Matrix metalloproteinases (MMPs) constitute a large group of endoproteases that play important functions during embryonic development, tumor metastasis and angiogenesis by degrading components of the extracellular matrix. Within this family, we focused our study on Mt4-mmp (also called Mmp17) that belongs to a distinct subset that is anchored to the cell surface via a glycosylphosphatidylinositol (GPI) moiety and with the catalytic site exposed to the extracellular space. Information about its function and substrates is very limited to date, and little has been reported on its role in the developing embryo. Here, we report a detailed expression analysis of Mt4-mmp during mouse embryonic development by using a LacZ reporter transgenic mouse line. We showed that Mt4-mmp is detected from early stages of development to postnatal stages following a dynamic and restricted pattern of expression. Mt4-mmp was first detected at E8.5 limited to the intersomitic vascularization, the endocardial endothelium and the dorsal aorta. Mt4-mmpLacZ/+ cells were also observed in the neural crest cells, somites, floor plate and notochord at early stages. From E10.5, expression localized in the limb buds and persists during limb development. A strong expression in the brain begins at E12.5 and continues to postnatal stages. Specifically, staining was observed in the olfactory bulb, cerebral cortex, hippocampus, striatum, septum, dorsal thalamus and the spinal cord. In addition, LacZ-positive cells were also detected during eye development, initially at the hyaloid artery and later on located in the lens and the neural retina. Mt4-mmp expression was confirmed by quantitative RT-PCR and western blot analysis in some embryonic tissues. Our data point to distinct functions for this metalloproteinase during embryonic development, particularly during brain formation, angiogenesis and limb development.

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The distribution of the mRNA of different C‐terminal splice variants of the μ‐opioid receptor in rat CNS was assessed by RT‐PCR. The mRNA species for MOR1, MOR1A and MOR1B were readily detectable and distributed widely throughout the rat CNS, with levels of MOR1 and MOR1A mRNA being overall greater than for MOR1B. We did not find convincing evidence that significant levels of MOR1C, MOR1C1, MOR1C2 and MOR1D are present in rat CNS. To examine possible differences in the agonist‐induced regulation of MOR1, MOR1A and MOR1B, we expressed these constructs in HEK293 cells along with G‐protein‐coupled inwardly rectifying K+ channel subunits and measured the rate and extent of desensitisation of (d‐Ala2,N‐Me‐Phe4,glycinol5)‐enkephalin (DAMGO)‐ and morphine‐induced G‐protein‐coupled inwardly rectifying K+ currents. Morphine‐induced desensitisation was rapid for all three splice variants (t½: 1.2–1.7 min) but DAMGO‐induced desensitisation was significantly slower for MOR1B (t½ 4.2 min). Inhibition of endocytosis by expression of a dynamin‐dominant negative mutant increased the rate of DAMGO‐induced desensitisation of MOR1B. These data show that some splice variants of μ‐opioid receptor are widely expressed in rat CNS but question the existence of others that have been reported in the literature. In addition, whereas the rate of desensitisation of MOR1 and MOR1A is agonist‐independent, that for MOR1B is agonist‐dependent.

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