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Figure 5: Kmt2d loss of function inhibits MHC expression at tailbud stages.
Embryos were injected with 2.5 ng MO (Kmt2d or 5 bp mismatch control) in
combination with 100 pg LacZ RNA in one dorsal blastomere at the 4-cell stage. At
stage 29 MHCexpression was analyzed by whole mount in situ hybridization.
Embryos are shown from the ventral side; asterisks indicate the injected side. A
Schematic drawing of MHCexpression at tailbud stages. First heart field (FHF) and
second heart field (SHF) are indicated. B Uninjected control embryo. C Embryo
injected with mismatch control MO. D Embryo injected with the Kmt2d MO showing a
reduction in MHCexpression (arrow). E Kmt2d morphant embryo showing a loss of MHCexpression on the injected side (arrow). F Graph summarizing three
independent injection experiments, standard errors of the means and numbers of
analyzed embryos are indicated for each column. * P-value in a Student’s t-test <
0.05 |
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Figure 2: Loss of Kmt2d affects Xenopus heart morphology at tadpole stages.
Embryos were injected at the 4-cell stage into one dorsal blastomere with 2.5 ng MO
(Kmt2d or the 5 bp mismatch control) in combination with 100 pg LacZ RNA. At stage
42 MHC expression was analyzed by in situ hybridization. Injected side of embryos
(blue ß-galactosidase staining) is marked with an asterisk; all embryos are shown
from the ventral side. A Uninjected control embryo. B Embryo injected with the
mismatch MO (mis). C,D Embryo injected with the Kmt2d MO (MO) showing a
reduction in heart size and defects in chamber formation. Arrow in D marks the heart.
E Magnification of the heart of the control embryo shown in A. F Magnification of the
heart of the Kmt2d morphant embryo shown in D. Dashed line separates the staining
in the heart from the staining in the jaw muscle. G Histological sagittal section of the
control heart shown in A. The section is orientated like the embryo seen in A. The
two atria and the ventricle can be distinguished. H Section through the heart region of
the mismatch control embryo shown in B. The two atria and ventricle are visible. I
Section through the Kmt2d morphant heart seen in D. Arrowhead indicates the tubelike
heart. J Graph summarizing the defects in heart morphology assessed by MHC
in situ hybridization of three independent experiments; standard errors of the means
and the number of analyzed embryos are indicated for each column. ** P-value in a
Student’s t-test < 0.01. Abbreviations: (a) atrium, (e) eye, (jm) jaw muscle, (ra) right
atrium, (la) left atrium, (v) ventricle. Scale bars indicate different magnifications for AD,
compared to E,F or G-I, respectively. |
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Figure 3: Design and heart phenotype of a Kmt2d splice MO. A Design of the
splice MO (spMO)and possible splicing outcomes. Xenopus laevis Kmt2d consists of
54 coding exons. The splice MO (red dashed square) targets the 5’ splice junction
representing the boundary between exon 53 and the last intron of the precursor
mRNA, spanning 10 nucleotides of exon 53 and 15 of the last intron. Different
splicing outcomes are shown. (1) The splice junction is skipped by the spliceosome
leading to intron inclusion. (2) Wild-type transcript, correctly spliced. (3) Exon 53 as
well as the following intron are deleted. Sizes (in bp) of the fragments, expected to be
amplified by RT-PCR using the indicated primer combination (blue arrows), are
indicated. B Lysates of embryos injected either with 7.5 ng control MO or splice MO,
as well as uninjected control embryos were analyzed by RT-PCR at neurula stage 17
using forward and reverse primers as indicated by blue arrows in A. Image shows the
result of the agarose gel electrophoresis of the different fragments obtained.
Numbers in brackets indicate splicing variants as predicted in A. C-F Ventral view of
embryos injected with the splice MO (5 ng, E,F), control MO (5 ng, D) and 150 pg
LacZ RNA or uninjected controls (C) analyzed by MHCin situ hybridization at
tadpole stage 42. Injected side of embryos (blue ß-galactosidase staining) is marked
with an asterisk. E Morphant with a tube-like misplaced heart. F Morphant with a
malformed heart. C’-F’ Higher magnification of the heart region of embryos shown in
C-F. G Graph summarizing the defects in heart morphology assessed by MHCα in
situ hybridization of three independent experiments; standard errors of the means and the number of analyzed embryos are indicated for each column. *** P-value in a
Student’s t-test < 0.001. |
