scale bar, 50 μm. (J) Schematic drawing of the bite force measurement instrument. (K) Scatter blots of the maximal bite force in Newton (N) of Npr2^flox/flox (control) and Npr2^flox/flox;Engr1^Cre mutant mice (15 weeks old). The maximal bite force of males was greater than in females [F_{(1, 22)} = 6.61; P = 0.017]. Bite force also differed between mutants and control groups [F_{(1, 22)} = 4.65; P = 0.042] with bite force being reduced in conditional Npr2 mutants. Interaction effects were not significant, however [F_{(1, 22)} = 0.02; P = 0.88]. Npr2^flox/flox, n = 6 male and n = 6 female; Npr2^flox/flox;Engr1^Cre, n = 8 male and n = 6 female.