ABSTRACT
Respiratory proteins are responsible for transport and storage of oxygen. It is well established that specific requirements for oxygen during vertebrate ontogeny cause switches of hemoglobin chain expression. Here, we characterize the developmental profiles of zebrafish (Danio rerio) globins by means of quantitative real-time reverse transcription PCR. The total mRNA levels of the hemoglobin chains, including a newly identified embryonic α-chain, as well as myoglobin, neuroglobin, cytoglobin 1 and 2, and globin X were estimated. mRNAs of all globins were detectable in unfertilized eggs, suggesting maternal storage. Embryonic α- and ß-hemoglobin mRNA peaked at hatching and the switch to adult hemoglobin expression occurred 16 dpf. Enhanced myoglobin mRNA levels were detected ~31 h post-fertilization (hpf), coinciding with the heart and the muscle development, while neuroglobin mRNA expression pattern correlates with the formation of the nervous system. Amounts of myoglobin and neuroglobin mRNA were similar within an order of magnitude throughout the ontogeny, tentatively supporting a respiratory role of neuroglobin. Cytoglobin 2 mRNA levels increased gradually, whereas cytoglobin 1 mRNA levels increased strongly after ~31 hpf, which is in agreement with a function in cell proliferation. Globin X mRNA level was highest in oocytes, but low in later stages. Together, these data suggest a specific role for each globin, which are also associated with certain events in fish development.
Subject(s)
Gene Expression/genetics , Globins/genetics , Zebrafish Proteins/genetics , Zebrafish/embryology , Zebrafish/metabolism , Animals , Cytoglobin , Embryo, Nonmammalian/metabolism , Embryonic Development/genetics , Hemoglobins/genetics , Larva/metabolism , Myoglobin/genetics , Nerve Tissue Proteins/genetics , Neuroglobin , Oocytes/metabolism , Ovum/metabolism , Phylogeny , RNA, Messenger/analysis , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Zygote/metabolismABSTRACT
Goldfish (Carassius auratus) may survive in aquatic environments with low oxygen partial pressures. We investigated the contribution of respiratory proteins to hypoxia tolerance in C. auratus. We determined the complete coding sequence of hemoglobin alpha and beta and myoglobin, as well as partial cDNAs from neuroglobin and cytoglobin. Like the common carp (Cyprinus carpio), C. auratus possesses two paralogous myoglobin genes that duplicated within the cyprinid lineage. Myoglobin is also expressed in nonmuscle tissues. By means of quantitative real-time RT-PCR, we determined the changes in mRNA levels of hemoglobin, myoglobin, neuroglobin and cytoglobin in goldfish exposed to prolonged hypoxia (48 h at Po(2) ~ 6.7 kPa, 8 h at Po(2) ~ 1.7 kPa, 16 h at Po(2) ~ 6.7 kPa) at 20 degrees C. We observed small variations in the mRNA levels of hemoglobin, neuroglobin and cytoglobin, as well as putative hypoxia-responsive genes like lactate dehydrogenase or superoxide dismutase. Hypoxia significantly enhanced only the expression of myoglobin. However, we observed about fivefold higher neuroglobin protein levels in goldfish brain compared with zebrafish, although there was no significant difference in intrinsic myoglobin levels. These observations suggest that both myoglobin and neuroglobin may contribute to the tolerance of goldfish to low oxygen levels, but may reflect divergent adaptive strategies of hypoxia preadaptation (neuroglobin) and hypoxia response (myoglobin).
