Arterial P CO 2 , a major determinant of breathing, is detected by chemosensors located in the brainstem. These are important for maintaining physiological levels of P CO 2 in the blood and brain, yet the mechanisms by which the brain senses CO 2 remain controversial. As ATP release at the ventral surface of the brainstem has been causally linked to the adaptive changes in ventilation in response to hypercapnia, we have studied the mechanisms of CO 2 -dependent ATP release in slices containing the ventral surface of the medulla oblongata. We found that CO 2 -dependent ATP release occurs in the absence of extracellular acidification and correlates directly with the level of P CO 2 . ATP release is independent of extracellular Ca 2+ and may occur via the opening of a gap junction hemichannel. As agents that act on connexin channels block this release, but compounds selective for pannexin-1 have no effect, we conclude that a connexin hemichannel is involved in CO 2 -dependent ATP release. We have used molecular, genetic and immunocytochemical techniques to demonstrate that in the medulla oblongata connexin 26 (Cx26) is preferentially expressed near the ventral surface. The leptomeninges, subpial astrocytes and astrocytes ensheathing penetrating blood vessels at the ventral surface of the medulla can be loaded with dye in a CO 2 -dependent manner, suggesting that gating of a hemichannel is involved in ATP release. This distribution of CO 2 -dependent dye loading closely mirrors that of Cx26 expression and colocalizes to glial fibrillary acidic protein (GFAP)-positive cells. In vivo, blockers with selectivity for Cx26 reduce hypercapnia-evoked ATP release and the consequent adaptive enhancement of breathing. We therefore propose that Cx26-mediated release of ATP in response to changes in P CO 2 is an important mechanism contributing to central respiratory chemosensitivity.

We have previously shown connexin mediated CO 2 -dependent ATP release from the surface of the medulla oblongata. Given the localization of connexin 26 (Cx26) to the chemosensing areas of the medulla, we have tested in a heterologous expression system (HeLa cells) whether Cx26 may be sensitive to changes in P CO 2 . Cx26 responded to an increase in P CO 2 at constant extracellular pH by opening and to a decrease in P CO 2 by closing. Furthermore, Cx26 was partially activated at a physiological P CO 2 of around 40 mmHg. Cx26 in isolated patches responded to changes in P CO 2 , suggesting direct CO 2 sensitivity of the hemichannel to CO 2 . Heterologous expression of Cx26 in HeLa cells was sufficient to endow them with the capacity to release ATP in a CO 2 -sensitive manner. We have examined other heterologously expressed connexins for their ability to respond to changes in P CO 2 . The closely related β connexins Cx30 and Cx32 also displayed sensitivity to changes in P CO 2 , but with slightly different characteristics from Cx26. The more distant Cx43 exhibited CO 2 -dependent closing (possibly mediated through intracellular acidification), while Cx36 displayed no CO 2 sensitivity. These surprising findings suggest that connexins may play a hitherto unappreciated variety of signalling roles, and that Cx26 and related β connexins may impart direct sensitivity to CO 2 throughout the brain.

A conserved network of eye field transcription factors (EFTFs) underlies the development of the eye in vertebrates and invertebrates 1 . To direct eye development, Pax6, a key gene in this network 2,3 , interacts with genes encoding other EFTFs such as . However, the mechanisms that control expression of the EFTFs remain unclear 7 . Here we show that purine-mediated signalling triggers both EFTF expression and eye development in Xenopus laevis. Overexpression of ectonucleoside triphosphate diphosphohydrolase 2 (E-NTPDase2) 8 , an ectoenzyme that converts ATP to ADP 9 , caused ectopic eye-like structures, with occasional complete duplication of the eye, and increased expression of Pax6, Rx1 and Six3. In contrast, downregulation of endogenous E-NTPDase2 decreased Rx1 and Pax6 expression. E-NTPDase2 therefore acts upstream of these EFTFs. To test whether ADP (the product of E-NTPDase2) might act to trigger eye development through P2Y1 receptors, selective in Xenopus for ADP 10,11 , we simultaneously knocked down expression of the genes encoding E-NTPDase2 and the P2Y1 receptor. This could prevent the expression of Rx1 and Pax6 and eye formation completely. We next measured ATP release 12-14 in the presumptive eye field, demonstrating a transient release of ATP at a time that could plausibly trigger (once converted to ADP) expression of the EFTFs. This surprising role for transient purinemediated signalling in eye development may be widely conserved, because alterations to the locus of E-NTPDase2 on human chromosome 9 cause severe head and eye defects, including microphthalmia [15][16][17][18] . Our results suggest a new mechanism for the initiation of eye development.To assess the developmental functions of the E-NTPDases, we simultaneously injected the mRNA for the closely related ENTPDases1-3 (ref. 8) with lineage tracer into a dorsal animal blastomere at the eight-cell stage to target overexpression of this gene to one side of the nervous system. Overexpression of E-NTPDase2 affected eye development in 27 of 41 embryos, causing in some cases complete duplication of the eye on the injected side (Fig. 1A, a). In contrast, overexpression of E-NTPDase1 decreased eye size (11 of 44 embryos; Fig. 1A, b), whereas overexpression of E-NTPDase3 gave a weaker phenotype somewhat similar to that of E-NTPDase2 (4 of 42 embryos, Fig. 1A, c, Supplementary Tables 1a and 2). E-NTPDases differ in their catalytic activity. Like their mammalian orthologues, E-NTPDase1 can metabolize ATP and ADP with roughly equal efficacy, E-NTPDase2 is highly selective for ATP and hardly metabolizes ADP, and E-NTPDase3 has intermediate selectivity for ATP and ADP ( Supplementary Fig. 2). The phenotypes elicited by overexpression of these membrane-bound E-NTPDases correlate with their capacity to metabolize ADP.The eye phenotypes caused by overexpression of E-NTPDase2 (Supplementary Table 1b) included the following: disrupted eye development (Fig. 1A, d); ectopic retinal pigment epithelium (RPE) (Fig. 1A, e); RPE extensions (Fig. 1A, f); and ectopic...