The expression of tissue inhibitor of metalloproteinase 2 (TIMP-2) is required for normal development of zebrafish embryos.
MMP activities are controlled by a combination of proteolytic pro-enzyme activation steps and inhibition by endogenous inhibitors like alpha2-macroglobulin and the tissue inhibitors of metalloproteinases (TIMPs). TIMPs are the key inhibitors in tissue. The expression of both MMPs and TIMPs is controlled during tissue remodeling to maintain a balance in the turnover of extracellular matrix. Disruption of this balance may result in a broad spectrum of diseases. Additionally, TIMP-2 has been reported to have growth factor activities. To further study the function of TIMP-2 in development, we utilized zebrafish as an experimental model system. We have successfully isolated a TIMP-2 homologue from zebrafish (zTIMP-2). This zebrafish TIMP-2 showed high similarity to human TIMP-2 with all critical features conserved. Whole-mount in situ analysis showed that zTIMP-2 was expressed as early as the one-cell stage indicating a maternal origin. This expression continued through later stages of development. RT-PCR analysis confirmed the early expression pattern from the 16-cell stage through blastula, gastrula and 24-h stages. In addition, at the protein level, immunoreactive zTIMP-2 was detected using antibody against recombinant human TIMP-2. RFP-reporter analysis indicated that TIMP-2 can be secreted into the extracellular space where ECM is forming. Functional studies showed that the balance of TIMP-2 expression is important to normal development as reflected by the fact that both blockage of TIMP-2 translation using antisense morpholino oligonculeotides or increased translation of TIMP-2 using a mRNA microinjection approach resulted in abnormal zebrafish development. This is in contrast to murine knockout studies that indicate that TIMP-2 does not have a major role in mouse embryogenesis.
Zhang
Jinsong
J
Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160, USA.
Bai
Shan
S
Tanase
Carmen
C
Nagase
Hideaki
H
Sarras
Michael P
MP
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004625:Embryo, Nonmammalian / Q000502:physiology; D023421:Models, Animal; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000096:biosynthesis* / Q000302:isolation & purification; D015027:Zebrafish / Q000196:embryology* / Q000378:metabolism
D019716:Tissue Inhibitor of Metalloproteinase-2