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We have identified a novel Tolloid-like metalloprotease, called Xolloid-related (Xlr), that is expressed during early Xenopus development. Transcripts for xlr are localized to the marginal zone of mid-gastrulae and are most abundant in ventral and lateral sectors. At neurula stages xlr is strongly expressed around the blastopore and in the pharyngeal endoderm, and more weakly expressed throughout the ventral half of the embryo. Transcripts are detected in the nervous system, particularly the hindbrain and spinal cord, and tailbud of tailbud stage embryos, with weaker expression in the anterior nervous system, otic vesicle, heart, and pronephric duct. Transcription of xlr is increased by BMP4 and decreased by Noggin and tBR, indicating that xlr is regulated by BMP signalling. Injection of xlr mRNA inhibits dorsoanterior development and the dorsal axis inducing ability of coinjected chordin, but not noggin or tBR, mRNA. Xlr conditioned media cleaves Chordin in vitro, indicating that this protease may regulate the availability of Chordin in vivo.
Fig. 3. Whole mount in situ hybridization analysis of xlr expression in Xenopus embryos. (A) Mid-gastrulae (stage 11.5) viewed from the vegetal pole (AP), dorsal surface uppermost. Transcripts are localized to the lateral and ventral marginal zone and a dorsal-anterior (ant) patch. (B) Neurula stage embryo (stage 18) showing strong expression of xlr around the blastopore (post) and an anterior-ventral (ant) patch close to the cement gland. Weaker expression is also detected throughout the ventral half of the embryo. (C) Sagital section through neurula stage embryo (stage 18) showing that the strong anterior-ventral expression of xlr is localized to the pharyngeal endoderm (pe), while the weaker ventral expression is localized to the ventral mesoderm (vm). (D) Early tailbud stage embryo (stage 26) showing strong expression of xlr in the tailbud (tb) and posterior neural plate. Expression is also detected in the proctodeum (pr) and hindbrain (hb). (E) Tailbud stage embryo (stage 32) showing strong expression of xlr in the tailbud (tb) and hindbrain (hb). Note that hindbrain expression spreads into anterior portions of the spinal cord. Weaker expression can also be seen in the head, heart (he), and pronephric duct (pnd). (F–I) Transverse histological sections through a stage 32 embryo, similar to that shown in (E), showing xlr expression in the neural tube (nt). no, notochord; so, somite; en, endoderm. (F) Section through the anterior hindbrain. (G) Section through the posterior hindbrain/anterior spinal cord. (H) Section through the posterior spinal cord. (I) Section through the posterior tail.
Fig. 5. Anterior defects in Xenopus embryos injected with 1.5 ng of xlr mRNA. (A) Uninjected tailbud stage embryos. (B) Weakly affected xlr injected embryo, note reduction in size of anterior structures. (C) Strongly affected xlr injected embryo, (D) Uninjected embryo showing xbf1 expression in the telencephalon and neural crest migrating into the branchial arches. (E) Weakly affected xlr injected embryo showing reduced xbf1 expression, indicating a reduced telencephalon. (F) Strongly affected xlr injected embryo with little xbf1 expression, indicating a dramatic reduction in the size of the telencephalon. (G) Uninjected embryo showing otxA expression in the forebrain and retina, and krox20 expression in rhombomeres 3 and 5 of the hindbrain. (H) Weakly affected xlr injected embryo showing reduced expression of otxA, indicating a reduction in the size of the forebrain and retina. Although two stripes of krox20 expression are evident, the gap between them is less distinct. (I) Strongly affected xlr injected embryo with greatly reduced otxA expression and a single krox20 stripe, indicating a dramatic reduction in the size of the forebrain, midbrain and hindbrain. (J) Uninjected embryo showing engrailed (en) expression at the midbrain-hindbrain boundary. (K) Weakly affected xlr injected embryo showing little affect on en expression. (L) Strongly affected xlr injected embryo with no en expression, indicating an absence of the midbrain and perhaps anterior portions of the hindbrain. The results suggest that injection of xlr mRNA causes severe reductions in anterior neural structures, including the forebrain, midbrain, and parts of the hindbrain.
Fig. 6. Ventralization of Xenopus gastrulae by Xlr. Xenopus embryos were injected with 1.5 ng of xlr mRNA and analysed at stage 11–12, by whole mount in situ hybridization. In all cases embryos are viewed from the vegetal pole and the dorsal axis is uppermost. (A) Uninjected embryo showing that not1 expression is localized to the dorsal midline (the notochord), extending anteriorly from the blastopore lip. (B) xlr injected embryo showing that not1 expression is retained, but is confined to the blastopore lip. (C) Uninjected embryo showing that myf5 expression is localized to dorsal-lateral mesoderm (somites) and is absent from the dorsal midline. (D) xlr injected embryo showing that myf5 expression expands into the dorsal midline of xlr injected embryos. As a consequence, and in contrast to control embryos, some dorsal midline cells will express both not1 and myf5. (E) Uninjected embryo showing that wnt8 expression is localized to lateral and ventral mesoderm. (F) xlr injected embryo showing a dorsal shift in wnt8 expression, although it is not expressed in the dorsal midline, and a reduction in ventral expression.