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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.
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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
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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.
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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
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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.
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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.
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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).
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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