Fig 1.
Mt4-mmp expression during early mouse embryonic development.
(A-E) Whole-mount β-gal staining in 6–10 somite stage Mt4-mmpLacZ/+ embryos. In a ventral view of an 8 somite-stage embryo, it becomes apparent the staining in the endocardial tissue of the primitive heart tube (asterisks in A, B). Staining is observed in the premigratory neural crest cells of a 6 somite-stage embryo (arrow in C). The premigratory and migratory neural crest cells (arrow and asterisk in D) express Mt4-mmp in 10 somite-stage embryos. Somites also show β-gal positive cells (E) in a pattern compatible with intersomitic arteries (arrow in E) beginning to branch from the dorsal aorta. (F-I) β-Gal staining of coronal sections from Mt4-mmpLacZ/+ embryos highlights Mt4-mmp distribution in neuroepithelial cells of the neural tube (F), somites (G), floor plate (arrow in H) and dorsal aorta (H) at E8.5 embryos (n = 8). Localization of β-galactosidase was detected in the intersomitic vasculature (arrow in I) and in the perineuronal vascular plexus (arrowheads in I). Detail of LacZ staining in the intersomitic blood vessels (arrows in J). Double-labelled cells for the endothelial markers ERG (red, arrows in K) or CD31 (magenta, arrows in L) and β-gal (green) demonstrate that cells expressing Mt4-mmp in this location are endothelial cells. Abbreviations: da, dorsal aorta; fp, floor plate; s, somite; NCC, neural crest cells; NT, neural tube. Scale bars: 200 μm (A-C), 100 μm (D, E), 50 μm (F-I), 30 μm (J-L).
Fig 2.
Whole-mount β-gal stained embryos from E9.5 to E14.5.
(A-C) E9.5 embryos (n = 7) show reporter expression in the allantois (A), the intersomitic arteries sprouting from the dorsal aorta (arrow in B) and in the posterior portion of the somites (arrow in C). (D-F) This vascular pattern of expression persists during somite development as shown in D and F. Other embryonic tissues like the limb buds, the mesenchymal tail tip (arrow in D and G), the allantois, the atrium and aorta (E) show β-gal staining at E10.5 (n = 5) and E11.5 (n = 3). At later stages of development, expression persists in the limbs (G-I) and appears in the primordium of follicle of vibrissa (arrows in H and I), lens (white arrow in H), eyelid (empty arrow in I), nose, pinna of the ear and brain (n = 6 for E12.5 and E14.5 embryos). Abbreviations: a, atrium; al, allantois; da, dorsal aorta; FL, forelimb; HL hindlimb; v, ventricle. Scale bars: 1 mm (D, F-I), 500 μm (A, E) and 250 μm (B, C).
Fig 3.
Mt4-mmpLacZ/+ expression during cardiovascular development.
(A-C) β-gal staining at distinct stages of heart development reveals the expression of Mt4-mmp in the endocardial endothelium of the primitive heart tube at E8.5 (arrows in A) and the epicardium of the atrium and ventricle (arrows in B and C) at E14.5 (B) and E18.5 (C). Expression was also detected in other vascular structures as the branch from internal carotid artery at E11.5 (D) and E14.5 (E) and the allantois at E9.5 (F). By E8.5 β-gal positive cells were detected in the posterior branch of primary head vein (arrow in G) and the dorsal aorta (arrow in H). Labelling in the dorsal aorta persists later in development and by E14.5 Mt4-mmp expression is not only restricted to the endothelium but also located in smooth muscle cells of the aortic wall (I). Double labelling immunohistochemistry for CD31 (J,K) and β-gal (J,L) confirmed the expression of Mt4-mmp in endothelial cells of the dorsal aorta at E10.5 (arrows in K and L). Abbreviations: a, atrium; al, allantois; ca, carotid artery; da, dorsal aorta; ec, endothelial cells; NT, neural tube; v, ventricle. Scale bars: 200 μm (B, C), 50 μm (A, E-H, J-L) and 20 μm (D, I).
Fig 4.
Real-time PCR and western blot analysis of Mt4-mmp expression in mouse embryonic tissues.
(A) Real-time PCR analysis was performed using cDNA prepared from E10.5, E12.5 and E14.5 WT, HT and KO mouse embryonic tissues. Mt4-mmp expression increases as development proceeds and particularly, high RNA levels were detected at E14.5 in distinct brain regions as the cerebral cortex, olfactory bulb, mesencephalon, hindbrain and spinal cord. Mt4-mmp RNA levels of were normalized to the housekeeping GAPDH levels and relative to expression levels in the adult cerebral cortex. Data shown are representative of three independent experiments and expressed as the mean ± SEM. In all cases, mRNA levels were significantly reduced in the HT compared to the WT tissues at the distinct embryonic stages analysed. Different letters indicate significant differences (p< 0.05) among distinct tissues of the same embryonic stage by ANOVA and post-hoc analysis (SNK). (B) Total cell lysates from forelimbs (FL), spinal cord (SC) and cerebral cortex (Ctx) of E14.5 WT, HT and KO embryos were analysed by western blotting with the anti-MT4-MMP antibody and anti-α-tubulin antibody for loading control. Specific bands correspond to the precursor of the GPI-anchored form (63 kDa, closed arrow) and the GPI-anchored latent and active forms (55 and 50 kDa, opened arrow) of MT4-MMP. Quantification of Western blot shows significant higher levels of protein expression in the cerebral cortex compared to the spinal cord and the forelimb of WT embryos. MT4-MMP protein levels were significantly decreased in the HT cerebral cortex compared to the WT embryos and were totally absent in all KO embryonic tissues analysed (**, indicates significant differences at p< 0.01).
Fig 5.
Mt4-mmpLacZ/+ distribution during mouse limb development.
Mt4-mmpLacZ+/- expression pattern was studied in whole mount limbs at distinct embryonic stages. (A, B) Distal (A) and proximal regions (B) of the limb buds as well the ZPA region show β-gal staining at E10.5. (C, D) Mt4-mmp expression was detected in distal regions of the hand and footplate (asterisks in C,D) as well as in the developing stylopodial and zeugopodial segments (thick and thin arrows in C). (E-J) β-gal staining from E12.5 to E14.5 appears in the stylopodium, zeugopodium and proximal regions of the handplate (E, G-J). In addition, anterior mesenchymal expression is observable in the E12.5 footplate (F). Mt4-mmp expression was first observed in digits at E12.5 (arrows in E) evolving to a pattern restricted to metacarpals and metatarsals of digits II, III, IV and V, either in dorsal (G, H) and ventral side (I, J) at E14.5. (K-N) Paraffin cross sections though hindlimb (K, L) and forelimb (M, N) showed β-gal positive cells in the mesenchyme (arrow in N) and in endothelial cells (ec) of blood vessels entering the limbs (L). The asterisk corresponds to the AER. (O-T) E14.5 limb crossed-sections from distal (O, P) to proximal (Q-T) levels evidence β-gal staining in the tendon blastemas (arrows in P), in subepidermal mesenchymal cells (arrows in Q, S) and in ventral muscle blocks (R) as well as in blood vessels entering the muscles (empty arrows in T). In all sections dorsal is up; ventral is down. Abbreviations: a, anterior; AER, apical ectodermal ridge; ec, endothelial cells; p, posterior; r, radius; u, ulna; v, blood vessel; ZPA, zone of polarizing activity. Scale bars: 500 μm (C-J), 250 μm (A, B, O, Q, S), 100 μm (M, P, T), 50 μm (K, R) and 25 μm (L, N).
Fig 6.
Expression of Mt4-mmp in the developing mouse brain.
β-gal staining of coronal sections of embryonic E11.5 (A) and longitudinal sections of E18.5 Mt4-mmpLacZ/+ embryos (B) (n = 3) revealed expression of Mt4-mmp in the ventral column of motoneurons in the spinal cord. In addition, β-gal positive cells were detected in the floor plate of the mesencephalon at E11.5 (C). LacZ-positive cells were located in the granular cell layer of the olfactory bulb at embryonic stage E14.5 (D) and postnatal stages (E). Reporter expression was also detected in the septum, striatum, at deep and superficial layers of the cerebral cortex, the hippocampus and the dorsal thalamus and dorsal lateral geniculate nucleus at E14.5 (H) and P1 (F,G,I; n = 3). Mt4-mmp expression was located at the rhombic lip at E12.5 (arrow in J) and the cerebellum primordium at E14.5 (K). By postnatal stages, many β-gal-positive cells were located at the cerebellum possibly corresponding to Purkinje cells and in the external granule layer (arrow in L). At this level, staining was also detected in the choroid plexus of the IV ventricle (empty arrow in L). Abbreviations: Cb, cerebellum; cp, choroid plexus; CP, cortical plate; CPu, caudate putamen; gcl, granular cell layer; dLGN, dorsal lateral geniculate nucleus; dTh, dorsal thalamus; Hp, hippocampus; MZ, mantle zone; NCx, neocortex; ob, olfactory bulb; rl, rhombic lip; S, septum; SVZ, subventricular zone; IVv, IV ventricle. Scale bars: 500 μm (F, G, I), 200 μm (B, D, E, H, K, L) and 100 μm (A, C, J).
Fig 7.
Mt4-mmp expression in sensory organs of the developing mouse embryo.
(A-D) Coronal sections through the embryo show the expression of scattered β-gal positive cells dorsally in the olfactory epithelium at the entrance to the nasal cavity at E12.5 (A) and E14.5 (B,D) as well as in the surrounding mesenchyme (arrow in B). High expression of Mt4-mmp was also detected in mesenchymal cells adjacent to the primordium of the follicle of vibrissae (B,C). (E-F) At this embryonic stage, Mt4-mmp expression was also detected in the ventral extremity of the upper jaw (asterisk in E), epithelium of the nose (arrows in E) and median fibrous septum (arrow) and intrinsic muscle of the tongue (F). (G-H) LacZ-positive cells were distributed in the pinna of the ear at E14.5 and E16.5 stages. Empty arrows indicate mesenchymal cells in the pinna. (I-K) From E10.5, the first Mt4-mmplacZ/+ cells were detected in the otic vesicle (arrow in I) and persists at E12.5, in the caudal portion of the saccule (arrow in J), the vestibulocochlear (VIII) ganglion complex (arrowheads in J) as well as in the semicircular canal (arrow in K). Abbreviations: d, dorsal; EAC, external auditory canal; es, endolymphatic sac; N, nasal cavity; oe, olfactory epithelium; otv, otic vesicle; Pi, pinna; s, saccule; sc, semicircular canal; tg, tongue; v, ventral; vb, sensory vibrissae; VIIIgc, vestibulocochlear (VIII) ganglion complex. Scale bars: 200 μm (B, E, F, G, H), 100 μm (A, C, D, J, K) and 50 μm (I).
Fig 8.
Expression of Mt4-mmpLacZ/+ during mouse eye development.
β-gal staining of coronal sections through the eye at different stages of development revealed Mt4-mmp expression in the hyaloid artery from E10.5 to E12.5 embryos (arrows in A-C). Scattered LacZ-positive cells were first detected in the lens by E12.5 and persist at E14.5 (opened arrows in C, D). A strong Mt4-mmp expression in the eyelids (arrow in D) and some β-gal positive cells was shown in the neural retina at E14.5 (D). At postnatal stages, expression of Mt4-mmp was distributed particularly in the retinal ganglion cell layer (E, F) of the neural retina and persists in the eyelids (arrow in E). Abbreviations: ha, hyaloid artery; ln, lens; NR, neural retina; RGC, retinal ganglion cells; RPE, retinal pigmented epithelium. Scale bars: 200 μm (D, E), 100 μm (B, C) and 50 μm (A, F).