Identification of LPIN1 mutations from a family presenting with adult-onset muscle weakness. (A) Pedigree of the investigated family. The proband (II:5) was indicated by black arrow. (B) Schematic representation of the localization of identified LPIN1 mutations c.2047A>C (p.I683L) and c.2201G>A (p.R734Q) in the genome structure (top) and protein structure (bottom). (C) Sanger sequencing chromatographs showing two compound heterozygous mutations of c.2047A>C and c.2201G>A presenting in patients. (D-G) Crystal structures of human LPIN1 wild type and mutants carrying p.I683L or p.R734Q, or p.I683L/p.R734Q. (H) LPIN1was highly conserved across different vertebrate species; the conserved I683L and R734Q are highlighted with a star (*).

The spatial and temporal expression patterns of lpin1 during zebrafish early development. The expression of lpin1 mRNA was ubiquitous during cleavage and gastrula stages (A-C), restricted to paraxial mesoderm at the bud stage (D), and abundant at somites from the 8-somite stage onwards (E-H'). The dynamic lpin1 mRNA expression was detected in the retina (red arrows in F and F'), pectoral fins (pf, black arrows in G and G') and the cephalic musculature (cm, green arrows in G and G'). (I-J') IHC staining showed the Lpin1 protein expression in myotomes and the retina (yellow arrows), acetylated α-tubulin was stained in green color. (F-J) Lateral views of embryos, (F'- J') Dorsal views of embryos. Scale bar: 100 µm.

Zebrafish lpin1 morphant embryos showed defects in the skeletal muscle at 4 dpf. (A-C') Classification of the lpin1 morphants was based on the trunk phenotype (A-C) and the corresponding reduction in birefringence observed in skeletal muscles (A'-C'). Class I (A, A') represents the wild-type-like phenotype with neither defect in embryonic trunk nor reduced birefringence in the skeletal muscle. Scale bar: 200 μm. (D) The phenotype of lpin1 morphants was rescued by the co-injected mRNA of human LPIN1^wt but not LPIN1^p.I683L, LPIN1^p.R734Q or LPIN1^{p.I683L& R734Q}. (E) Quantification data are calculated by the mean intensity of 10 somites (from somite 5 to 15) for each embryo using Image J, which is normalized to that of class I (control) to obtain percentage values. n = 10 embryos for each class. *** indicates p < 0.001 by Student's t-test.

Knockdown of lpin1 in zebrafish embryos affected neuron development and AChR clustering. Znp1 antibody staining (A-B) and α-BTX labeling (A'-B') of control embryos (A-A'') and lpin1 morphants (B-B'') at 26 hpf. Zn8 antibody staining (C-D) and α-BTX labeling (C'-D') of control embryos (C-C'') and lpin1 morphants (D-D'') at 60 hpf. Scale bar: 100 µm. Measurements of the length of motor neurons (E, n = 6 motor neurons/larvae), the distance between two adjacent motor neurons (F, n = 6 segments/larvae), the angle of AChR clusters (G, n = 6 AChR clusters/larvae), and the density of post-synapses (H, n = 6 areas of myotome/larvae), were carried out in 9 larva under each treatment. (I) Schematic representations of the parameters in E, F, G and H. *** indicates p < 0.001 by Student's t-test.

Lpin 1 deficiency impaired the maturation of schwann cells and myelin synthesis. (A-B') GFP signals of 5 dpf Tg(mbp:eGFP) larvae was visualized by fluorescent microscope, yellow arrows indicated GFP signal in the spinal cord, and red arrows indicated GFP signal motor neurons, lateral views, scale bar: 100 μm. (C) Mbp fluorescence signals of control embryos and lpin1 morphants were analyzed by Image J software. Ten independent samples were evaluated. (D) qRT-PCR measurements of immature SC markers, mature SC markers, and myelin gene markers in lpin1 morphants and STD-MO injected embryos at 3dpf. (E-G) qRT-PCR measurements of pou3f1, egr2b, and mpz dynamic expression at 1 dpf, 3 dpf, and 5 dpf respectively. Values in (D-G) represented means ± SE of data from three independent experiments, **p< 0.01, and ***p < 0.001 (Student's t-test).

Locomotor deficits in lpin1 morphants. (A-C)lpin1 morphant embryos exhibited reduced touch-evoked response at 4 dpf (B) while control embryos swam away rapidly after mechanosensory stimulation (A). The reduced touch-evoked response of lpin1 morphant could be rescued by co-injected hLPIN1^wt mRNA (C). Scale bar: 200 µm. (D) Cumulative plots of the position and velocity of control larva, lpin1 morphants, and lpin1 morphants co-injected with hLPIN1^wt mRNA; during 5 min of behavioral recording. 8 representative individuals were recorded for each treatment group. (E) Representative heat map plots. Red dots indicate the long rest time without any movement. (F) Cumulative duration records showed that the lpin1 morphants were less active. (G) Velocity was dramatically decreased in lpin1 morphants, but can be rescued by the hLPIN1^wt mRNA. (H) Total movement distance during a 5 min startle response analysis as follows: 20 s spontaneous movement tracking followed by cycle stimulation with light on 5 s, interval 1 s, trigger tap 1 s, and interval 48 s; repeated 5 cycles. Data shown are means of distance moved in 5 s ± SE (n = 24 for each group at 5 dpf).

lpin1 knockdown affects gene expressions in the muscle and neuron development of zebrafish larva. The expression of somite marker deltaC in control embryos (A) and lpin1 morphant (A') embryos at 8-somite stage. Red arrows indicate newly formed somites and red bars indicate the length of presomitic mesoderm. Control embryos (B) and lpin1 morphant (B') embryos at 25 hpf were stained for actn3a. Note the shape of myotome, indicated by red angle, was changed in lpin1 morphants. The expression of neuron markers neurog1(C, C') at 50 hpf and isl1(D, D') at 25 hpf in control embryos (C, D) and lpin1 morphants (C', D'). In lpin1 morphants, the expression of neurog1 was reduced in the midbrain (mb), hindbrain (hb), and spinal cord (sp) (pink arrows); and that of islet1 was reduced in PMNs (yellow arrows). Scale bar: 100 μm. The expressions of muscle (E) and neuron (F) markers, as determined by qRT-PCR, were significantly changed in lpin1 morphants at 4 dpf and 1 dpf respectively. Lpin1 siRNA knockdown in C2C12 (G) and U87 cells (H) resulted in abnormal expressions of muscle and neuron markers. Values in (E, F, G and H) represent means ± SE of data from three independent experiments, *p< 0.05, **p< 0.01, and ***p< 0.001 (Student's t-test).

Loss of lipin 1 function results in activation of Notch signaling in zebrafish and mammalian cells. (A) Dual luciferase reporter assay to quantify the Notch signaling activity as her1 was coinjected with lpin1 morpholino into zebrafish embryos. (B) Lpin1-deficient zebrafish embryos showed upregulated gene expressions of Notch signaling components, which can be partially rescued by Notch inhibitor DAPT. (C) Dual luciferase reporter assay to quantify the Notch signaling activity as HES1 was coexpressed with the indicated LPIN1 cDNA in HEK293T cells. (D) Western blot and quantification analysis of LPIN1 wild type and mutant proteins. (E)LPIN1 siRNA in HEK293T cells achieved 65% knockdown with upregulated gene expressions of NOTCH signaling components. The abnormal upregulation of NOTCH signaling can be rescued by NOTCH inhibitor DAPT. Values in (A-E) represent means ± SE of data from three independent experiments, *p< 0.05, **p< 0.01, and ***p< 0.001 (Student's t-test).

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.