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Abstract
Adrenaline (also known as Epinephrine) is a hormone, which works as major regulator of various biological events such stages of vertebrate, the role of adrenaline for early embryogenesis has been as heart rate, blood vessel and air passage diameters, and metabolic shifts. Although its specific receptors are expressing at the early developmental stage those functions are poorly understood. Here, we show that loss-of-functional effects of adrenergic receptor β-2 (Adrβ2), which was known as the major receptor for adrenaline and highly expressed in embryonic stages, led posterior defects at the tadpole stage of Xenopus embryos, while embryos injected with Adrβ2 mRNA or treated with adrenaline hormone adversely lost anterior structures. This posteriorization effect by adrenaline hormone was dose-dependently increased but effectively rescued by microinjection of antisense morpholino oligomer for Adrβ2 (Adrβ2-MO). Combination of adrenaline treatments and microinjection of Adrβ2 mRNA maximized efficiency in its posteriorizing activity. Interestingly, both gain- and loss-of-functional treatment for β-adrenergic signaling could not influence anterior neural fate induced by overexpression of Chordin mRNA in presumptive ectodermal region, meaning that it worked via mesoderm. Taken together with these results, we conclude that adrenaline is a novel regulator of anteroposterior axis formation in vertebrates.
Fig. 1. Expressions of Adrb1, Adrb2, and PNMT mRNA during the Xenopus embryonic period. In all stages expression of Adrb2 were detected, while maternal or zygotical expressions were observed in Adrb1 and PNMT mRNAs, respectively. ODC was used for loading control.
Figure 2. Loss-of-functional effects by antisense morpholino oligomer for Adrβ2 (Adrβ2-MO) for Xenopus embryogenesis. (A) Experimental design using Adrβ2-MO. Adrβ2-MO was designed to target the translation initiation site (blue bar). This MO does theoretically not bind to artificially created mRNA containing five mismatched nucleotides (5mis) shown below. Red and green letters indicate start codon and changed nucleotides, respectively, without changing coding information. (B) Control embryo at stage 38. (C) Adrβ2-MO injected embryos. 75% (6/8) of embryos lost tail structures. (D) Adrβ2 mRNA injected embryos. 89.5% (17/19) of embryos lost eye structures. (E) 5mis mRNA injected embryos.88.2% (15/17) of them lost eye structures. (F) Adrβ2-MO and Adrβ2 mRNA coinjected embryos. 9.5% (2/21) of embryos lost tail structures. (G) Adrβ2-MO and 5mis mRNA coinjected embryos. No embryo lost tail structures, and 53.6% (15/28) of embryos looked almost normal. Scale bars represent 1 mm.
Fig. 3. Posteriorizing effects by adrenaline treatments. (A) Experimental procedures. Embryos injected with/without antisense morpholino oligomer for Adrb2 (Adrb2-MO) or with/without Adrb2 were cultured. Animal cap regions (ACs) were cut at stage 8 and then treated with various concentrations of adrenaline solutions (0.05-0.5 mmol/L). (B) Control embryos at stage 38. (C) Embryos treated with acetic acid solutions were neutralized with NaOH. All embryos (13/13) developed normally. (D�G) Embryos treated with 0.05, 0.1, 0.25, or 0.5 mmol/L of adrenaline solutions. 17.4% (4/23), 14.3% (4/28), 23.1% (6/26), or 27.6% (8/29) of embryos, lost eye structures. (H) Embryos treated with 0.5 mmol/L of adrenaline solution following microinjection of Adrb2-MO. 45.6% (5/11) of embryos developed nor- mally. Compared to G, it indicated that posteriorizing effects by adrenaline hormone were clearly rescued by Adrb2-MO. (I) Embryos treated with 0.05 mmol/L of adrenaline solution following microinjection of Adrb2 mRNA. 80.0% (8/10) lost eye structures. Compared to D, it indicated that posteriorizing effects by adrenaline hormone were clearly amplified by Adrb2 mRNA. Scale bars represent 1 mm
Fig. 4. The effects of b-adrenergic sig- naling on anterior and posterior neural marker expressions. (A, C, E, G, I) Lat- eral views of embryos. (B, D, F, H, J) Dorsal views of embryos. (A, B) Control embryos. (C, D) Embryos injected with Adrb2 mRNA. (E, F) Embryos injected with antisense morpholino oligomer for Adrb2 (Adrb2-MO). (G, H) Embryos treated with adrenaline solutions. (I, J) Embryos treated with adrenaline solu- tions following microinjection of Adrb2- MO at 2-cell stage. Scale bars repre- sent 0.5 mm.
Fig. 5. The effects of b-adrenergic signaling on mesoderm-inde- pendent neural marker expressions. (A) The experimental proce- dure for B and C. Animal cap regions (ACs) were cut from embryos injected with/without mRNAs and/or Adrb2-MO, cul- tured with/without 1 lM of adrenaline solution until stage 26, and used for reverse transcription-polymerase chain reaction (RT- PCR) analyses. More than 0.01 mmol/L of adrenaline solution killed AC cells for unfathomable reasons. (B, C) Inhibition of anterior neural marker expression by up- or downregulation of b- adrenergic signaling. Anterior neural markers, Six3 and Rx2a, mid-hindbrain boundary marker, En2, posterior neural (spinal cord) marker, HoxB9, and pan-neural markers, NCAM and N- tubulin, were not affected in both cases. a-actin and ODC were respectively used for showing no contamination of mesoderm and loading control. (D) Downstream model of Adrb2. (E) Wes- tern blotting analysis of phospho-ERK in adrenaline treated embryos. Embryos were treated from 2-cell stage with 0.5 mmol/ L of adrenaline solution and fixed at stage 26.
Fig. 6. The late effect of b-adrenergic signaling on Xenopus embryogenesis. (A, E, I, M) dorsal, (B, F, J, N) lateral, and (C, G, K, O) ven- tral and enlarged view of embryos. (D, H, L, P) Histology of gastrointestinal regions. Sectioned sites were indicated in C, G, K, O by dot- ted lines. (A-D) Control embryo. (E-H) 100% (3/3) of embryos treated with acetic acid solution neutralized with NaOH. (I-L) 100% (3/3) embryos treated with 0.5 mmol/L of adrenaline solutions. (M-P) 100% (4/4) of embryos treated with 1 mmol/L of adrenaline solutions. bd, bile duct; i, intestine; li, large intestine; lu, lungs; pa, pancreas; si, small intestine; st, stomach. Scale bars represent 1 mm.
