Expression analysis of the lncRNA LncRNA-HIT.

(A)In situ hybridization using an antisense LncRNA-HIT riboprobe detects the transcript as early as E 10.5 in the distal limb which expands throughout the limb bud at E 11.5 (B). (C) In situ hybridization using a sense orientation LncRNA-HIT riboprobe detects no LncRNA-HIT transcripts, confirming the unidirectional transcription of LncRNA-HIT in the same orientation as the 5’ HoxA genes in the developing limb. (D and E) Analysis of Hoxa13 expression shows a high level of overlap with LncRNA-HIT in the distal limb. (F-H) Analysis of Hoxa9-Hoxa11 expression in the E 11.5 distal limb reveals some overlap with LncRNA-HIT in the limb bud. (I-K) Analysis of Hoxd11-Hoxd13 expression in the E 11.5 distal limb reveals some overlap with LncRNA-HIT. (L) LncRNA-HIT is also expressed in the developing genital tubercle, gut epithelium, urogenital sinus, spinal cord, and vertebral bodies at E 13.5. (M-N) LncRNA-HIT expression is detected in the digit perichondrial tissues, digit joint fields, and in the developing carpal/tarsal skeletal elements in E 13.5 forelimbs and hindlimbs. (O) Relative fold expression of LncRNA-HIT and Hottip expression in the E 11.5 limbs as determined by qRTPCR. Values represent the Gapdh normalized average expression of LncRNA-HIT and Hottip in the limb calculated from three independent analyses. Bars represent the standard deviation of the mean from the three independent assays. UGS = urogenital sinus, VB = vertebral body, GT = genital tubercle, GE = gut epithelium, SC = spinal cord.