PMC

Increased levels of Mcrs1 expands neural plate and neural crest domains. (A) sox2 expression in the neural plate is expanded on the mcrs1 injected side of the embryo (red bar), compared to control, uninjected side (black bar). Pink dots are βGal-lineage labeled cells that received the injected mRNAs. (B) sox11 expression in the neural plate is expanded on the mcrs1 injected side of the embryo (red bar), compared to control, uninjected side (black bar). In this embryo, the sox11 PPE domain on the mcrs1 injected side (between red arrows) is expanded compared to the control, uninjected side (between black arrows). (C) zic1 expression in the lateral neural plate is expanded on the mcrs1 injected side of the embryo (red bar), compared to control, uninjected side (black bar). The neural crest domain (red bracket) also is expanded. (D) zic2 expression in the lateral neural plate is expanded on the mcrs1 injected side of the embryo (red bar), compared to control, uninjected side (black bar). The neural crest domain (red bracket) also is expanded. (E) msx1 expression along the border of the neural plate is expanded on the mcrs1 injected side of the embryo (red bar in right image), compared to control, uninjected side of the same embryo (black bar in left image). (F) pax3 expression along the border of the neural plate is expanded on the mcrs1 injected side of the embryo (red bar), compared to control, uninjected side (black bar). (G) foxd3 expression is expanded on the mcrs1 injected side of the embryo (red bar) compared to control, uninjected side (black bar). (H) Two examples of sox9 expression after increased Mcrs1. In both embryos, the neural crest domain is broader on the mcrs1 injected side (red bars) compared to control, uninjected side (black bar). In the left image, otic placode domain (red arrow) is smaller, whereas in in the right image it is larger (red arrow), compared to control, uninjected sides (black arrows). (I) six1 expression is reduced on the mcrs1 injected side of the embryo (red arrow) compared to control side (black arrow). (J) Two examples of irx1 expression after increased Mcrs1. In both cases, the neural plate expression is expanded on the mcrs1 injected side of the embryo (red bar), compared to control, uninjected side (black bars). In the left image, irx1 expression in the PPE on the mcrs1 injected side of the embryo is expanded (red bar) compared to the control, uninjected side (black ba). In the right image, it is reduced. .

Mcrs1 is required for properly proportioning the cranial neural gene expression domains. (A) sox2 expression in the neural plate is expanded on the S-MO injected side of the embryo (red bar), compared to control, uninjected side (black bar). (B) sox11 expression in the neural plate is expanded on the S-MO injected side of the embryo (red bar), compared to control, uninjected side (black bar). In this embryo, the sox11 PPE domain on the S-MO injected side (between red arrows) is reduced compared to the control, uninjected side (between black arrows). (C) zic1 expression in the lateral neural plate is expanded on the S-MO injected side of the embryo (red bar), compared to control, uninjected side (black bar). In this embryo, the neural crest domain (red bracket) also is expanded compared to control, uninjected side (black bracket). (D) zic2 expression in the lateral neural plate is expanded on the S-MO injected side of the embryo (red bar), compared to control, uninjected side (black bar). In this embryo, the neural crest domain (red bracket) also is expanded compared to control, uninjected side (black bracket). (E) Two examples of the effect of Mcrs1 knock-down by S-MOs on msx1 expression. In the left image, knock-down results in expansion of the msx1 domain (red arrow) and in the right image, in the diminution of the msx1 signal (red arrow). (F) In this example, tfap2α expression is reduced on the S-MO injected side of the embryo (red arrow). (G) foxd3 expression is reduced on the S-MO injected side of the embryo (between red arrows) compared to control side (between black arrows). (H) Two examples of the effect of Mcrs1 knock-down by S-MOs on sox9 expression. In the left image, knock-down results in a smaller neural crest domain of sox9 (between red arrows), and in the right image, in the diminution of the sox9 signal (between red arrows) compared to control, uninjected sides (between black arrows). In the left image, sox9 otic placode expression (small green arrows) is the same on both sides of the embryo, whereas in the right image it is diminished on the S-MO side (right side of image). (I) six1 expression is reduced on the S-MO injected side of the embryo (red arrow) compared to control side (between black arrows). (J) Two examples of the effect of Mcrs1 knock-down on irx1 expression. In both cases, the neural plate expression is expanded on the S-MO injected side of the embryo (red bar), compared to control, uninjected side (black bar). In the left image, irx1 expression in the PPE on the S-MO injected side of the embryo (between red arrows) is expanded compared to the control, uninjected side (between black arrows). In the right image, it is reduced. (K) Mcrs1 knock-down by S + L MOs results in a broader sox11 neural plate domain (red bar) compared to control side (black bar) and a narrower PPE domain (between red arrows) compared to control side (between black arrows). These are the same effects as with S-MOs (cf. ). (L) Mcrs1 knock-down by S + L MOs results in a smaller foxd3 neural crest domain (between red arrows) compared to control side (between black arrows). This is similar to the effect of S-MOs (cf. ). (M) Mcrs1 knock-down by S + L MOs results in a fainter six1 PPE domain (red arrow) compared to control side (between black arrows). This is similar to the effect of S-MOs (cf. ). (N) Mcrs1 knock-down primarily results in expansion of neural plate gene domains. For sox11 and irx1, the S + L MOs (S + L) resulted predominantly in broader phenotypes, similar to S-MOs (S), but significantly more embryos showed no change (*, p < 0.05, Chi-square test). Numbers in each bar indicates sample sizes in N-Q. (O) Mcrs1 knock-down results in about 50% of embryos showing reduced domains of neural border genes, with a smaller frequency of embryos showing broader domains. For msx1, there was no significant difference between frequencies resulting from S-MOs compared to S + L MOs (p > 0.05, Chi-square test). (P) Mcrs1 knock-down results in broader zic1 and zic2 neural crest domains, but primarily reduction of foxd3 and sox9 neural crest domains. For foxd3 and sox9, there were no significant differences between frequencies resulting from S-MOs compared to S + L MOs (p > 0.05, Chi-square test). (Q) Mcrs1 knock-down results in about 50% of embryos showing reduced PPE domains of six1, sox11 and irx1, with a smaller frequency of embryos showing broader domains. In contrast, it results in a high frequency of reduction of the sox9 otic placode domain. Only for irx1 was there a significant difference between frequencies resulting from S-MOs compared to S + L MOs. (*, p < 0.05, Chi-square).(For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

CT 3D image of P1, P2, and P3. (A) CT 3D reconstruction image of P1 sample, 1 month after unilateral (left) alveolar defect created ① lateral view of right side. ② lateral view of left side, bony defect would be observed cleared (black arrow pointed). ③ palatal view of P1 skull, there is a bony defect on the left side (black arrow pointed). (B) CT 3D reconstruction image of P2 sample, 1 month after unilateral (left) alveolar defect created. ① lateral view of right side. ② lateral view of left side, bony defect would be observed cleared (black arrow pointed) with irregular bony margin. ③ palatal view of P2 skull, there is a Bony defect on the left side (black arrow pointed). (C) CT 3D reconstruction image of P3 sample, CT was taken immediately after operation. ① lateral view of right side, bony defect would be observed cleared (black arrow pointed). ② lateral view of left side, bony defect would be observed cleared (black arrow pointed). ③ palatal view of P3 skull there are bony defect regions on both sides (black arrow pointed). (D) CT 3D reconstruction image of P3 sample, CT was taken 1 month after operation. ① lateral view of right side, bony defect would be observed cleared (black arrow pointed). ② lateral view of left side, bony defect would be observed cleared (black arrow pointed). ③ palatal view of P3 skull there are bony defect regions on both sides (black arrow pointed). 3D, three-dimensional; CT, computed tomography.

Hydrogen-bonding (black dashed lines), π–π (orange dashed lines), and cation−π (pink dashed lines) interactions of R61 or K61 with dATP and TTD. Each frame shows the WC base pairing interactions between the dATP and the 3′T of TTD as black dashed lines. (a) The guanidinium side chain of R61 interacts with one of the α-phosphate oxygens through charge–charge and hydrogen-bonding interactions (black dashed line, circled). This configuration displays a T-shaped π–π (not indicated here) and a cation−π (pinked dashed line) interaction between the R61 side chain and dATP(N7). (b) The guanidinium side chain of R61 interacts with the dATP base through π–π (orange dashed lines) and cation−π (pink dashed line, virtually indistinguishable from orange lines) interactions. This side chain configuration of R61 also allows hydrogen-bonding interactions with the 5′T of TTD (black dashed lines). (c) The guanidinium side chain of R61 interacts with the dATP(N7) through T-shaped π–π (not indicated here), cation−π (pink dashed line), and hydrogen-bonding (black dashed line) interactions. (d) The amine side chain of K61 interacts with the dATP(N7) through cation−π (pink dashed line) and hydrogen-bonding (black dashed line) interactions. This side chain configuration of K61 also allows the formation of charge–charge and hydrogen-bonding interactions with one of the α-phosphate oxygens (black dashed line). (e) The amine side chain of K61 interacts with the dATP base through cation−π interactions (pink dashed line). This side chain configuration of K61 also allows hydrogen-bonding interactions with the 5′T(O4) of TTD (black dashed line). (f) The A61 side chain opens up more room for incoming water and Na⁺ ions in the active side, which compensate for the absence of a charged and bulky protein side chain. The hydrogen-bonding interactions between the dATP(N7) and nearby water, as well as 5′T of TTD and water, are shown as black dashed lines. A Na⁺ ion comes into the active site occasionally and forms a cation−π interaction with the dATP(N7) (pink dashed line).