Roberts NA, Woolf AS, Stuart HM, Thuret R, McKenzie EA, Newman WG, Hilton EN.

MR/L002744/1 Medical Research Council , Wellcome Trust

             post-anal tail morphogenesis
             digestive tract morphogenesis

Xtr Wt + hpse2 MO(fig.4.e, f)
Xtr Wt + hpse2 MO(fig.5.b, d, f, h)

	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 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).
	Figure 2. Expression of UFS genes and immunolocalization of UFS proteins. (A) RT-PCR analysis of a developmental stage series derived from whole-embryo RNA. hpse2 was absent at Nieuwkoop–Faber Stage 1 but was detected from Stages 15 to 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 immunizing 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 localization 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 localization is indicated in red while other proteins are indicated in green. (A–C) Heparanase 2 colocalized with acetylated α-tubulin (AcTubulin) in the lateral zones of the Stage 30 neural tube. The flanking myotomes were also positive for heparanase 2. (D–F) At Stage 42, heparanase 2 was absent in the neural tube but myotomes, co-immunostained with the muscle-marker antibody 12/101 (it is the name of an antibody), remained positive. (G–I) The Stage 42 pronephric tubule did not display a specific IHC signal for heparanase 2; note that here, the weak signal is background autofluorescence. The two arrows indicate a proximal tubule which in H and I is seen to be reactive with Na+/K+-ATPase antibody. The set of three arrowheads in (G)–(I) demonstrate specific heparanase 2 immunostaining in the apical zone of epithelia lining the gut lumen. Scale bars are 50 µm.
	Figure 4. Morpholino knockdown of heparanase 2. (A) Schematic diagram of the X. tropicalis hpse2 gene, showing: exons (blue blocks); ATG MO and 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 missplicing 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 pre-mature 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. Visualization of motor neurons in parasagittal imaging plane. (A)–(F) whole-mounts 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 μm. (A–F) Across the top of each frame, a longitudinal section of the neural tube is evident, with the anterior to the left. In morphants, at Stages 36, 38 and 41, neurons which had emanated from the neural tube were regularly spaced but their axons lacked compact bundling and coherent directional extension seen in controls. The irregular topography of certain morphant nerves is indicated by arrowheads in (B), (D) and (F). (G and H) Morphants showed lack of clear separation of skeletal muscle blocks at somitic boundaries (arrowheads in H). (I and J) Nerve lengths (means ± SD), as assessed by determining the shortest distance between where they exited the neural tube and their overt termini (I) or by tracing individual nerves (J). The average length of 4–6 nerves per embryo was used to generate a value for each embryo, with 3–10 embryos in each experimental group, as indicated.
	Figure 6. Expression analyses in heparanase 2 knock-down embryos. RNA from pools of Stages 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 wt 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 knock-down embryos versus CTL MO embryos between Stages 30 and 36. This effect was no longer apparent at Stages 40 and 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 diamindino-2-phenylindole (DAPI), (C) heparanase 2 immunoreactivity, (D) pERK immunoreactivity, and (E) merged image. pERK+ nuclei (two are 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.

Al Badr, Exome capture and massively parallel sequencing identifies a novel HPSE2 mutation in a Saudi Arabian child with Ochoa (urofacial) syndrome. 2011, Pubmed

Al Badr, Exome capture and massively parallel sequencing identifies a novel HPSE2 mutation in a Saudi Arabian child with Ochoa (urofacial) syndrome. 2011, Pubmed
Arvatz, The heparanase system and tumor metastasis: is heparanase the seed and soil? 2011, Pubmed
Baldwin, Regulation of acetylcholine receptor transcript expression during development in Xenopus laevis. 1988, Pubmed , Xenbase
Bertolesi, Two heparanase splicing variants with distinct properties are necessary in early Xenopus development. 2008, Pubmed , Xenbase
Bertolesi, Two promoters with distinct activities in different tissues drive the expression of heparanase in Xenopus. 2011, Pubmed , Xenbase
Bilican, Induction of Olig2 precursors by FGF involves BMP signalling blockade at the Smad level. 2008, Pubmed
Bronchain, The olig family: phylogenetic analysis and early gene expression in Xenopus tropicalis. 2007, Pubmed , Xenbase
Cruz, Spinal ERK activation contributes to the regulation of bladder function in spinal cord injured rats. 2006, Pubmed
Dali, Signals that instruct somite and myotome formation persist in Xenopus laevis early tailbud stage embryos. 2002, Pubmed , Xenbase
Daly, Mutations in HPSE2 cause urofacial syndrome. 2010, Pubmed
Del Rio, In vivo analysis of Lrig genes reveals redundant and independent functions in the inner ear. 2013, Pubmed
Dichmann, Nkx6 genes pattern the frog neural plate and Nkx6.1 is necessary for motoneuron axon projection. 2011, Pubmed , Xenbase
Elejalde, Genetic and diagnostic considerations in three families with abnormalities of facial expression and congenital urinary obstruction: "The Ochoa syndrome". 1979, Pubmed
Fowler, The neural control of micturition. 2008, Pubmed
Gandhi, Computational analyses of the catalytic and heparin-binding sites and their interactions with glycosaminoglycans in glycoside hydrolase family 79 endo-β-D-glucuronidase (heparanase). 2012, Pubmed
Ganesan, More than meets the smile: facial muscle expression in children with Ochoa syndrome. 2011, Pubmed
Garcia-Minaur, Three new European cases of urofacial (Ochoa) syndrome. 2001, Pubmed
Guo, The LRIG gene family has three vertebrate paralogs widely expressed in human and mouse tissues and a homolog in Ascidiacea. 2004, Pubmed
Havis, Sim2 prevents entry into the myogenic program by repressing MyoD transcription during limb embryonic myogenesis. 2012, Pubmed , Xenbase
Hilton, Left-sided embryonic expression of the BCL-6 corepressor, BCOR, is required for vertebrate laterality determination. 2007, Pubmed , Xenbase
Huang, BDNF promotes target innervation of Xenopus mandibular trigeminal axons in vivo. 2007, Pubmed , Xenbase
Hufton, Genomic analysis of Xenopus organizer function. 2006, Pubmed , Xenbase
Jones, Xenopus: a prince among models for pronephric kidney development. 2005, Pubmed , Xenbase
Levy-Adam, Heparanase 2 interacts with heparan sulfate with high affinity and inhibits heparanase activity. 2010, Pubmed
Levy-Adam, Tumorigenic and adhesive properties of heparanase. 2010, Pubmed
Li, Reciprocal regulation of axonal Filopodia and outgrowth during neuromuscular junction development. 2012, Pubmed , Xenbase
Li, Axonal filopodial asymmetry induced by synaptic target. 2011, Pubmed , Xenbase
Lu, Expression of synapsin I correlates with maturation of the neuromuscular synapse. 1996, Pubmed , Xenbase
Mahmood, First HPSE2 missense mutation in urofacial syndrome. 2012, Pubmed
McCoy, Collectrin/tmem27 is expressed at high levels in all segments of the developing Xenopus pronephric nephron and in the Wolffian duct. 2008, Pubmed , Xenbase
McKenzie, Cloning and expression profiling of Hpa2, a novel mammalian heparanase family member. 2000, Pubmed
Mermerkaya, Nocturnal lagophthalmos in children with urofacial syndrome (Ochoa): a novel sign. 2014, Pubmed
Myler, Heparanase and platelet factor-4 induce smooth muscle cell proliferation and migration via bFGF release from the ECM. 2002, Pubmed
Ochoa, Can a congenital dysfunctional bladder be diagnosed from a smile? The Ochoa syndrome updated. 2004, Pubmed
Pang, Loss-of-function mutations in HPSE2 cause the autosomal recessive urofacial syndrome. 2010, Pubmed
Rafidi, Leucine-rich repeat and immunoglobulin domain-containing protein-1 (Lrig1) negative regulatory action toward ErbB receptor tyrosine kinases is opposed by leucine-rich repeat and immunoglobulin domain-containing protein 3 (Lrig3). 2013, Pubmed
Reiland, FGF2 binding, signaling, and angiogenesis are modulated by heparanase in metastatic melanoma cells. 2006, Pubmed
Ribes, Establishing and interpreting graded Sonic Hedgehog signaling during vertebrate neural tube patterning: the role of negative feedback. 2009, Pubmed
Rondahl, Lrig2-deficient mice are protected against PDGFB-induced glioma. 2013, Pubmed
Rudnicki, The molecular regulation of muscle stem cell function. 2008, Pubmed
Stuart, LRIG2 mutations cause urofacial syndrome. 2013, Pubmed
Takeda, Differential expression of ionic conductances in interstitial cells of Cajal in the murine gastric antrum. 2008, Pubmed
Tseng, Function and regulation of FoxF1 during Xenopus gut development. 2004, Pubmed , Xenbase
van Mier, Structural and functional properties of reticulospinal neurons in the early-swimming stage Xenopus embryo. 1989, Pubmed , Xenbase
van Mier, Development of early swimming in Xenopus laevis embryos: myotomal musculature, its innervation and activation. 1989, Pubmed , Xenbase
Vlodavsky, Extracellular matrix-resident basic fibroblast growth factor: implication for the control of angiogenesis. 1991, Pubmed
Wang, Downregulation of LRIG2 expression by RNA interference inhibits glioblastoma cell (GL15) growth, causes cell cycle redistribution, increases cell apoptosis and enhances cell adhesion and invasion in vitro. 2009, Pubmed
Weber, Muscarinic Acetylcholine Receptor M3 Mutation Causes Urinary Bladder Disease and a Prune-Belly-like Syndrome. 2011, Pubmed
Wong, Lrig1 controls intestinal stem-cell homeostasis by negative regulation of ErbB signalling. 2012, Pubmed
Woolf, Urofacial syndrome: a genetic and congenital disease of aberrant urinary bladder innervation. 2014, Pubmed
Zhou, The bHLH transcription factors OLIG2 and OLIG1 couple neuronal and glial subtype specification. 2002, Pubmed