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. 2018 Feb 8;11:19. doi: 10.3389/fnmol.2018.00019

Figure 1.

Figure 1

Generation of a conditional Npr2-deficient mouse mutant and analysis of sensory axon bifurcation at the DREZ. (A) Targeting strategy for the generation of the floxed Npr2 allele and inactivation by Wnt1-Cre. (B) Southern blotting of the wild type, heterozygotes, and homozygous floxed allele using genomic DNA isolated from liver tissue. (C) PCR genotyping of wild type, heterozygotes and homozygous allele. Upper panel primer P1 and P2 flanking the loxP1 site, lower panel primer P3 and P4 flanking loxP2. (D) Western blotting of membrane fractions from E13.5 DRGs demonstrating the absence of Npr2 protein after recombination with Wnt1-Cre. The heavy chain of clathrin served as loading control. (E) Depiction of wild type, Npr2LacZ/lacZ, Npr2fl/fl or Npr2fl/fl;Wnt1Cre mice of mature stages (see also Figure S1). (F) Survival rate of Npr2fl/fl or Npr2fl/fl;Wnt1Cre mice at different ages. Numbers of animals inspected are given in parenthesis. (G–J) Naso-anal length and body weight of Npr2fl/fl or Npr2fl/fl;Wnt1Cre males and females at different ages. (K) Analysis of DRG axon bifurcation by DiI tracing in Npr2fl/fl or Npr2fl/fl;Wnt1Cre E12.5 embryos (see also Figure S2). Arrow heads mark bifurcations or turns of sensory axons in wild type or mutants, respectively. Bar, 25 μm. (L) Quantification of bifurcation errors demonstrated the complete absence of DRG axon bifurcation in Npr2fl/fl;Wnt1Cre E12.5 embryos. The numbers of counted axons and embryos are indicated below the columns. (M) Bifurcation errors persisted at mature stages (P15) as indicated by the reporter Thy1-YFP-H in the Npr2fl/fl;Wnt1Cre;Thy1YFP−H mouse. Arrow heads mark bifurcations or turns of sensory axons in wild type or mutants, respectively. Bar, 250 μm. (N) Analysis of grip strength revealed indistinguishable maximal muscle strength (mean ± SEM) of the forelimbs between mature Npr2fl/fl (n = 6 males and 6 females) and Npr2fl/fl;Wnt1Cre (n = 8 males and 7 females) of both sexes (paired t-test; p = 0.90 and 0.72 in males and females, respectively).