Defective respiratory rhythmogenesis and loss of central chemosensitivity in Phox2b mutants targeting retrotrapezoid nucleus neurons

Abstract

The retrotrapezoid nucleus (RTN) is a group of neurons in the rostral medulla, defined here as Phox2b-, Vglut2-, neurokinin1 receptor-, and Atoh1-expressing cells in the parafacial region, which have been proposed to function both as generators of respiratory rhythm and as central respiratory chemoreceptors. The present study was undertaken to assess these two putative functions using genetic tools. We generated two conditional Phox2b mutations, which target different subsets of Phox2b-expressing cells, but have in common a massive depletion of RTN neurons. In both conditional mutants as well as in the previously described Phox2b(27Ala) mutants, in which the RTN is also compromised, the respiratory-like rhythmic activity normally seen in the parafacial region of fetal brainstem preparations was completely abrogated. Rhythmic motor bursts were recorded from the phrenic nerve roots in the mutants, but their frequency was markedly reduced. Both the rhythmic activity in the RTN region and the phrenic nerve discharges responded to a low pH challenge in control, but not in the mutant embryos. Together, our results provide genetic evidence for the essential role of the Phox2b-expressing RTN neurons both in establishing a normal respiratory rhythm before birth and in providing chemosensory drive.

Figure 1.

Characterization of Atoh1-expressing neurons in the RTN. A–J, Transverse sections through E18.5 (A–D) and E15.5 (E–J) medulla. A–D, ISHs with Atoh1 (A, green) and Vglut2 (C, red) probes combined with anti-Phox2b immunofluorescence (B, blue). All Atoh1-positive cells express Phox2b and Vglut2. The sections shown in A–D are through the rostral end, those shown in E–J through the center of nVII. E–J, ISH with an Atoh1 probe (E and H, green) followed by anti-NK1R (F) or anti-TH (I) immunofluorescence (red). Atoh1-positive cells express NK1R (G) but not TH (J). A white arrow points to a TH⁺; Atoh⁻ cell. A white line delimits nVII, and a dotted line marks the ventral medullary surface; lateral is to the right. Asterisks, Nonspecific signal from blood vessels. K–N, Transverse sections through r6 at E11.5 (K), E12.5 (L), E13.5 (M), and E15.5 (N) labeled by Atoh1 ISH (blue) and anti-Phox2b immunohistochemistry (orange). The insets show enlargements of the framed regions. Note the absence of double-positive cells at E11.5 (K). A black arrowhead points to the Phox2b⁺ dB2 progenitor domain (K). Open arrows indicate the nVII precursor migratory stream to the pial surface of r6. O, Schematic representation of Atoh1 expression in transverse sections through the medulla of an E14.5 embryo. The red dots represent the parafacially located Atoh1⁺ cells in the ventral medulla, the red areas the strongly Atoh1⁺ cells of the rhombic lip and its derivatives. The distance from the rostral end of nVII is given in μm below each section. nA, Nucleus ambiguus.

Figure 2.

The Atoh1-expressing RTN neurons are preserved in Islet1^cre/+ Phox2b conditional null mutants and lost in Phox2b^27Ala/+ mutants. A–H, ISH with an Atoh1 probe (blue) followed by anti-Phox2b immunohistochemistry (orange) on transverse sections through E13.0 (A, B) and E16.5 (C, D) brains of Phox2b^lox/+; Islet1^cre/+ (control) (A, C) and Phox2b^lox/lox; Islet1^cre/+ (B, D) embryos and through E12.5 (E, F) and E15.5 (G, H) brains of wild-type (E, G) and Phox2b^27Ala/+ (F, H) brains. The ventral medullary surface is down, and lateral is to the right.

Figure 3.

RTN neurons originate from Lbx1- and Egr2-expressing precursors. A–J, Transverse sections through E15.5 brains of Egr2^cre/+; Tau^GFPnLacZ (A–E) and Lbx1^cre/+; Tau^GFPnLacZ (F–J) embryos. A–D, F–I, Anti-β-galactosidase (blue, A and F), combined anti-Islet1,2 (nuclear labeling) and anti-TH (cytoplasmic labeling) (green, B and G), and anti-Phox2b (red, C and H) immunofluorescence. A white line delimits nVII, and a dotted line marks the ventral medullary surface. There are a few β-galactosidase⁺;Phox2b⁺;TH⁺ cells in the RTN from Egr2^cre/+; Tau^GFPnLacZ embryos (white arrows) and one Phox2b⁺; TH⁻;Islet1/2⁻ cell that is β-galactosidase-negative in the RTN from Lbx1^cre/+; Tau^GFPnLacZ embryo (white arrowhead). Asterisks, nonspecific signal from blood vessels. E, J, ISH with an Atoh1 probe (blue) followed by anti-β-galactosidase immunohistochemistry (orange). A black dotted line marks the ventral medullary surface, lateral is to the right.

Figure 4.

Loss of RTN neurons in Phox2b conditional null mutants. A, B, anti-Phox2b (green) and anti-NK1R (red) immunofluorescence on transverse sections through E15.5 brains of Phox2b^lox/+; Lbx1^cre/+ (A) and Phox2b^lox/lox; Lbx1^cre/+ (B) embryos. NK1R-positive cells and fibers at the ventral medullary surface beneath nVII are absent in the mutant embryo (B). A dotted line marks the ventral medullary surface. Asterisks, Nonspecific signal from blood vessels. C–F, ISH with an Atoh1 probe (blue) and anti-Phox2b immunohistochemistry (orange) on transverse sections through E15.5 brains of control (C, E), Phox2b^lox/lox;Lbx1^cre/+ (D), and Phox2b^lox/lox; Egr2^cre/+ (F) conditional mutants. Note the absence of Atoh1 staining in the conditional null mutants (D, F).

Figure 5.

Loss of rhythmic activity in the parafacial area of brainstem preparations from Phox2b mutant embryos. Left panels, Photomicrographs of whole-hindbrain (WHB) preparations from E14.5 mouse embryos loaded with Calcium Green-1 AM (left) and images of spontaneous calcium transients recorded in the facial area (delimited by the rectangle in the leftmost panel) are shown (right). One image taken during a facial burst of activity (appearing in white on the ΔF/F panel) was used to position the nVII outlined by a white line. The calcium transients integrated over the e-pF/RTN area outlined in red are shown as relative changes in fluorescence (ΔF/F). Middle panels, Tracings of the calcium transients integrated over the e-pF/RTN area (outlined in red in the left panels) and of the rhythmic motor activity recorded from nVII and the root of the facial nerve (7n) at pH 7.4 and pH 7.2. Right panels, Quantification of the frequency of rhythmic bursts of the e-pF (pF) and 7n as indicated. p values are 8 × 10⁻⁴ and 8 × 10⁻⁵ for the differences in e-pF and 7n burst frequencies, respectively, between the two pH conditions in the control embryos. ns, p > 0.1; n, number of hindbrain preparations analyzed. A, Calcium transients and motor bursts in wild-type embryos. B–D, Same as A for Phox2b^27Ala/+ (B) and the conditional Phox2b mutant embryos with the genotypes Phox2b^lox/lox;Egr2^cre/+ (C) or Phox2b^lox/lox;Lbx1^cre/+ (D). In the mutants, there is a complete absence of rhythmic activity in the e-pF/RTN that is not restored by low pH and a markedly reduced frequency of motor bursts that is equally pH unresponsive.

Figure 6.

Emergence of a functional preBötC in Phox2b^27Ala embryos. A, Photomicrograph of an E15.5 transverse medullary slice through the preBötC loaded with Calcium Green-1 AM. The positioning of the electrode for population recording from the preBötC (preBötC int) and the area (preBötC Ca²⁺) in which spontaneous calcium transients were measured are indicated. B, Combined calcium imaging and recording of population activity in Phox2b^+/+ and Phox2b^27Ala/+ embryos as indicated. Shown are images of the relative changes in fluorescence (ΔF/F) and the calcium transients recorded over the preBötC area (upper trace). The bottom traces show the integrated electrical activity simultaneously recorded from the opposite preBötC. C, Quantification of the frequency of rhythmic bursts in Phox2b^+/+ and Phox2b^27Ala/+ embryos at pH 7.4 and pH 7.2. ns, p > 0.1. n, number of slice preparations analyzed. Significant differences in burst frequencies were observed neither between wild-type and mutant embryos nor between the two pH conditions.

Figure 7.

Slowed-down respiratory-like rhythm unresponsive to a low pH challenge in Phox2b E16.5 mutant embryos. A, Integrated phrenic nerve discharges (C4 int) at pH 7.4 (upper trace) and pH 7.2 (lower trace) for a Phox2b^lox/+ control embryo. B–D, Same as A for a Phox2b^27Ala/+ (B), a Phox2b^lox/lox;Lbx1^cre/+ (C), and a Phox2b^lox/lox;Egr2^cre/+ (D) mutant embryo. E, Quantification of the burst frequencies for control and mutant embryos as indicated. p values are 3 × 10⁻⁸ for the effect of lowering the pH in the controls, and 2 × 10⁻⁴ to 5 × 10⁻⁵ for the differences between burst frequencies in control and mutant preparations measured at pH 7.4. ns, p > 0.1; n, number of hindbrain preparations analyzed. The motor output of the respiratory network recorded from C4 nerve roots was substantially lower in the mutants. In contrast to the marked acceleration seen in control embryos, the frequencies of the rhythmic bursts were not changed by low pH in the mutants.