Excitatory neurons of the proprioceptive, interoceptive, and arousal hindbrain networks share a developmental requirement for Math1

Abstract

Hindbrain networks important for sensation and arousal contain diverse neuronal populations with distinct projections, yet share specific characteristics such as neurotransmitter expression. The relationship between the function of these neurons, their developmental origin, and the timing of their migration remains unclear. Mice lacking the proneural transcription factor Math1 (Atoh1) lose neurons essential for hearing, balance, and unconscious proprioception. By using a new, inducible Math1(Cre*PR) allele, we found that Math1 is also required for the conscious proprioceptive system, including excitatory projection neurons of the dorsal column nuclei and for vital components of the interoceptive system, such as Barrington's nucleus, that is closely associated with arousal. In addition to specific networks, Math1 lineages shared specific neurotransmitter expression, including glutamate, acetylcholine, somatostatin, corticotropin releasing hormone, and nitric oxide. These findings identify twenty novel Math1 lineages and indicate that the Math1 network functions partly as an interface for conscious (early-born) and unconscious (late-born) proprioceptive inputs to the cortex and cerebellum, respectively. In addition, these data provide previously unsuspected genetic and developmental links between proprioception, interoception, hearing, and arousal.

Fig. 1.

Novel early-forming Math1 lineages in the medulla and caudal pons. (A) Time course (E10.5–16.5) of LacZ expression in the medulla of Math1^LacZ/+ mice (approximately level 5 in B′). An early migration (yellow arrows) from the cRL is seen several days before the PES (black arrowheads), giving rise to novel Math1 lineages (yellow arrowheads). (Inset) cRL and migrating cells (yellow arrow) at E10.5. (B) Brainstem schematic. Lines 1–6 indicate coronal hemisection levels shown in (B′) depicting nuclei that contain new (dark blue) and known (light blue) Math1 lineages. (C–F) Side views of whole-mount E18.5 Math1^Cre*PR/+;ROSA^LacZ/+ hindbrains induced on E9.5, E10.5, E12.5, or E13.5, showing LacZ-labeled cell somas (blue) of new (yellow arrowheads) and known (black arrowheads) lineages. (G) A Math1^Cre*PR/−;ROSA^LacZ/+ (Math1-null) hindbrain induced daily (E9.5 to E17.5) had staining mainly at the cRL (yellow arrowhead). (Some background fourth-ventricle staining was visible due to tissue clearing). (C′–G′) Ventral surfaces of C–G. (H and I) Coronal sections from E18.5 Math1^Cre*PR/+; ROSA^EYFP/+ (H) and Math1^Cre*PR/−; ROSA^EYFP/+ (I) null hindbrains induced at E10.5, showing loss of most staining from the medulla in the Math1-null. (H′ and I′) Magnified boxed regions in H and I, from slightly more rostral positions, show primarily labeled DC neurons in Math1^Cre*PR/+ and CP cells in Math1^Cre*PR/− (yellow arrowhead), adjacent to the cRL. Lateral to left in A and B′ and H–I′; rostral to left in B and C–G; rostral up in C′–G′. Abbreviations: cRL, caudal rhombic lip; CP, choroid plexus. See Table 1 for other abbreviations. (Scale bar: A, 290 μm; A Inset, 60 μm; C–G, 1,000 μm; C′–G′, 900 μm; H and I, 400 μm; H′ and I′, 100 μm.)

Fig. 2.

Temporally similar Math1-dependent neurons share marker expression. (A) Schematics of hindbrain nuclei containing early- (yellow) and late-forming (black) Math1 lineages (listed to right in corresponding colors) on coronal hemisections, rostral to caudal (levels 1–6, Fig. 1B). (B and C) Magnified boxed regions in A from corresponding sections of E18.5 Math1^Cre*PR/+;ROSA^EYFP/+ and Math1^Cre*PR/−;ROSA^EYFP/+ null mice induced at E10.5 (B) or E12.5 (C). (Dashed line at level 2 marks the midbrain/hindbrain junction.) The regions shown contain mainly E10.5 populations, so E12.5 populations are marked by gray arrowheads. (D and E) Corresponding regions to B and C from E18.5 WT and Math1-null hindbrains were stained either with Lhx9 (D) or Barhl1 (E) (gray arrowheads) ISH probes. Most Lhx9 and Barhl1 expression was lost in Math1-null hindbrains, whereas some expression persisted in non-Math1 lineages of the midbrain. (F and G) Colabeling of anti-GFP (green) with anti-Lhx9/Lhx2 (red) or anti-Barhl1 (red) when induced at E10.5 (F) or E12.5 (G), respectively, in select Math1^Cre*PR/+;ROSA^EYFP/+ hindbrain nuclei. These regions, labeled by nucleus, do not correspond to the boxes in A. Higher magnifications below each region show that Lhx9 is expressed in early-born neurons and Barhl1 is expressed in later-born neurons. Abbreviations: Please see Table 1 for list of nuclei. (Scale bar: B–E, 500 μm; F and G, 83 μm; F and G Insets, 28 μm.)

Fig. 3.

Subsets of glutamatergic, somatostatin, CRH, nitric oxide, cholinergic, and L-dopa neurons require Math1. (A) Schematics of hindbrain nuclei containing Math1 lineages (blue, listed to right) on coronal hemisections, rostral to caudal (levels 1–6, Fig. 1B). (B–G) Magnified boxed regions in A from corresponding E18.5 WT and Math1-null sections stained with ISH probes for glutamate (Vglut2), somatostatin (SST: Sst), corticotropin releasing hormone (CRH: Crh), nitric oxide (NO: Nos1), acetylcholine (ACh: Vacht), and levodopa (L-Dopa: tyrosine hydroxylase, Th) neurons. Nuclei containing Math1-dependent Vglut2 or Sst are marked by dotted outlines, whereas other Math1-dependent neurotransmitters are marked by gray arrowheads. Abbreviations: Please see Table 1 for list of nuclei. (Scale bar: B–G, 500 μm.)

Fig. 4.

Nitric oxide, CRH, and somatostatin neurons of the arousal, interoceptive, and conscious proprioceptive systems arise from the RL. (A) Schematic of coronal hemisection with nuclei containing Math1 lineages (blue); boxed region magnified above shows the LDT and BN (green) medial to the LC (orange). (B and C) Dotted outlines mark approximate boundaries of these three nuclei on adjacent sections from a Math1^Cre*PR/+;ROSA^EYFP/+ hindbrain (E18.5) induced at E10.5 and stained for EYFP (green) and Nos1 (red) with either Crh (B) (blue) or Th (C) (blue). (B′ and C′) Regions magnified from yellow boxes in A and B with yellow dashed boxes further magnified below each. EYFP colabeled with Crh and Nos1 but not with Th. (D) Schematic showing nuclei containing Math1 lineages (blue) on a coronal hemisection from the caudal medulla. (E) Boxed region from D shows EYFP and Sst expression in the Cu/Gr nuclei. Dashed box, magnified below, shows colabeling of EYFP (somas) with Sst (cytoplasmic + extracellular). Abbreviations: CRH, corticotropin releasing hormone; Nos1, nitric oxide synthase 1; Sst, somatostatin. See Table 1 for other abbreviations. (Scale bar: B and C, 250 μm; B′, C′, and E′, 80 μm; Lower frames of B, C, and E, 40 μm.)

Fig. 5.

Early Math1 lineages project in the medial and lateral lemnisci and the superior cerebellar peduncle. Math1^Cre*PR/+; ROSA^EYFP/+; Tau^mGFP_nLacZ/+ hindbrains (P0) induced at E10.5 (A–B′), E12.5 (C), or E14.5 (D). (A and A′) Lateral (A) and medial (A′) sagittal sections showing mGFP-labeled projections (black) with indicated sources (gray arrowheads) and known targets (black arrowheads) of selected projections (yellow arrowheads), including those in the ll, ml, and scp. Insets (from yellow box) show overlapping expression of mGFP and Sst processes in the VPL thalamic nucleus. (Although somas also expressed EYFP, primarily mGFP processes were visible at this magnification.) (B) Coronal sections from positions 1–5 (dotted lines on A′). Section level 1 is a hemisection (dashed line). (B′) Magnified boxed regions from B show mGFP processes (green) and βgal-labeled somas (red) (from the Tau^mGFP_nLacZ/+), with selected projections (yellow arrowheads) and nuclei (white arrowheads) indicated. (C–D′) Lateral (C and D) and medial (C′ and D′) sagittal sections showing projections in the icp (C) and mcp (D) when induced at E12.5 and E14.5, respectively. Abbreviations: Sst, somatostatin; R, red nuclei; VPL, ventral posterolateral nuclei; IC, inferior colliculus; ctx, cortex; ia, internal arcuate fibers; ml, medial lemnesci; ll, lateral lemnesci; scp, superior cerbellar peduncles; icp, inferior cerebellar peduncles; mcp, middle cerebellar peduncles. See Table 1 for other abbreviations. (Scale bar: A, A′, and C–D′, 1,100 μm; B, 800 μm; B′, 215 μm.)