(A) Pedigree of the family; arrow points to the proband; triangular shapes indicate instances of spontaneous abortion. (B-F) MRI of upper and lower extremities demonstrates enlarged nerves. (B) Image showing greatly enlarged brachial plexus. Arrows indicate the thick peripheral nerves. (C) Sciatic nerve at the level of the femoral head (arrows). (D) Sciatic nerve at the level of the mid-thigh (arrows). (E) Posterior tibial (double arrows) and peroneal (single arrows) nerves at their origins. (F) Posterior tibial (double arrows) and peroneal (single arrows) nerves at the level of the fibular head. Scale bars, 3 cm. (G) Representative brain MRI (axial, T2-weighted) showing poor delineation of white and gray matter, indicating reduced myelination, when compared to age-matched control (H). Note the delineation of white and gray matter in control (arrows) that is not seen in the patient.

(A-C) ypel3 is maternally deposited and expressed during neural development in zebrafish. (A) Two-cell stage embryo. (B-C) Transverse sections. (B) At 24 hours postfertilization (hpf), ypel3 is expressed broadly in the spinal cord and sparsely in the somites (red arrows). (C) At 56 hpf, ypel3 is expressed in the spinal cord and in the region where the motor roots develop (red arrows). (D) Diagram of spinal cord domains, their derivatives, and molecular markers. The pMN domain (olig 2+) gives rise first to motoneurons, and then later oligodendrocytes (Briscoe et al., 2000; Jessell, 2000; Danesin and Soula, 2017). The p3 domain (nkx2.2+) is localized ventral to the pMN domain and gives rise to oligodendrocytes and a subset of perineurial cells (Clark et al., 2014; Kucenas et al., 2008b). Schwann cells (SC) are neural crest derivatives. Scale bar: 250 μm in A, 25 μm in B and C.

(A-F) Analysis of the pMN domain and its derivatives. Transverse sections. (A-C) WT. (D-F) maternal zygotic (mz) ypel3^-/-. (A, D) pMN domain and its derivatives labeled by olig2:kaede transgene. (B, E) Topro nuclear labeling. (C, F) Merge. (G-J) Analysis of motoneuron development in mz ypel3 mutants. Lateral views. Motoneurons are labeled by the mnx1:gfp transgene. (G-H) WT embryo at 26 hpf (G) and larva at 52 hpf (H). (I-J) mz ypel3 mutant at 26 hpf (I) and larva at 52 hpf (J). Images were taken at the level of somites 6 to10. (K-L) Quantification of the number of motoneurons at 26 hpf (K) and at 52 hpf (L). Bars show +/- SEM. Scale bars: 5 μm in A-F, 25 μm in G-J.

(A-B) WT larvae at 52 hpf (A) and at 72 hpf (B). (C-D) mz ypel3 mutants at 52 hpf (C) and at 72 hpf (D). Arrows indicate myelinating oligodendrocytes (double mRFP, mGFP positive cells) in (A) and (C). Black arrowheads show myelinating oligodendrocytes expressing mbpb in (B and D). Scale bars: 25 μm. Images are lateral views. (E-H) Quantification of the number of myelinating oligodendrocytes at 52 hpf (E), mbpb expressing cells at 72 hpf (F), EdU positive cells at 52 hpf (G) and 72 hpf (H). Bars represent +/- SEM.

(A-B) sox10 mRNA in situ hybridization (ISH) in WT (A) and mz ypel3 mutant (B) showing maintained sox10 expression (red arrows) in the mz ypel3 mutant at 26 hpf. (C-D) Schwann cell precursors migrate correctly in the region of the developing motor nerves in the mz ypel3 mutant. WT (C) and mz ypel3 mutant (D) embryos at 26 hpf. Arrowheads indicate Schwann cell precursors. (E-F) Initial ensheathing of motor axons by Schwann cells occurs properly in the mz ypel3 mutant. WT (E) and mz ypel3 mutant (F) larvae at 56 hpf. Arrowheads indicate motor nerves. Arrow indicates ventral Schwann cells ensheathing motor nerve. (G-H) Schwann cells fail to form normal structures in the mz ypel3 mutant. At 5 days postfertilization (dpf), in WT (G), Schwann cells form slender rod-like structures (arrows). At this stage, the mRFP signal has cleared from the ventral myotome. In the mz ypel3 mutant (F), Schwann cells fail to wrap the motor axon tightly and sox10 expression as indicated by mRFP is maintained within the ventral region of the myotome. Yellow dashed lines outline melanocytes. Green arrowheads indicate blood vessels. All images are lateral views. Scale bars: 25 μm. (I) Quantification of the average number of motor nerves per somite labeled with sox10:mRFP at 56 hpf. (J) Quantification of the average number of motor nerves per somite labeled with sox10:mRFP at the level of ventral myotome. Bars represent +/-SEM.

(A) WT larva at 56 hpf. Perineurial glia (expressing nkx2.2a:GFP, green) have migrated out of the CNS and begun to ensheath the motor nerve. White arrows indicate perineurial glia associated with the immature motor nerve. (B) mz ypel3^-/- mutant at 56 hpf. At this stage, perineurial glia are largely absent, and the few that migrate out of the CNS appear wispy and thin (yellow arrows). (C) In WT larvae at 5 dpf, perineurial glia are tightly associated with Schwann cells (expressing sox10:mRFP, magenta) forming slender tubes (white arrows). A small percentage of WT perineurial cells have not formed these slender tubes (arrowhead). (D) In mz ypel3^-/- mutants, at 5 dpf, perineurial glia are overgrown and fail to associate with the Schwann cells. Yellow dashed lines label the somite boundaries. Scale bar: 25 μm. (E-F) Quantification of the average number of motor nerves per somite labeled with GFP (E) and the average number of motor nerves per somite with slender tube morphology (F). (G-G”) WT larvae at 7 dpf. Perineurial glia have completely wrapped the Schwann cells forming a tubular structure (arrows). Some mRFP expression remains within the Schwann cells. (G) Merge. (G’) mRFP expression is clustered within the GFP positive domain (outlined with white dashed lines), which is composed of perineurial cells. (G”) Perineurial cells form a tubular structure (outlined with white dashed lines). (H-H”) mz ypel3 mutant at 7 dpf. Perineurial glia are overgrown and form a defasciculated structure. (H) Merge. (H’) mRFP signal is elevated in the mz ypel3 mutant Schwann cells and delineates three defasciculated structures (1, 2, 3). (H”) mz ypel3 mutant perineurial glia fail to wrap Schwann cells correctly resulting in an enlarged nerve (outlined with white dashed lines). White horizontal line in (G-H”) spinal cord ventral border. White horizontal dashed line (A-D) horizontal myoseptum. SC: spinal cord. *: loose perineurial cells. All images are lateral views. Scale bars: 25 μm in A-D, 5 μm in G-H”.

(A-D) Myelin basic protein b (Mbpb) expression. Lateral views of embryos labeled for Mbpb at 7 dpf. (A) WT. (B-D) mz ypel3 mutants. Mbpb levels are undetectable in the spinal cord (sc) and variably reduced in the motor nerves (arrows). Yellow arrow shows a fasciculated nerve with a homogenous distribution of the Mbpb signal in WT. White arrows show defasciculated nerves in mz ypel3 mutants. Mutant nerves have variable levels and an uneven distribution of Mbpb. White horizontal dashed lines indicate the spinal cord ventral border in (A-B) and the horizontal myoseptum in (C-D). Yellow dashed chevron: somite borders. Scale bar: 25 μm. (E) Lipidomics panel showing an increase in phosphatic acids (PA) and ceramides (Cer) except Cer(d18:1/20:0) in the mz ypel3 mutant compared to WT. (F-K) Adult mz ypel3^-/- mutants have behavioral and locomotion defects. (F-H) Representative swimming traces of WT (F), heterozygous ypel3^b1309 (G), and homozygous mz ypel3^b1309 mutants (H). (I-K) Quantification of total distance traveled (I), average relative distance from the surface (J), and percent time spent in motion (K). +/+: WT. +/-: heterozygous. -/-: mz ypel3 mutants. Bars represent +/- standard error of the mean (+/- SEM).

YPEL3 is evolutionarily conserved and expressed within the developing CNS and in the motor nerve. (A) Alignment of human and zebrafish YPEL3 protein variants. The YPEL3 gene encodes two transcript variants: a long variant (NM_031477) that gives rise to a protein composed of 157 amino acids (NP_113665.3) and a short variant (NM_001145524) that encodes a protein of 119 amino acids (NP_001138996.10. One zebrafish ypel3 transcript variant (NM_212790.1) has been characterized to date. It encodes a protein of 119 amino acids (NP_997955.1). TV1: Long human YPEL3 protein variant. TV2: Short human YPEL3 protein variant. danio: Ypel3 zebrafish protein. Blue arrowhead indicates the location of Val92 in the long human isoform. (B) Section of 80 hpf zebrafish larva showing ypel3 expression at the level of the spinal cord and motor nerve (red arrows). (C) Section of the same specimen at a more rostral level of the spinal cord. Note the absence of ypel3 in the ventral medial region. Scale bar: 25 μm.

ypel3b1309 and ypel3b1310 CRISPR engineered alleles are equivalent to the YPEL3 variant. (A) Zebrafish mutant alleles created by CRISPR/Cas9 technology. Two alleles were generated using the same single guide RNA. Both ypel3b1309 and ypel3b1310 are frameshift mutations, p.Asp56Glyfs*1 and p.Gly60Argfs*4, respectively. (B) Schematic representations of WT human and zebrafish YPEL3 variants. WT YPEL3: WT human short isoform of 119 amino acids (aa). WT Ypel3: wild-type zebrafish isoform of 119 aa. (C) Schematic representation of mutant YPEL3 variants based on the short isoform annotations. UDN variant: predicted YPEL3 variant found in the affected person. B1309, B1310: mutant zebrafish protein variants. Light blue: endogenous amino acids. Green: Exogenous amino acids introduced by the frameshift mutation.

Loss of Ypel3 does not affect motor axon development. (A-B) Quantification of motor nerve width or thickness in WT and mz ypel3 mutants at 26 (A) and 52 (B) hpf. (C-D) Quantification of average motor nerve length (C) and distance between motor nerves in WT and mz ypel3 mutants at 80 hpf.

Loss of Ypel3 transiently depletes OPC population and results in oligodendrocyte filopodial defects. (A) Average number of GFP+, mRFP+ double positive oligodendrocytes per somite as a function of developmental stage. (B) Average number of filopodia per myelinating oligodendrocyte. (C) Average filopodial length.

sox10+ olig2+ oligodendrocyte population recovers in mz ypel3 mutants. (A, B) Lateral views at the level of the spinal cord from somites 8 to 11 of 80 hpf larvae. WT and mz ypel3 mutant embryos were photoconverted at 26 hpf and then imaged at 80 hpf. As expected, primary motoneurons are labelled magenta (arrowhead). Newly added cells are labeled green (Tg[olig2:kaede]). Oligodendrocytes are colabeled with Tg[olig2:kaede]) and Tg[sox10:mRFP] (arrows in A and B). (C) Quantification of the average total number of sox10+, olig2+ oligodendrocytes at 80 hpf in WT and mz ypel3-/- mutants. (D) Quantification of the average total number of nkx2.2+, sox10+ oligodendrocytes at 7 dpf.

Quantification of the number of sox10:mRFP cells at the level of the motor nerves at 28 hpf.

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.