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BACKGROUND: Kabuki syndrome is a haploinsufficient congenital multi-organ malformation syndrome, which frequently includes severe heart defects. Mutations in the histone H3K4 methyltransferase KMT2D have been identified as the main cause of Kabuki syndrome, however, the role of KMT2D in heart development remains to be characterized.
RESULTS: Here we analyze the function of Kmt2d at different stages of Xenopus heart development. Xenopus Kmt2d is ubiquitously expressed at early stages of cardiogenesis, with enrichment in the anterior region including the cardiac precursor cells. Morpholino-mediated knockdown of Kmt2d led to hypoplastic hearts lacking the three-chambered structure. Analyzing different stages of cardiogenesis revealed that development of the first and second heart fields as well as cardiac differentiation were severely affected by loss of Kmt2d function.
CONCLUSION: Kmt2d loss of function in Xenopus recapitulates the hypoplastic heart defects observed in Kabuki syndrome patients and shows that Kmt2d function is required for the establishment of the primary and secondary heart fields. Thus, Xenopus Kmt2d morphants can be a valuable tool to elucidate the etiology of the congenital heart defects associated with Kabuki syndrome.
Figure 1: Kmt2d is expressed at early stages of Xenopus development. Kmt2d
expression was analyzed by in situ hybridization. A 2-cell stage embryo. B Embryo at
blastula stage 8. C Embryo at gastrula stage 10.5. D Embryo at neurula stage 14,
anterior view. Arrow marks Kmt2d expression in the anterior region. E Embryo at
stage 17, anterior view. F Same embryo as in E, dorsal view. G Embryo at stage 22,
lateral view. H Embryo at stage 29 lateral view. I Embryo at neurula stage 14, the
dashed line indicates the plane of the sagittal section shown in J. Abbreviations: a
(anterior), e (eye), ea (eye anlage), ect (ectoderm), en (endoderm), m (mesoderm), s
(somites), p (posterior), pn (premigratory neural crest).
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.
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.
Figure 4: Kmt2d knockdown does not affect heart rate of Xenopus tailbud
stage embryos. Embryos were injected with 2.5 ng MO Kmt2d (or 2.5 ng 5 bp
mismatch MO) or 5 ng splice MO (or 5 ng control MO) in one dorsal blastomere at
the 4-cell stage. 50 pg mGFP mRNA were co-injected as a lineage tracer. The
beating frequency of the embryos’ hearts was analyzed at stage 44. Representative
embryos (A-E) are shown from the ventral side; the respective heart rate is indicated.
Asterisks mark the injected side. A'-E' show a higher magnification of the heart area.
Ventricle and outflow tract are indicated by dashed circles. Movies of the beating
hearts of the presented embryos can be found in the supplement. A,B,C Uninjected
(A,A') as well as mismatch MO (mis) (B,B') and control MO (coMO) (C,C') injected
embryos showing a normal heart morphology. D,E Embryos injected with Kmt2d MO
(MO) (D,D') and splice MO (spMO) (E,E') displaying reduced ventricle size. F Boxplot
showing the heart rates of 30 embryos per condition from two independent
experiments.
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
Figure 6: The expression of first and second heart field markers is affected by
Kmt2d loss of function. 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 and analyzed by Tbx20 or Isl1 in situ hybridization. A Schematic
drawing of Tbx20 expression in the first heart field at stage 29. B-E Embryos
analyzed by Tbx20 in situ hybridization shown from the ventral side; asterisks
indicate the injected side. B Uninjected control embryo. C Embryo injected with
mismatch control MO. D,E Kmt2d morphant embryos showing a reduction as well as
a posterior shift of Tbx20 expressing cells on the injected side. 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 <
0.001. G Schematic drawing of Isl1 expression in the second heart field at stage 29.
H-K Embryos analyzed by Isl1 in situ hybridization. H Uninjected control. I Mismatch
control. J,K Kmt2d morphants with reduced expression of Isl1. L Graph summarizing
three independent experiments, s.e.m as well as numbers of analyzed embryos are
indicated. ** P-value in a Student’s t-test < 0.01.
Figure 7: Nkx2.5 expression is downregulated in Kmt2d morphant embryos.
Embryos were injected with 2.5 ng MO Kmt2d (or 2.5 ng 5 bp mismatch control) or 5
ng splice MO (or 5 ng control MO) in combination with 100 pg LacZ RNA in one
dorsal blastomere at the 4-cell stage. Whole mount in situ hybridization of st. 28
embryos was performed for the cardiac lineage marker Nkx2.5. Asterisks mark the
injected side; all embryos are shown from the ventral side. A,B,C Uninjected control and embryos injected with 2.5 ng mismatch MO (B) or 5 ng control MO (C) showing
normal expression of Nkx2.5 on both sides. D,E Kmt2d MO (D) and splice MO (E)
injected embryos displaying a strongly reduced expression of Nkx2.5 (arrow; dashed
line marks ventral midline). F Schematic drawing of Nkx2.5 expression in a st. 28
embryo. G Graph summarizing three independent experiments with the number of
analyzed embryos and s.e.m. indicated for each condition. ** P-value in a Student’s ttest
< 0.01; * P-value in a Student’s t-test < 0.05.
Figure 8: The development of Isl1-positive cardiac progenitor cells is largely
unaffected in Kmt2d morphants. Embryos were injected with 2.5 ng MO Kmt2d (or
2.5 ng 5 bp mismatch control) or 5 ng splice MO (or 5 ng control MO) in combination
with 100 pg LacZ RNA in one dorsal blastomere at the 4-cell stage. At early neurula
stage 14/15 embryos were analyzed for Isl1 expression by in situ hybridizationA
Uninjected control embryo; left (anterior view), right (dorsal view) of the same
embryo. B Mismatch MO injected embryo. C Kmt2d MO injected embryo. D Graph
summarizing three independent experiments. Standard errors of the means and
numbers of analyzed embryos are shown for each condition; n.s. not significant in a
Student’s t-test. E Uninjected control embryo. F Control MO injected embryo. G
Splice MO injected embryo. H Graph summarizing three independent experiments.
Standard errors of the means and numbers of analyzed embryos are shown for each
column; n.s. not significant in a Student’s t-test.
kmt2d ( lysine methyltransferase 2D ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anteriorleft, dorsal up.
