Hum Mol Genet
August 15, 2014;
Heparanase 2, mutated in urofacial syndrome, mediates peripheral neural development in Xenopus.
Urofacial syndrome (UFS; previously Ochoa syndrome) is an autosomal recessive disease characterized by incomplete bladder
emptying during micturition. This is associated with a dyssynergia in which the urethral walls contract at the same time as the detrusor smooth muscle
in the body of the bladder
. UFS is also characterized by an abnormal facial expression upon smiling, and bilateral weakness in the distribution of the facial nerve
has been reported. Biallelic mutations in HPSE2
occur in UFS. This gene encodes heparanase
2, a protein which inhibits the activity of heparanase
. Here, we demonstrate, for the first time, an in vivo developmental role for heparanase
2. We identified the Xenopus orthologue of heparanase
2 and showed that the protein is localized to the embryonic ventrolateral neural tube
where motor neurons arise. Morpholino-induced loss of heparanase
2 caused embryonic skeletal muscle
paralysis, and morphant motor neurons had aberrant morphology including less linear paths and less compactly-bundled axons than normal. Biochemical analyses demonstrated that loss of heparanase
2 led to upregulation of fibroblast
growth factor 2/phosphorylated extracellular signal-related kinase signalling and to alterations in levels of transcripts encoding neural- and muscle
-associated molecules. Thus, a key role of heparanase
2 is to buffer growth factor signalling in motor neuron
development. These results shed light on the pathogenic mechanisms underpinning the clinical features of UFS and support the contention that congenital peripheral neuropathy is a key feature of this disorder.
Hum Mol Genet
Disease Ontology terms:
UROFACIAL SYNDROME 1; UFS1
[+] show captions
Figure 2. Expression of UFS genes and immunolocalisation of UFS proteins. (A) RT- PCR analysis of a developmental stage series derived from whole embryo RNA. hpse2 was absent at Nieuwkoop-Faber (NF) stage 1 but was detected from stage 15 to stage 45. lrig2 was expressed at stage 1, then weakly at stages 15/20, after which transcript levels became prominent. hpse1 was detected at all stages analysed. All three transcripts were present in adult urinary bladder (Blad). gapdh was included as a cDNA loading control. In the H2O column, no cDNA was used. (B) Transverse section through the trunk of a stage 40 embryo, with the dorsal surface uppermost. Brown colour shows positive IHC signal for heparanase 2, most prominent in the myotomes flanking the central neural tube, and fainter expression in the ventrolateral neural tube. (C) Adjacent section to (B), with the same orientation, showing minimal IHC signal after preincubating primary antibody with the immunising peptide. (D) Transverse section through the trunk at stage 30, with the dorsal surface uppermost. Prominent heparanase 2 immunoreactivity in detected in the ventrolateral regions of the neural tube (NT), in the notochord (Nc) and in the flanking skeletal muscle myotomes (My). (E) Adjacent section to (D), with the same orientation, immunostained for LRIG2. A similar pattern to that of heparanase 2 is observed, extending into the dorsal half of the neural tube. (F) Transverse section from a stage 42 embryo, with the left side uppermost. Three sections of gut are apparent, the archenteron having undergone coiling. Heparanase 2 immunoreactivity is observed in the luminal surface of the gut epithelium, and shown in closer detail in (G). B-G were counterstained with haematoxylin, rendering nuclei blue. Scale bars are 50 μm.
Figure 3. Tissue-specific localisation of heparanase 2. Transverse sections through the trunk of Xenopus embryos, as indicated in the diagrams shown on the right, imaged by immunofluorescence, with the neural tube outlined by blue dashed circles. In merged images, heparanase 2 localisation is indicated in red while other proteins are indicated in green. (A-C) Heparanase 2 colocalised with acetylated
Figure 4. Morpholino knockdown of heparanase 2. (A) Schematic diagram of the Xenopus tropicalis hpse2 gene, showing: exons (blue blocks); splice MO targets (red blocks) at the splice acceptor (MO1) and splice donor (MO2) sites of exon 2; PCR primers flanking exon 2 (black arrows); and the premature stop codon in exon 3 (red asterisk) generated by MO1. (B) Injection of increasing amounts of MO1 induced mis-splicing of hpse2, with 5 ng abolishing expression of wild type (wt) hpse2 mRNA in favour of a shorter mRNA (exon 2δ). (C) Sanger sequencing of the shorter PCR product confirmed absence of exon 2 (indicated by dotted vertical line). A novel, in-frame premature stop codon was generated in exon 3 (asterisk). (D) Injection of 5 ng MO1 led to near complete loss of heparanase 2 neural tube and myotome immunoreactivity, as demonstrated in this stage 40 embryo; CTL MO on left and MO1 on right. (E) Frequency (%) of the hypomotility, lack of gut looping (gut defect) and tail curvature (tail defect) phenotypes associated with administration of CTL MO or MO1, with total numbers of embryos assessed indicated by ‘n’. (F) The upper two images depict CTL MO-administered embryos and the lower two images show effects of MO1. Left- hand section depicts embryos viewed from the side; note the protruding proctodeum (white arrow) and tail curvature in the morphant. The two frames on the right depict the embryos viewed from their ventral aspects; note that gut coiling is present in the control embryo but not in the morphant.
Figure 5. Visualisation of motor neurons in parasagittal imaging plane. A-F were wholemounts immunostained with antibody to acetylated α-tubulin. This labels axons and also multiciliated round organs in the skin, the latter appearing as white ovals. G and H were probed with the muscle antibody 12/101. A, C, E and G are from CTL MO-administered embryos, while B, D, F and H are from MO1-administered embryos. Scale bars are 50
Figure 6. Expression analyses in heparanase 2 knockdown embryos. RNA from pools of stage 32 and 40 control (CTL MO) and morphant (hpse2 MO1) embryos was subjected to RT-PCR, with serial dilutions of cDNA depicted on the right of each row. drosha, which encodes an RNase III enzyme, was used as a housekeeping control. Morphants had: downregulated wild type hpse2 exon 2 and the appearance of the shorter exon 2δ amplicon; increased levels of hpse1 and lrig2; increased levels of fgf2 and of the neuronal precursor markers olig2 (at stage 40) and nkx6.1; upregulated myod1, encoding a skeletal muscle transcription factor, and downregulated myh11, encoding a smooth muscle myosin (at stage 40). Levels of syn1 and chrnb2, encoding synaptic molecules, were similar in morphants and controls.
Figure 7. pERK detected by Western blotting and IHC. (A) Western blots for pERK and tERK in sets of pooled embryos collected, as indicated, between stages 30 and 41. Note that the signal for pERK was clearly increased in heparanase 2 knockdown embryos versus CTL MO embryos between stages 30 and 36. This effect was no longer apparent at stage 40/41 (B- E) Transverse section of the neural tube of stage 32 uninjected embryo, with the dorsal surface uppermost, showing (B) all nuclei stained with DAPI (C) heparanase 2 immunoreactivity (D) pERK immunoreactivity (E) merged image. pERK+ nuclei (two are
35& 36 indicated by arrows) and heparanase 2+ cells (two are indicated by arrowheads) are detected in the lateral zones of the tube. Note that the cells with strong nuclear pERK immunostaining and those with strong heparanase 2 signals tend to be mutually exclusive. The image is representative of experiments on three embryos.
Figure 8. Schematic diagram of proposed heparanase 2 function in FGF signalling and motor neuron development. (A) In normal development, heparanase 2 inhibits the activity of heparanase 1, both biochemically by sequestering HSPGs (17) and possibly transcriptionally (this study). This inhibition maintains normal levels of FGF signalling and downstream genes within the embryo, allowing correct motor neuron functional development. (B) Loss of heparanase 2 causes upregulation of FGF signalling (and subsequent deregulation of downstream pathway components), perturbing expression of genes required for motor neuron development.
Figure 1. Alignment of the Xenopus heparanase 2 protein sequence with human and mouse orthologues. Xenopus heparanase 2 shares 80% identity (black highlight) and 88% similarity (black plus grey highlight) with human heparanase 2. The majority of non-conserved amino acids are in the N-terminal region (black bar), predicted to constitute a signal peptide. Putative HS-binding motifs are indicated by orange bars. Glycosylated amino acids are indicated by blue diamonds. Asn543, which undergoes a missense mutation in UFS (10), is conserved between species. The immunizing peptide used to generate the heparanase 2 antibody used in this study is indicated in green as is the nonsense mutation created by injection of the splice acceptor morpholino (MO1).