Supplementary MaterialsS1 Fig: Strategy for the identification of Bmp2b downstream genes from the liver organ versus pancreas destiny decision. protein are detected in the Prox1-positive pancreas cells weakly. To raised imagine hepatic and pancreatic Fhl1 manifestation, magnified images for Prox1 (red; top panel), Fhl1 (green; middle panel), and a merged view (bottom panel) are shown in insets in D-D and E-E, respectively.(TIF) pgen.1005831.s002.tif (4.5M) GUID:?9F5DC64B-4027-4D9B-ADF4-AE8E95C0DE6E S3 Fig: Specificity of morpholinos. (A) Schematic diagram of genomic structure and targeting positions of MOs (red lines). Black arrows indicate the position of primers (F and R) used for RT-PCR analysis shown in (B). E1-E6: exon 1 to exon 6. Dark grey, coding regions; Light grey, untranslated regions. (B) RT-PCR analysis of knockdown efficiency. Both MO 1 and MO 2 blocked the endogenous splice site of and, as a result, either a deletion of exon 2 (MO 1, white asterisk) or a formation of a cryptic splice form of exon 3 (MO 2, white asterisk) occurred, while a combination of MO 1 and 2 led to deletion of both exon 2 and 3 (MO 1 & 2, white asterisk). (C) The percentages of embryos are given for each single MO or combination of MOs based upon the expression domain of in the pancreas and Prox1 in the liver at 55 hpf. The embryos were scored as having a reduced or increased expression domain when the expression area of each marker was distinctly ( 25%) smaller or larger than that of the control embryos based upon the calculation using ImageJ. (D-F) Fluorescent images of and expression showing that the developmental defects of the liver (white dotted circles) and -cell formation in single morphants (E) was comparable to double morphants (F) at 55 hpf (n = 52, control; n = 64, single morphants; n = 72, double morphants). (G-I) Bright-field images combined with fluorescent images showing the overall morphology of embryos and expression (red) in control (G), single morphants (H), and double morphants (I) at 5 dpf. The enlarged -expressing cell population (white squares and insets) in single morphants (H) was similar to that in embryos co-injected with and MOs (I). Note that potential off-target ventricle lumen inflation defects in the brain of Fabomotizole hydrochloride single morphants Fabomotizole hydrochloride were attenuated by co-knockdown of (black arrows), whereas pericardial edema persisted both in single morphants and double morphants (black arrowheads). (J) Quantification of the results in G-I. The embryos were scored as having an increased expression domain when the expression area of was distinctly ( 25%) larger than that of the control embryos based upon the calculation using ImageJ. (K) Quantification of the number (meanSD) of Prox1-positive cells in the liver at 55 hpf. 252.611.5 cells were Prox1-positive in control embryos, while 151.316.2 and 142.317.4 cells expressed Prox1 in single morphants and double morphants, respectively (= 0.0009 and = 0007, respectively). Cells in 20 planes of confocal images from 5 individual embryos were counted. Asterisks indicate statistical significance: ***, 0.001. (L-N) Confocal images of control embryos (L), single morphants (M), and double morphants (N) at 55 hpf, stained for Prox1 (blue). The reduced Prox1-expressing cell population in single morphants (M) was similar to that in embryos co-injected with and MOs (N). D-F, dorsal views, anterior to the left. G-I, lateral views, anterior to the right. L-N, confocal projection images, ventral views, anterior to the top. Scale bars: D-F and L-N, 20 M; G-I, 100 M.(TIF) pgen.1005831.s003.tif (8.6M) GUID:?85234E2C-03E3-405B-BEAA-E8244F54B53B S4 Fig: Lack Fabomotizole hydrochloride of Fhl1b activity compromises liver organ specification and enhances induction of Pdx1-positive cells in the dorsal pancreatic bud. (A-B) Confocal pictures of control embryos (A and A) and morphants (B and B) at 30 hpf, stained for Pdx1 (reddish colored; dorsal pancreatic bud can be defined by white dotted circles) and Prox1 (blue Fabomotizole hydrochloride inside a and B; gray in B) and A. The somites are Pdx1 positive also. In comparison to control embryos (A and A), in morphants LAMP3 (B and B), the Pdx1 manifestation site Fabomotizole hydrochloride in the dorsal pancreatic bud was extended, as the Prox1 expression domain was decreased. (C-D) Quantification of the quantity (meanSD) of Pdx1-positive cells in the pancreas at 30 hpf. Cells in 20 planes.