Fig 2: Temporal delay of Hox protein expression in postmitotic motor neurons.(a) Schematic illustration of protocol to generate spinal motor neurons from embryonic stem cells (ESCs). RA: retinoic acid. SAG: smoothened agonist. NE: neural epithelium. pMN: motor neuron progenitors. MNs: motor neurons. (b) Rapid induction of Hoxa1–Hoxa5 gene expression upon RA/SAG induction. Heat map represents the intensity of gene expression along the Hoxa cluster at five time-points during motor neuron differentiation by Quantile analysis. (c) Quantitative PCR (qPCR) analysis of Hoxa5 during MN differentiation. mRNA levels were normalized against Day4 pMN cells (mean±s.d., n=3 independent experiments). FC-fold change. (d) Immunodetection of Olig2, Hb9 and Hoxa5 in EBs under RA/SAG conditions. Scale bar represents 50 μm. (e,f) Comparisons of Olig2, Hb9 and Hoxa5 mRNA and protein expression by qPCR and immunostaining. mRNA/protein levels were normalized against Day 3 EBs (mean±s.d., n=3 independent experiments). Only Hoxa5 exhibits a significant delay of ∼72 h between gene and protein expression (f). FC-fold change, RO-ratio.

Fig 3: The down-regulation of HOXA5 promotes HCC tumorigenicity and angiogenesis in vivo. (A) HOXA5 down-regulation promoted the growth of Huh7 xenograft tumors. HOXA5 down-regulated and control Huh7 cells were subcutaneously injected into nude mice (n = 8). The nude mice were sacrificed on day-28 after inoculation and the tumor were harvested. (B) The tumor weight on day-28 after inoculation are presented. (C) The tumor growth curve was measured every week after inoculation. The mean tumor volume was significantly increased in the HOXA5-downregulated group. (D) HOXA5 down-regulation elevated the expression of angiogenesis markers. Immunohistochemical expression of angiogenesis markers CD31, CD34 and VEGF were detected on serial sections of tumor samples. Scale bar, 100 µm (100x), 25 µm (400x). (E) The staining intensity of CD31, CD34, and VEGF from tumor samples were shown.Data are presented as their mean ± SD. Statistical analysis was performed with Student's t-test. *p < 0.05; ***p < 0.001.

Fig 4: Strong heterogeneous expression levels of Hoxa5 mRNAs in the pMNs.(a) Schematic illustration of single molecule RNA FISH (smRNA FISH) of Hoxa5 transcripts in vitro and in vivo. (b,c) smRNA FISH of Hoxa5 transcripts on Hb9::GFP ESC-derived motor neurons (b) or on sagittal sections (5–10 μm) of mouse spinal cord of E9.5 Hb9::GFP mouse embryos (c). Scr: scrambled sequence control probe. Scale bar represents 20 μm. (d,e) smRNA FISH signal quantifications show strong cellular variance of Hoxa5 mRNA number in the pMNs (Olig2on, GFPoff) compared to robust and steady Hoxa5 levels in postmitotic MNs (GFPon). N-cadherin (NCAD) is used to demarcate cell boundaries and nuclei are counterstained with DAPI. CV: coefficient of variation.

Fig 5: mir-27 controls robustness of the Hoxa5-Hoc8 boundary.(a) Generation of inducible ESC lines expressing eight repetitive mir-27b sponge sequences inserted into the GFP 3′UTR. ESCs were differentiated under RA/SAG conditions with Dox treatment on Day 2 of differentiation. A scrambled sequence was inserted as a control. Scale bar represents 50 μm. (b,c) Expression of Hoxa5 and Olig2 in EBs from control (iScrmSP) and mir-27b sponge (iMir-27SP) cells. Induction of mir-27b sponge on Day 2 of differentiation under RA/SAG conditions led to precocious Hoxa5 expression, while control cells had no detectable Hoxa5. In contrast, induction of mir-27bSP had no discernible effect on Olig2 expression (n=3 independent experiments, mean±s.d., P<0.01). (d) Schematic illustration of the stages in performing in ovo electroporation in the embryonic spinal cord. (e,f) Immunostaining of brachial spinal cord sections demonstrates noisy co-expressed Hoxa5/Hoxc8 at electroporated side of mir-27SPelect spinal cords, whereas mutually exclusive Hoxa5/Hoxc8 expression is present in the control mir-ScrmSPelect spinal cords. Generic Isl1(2)on MNs are unaffected. Scale bar represents 50 μm. (g) Quantification of percentage of co-expressed Hoxa5onHoxc8on cells against Hoxa5on cells at brachial spinal cord (number of positive cells per 20 μm ventral spinal cord section) in mir-ScrmSPelect and mir-27SPelect chicken embryos (mean±s.d., n=3 embryos, *P<0.01).