Identification of dlx4a as putative target of Kdm6b.1. (A) Control or Kdm6b.1 morpholino injected animals were amputated and allowed to regenerate for 48 h at which point they were fixed and stained with hematoxylin. The amputation planes are similar to that shown above. (B) Proliferating cells in control and Kdm6b.1 morphants. Control (left panel) or Kdm6b.1 MO (right panels) for 48 h, at which point they were stained with PCNA antibodies (PCNA+ cells are stained brown). n, notochord. The amputation planes are similar to that shown above. (C) dlx4a is downregulated in Kdm6b.1 MO animals. The expression levels of various candidate genes were tested in Kdm6 b.1 MO4 and control animals at 96 hpf (24 hpa) and normalized to Ribosomal Protein L18 expression. Data are expressed as average expression levels in the morphants relative to control animals. Error bars, the standard deviation and an asterisk (*) indicates a P value of 0.02 (n = 6 animals for each). (D) Jmjd3 associates with the dlx4 promoter in developing limb buds. Limb buds from approximately 20 pups were harvested at day e10.5 and processed for ChIP with control or Jmjd3 antibodies, followed by qPCR using primers at the transcription start sites of dlx4 or fgf2. Shown are the data from one experiment; error bars indicate the deviation from the mean duplicate ChIPs. Similar data were obtained in another experiment from different limb buds.