Sequences surrounding proteolytic cut sites are conserved in the propeptide of gelatinase A orthologues. Blue arrow indicates the N-terminus created by removal of the secretory signal during translation, green indicates the N-terminus of the intermediate created by MT1-MMP and red indicates the active MMP-2 terminus thought to be an autocatalytic cleavage site (Strongin et al., 1993). Grey shading indicates the novel cut site detectable by thrombin/factor Xa when mutagenesis (†, ‡) blocks the recognition of the other two cut sites (Koo et al., 2009). Asterisk marks the cysteine of the cysteine switch. Sequence coordinates marked are alignment coordinates, not protein coordinates.

EMMA-Mmp2 is proteolytically processed by endogenous Mmp2 activating mechanisms in vivo to yield the expected products. Immunoblot analysis reveals four bands, one that has maintained both C and N-termini (a), two that retain their C-terminus (b, c) and one that only retains its N-terminus (d). Indicated molecular weights are based on conserved cut sites in the pro-peptide (I, II from (Strongin et al., 1993) and III from (Koo et al., 2009)).

Mmp2 is activated in the notochord and EVL shortly after the onset of somitogenesis. Epifluorescence and bright field imaging of early somitogenesis embryos expressing EMMA-Mmp2 (A,B) and whole mount immunostains (C–E) at 4 hpHS. EMMA-Mmp2 is expressed in response to heat shock embryos during early somitogenesis in both embryos at 1–2 somites (A) and embryos of 5–8 somites (B). Expression is prominent anteriorly (I) and in the notochord (II). EMMA-Mmp2 is not activated in 1–2 somite embryos (C), but is activated in 3–8 somite embryos (D) in the EVL (D) and notochord (D′, D″). Localization and activation of EMMA-Mmp2 in the EVL occurs in a patchwork pattern (D). The lowest amount of activation is found in the mesoderm (V) and the highest in the notochord (VI). Signal to noise ratio in the EVL decreases with time post heat shock, but patterns remain similar. Calibration bar at the right relates the heat map to the extent of activation, with H indicating high levels of activation (GFP/(GFP ​+ ​HA) ratio approaching 1), and L indicating low levels of activation (GFP/(GFP ​+ ​HA) ratio approaching 0). The patterns of activation illustrated were consistent in all embryos imaged at this stage (n ​= ​3).

Activated Mmp2 co-localizes with denatured collagen in early development. CHP-mediated detection of total collagen (A, A′) consistently reveals abundant collagen in the notochord sheath and notochord cells (n ​= ​10). The collagen in the notochord sheath at the yolk extension is 1–2 ​μm thick and fibrous (A′). Endogenously denatured collagen is found in the notochord (B), especially in the sheath (I), but also in between the notochord cells (B′). Activation of EMMA-Mmp2 in the notochord (C) was visible in isolated 'hot spots' along the trunk in 18 of 96 embryos imaged (II), that were in register with myotome boundaries in 9 of 25 instances. Activation of EMMA-Mmp2 is consistently observed in the notochord sheath (III), especially in the posterior tail (C′). Collagen is always present in somite boundaries almost as soon as they are distinguishable (D), and includes a small amount of denatured collagen at this stage (E). Activation of EMMA-Mmp2 is usually (74%) associated with the gut epithelium (IV) and we observed activation in ventral caudal hematopoietic tissue (V) in 80% of embryos. In all embryos examined (n ​= ​27) we see activation in somite boundaries (VI), which is always stronger in anterior somite boundaries (VII) compared with posterior somites (VIII).

Collagen remodelling occurs during fin development, as does Mmp2 activation. Collagen is a constitutive component of developing fins (A), found perichordally (I) and along the developing fin rays (II). Denatured collagen is found along the notochord sheath (I), as well as, to a lesser extent, in the interior of the notochord, evident most strongly at the anterior tip (III). Denatured collagen is also found between where the layers of actinotrichia would be found, on podosome-like cell processes (IV). Activation of EMMA-Mmp2 (E,F) is not strongly associated to the notochord sheath, but instead is concentrated in the neural tube (V) and in a patchwork pattern of epithelial cells (VI) at these stages. Patterns are representative of at least 10 embryos imaged for each assay.