Changes in hemoglobin function and isoform expression during embryonic development in the American alligator, Alligator mississippiensis

Abstract

In the developing embryos of egg-laying vertebrates, O2 flux takes place across a fixed surface area of the eggshell and the chorioallantoic membrane. In the case of crocodilians, the developing embryo may experience a decrease in O2 flux when the nest becomes hypoxic, which may cause compensatory adjustments in blood O2 transport. However, whether the switch from embryonic to adult hemoglobin isoforms (isoHb) plays some role in these adjustments is unknown. Here, we provide a detailed characterization of the developmental switch of isoHb synthesis in the American alligator, Alligator mississippiensis. We examined the in vitro functional properties and subunit composition of purified alligator isoHbs expressed during embryonic developmental stages in normoxia and hypoxia (10% O2). We found distinct patterns of isoHb expression in alligator embryos at different stages of development, but these patterns were not affected by hypoxia. Specifically, alligator embryos expressed two main isoHbs, HbI, prevalent at early developmental stages, with a high O2 affinity and high ATP sensitivity, and HbII, prevalent at later stages and identical to the adult protein, with a low O2 affinity and high CO2 sensitivity. These results indicate that whole blood O2 affinity is mainly regulated by ATP in the early embryo and by CO2 and bicarbonate from the late embryo until adult life, but the developmental regulation of isoHb expression is not affected by hypoxia exposure.

Evolution and molecular basis of a novel allosteric property of crocodilian hemoglobin

“…Crocodilians possess multiple copies of β-type globin genes (25) and express structurally distinct Hb isoforms (isoHbs) at different stages of prenatal development, as is the case with all other amniotes (22, 26-28). We previously discovered that crocodilian embryos express two structurally distinct isoHbs: an embryonic isoHb that incorporates the β-chain product of HBB-T1 (HbI) and the normal adult isoHb that incorporates the β-chain product of HBB-T4 (HbII); both isoHbs share the same α-chain subunit (20)(Fig. 3 A ).…”

“…We expressed and purified each of the three recombinant Hbs (rHbs), and we then measured oxygenation properties in vitro. The reconstructed AncArchosaur and AncCroc rHbs exhibited intrinsic O2-affinities, cooperativities, and pH sensitivities (Bohr effect) that are typical of native Hbs from modern crocodilians and non-avian reptiles (4,6,(17)(18)(19)(20)(Table S1). We used an experimental approach to confirm that all functional inferences are robust to uncertainty in ancestral sequence estimates (21)(Supplementary Text and Fig.…”

“…Collection of blood samples and purification of native isoHbs. We sampled blood of alligator embryos at the 40% stage of pre-hatching development by catheterizing the major artery of the chorioallantoic membrane, as described by Bautista et al (20). See Supplementary Material for details regarding hemolysate preparation and protein purification via anion-exchange chromatography.…”