Fig. 3
Loss of Dyrk1a affects kidney development inXenopus laevis. (a'–e’) Embryos were unilaterally injected at the 8-cell stage with 10 ng of Dyrk1a morpholino (MO) or standard MO (Std MO) along with 50-pgβ-gal, wild-type,DYRK1AR205*, or DYRK1AL245R RNA. Stage 40 tadpoles were stained with kidney antibodies 3G8, which labels the proximal tubules, and 4A6, which labels the distal and connecting tubules. Letters without apostrophes (a–e) represent the injected side, whereas letters with apostrophes (a’–e’) represent the uninjected side. (b) Knockdown with a translation-blocking Dyrk1a MO disrupts kidney development, which can be partially rescued (c) by coinjecting with wild-type human DYRK1A RNA but not (d–e)DYRK1AR205* or DYRK1AL245R RNA. (a) Coinjection of a standard MO and β-gal serves as a negative control. Scale bars represent 100 µm. (f) The graph demonstrates a significant difference between embryos injected with either Dyrk1a MO + β-gal or Dyrk1a MO + DYRK1AR205* versus with Dyrk1a MO + DYRK1A suggesting successful rescue with human DYRK1A but not with the nonsense RNA. (g) The second graph demonstrates a significant difference between embryos injected with Dyrk1a MO + DYRK1AL245R versus with Dyrk1a MO + DYRK1A, which suggests that the missense RNA also fails to rescue. Significance was established against embryos that had a moderate or severe kidney phenotype (orange bar) and excluded embryos that had a weak phenotype (yellow bar). (f) Asterisk (*) indicates p < 0.001 comparing individual experimental groups with standard MO + β-gal. Pound sign (#) indicates p < 0.006 comparing Dyrk1a MO + DYRK1A with Dyrk1a MO + DYRK1AR205*. (g) Asterisk (*) indicates p < 0.001 comparing standard MO + β-gal with Dyrk1a MO + β-gal or Dyrk1a MO + DYRK1AL245R. Pound sign (#) indicates p < 0.05 comparing Dyrk1a MO + DYRK1A with Dyrk1a MO + DYRK1AL245R. For edema assays, embryos were injected at the 4-cell stage in both ventral cells to target both kidneys while avoiding the dorsal cells fated to become the heart and liver, which can also lead to edema. (h) Embryos injected with the standard MO did not develop edema while embryos injected (i) with the Dyrk1a MO did develop edema and also suffered from abnormal kidney formation. (j) The graph demonstrates a significant difference in edema and kidney abnormalities in embryos injected with either standard MO or Dyrk1a MO. Asterisk (*) indicates p < 0.008 comparing standard MO with Dyrk1a MO embryos with edema, defects in one, and defects in both kidneys. Error bars represent standard error. For ease of comparison of (n) and p values across conditions, please refer to Table S1–3.
