**Background:** Mutations in PRDM15 cause a syndromic form of holoprosencephaly (HPE) known as Galloway–Mowat syndrome (GAMOS), characterized by early-onset steroid-resistant nephrotic syndrome and brain anomalies, often accompanied by ocular defects such as anophthalmia, microphthalmia, and coloboma. Previous work showed that Prdm15 regulates WNT/PCP signaling in mouse embryos, but the role of Prdm15 in anterior neural development and its relationship with Wnt4 signaling remained unclear. This study uses Xenopus laevis as a vertebrate model to investigate the function of Prdm15 in eye, head, and brain development and to test whether Wnt4 acts downstream of Prdm15.
**Methods:** The spatiotemporal expression of prdm15 was analyzed by whole-mount in situ hybridization (WMISH) from cleavage stages (stage 5) through organogenesis (stage 43). A morpholino oligonucleotide (MO) targeting prdm15 was injected unilaterally into one dorsal animal blastomere of eight-cell-stage X. laevis embryos (15 ng per embryo). Co-injection of GFP RNA (0.5 ng) was used to monitor injection efficiency. Control MO (CoMO) was used as a negative control. Rescue experiments were performed by co-injecting human full-length PRDM15 wild-type (WT) RNA (0.5 ng), or human PRDM15 RNA carrying the PR/SET domain mutation M154K (c.461T>A) or the zinc finger domain mutation C844Y (c.2531G>A). Additional rescue experiments used wnt4 RNA (50 pg), constitutive active JNK1 (caJNK1) RNA (1 ng), or disheveled deletion constructs dvlΔDIX and dvlΔDEP (100 pg each). Phenotypes were assessed by measuring eye area, coloboma angle, head area, head width, and interocular distance. Retinal lamination was examined by WMISH with cell-type-specific markers (rho, prox1, vsx1, pax6, pou4f1) and lens markers (celf1, cryba1). Cranial cartilage was visualized by Alcian blue staining, and cranial nerves by 3A10 antibody staining. Gene expression changes were quantified by WMISH and computer-based area/intensity measurements using ImageJ2. Statistical significance was determined by Mann–Whitney rank-sum test; error bars represent SEM.
**Key Results:** prdm15 transcripts were detected in the animal pole at stage 5, and from stage 13 onward in the anterior neural plate, neural tube, eye vesicle, developing retina, lens, retinal pigment epithelium, ciliary marginal zone, somites, pronephric anlage, brain, and migrating neural crest cells (mandibular, hyoid, and branchial arches). Prdm15 MO injection caused a spectrum of anterior defects: microphthalmia (smaller eye area), coloboma, disordered retinal lamination (displaced photoreceptor, horizontal, bipolar, amacrine/ganglion, and ganglion cells), and smaller lens (reduced celf1 and cryba1 expression area). Head area, head width, and interocular distance were significantly reduced. Cranial cartilages (especially branchial arches) were narrowed or deformed, and cranial nerves were shortened or disorganized. NCC marker genes (snai2, egr2, twist1, foxd3) were significantly reduced at stages 20–23. Brain-specific markers (rgma, otx2, pax6, egr2) showed reduced expression mainly in midbrain and hindbrain; forebrain marker emx1 was not significantly affected. Proliferation (pH H3 staining) was decreased in anterior neural tissue. Co-injection of human PRDM15 WT RNA rescued eye and head phenotypes in most embryos. The PR/SET domain mutant (M154K) also rescued, but the zinc finger mutant (C844Y) only partially rescued (smaller eyes, more coloboma). wnt4 expression area and intensity were significantly reduced in Prdm15 morphants at stages 20 and 28, and its direct target alcam was also reduced. Co-injection of wnt4 RNA partially rescued the Prdm15 MO-induced reduction of rax, pax6, snai2, and foxd3 expression at stages 13–23, and significantly rescued the eye and head phenotypes. Furthermore, co-injection of caJNK1, dvlΔDIX (non-canonical pathway activators), or dvlΔDEP (canonical pathway activator) all significantly rescued the Prdm15 MO-induced eye/head phenotype.
**Clinical Implications:** This study demonstrates that Prdm15 is essential for anterior neural development in Xenopus laevis, and its depletion recapitulates key features of GAMOS/HPE, including microphthalmia, coloboma, microcephaly, and craniofacial defects. The finding that Prdm15 acts upstream of both canonical and non-canonical Wnt4 signaling provides a mechanistic explanation for the ocular and neural anomalies in patients with PRDM15 mutations. The partial rescue by the C844Y variant confirms its pathogenicity in anterior neural tissues, while the M154K variant appears less deleterious in this context. These results suggest that Wnt4 pathway components could be potential therapeutic targets for GAMOS-associated neural defects. The study also highlights the value of X. laevis as a model for functional evaluation of human PRDM15 variants.