A homolog of Subtilisin-like Proprotein Convertase 7 is essential to anterior neural development in Xenopus

Abstract

Background: Subtilisin-like Proprotein Convertase 7 (SPC7) is a member of the subtilisin/kexin family of pro-protein convertases. It cleaves many pro-proteins to release their active proteins, including members of the bone morphogenetic protein (BMP) family of signaling molecules. Other SPCs are known to be required during embryonic development but corresponding data regarding SPC7 have not been reported previously.

Methodology/principal findings: We demonstrated that Xenopus SPC7 (SPC7) was expressed predominantly in the developing brain and eye, throughout the neural plate initially, then more specifically in the lens and retina primordia as development progressed. Since no prior functional information has been reported for SPC7, we used gain- and loss-of-function experiments to investigate the possibility that it may also convey patterning or tissue specification information similarly to Furin, SPC4, and SPC6. Overexpression of SPC7 was without effect. In contrast, injection of SPC7 antisense morpholino oligonucleotides (MO) into a single blastomere at the 2- or 4-cell stage produced marked disruption of head structures; anophthalmia was salient. Bilateral injections suppressed head and eye formation completely. In parallel with suppression of eye and brain development by SPC7 knockdown, expression of early anterior neural markers (Sox2, Otx2, Rx2, and Pax6) and late eye-specific markers (β-Crystallin and Opsin), and of BMP target genes such as Tbx2 and Tbx3, was reduced or eliminated. Taken together, these findings suggest a critical role for SPC7-perhaps, at least in part, due to activation of one or more BMPs-in early patterning of the anterior neural plate and its derivatives.

Conclusion/significance: SPC7 is required for normal development of the eye and brain, possibly through processing BMPs, though other potential substrates cannot be excluded.

Figure 1. Sequence of SPC7.

The amino acid sequence is aligned with that of human SPC7. Identities are highlighted in yellow, similarities in green, the catalytic domain in magenta, and the proprotein convertase domain in cyan.

Figure 2. Expression of SPC7 mRNA in Xenopus embryos at different developmental stages.

(A) Real-time PCR. (B-N) Whole mount hybridization in situ (in panels B, C, E, and I-L, anterior is to the left. Dotted lines in C, E, G, and L indicate the approximate plane for images in D, F, H, and M, respectively). (B-C) Lateral (B) and dorsal (C) views of a stage 13 embryo showing staining in the early neural plate. (D) Hemisected stage 13 embryo displaying detectable expression of SPC7 in the sensorial layer of the neuroectoderm, but not in the underlying somitogenic mesoderm. (E) Dorsal view of stage 16 embryo showing SPC7 staining in the neural folds and presumptive eye field. (F) hemisected midneurula (stage 16) showing staining in the sensorial ectoderm (arrow). (G) Antero-dorsal view of a late neural fold (stage 18) embryo showing staining in the anterior part of neural plate and anterolateral edges. (H) Plastic section through over-stained embryo showing staining in neuroectoderm. Lateral (I) and dorsal (J) views of a stage 21–22 embryo showing prominent staining in the eye, cement gland, brain, and neural tube. (K) Lateral view of stage 26 embryo showing SPC7 expression in the retina, lens, cement gland, otic vesicle, and throughout the head and somites. (L) At stage 32, staining in the lens became more prominent, primarily because expression in the retina decreased substantially. (M) Plastic section of over-stained stage 35 embryo showing specific SPC7 localization in the lens. (N) Enlarged view of boxed area shown in M. (O-R) Stage 30–32 (O, P) and stage 35–37 (Q, R) paraffin sections analyzed for SPC7. (O) Total fluorescence. (P) Same section as in O showing specific SPC7 staining in retina and lens (Fast Red). (Q) Total fluorescence. (R) Same section as in Q showing specific SPC7 fluorescent staining localized to the lens. (NC = Notochord; EE = Epithelial Layer of Neuroectoderm; SE = Sensorial Layer of Neuroectoderm; SM = Somitogenic Mesoderm).

Figure 3. SPC7 antisense morpholino oligonucleotide causes dose-dependent disruption of eye and brain development.

(A, B) Embryos injected unilaterally with antisense SPC7-MO at the two cell (A) or four cell stage (B). Injected side is shown on the left. Both the two cell and four cell stage loss-of-function SPC7 experiments resulted in the same phenotype: lack of eye, lack of branchial arches, and diminished head structures on the MO-injected side. The morphology of the rest of the embryo appeared normal. (C) Dorsal view of tadpole-stage embryo injected at the four cell stage with 90 ng MO; the eye appears to be completely absent on the injected side. (D) Same embryo as in C showing MO location on the left. (E) Frontal section of embryo in C and D showing total loss of eye and mesencephalon on the SPC7-MO injected side. The uninjected side was unaffected. Arrows 1 and 2 show presumptive eye field and mesencephalon, respectively, on the MO injected side. In each experiment, embryos injected with control MO developed normally (not shown). (F, G) Histological analysis of control-MO injected (F) and SPC7-MO unilaterally-injected (G, injected side to the left) stage 35 embryos. The planes of section presented are shown diagrammatically at the top. As noted previously, the eye and brain are severely dysmorphic on the injected side; the otic vesicle and pronephros appear normal. (L =  Lens; R = Retina; NC = Notochord; MC = Mesencephalon; RC = Rhombancephalon; SC =  Spinal Cord; OV = Otic Vesicle; PN = Pronephros; EF = Eye Field).

Figure 4. 5′ sequence of SPC7.

(A) 5′ sequence of SPC7 and antisense morpholino oligonucleotides. Sequences corresponding to SPC7 MOs 1 and 2 are shown in green and magenta, respectively. Start codon is underlined. Arrow denotes 5′ primer used to construct synthetic mRNA used in rescue experiments. (B) Sequences of antisense morpholino oligonucleotides. (C, D) Bright and dark field images of stage 37 SPC7 antisense morpholino-only control embryo showing anophthalmic SPC7-MO phenotype on injected side. (E) Histological section through SPC7-MO embryo; note lack of distinguishable eye structures on the injected side and severe mesencephalic hypoplasia. (F, G) Bright and dark field images of stage 37 embryo coinjected with SPC7 antisense morpholino oligonucleotides and synthetic rescue mRNA, showing normal morphology on the injected side. For all rescue experiments, we used the 90 ng dose. Complete rescue as shown in (F-H) was observed in all embryos in each of three separate experiments (106 total embryos were scored); identical results were obtained with either SPC7-MO. (H) Histological section through coinjected embryo; both eyes appear normal.

Figure 5. Loss of SPC7 function does not affect mesodermal and posterior neuroectodermal marker expression patterns.

Whole mount hybridization in situ of embryos injected unilaterally with 90 ng SPC7 morpholino. Injected sides are shown to the left. (A, B) Lateral views of stage 35 embryo showing that muscle actin (dorsal mesoderm) staining was equally visible in both the injected and uninjected sides of the embryo. (C, D) Lateral views showing that HoxB9 (posterior neuroectoderm) expression was equally apparent in the posterior dorsal region of the embryo in both the injected and uninjected sides.

Figure 6. Loss of function of SPC7 diminishes expression of neural and eye markers.

(A-D) Whole mount hybridization in situ of embryos injected unilaterally with 90 ng SPC7 morpholino. Injected sides are shown to the right. (A) Frontal, dorsal, and lateral views showing Sox2 expression in control (left) and SPC7-MO unilaterally injected (right) embryos. Early- (top row) and mid-neurula (second row) embryos showing diminished Sox2 staining on the SPC7 morpholino injected side, most obviously in the eye anlage but apparent throughout the head folds (arrows). The loss of expression became more obvious at later stages. Row Three: antero-frontal view of stage 22 embryos. Sox2 staining was undetectable in retina, lens, and neural tube, and was reduced in the midbrain on the injected side. Row 4: dorsal view of the same embryos showing that Sox2 staining was absent in the neural tube on the injected side. Bottom row: Stage 35 embryo showed complete loss of Sox2 staining in the eye on the injected side. (B) Lateral views showing Rx2a expression was not detectable on the injected side (arrow). (C) Lateral views showing Pax6 expression pattern in control (left) and injected (right) stage 26–27 embryos. Pax 6 staining was absent in the eye field on the injected side (arrow). (D) Otx2 expression in uninjected control (left) and SPC7-MO unilaterally injected (right) embryos. Top row: antero-dorsal view of stage 14 embryos showing significantly diminished Otx2 expression on the injected side (arrow). Bottom row: Lateral views of control (left) and SPC7 MO-injected (right) sides of the same embryo showing barely detectable Otx2 expression on the injected side (arrow). (E, F) Frontal sections through the head of stage 35 embryos injected unilaterally (injected side to the right) with 30 ng SPC7-MO showing expression of β-Crystallin (E) and Opsin (F) expression on the non-injected side only.

Figure 7. BMP target genes Tbx2 and Tbx3.

Whole mount hybridization in situ of BMP target genes Tbx2 and Tbx3 in stage 33/34 embryos injected unilaterally with 90 ng SPC7 morpholino. Injected sides are shown to the right. (A, B) Lateral view of embryos showing Tbx2 expression was lost in the eye on the MO-injected side only (NR, neural retina; CG, cranial ganglion; OV, otic vesicle; BA, branchial arches; FN, frontonasal process). (C, D) Lateral view of stage 33/34 embryos showing a similar result for Tbx3.