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First published online 19 November 2003
doi: 10.1242/dev.00866

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Phox2b controls the development of peripheral chemoreceptors and afferent visceral pathways

Stéphane Dauger^1,*, Alexandre Pattyn^2,*, Frédéric Lofaso¹, Claude Gaultier¹, Christo Goridis², Jorge Gallego¹ and Jean-François Brunet^2,

¹ Laboratoire de Neurologie et Physiologie du Développement, INSERM EPI9935, Hôpital Robert Debré, 48 Bd Serurier, 75019 Paris, France
² CNRS UMR 8542, Département de Biologie, Ecole Normale Supérieure, 46 rue d'Ulm, 75005 Paris, France

View larger version (120K): [in a new window]   Fig. 1. The nTS and AP do not form in Phox2blacZ/lacZ mutants. (A) Position of the mature nTS in the dorsal medulla and connections with the area postrema (AP) and cranial sensory ganglia. A fraction of sensory cells in the petrosal ganglion are postsynaptic to chemosensors of the carotid body. Most sensory cells in the nodose ganglion innervate sub-diaphragmatic organs, such as the gut. (B-I) Development of the nTS from Phox2b+ dorsal precursors in the medulla of wild-type embryos. (B-E) Immunohistochemistry for Phox2b, Islet1 and Lmx1b (B-D) and in situ hybridization for Rnx (E) show that Phox2b+ precursors at the level of r7 can be subdivided into motoneuron and nTS precursors according to their co-expression of Islet1 (C), or Lmx1b (D) and Rnx (E), respectively. Red cells in C correspond to Islet1+/Phox2b-somatic motoneurons. (F-H) Immunohistochemistry for Phox2b and Lmx1b between E11.5 and E13.5 reveals the migration of both populations towards each other resulting in the formation of the dmnX (asterisk) and nA (arrowhead in F, not visible in G,H) (green), and of the nTS, whose cells co-express Phox2b and Lmx1b (yellow). Red cells correspond to several classes of Lmx1b+/Phox2b- interneurons. (I) Schematic representation of the origin and migratory behavior of the dmnX, nA (red) and nTS (blue) precursors. The boxed areas are those photographed in B-H. (J-U) The nTS and AP do not form in Phox2blacZ/lacZ mutants. (J-M) A normal complement of dorsal cells detected by lacZ in situ hybridization are born and migrate ventrally in a homozygous (K,M) compared with a heterozygous (J,L) mutant between E10.5 and E11.5. Note that ventrally, lacZ expression is preserved in the neuroepithelium of Phox2blacZ/lacZ embryos (asterisk), where it persists until at least E13.5 (see Q), but not in the mantle layer. This reflects the fact that no bm/vm motor neurons are born (Pattyn et al., 2000b). (N,O) The dorsal emergence of lacZ+ cells continues normally in the homozygous mutants at E12.5 but no ventral accumulation occurs, and the dmnX (arrowhead, N) does not form. (P,Q) The entire dorsal vagal complex is absent at E13.5, as assessed by lacZ expression. (R,S) Dorsal view of a hindbrain at E18.5 showing the obex of the fourth ventricle (red arrow) caudally displaced and wider in a homozygous (S) compared with a heterozygous (R) mutant. (T,U) Transverse sections caudal to the obex stained by immunohistochemistry for Phox2b and in situ hybridization for peripherin, showing a loss of tissue in the homozygote (U) affecting the region where the dmnX, nTS and area postrema (i.e. the dorsal vagal complex) are found in the heterozygote (T). Note that the peripherin+ somatic motoneurons of the hypoglossal nucleus are preserved in the mutants. AP, area postrema; dmnX, dorsal motor nucleus of the vagus nerve; nA, nucleus ambiguus; nTS, nucleus of the solitary tract.

View larger version (131K): [in a new window]   Fig. 2. Involution of the petrosal-nodose ganglionic complex and carotid body between E13.5 and E16.5 in Phox2blacZ/lacZ mutants. (A-F) At E13.5 the petrosal-nodose complex is already markedly atrophic, as assessed by peripherin in situ hybridization (A,B, quantified in I) and has lost expression of lacZ (C,D) and Phox2a (E,F), which was initially expressed in the mutant placodal precursors at E10.5 (Pattyn et al., 1999). (G,H) In situ hybridization with peripherin showing that, at E16.5, the involution is almost complete. (I) Quantification of the ganglionic atrophy at E13.5 and E16.5. Measurements of control ganglia were considered as 100±5.5% at E13.5 (n=6) and 100±3.7% at E16.5 (n=4). The relative measurements of mutant ganglia are 25.8±5.3% at E13.5 (n=4) and 7±2% at E16.5 (n=4). (J-O) The anlage of the carotid body is first detected at E13.5 in a heterozygous mutant (arrowhead in J) by immunohistochemistry against Phox2a (L) or in situ hybridization for lacZ (J) or Tbx20 (N). In a homozygous mutant, lacZ expression is already affected at this stage (arrowhead in K) and neither Phox2a (M) nor Tbx20 (O) expression occurs. (P-S) At E16.5 the carotid body, which still expresses lacZ (P) and has switched on Th (R) in the heterozygote, is no longer detectable in the homozygote (Q,S). ec, external carotid artery; ic, internal carotid artery.

View larger version (21K): [in a new window]   Fig. 3. Abnormal ventilatory response of heterozygous mutants to hypoxia and hypercapnia. (A) Ventilatory tracings in one Phox2b+/+ pup (top) and one Phox2blacZ/+pup (bottom). Both pups had similar baseline ventilation (air) and initial increase during hypoxia, but the Phox2blacZ/+ pup showed long post-hypoxic apneas (flat respiratory tracing). (B) Total duration of apneas (defined as respiratory pauses longer than twice the duration of the preceding breathing cycle) in air condition (3 minutes) and post-hypoxic condition (6 minutes). Apneas (0.7 and 2.8 apneas/minute in Phox2blacZ/+ mice; 0.8 and 2.8 apneas/minute in Phox2b+/+ mice) were strikingly longer in Phox2blacZ/+ mice (***P<0.001). Values are group means±s.e.m. (C) Ventilatory tracings in one Phox2b+/+ pup (top) and one Phox2blacZ/+ pup (bottom). Both pups had similar baseline ventilation but the Phox2blacZ/+ pup showed a lower ventilatory response to hypercapnia. (D) The ventilatory increase during hypercapnia (three minutes) in Phox2blacZ/+ pups was about half that in Phox2b+/+ pups, because of their lower increase in breathing frequency (not shown). *P<0.05; **P<0.01. Values are group means±s.e.m. (E) Ventilatory increase during hypercapnia at P10. The pups were exposed to 8% CO2 for 5 minutes (instead of 3 minutes as in D) to examine possible changes in the time course of the hypercapnic response. No difference were observed between Phox2b+/+ and Phox2blacZ/+ pups. Values are group means±s.e.m.

View larger version (69K): [in a new window]   Fig. 4. Decrease in Th expression in the petrosal ganglion of Phox2blacZ/+ E16.5 embryos and normalization at P10. (A,B) In situ hybridization for Th expression shows that the heterozygous mutant glomus expresses normal levels of Th. (C,D) The subpopulation of petrosal ganglionic neurons, positioned ventrally, which expresses Th seen in the wild type in C is sparser in heterozygous mutants (D). The contour of the ganglion is outlined in red. (E) Quantification of Th-positive cells in the petrosal ganglion of E16.5 wild-type (n=6 ganglia) and heterozygous mutant (n=6 ganglia) embryos and P10 wild-type (n=4 ganglia) and heterozygous mutant (n=4 ganglia) pups. Values are means±s.e.m. (**P<0.01).