During the early stages of development, most creatures are small enough to meet their metabolic demands by diffusion alone. However, as embryos grow they switch to circulatory systems and gas exchange to satisfy their metabolic requirements. A key molecular component of most circulator systems is the enzyme carbonic anhydrase (CA), which speeds up the conversion of carbon dioxide to soluble bicarbonate for transport. Katie Gilmour, from the University of Ottawa, explains that although the development of the oxygen delivery system is well understood, little is known about the development of the systems involved in carbon dioxide excretion. Intrigued by all aspects of carbon dioxide excretion involving CA, Gilmour and her collaborator Steve Perry were curious to know when a developing animal switches from gas exchange by diffusion to a circulatory system, and whether that is correlated with the expression of CA. Gilmour and Perry turned to the zebrafish to find the answer (p. 3837).
‘One of the exciting possibilities of working in zebrafish is the capacity to work in very young fish,’ explains Gilmour. Teaming up with Andrew Esbaugh, Gilmour and Perry set out to measure when CA expression kicked in by measuring the relative amount of CA mRNA in zebrafish embryos and larvae, ranging from 3 h to 5 days postfertilisation. Knowing that zebrafish produce various different forms of CA, each specialised for specific physiological functions, Gilmour and Perry focused on the form involved in CO2 excretion in red blood cells (CAb) and another form, specialised in acid—base regulation found in almost all tissues (CAc). Using real-time PCR, the team saw that the expression levels of the red blood cell form of CA (CAb) were always higher than the more general form of the enzyme (CAc), and there was a substantial increase in CAb at 8 h postfertilisation. But would that correlate with the amount of CO2 that the embryos and larvae were excreting? Gilmour and Perry teamed up with honours student Kelli Thomas to find out.
‘CO2 chemically reacts with water to give bicarbonate so you have to measure the total CO2 content, which is very finicky,’ says Gilmour. Thomas painstakingly sealed groups of embryos or larvae in a chamber for a period ranging from 20 to 90 min (depending on the age of the embryos/larvae), collected water samples at the beginning and end of the period and measured the total carbon dioxide levels with Gilmour's cantankerous Capni-Con 5. Calculating the CO2 levels relative to the water's oxygen levels, the team could see that the fish began excreting large amounts of CO2 at 48 h, around the time that they hatch. In fact the team were surprised to realise that, prior to hatching, CO2 excretion by the eggs was far less than O2 uptake. They must be storing CO2, although Gilmour and Perry are unsure where.
Having found the point at which the larvae begin excreting CO2, Gilmour and Perry were keen to find out which type of CA was responsible for the massive increase in CO2 excretion. Using a molecular technique only available in zebrafish, the team were pleased to see that CAb was partially responsible for the increase in CO2 excretion. But they were amazed to see that CAc was involved in CO2 excretion too. ‘That isn't the case in adult animals,' says Gilmour.
Having found that CAb is a key player in CO2 excretion, Perry and Gilmour are keen to discover whether red blood cells are actively involved in CO2 excretion at 48 h, which is long before they are essential for oxygen transport, at 14 days.