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Fig. 2. 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. Fig. 2B). (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. Fig. 2G). (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. Fig. 2I). (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). |
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Fig. 7. Effects of altering Mcrs1 levels on otic vesicle gene expression and size. (A) Otic vesicles in larvae injected with Mcrs1 S-MOs primarily displayed lower intensity of six1 ISH signal. Black arrow = control otic vesicle; red arrow = Mcrs1 KD otic vesicle of same embryo. (B) Otic vesicles in larvae injected with Mcrs1 S-MOs primarily displayed lower intensity of irx1 ISH signal. Black arrow = control otic vesicle; red arrow = Mcrs1 KD otic vesicle of same embryo. (C) Otic vesicles in larvae injected with Mcrs1 S-MOs primarily displayed lower intensity of sox9 ISH signal. Black arrow = control otic vesicle; red arrow = Mcrs1 KD otic vesicle of same embryo. (D) The percent of embryos in which otic vesicle gene expression on the knock-down side was different from the control side of the same embryo. The otic expression of all three genes (six1, irx1, sox9) is primarily reduced (blue). Orange = percent increased; gray = percent no change. Numbers within the bars indicate sample sizes. (E) In tailbud embryos, sox9 expression indicates that the otic tissue is smaller on the Mcrs1 knock-down side (red bar) compared to control side (black bar). (F) Otic vesicle volumes (top) were calculated from serial sections of larvae (bottom) at the same stages as in A-C. Total volume was calculated within the outer dashed line, lumen (L) volume within the inner dashed line, and tissue volume between the two dashed lines. For each measurement, the volume of the Mcrs1 knock-down side (MO, red dashed lines) was significantly smaller compared to the control side (black dashed lines) of the same embryo. (total, p = 0.00002; tissue, p = 0.00004; lumen, p = 0.0009; two-tailed, paired t-test, n = 13).(G) An example of six1 otic vesicle expression that is more intense on the mcrs1-injected side (red arrow; 100 pg mRNA) compared to the control side (black arrow) of the same larva.(H) An example of irx1 otic vesicle expression that is similar on the mcrs1-injected side (red arrow; 200 pg mRNA) and control side (black arrow) of the same larva. (I) An example of sox9 otic vesicle expression that is similar on the mcrs1-injected side (red arrow; 100 pg mRNA) compared to the control side (black arrow) of the same larva. (J) The percent of embryos in which otic vesicle gene expression on the mRNA-injected side was different from the control side of the same embryo. The otic expression for all three genes (six1, irx1, sox9) are a mixture of reduced (blue), broader (orange) and no change (gray). Two different concentrations of mRNA (100 pg, 200 pg) were analyzed, and for each there were significant differences in the frequencies of the phenotypes (six1, p = 0.00002; irx1, p = 0.0015; sox9, p = 0.0117; Chi-square test). Numbers within the bars indicate sample sizes. (K) Otic vesicle volumes (top) were calculated from serial sections (bottom) of larvae (same stages as in G-I). There were no significant differences in total volume, tissue volume or lumen volume between mcrs1 mRNA-injected (pink lineage label) side versus control side of the same embryo (p < 0.05, paired, two-tailed t-test; n = 14 embryos). |
