Figure legends:
Figure 1: Pedigree, clinical, and variant information. (A)
Pedigree with two affected individuals from a second-cousin marriage.
(B) Sanger sequencing confirmed the heterozygous and homozygous missense
variant c.423G>A (NM_198679.1) in the RAPGEF1 gene
in the father III.4 and the patient IV.2, respectively, that had been
identified through whole exome sequencing. (C) Clinical information of
the two affected individuals in the format of Human Phenotype Ontology
(HPO) (Kohler et al., 2019).
Figure 2: Effects of RAPGEF1 variants. (A) Pathogenicity
prediction and population abundances of RAPGEF1 variant. (B)
Distribution of domains of RAPGEF1 and location of the variant. (C)
Multiple alignment of protein sequences in species indicated that the
variant altered a conserved amino acid. (D) Three-dimensional structural
model of RAPGEF1 and a close-up view of structural superposition of
RAPGEF1-WT (white) and RAPGEF1-M141I (orange), which were displayed with
transparent new cartoon representation. The Met141 and Ile141 residues
were shown with white and orange Licorice representation, respectively.
The change of Gibbs free-energy gap (ddG) and the stability upon
mutation were also indicated. (E) Modules of protein-protein interaction
network included three KEGG pathways, which linked RAPGEF1 (red) and
neurodevelopmental genes (green).
Figure 3: Custom-made morpholino nucleotides for zebrafishrapgef1 and the performance. (A) The zebrafish rapgef1agene was targeted by two specific morpholino antisense strategies to
prevent either the translation of the zebrafish gene (ATG-MO) or proper
splicing of exon 2 (E2I2-MO). Primers 1F and 3R interrogated the
presence of wildtype transcripts or those in which exon 2 was skipped.
(B) RT-PCR of rapgef1a transcript from Control-MO and E2I2-MO
morpholino-injected embryos 3 days after fertilization. Injection of 1
ng of rapgef1a morpholino altered the splicing between exon 2 and intron
2, as revealed by shift in PCR bands between control (315bp) and
morpholino-injected embryos (136bp). ef1a served as a negative control
to avoid unspecific effects.
Figure 4: Effects of rapgef1-knockdown on gross
morphology and survival rate. (A) Bright field images and hb9-EGF
fluorescent images showed the gross morphology of Control-MO, E2I2-MO
and ATG-MO processed zebrafish embryos at 6 hpf. Blue arrows pointed to
the abnormal brain patterns. (B) Bright filed images showed the brain
patterns of Control-MO, E2I2-MO and ATG-MO processed zebrafish embryos
at 6 hpf. Blue arrows pointed to the abnormal brain patterns. Red and
yellow arrows indicated the reduction of interorbital distance of therapgef1 knockdown zebrafish embryos. (C) Upper: percentage of
embryos with defects at 6-hpf in the groups of Control-MO, E2I2-MO and
ATG-MO. Lower: a time-course plot of survival rate in the groups of
Control-MO, E2I2-MO and ATG-MO for 5 days. (D) Bright field images and
hb9-EGF fluorescent images showed the gross morphology of Control-MO,
E2I2-MO, E2I2-MO plus mutant human RAPGEF1 mRNA (E2I2-MO+ MTRAPGEF1 mRNA), and E2I2-MO plus wildtype human RAPGEF1mRNA (E2I2-MO+ WT RAPGEF1 mRNA) processed zebrafish embryos at 36
hpf. Blue arrows pointed to the abnormal brain patterns. (E) Bright
filed images showed the brain patterns of Control-MO, E2I2-MO, E2I2-MO
plus mutant human RAPGEF1 mRNA (MO+MT RAPGEF1 ) , and
E2I2-MO plus wildtype human RAPGEF1 mRNA (MO+WT RAPGEF1 )
processed zebrafish embryos at 36 hpf. Blue arrows pointed to the
abnormal brain patterns. Red and yellow arrows indicated the reduction
of interorbital distance of the rapgef1 knockdown zebrafish
embryos. (F) Upper: percentage of embryos with defects at 36-hpf in the
groups of Control-MO, E2I2-MO, E2I2-MO plus mutant human RAPGEF1mRNA (E2I2-MO+ MT RAPGEF1 mRNA), and E2I2-MO plus wildtype humanRAPGEF1 mRNA (E2I2-MO+ WT RAPGEF1 mRNA). Lower: a
time-course plot of survival rate in the groups of Control-MO, E2I2-MO,
E2I2-MO plus mutant human RAPGEF1 mRNA (E2I2-MO+ MTRAPGEF1 mRNA), and E2I2-MO plus wildtype human RAPGEF1mRNA (E2I2-MO+ WT RAPGEF1 mRNA) for 5 days. hpf: hours post
fertilization.
Figure 5: Effects of rapgef1-knockdown on locomotor
capacity and motor neuron development. (A) Digital tracks and heatmap
images of zebrafish larvae at 5-dpf in the groups of Control-MO,
E2I2-MO, and ATG-MO. (B) Statistical analysis on the four parameters of
movement in the three aforementioned groups, namely, total distance,
velocity, mobility, and maximal acceleration. * P < 0.05, ** P
< 0.01, N = 4, ANOVA. (C) Digital tracks and heatmap images of
zebrafish larvae at 5-dpf in the groups of Control-MO, E2I2-MO, E2I2-MO
plus mutant human RAPGEF1 mRNA, and E2I2-MO plus wildtype humanRAPGEF1 mRNA. (D) Statistical analysis on the four parameters of
movement in the four aforementioned groups, namely, total distance,
velocity, mobility, and maximal acceleration. * P < 0.05, ** P
< 0.01, *** P < 0.001, ns: not significant, N = 6,
ANOVA. (E) Gross morphology of Tg(hb9:EGFP) zebrafish embryos at
36-hpf in the groups of Control-MO, E2I2-MO, and ATG-MO. The spinal
motor neurons were visualized by EGFP fluorescence. Irregular motor
neuron axons were labeled by asterisk. (F) Gross morphology ofTg(hb9:EGFP) zebrafish embryos at 48-hpf in the groups of
Control-MO, E2I2-MO, E2I2-MO plus mutant human RAPGEF1 mRNA
(E2I2-MO+ MT RAPGEF1 mRNA), and E2I2-MO plus wildtype humanRAPGEF1 mRNA (E2I2-MO+ WT RAPGEF1 mRNA). The spinal motor
neurons were visualized by EGFP fluorescence. Irregular motor neuron
axons were labeled by asterisk. (G) Upper: quantification of the average
length of motor neuron axon in the groups of Control-MO, E2I2-MO, and
ATG-MO. Lower: quantification of the average length of motor neuron axon
in the groups of Control-MO, E2I2-MO, E2I2-MO plus mutant humanRAPGEF1 mRNA (E2I2-MO+ MT RAPGEF1 mRNA), and E2I2-MO plus
wildtype human RAPGEF1 mRNA (E2I2-MO+ WT RAPGEF1 mRNA). **
P < 0.01, *** P < 0.001, N = 10, ANOVA. dpf: days
post fertilization. hpf: hours post fertilization.
Figure 6: Effects of rapgef1-knockdown on vascular
development and somitogenesis. (A) Bright field and fluorescent images
of Tg(fli1a:EGFP) zebrafish embryos at 72-hpf in the groups of
Control-MO, E2I2-MO, and ATG-MO. Vascular structure was visualized by
EGFP fluorescence. Labels were used for ISV (intersegmental vessel),
DLAV (dorsal longitudinal anastomotic vessel), DA (dorsal aorta), PCV
(posterior cardinal vein), and sprouts. (B) Gross somite morphology at
3-dpf in the groups of Control-MO, E2I2-MO, and ATG-MO. Dotted lines
delineated somite boundaries. (C) Bright field and fluorescent images of
zebrafish embryos at 72-hpf in the groups of Control-MO, E2I2-MO,
E2I2-MO plus mutant human RAPGEF1 mRNA (E2I2-MO+ MTRAPGEF1 mRNA), and E2I2-MO plus wildtype human RAPGEF1mRNA (E2I2-MO+ WT RAPGEF1 mRNA). Vascular structure was
visualized by EGFP fluorescence. Labels were used for ISV
(intersegmental vessel), DLAV (dorsal longitudinal anastomotic vessel),
DA (dorsal aorta), PCV (posterior cardinal vein), and sprouts. (D) Gross
somite morphology at 3-dpf in the groups of Control-MO, E2I2-MO, E2I2-MO
plus mutant human RAPGEF1 mRNA (E2I2-MO+ MT RAPGEF1 mRNA),
and E2I2-MO plus wildtype human RAPGEF1 mRNA (E2I2-MO+ WTRAPGEF1 mRNA). Dotted lines delineated somite boundaries. dpf:
days post fertilization. hpf: hours post fertilization.