Detection of gelatinolytic activity in developing basement membranes of the mouse embryo head by combining sensitive in situ zymography with immunolabeling

Abstract

Genetic evidence indicates that the major gelatinases MMP-2 and MMP-9 are involved in mammalian craniofacial development. Since these matrix metalloproteinases are secreted as proenzymes that require activation, their tissue distribution does not necessarily reflect the sites of enzymatic activity. Information regarding the spatial and temporal expression of gelatinolytic activity in the head of the mammalian embryo is sparse. Sensitive in situ zymography with dye-quenched gelatin (DQ-gelatin) has been introduced recently; gelatinolytic activity results in a local increase in fluorescence. Using frontal sections of wild-type mouse embryo heads from embryonic day 14.5-15.5, we optimized and validated a simple doublelabeling in situ technique for combining DQ-gelatin zymography with immunofluorescence staining. MMP inhibitors were tested to confirm the specificity of the reaction in situ, and results were compared to standard SDS-gel zymography of tissue extracts. Double-labeling was used to show the spatial relationship in situ between gelatinolytic activity and immunostaining for gelatinases MMP-2 and MMP-9, collagenase 3 (MMP-13) and MT1-MMP (MMP-14), a major activator of pro-gelatinases. Strong gelatinolytic activity, which partially overlapped with MMP proteins, was confirmed for Meckel's cartilage and developing mandibular bone. In addition, we combined in situ zymography with immunostaining for extracellular matrix proteins that are potential gelatinase substrates. Interestingly, gelatinolytic activity colocalized precisely with laminin-positive basement membranes at specific sites around growing epithelia in the developing mouse head, such as the ducts of salivary glands or the epithelial fold between tongue and lower jaw region. Thus, this sensitive method allows to associate, with high spatial resolution, gelatinolytic activity with epithelial morphogenesis in the embryo.Keywords Mouse embryo Á Craniofacial development Á Matrix metalloproteinase Á Basement membrane Á In situ zymography Á Dye-quenched gelatin Á Immunofluorescence

Site-Specific Expression of Gelatinolytic Activity during Morphogenesis of the Secondary Palate in the Mouse Embryo

“…2G–I), although activity was increased compared to E14.5. Furthermore, prominent activity was evident for maxillary bone, as has been shown before for mandibular bone [33]. The present findings by ISZ of a temporal increase in gelatinolytic activity during embryonic development of the craniofacial region confirm previous results obtained with extracts from mouse embryo heads, which were analyzed for MMP activity (by SDS-gel zymography), protein (by immunoblotting), and mRNA (by RT-PCR), respectively [38].…”

“…8B, D). MMP-2 colocalized with strong gelatinolysis at the basal side of the nasal septum, in the newly formed maxillary bone (as has been shown before for mandibular bone; [33]), and in the palatal mesenchyme adjacent to the nasopharyngeal fold. MT1-MMP immunoreactivity was also prominent at these sites (Fig.…”

“…The specificity of the ISZ protocol used here was thoroughly tested before [33] and confirmed in the present study through various types of control experiments. For negative controls, the DQ-gelatin was either omitted from the gelatinase reaction buffer, or replaced by 20 µg/ml unlabeled pig skin gelatin (Merck); in both cases, no signal was observed.…”

“…To combine ISZ with antibody labeling, we performed ISZ on unfixed cryosections, before continuing with immunolabeling as published previously [33]. Immediately after processing for ISZ, sections were fixed with cold acetone (−20°C) for 1 min, blocked with 3% bovine serum albumin in PBS (BSA/PBS) for 10 min, and then incubated with primary antibody diluted in BSA/PBS for 45 min at room temperature.…”

“…The major enzymes detected in tissues by gelatin zymography are MMP-2 (gelatinase B) and MMP-9 (gelatinase A); however other MMPs with weaker gelatinolytic activity might contribute to the signal [32]. This method is able to detect gelatinolytic activity associated with minute structures such as distinct basement membranes in the developing mouse embryo head [33]. Double labeling with antibodies to specific MMPs allows to reveal the relationship between active enzyme and protein distribution, which might differ due to the complex regulation of MMP activity.…”