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1.
Figure 3

Figure 3. Atoh1is cell–autonomously required for RTN ventral migration and differentiation. From: Atoh1 governs the migration of post-mitotic neurons that shape respiratory effectiveness at birth and chemoresponsiveness in adulthood.

(A) Expression pattern of the Phox2bCre line as assessed by RosaEYFP/EYFP reporter showing that the Cre (EYFP, green), Phox2b (blue), and Lbx1 (red) in the RTN neurons (yellow arrowheads)are highly overlapped.(B)The RTN neurons of WT and Atoh1Phox2bCKO brainstems express lineage markers Phox2b (red) and Lbx1 (green) in anAtoh1–independent manner (white arrowheads), despite the migration defect in the Atoh1Phox2bCKO brainstem. Images in A and B are sagittal sections from E18.5 brainstems.(C) Expression of RTN differentiation marker NK1R (red)is lost in Atoh1Phox2bCKO RTN neurons (LacZ, green)as shown in coronal sections from E16.5 brainstems. RTN neurons in the overlaid images (left) are outlined with yellow dotted box, and markers are shown individually (right). All scale bars represent 100 µm.

Wei-Hsiang Huang, et al. Neuron. ;75(5):799-809.
2.
Figure 6

Figure 6. Atoh1Phox2bCKO survivor mice develop abnormal chemosensory responses in adulthood. From: Atoh1 governs the migration of post-mitotic neurons that shape respiratory effectiveness at birth and chemoresponsiveness in adulthood.

Graphs show respiratory frequency (RF), tidal volume (VT) and minute ventilation (VE) of 3 month old Atoh1Phox2bCKO mice (n=9) as well as WT (n=21) when challenged with hypercapnia (5% CO2, A) or hypoxia (10% O2, B). Atoh1Phox2bCKO mice have a significantly weaker response to hypercapnia (A) but have a stronger response to hypoxia (B). Shown are the mean values ± SEM over 20 minutes of normoxic baseline, 5 minutes of gas challenge and 15 minutes of normoxic recovery phases.*p<0.05, ANOVA.

Wei-Hsiang Huang, et al. Neuron. ;75(5):799-809.
3.
Figure 5

Figure 5. Atoh1–mediated RTN development is involved inneonatal inspiratory rhythm generation. From: Atoh1 governs the migration of post-mitotic neurons that shape respiratory effectiveness at birth and chemoresponsiveness in adulthood.

(A)Neonatal Atoh1Phox2bCKO mice generate significantly slower inspiratory motor activity when compared to WT. The integrated suction electrode traces of E18.5 WT (n=9) and Atoh1Phox2bCKO (n=5) en bloc preparations represent the fictive respiratory motor rootlet activity, quantified in a bar graph(*p<0.05, mean values ± SEM, independent samples t–test).(B) Substance P (SP) does not trigger inspiratory–related motor activity in Atoh1Phox2bCKO brainstems, which is normally seen in WT preparations. Electrode traces were generated during baseline and brainstem application of 1µM SP in E18.5 WT and Atoh1Phox2bCKO en bloc preparations. The bursting frequency 5 minutes before and after SP treatment was quantified in the bar graph.
(C)Embryonic Atoh1Phox2bCKO mice show significantly slower inspiratory motor activity compared to WT under both baseline (pH 7.4)and acidosis (pH 7.2) environments. The integrated suction electrode traces of E16.5 WT (n=7) and Atoh1Phox2bCKO (n=11) en bloc preparations (left) represent the fictive respiratory motor rootlet activity, quantified in a bar graph (right, *p<0.001, mean values ± SEM, independent samples t–test).

Wei-Hsiang Huang, et al. Neuron. ;75(5):799-809.
4.
Figure 1

Figure 1. Brainstem neurons connect to the preBötC in an Atoh1–dependent manner. From: Atoh1 governs the migration of post-mitotic neurons that shape respiratory effectiveness at birth and chemoresponsiveness in adulthood.

(A) Schematic representationofAtoh1–expressingneurons (in blue) in a sagittal plane of the E18.5 hindbrain. Rostral is left; PB: parabrachial, pTRI: paratrigeminal, GC: granule cells, PN: pontine, nV: trigeminal motor, ECu: external cuneate, Sp5I: spinal trigeminal, RTN: retrotrapezoid, nVII: facial motor, BötC: Bötzinger complex, preBötC: preBötzinger Complex (orange dotted circle), rVRG: rostral ventral respiratory group, and LRt: lateral reticular nuclei.(B-G) Lineage (B–E) and neuronal projection (F, G) mapping of WT (Atoh1Cre/+; TaumGFP-nLacZ) and Atoh-null (Atoh1Cre/−; TaumGFP-nLacZ) brainstems at E18.5.The somas (nLacZ) of Atoh1 descendants in the Atoh1-null (C) brainstems are either mislocalized(RTN, white arrowhead)or lost(Sp5I and LRt nuclei). The expression of RTN neuronal lineage markersPhox2b and Lbx1 in WT (D) and Atoh1-null (E) are similar. The Atoh1-dependent projections (mGFP shown in green) originate from rostral (white open arrowheads) and caudal (white arrowheads) Atoh1 populations that innervate the preBötC (orange dotted circle, marked by somatostatin, Sst in red)are detected in WT(F) but lost in Atoh1-null (G).Neurites of the RTN neurons in Atoh1-null mice do not connect with the pre BötC and accumulate at the dorsal surface of nVII (G, yellow arrowhead). All images are shown in sagittal sections. Scale bars represent 100 µm.

Wei-Hsiang Huang, et al. Neuron. ;75(5):799-809.
5.
Figure 4

Figure 4. Phox2bCre allele selectively targets paramotor neurons and spares the rhombic lip (RL)–derived Atoh1populations. From: Atoh1 governs the migration of post-mitotic neurons that shape respiratory effectiveness at birth and chemoresponsiveness in adulthood.

(A)in situ hybridization of E14.5 WT and Atoh1Phox2bCKO sagittal brainstem sections (rostral to the left) confirming Phox2bCre selectively removes Atoh1 mRNA from the RTN (white arrowhead) and the pTRI (yellow arrowheads) neurons. The caudal rhombic lip (cRL, black arrowheads) progenitors retained Atoh1 expression in the Atoh1Phox2bCKO brainstem. (B)Side view of the whole mount X-gal staining (rostral to the left) to trace the Atoh1 lineages in WT (Atoh1LacZ/+) and Atoh1Phox2bCKO brainstems at E18.5. LacZ activity marks the Atoh1 descendants. The RTN neurons are mislocalized in Atoh1Phox2bCKO mice, while the RL lineages remain anatomically intact. (C) Schematics of the Atoh1 hindbrain lineages (shown in green, indicated by yellow arrowheads). All coronal hemisections (1–4, rostral to caudal) are oriented with lateral to the left. (D, E) Serial coronal sections from E16.5 Atoh1LacZ/+ (D) and Atoh1Phox2bCKO (E) brainstems at approximate levels 1–4 in (C). Sections were co-stained with LacZ (green, Atoh1 lineages indicated by yellow arrowheads) and Phox2b (red). The RL-derived Atoh1 populations remain anatomically intact in the Atoh1Phox2bCKO brainstems, and the RTN neurons are dorsally misplaced in the Atoh1Phox2bCKO brainstem (co-localization of LacZ and Phox2b double positive cells are shown in white). Abbreviations: AES: anterior precerebellar extramural stream, DC: dorsal cochlear, DLL: dorsal lateral lemniscal, DNs: deep cerebellar, ECu: external cuneate, EGL: external granule layer, MiTg: microcellular tegmental, MVe: medial vestibular, PB: parabrachial, PN: pontine, PPTg: pedunculopontine, Pr5: principal sensory trigeminal, pTRI: paratrigeminal, Rtgn: reticulotegmental, RTN: retrotrapezoid, Sp5I: interpolar division of the spinal trigeminal nucleus, VC: ventral cochlear, and X: vestibular nuclei. Scale bars represent 100 µm.

Wei-Hsiang Huang, et al. Neuron. ;75(5):799-809.
6.
Figure 2

Figure 2. Characterization of the HoxA4Cre allele and selective removal of Atoh1 from the caudal rhombomeres. From: Atoh1 governs the migration of post-mitotic neurons that shape respiratory effectiveness at birth and chemoresponsiveness in adulthood.

(A-G)Cre recombinase expression of the HoxA4Cre driver line was evaluated by crossing HoxA4Cre with RosaLacZ/LacZ reporter mice followed by sagittal (A) and coronal (B-G) sections of 3-week-old mice. Sections were co-stained with LacZ(green, indicates Cre activity) and neurokinin 1 receptor(NK1R marked in red as molecular landmark). (A) Sagittal sections showing that HoxA4Cre activities are restricted to structures posterior to rhombomere 7 and spared the NK1R positive RTN neurons. (B-F) Coronal sections correspond to the levels indicated by white dotted line in (A). A small HoxA4Cre–expressing subpopulation migrates anteriorly to the pontine nucleus (white arrowhead in B).(G) The HoxA4Cre allele also targets neurons within the spinal cord. (H, I)Side view of the whole mount X-gal staining (rostral to the left) to trace the Atoh1 lineages in WT(H, Atoh1LacZ/+) and Atoh1HoxA4CKO(I) brainstems at E18.5. LacZ activity marks the Atoh1 descendants. The Atoh1–dependent ECu (yellow arrowheads) and LRt nuclei (black arrowheads) fail to form due to the loss of Atoh1 in the HoxA4 lineages, while the RTN neurons are unaffected and migrate normally to the ventral medullar surface (white arrowheads). The anterior RL–derived Atoh1 neurons, such as cerebellar granule cells and pontine nucleus, are also preserved due to different rhombomeric origins, confirming the specificity of the transgenic Cre alleles. Abbreviations: CB: cerebellum, ECu: external cuneate, GC: cerebellar granule cells, LRt: lateral reticular, NA: nucleus ambiguous, nV: trigeminal motor, nVII: facial motor, PN: pontine, and RTN: retrotrapezoid nuclei. Scale bars represent 500 µm.

Wei-Hsiang Huang, et al. Neuron. ;75(5):799-809.

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