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.
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ABSTRACT: The cerebellum is a pre-eminent model for the study of neurogenesis and circuit assembly. Increasing interest in the cerebellum as a participant in higher cognitive processes and as a locus for a range of disorders and diseases make this simple yet elusive structure an important model in a number of fields. In recent years, our understanding of some of the more familiar aspects of cerebellar growth, such as its territorial allocation and the origin of its various cell types, has undergone major recalibration. Furthermore, owing to its stereotyped circuitry across a range of species, insights from a variety of species have contributed to an increasingly rich picture of how this system develops. Here, we review these recent advances and explore three distinct aspects of cerebellar development - allocation of the cerebellar anlage, the significance of transit amplification and the generation of neuronal diversity - each defined by distinct regulatory mechanisms and each with special significance for health and disease.Development 11/2014; 141(21):4031-4041. · 6.27 Impact Factor
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ABSTRACT: The delivery of luminal substances across the intestinal epithelium to the immune system is a critical event in immune surveillance, resulting in tolerance to dietary antigens and immunity to pathogens. How this process is regulated is largely unknown. Recently goblet cell-associated antigen passages (GAPs) were identified as a pathway delivering luminal antigens to underlying lamina propria (LP) dendritic cells in the steady state. Here, we demonstrate that goblet cells (GCs) form GAPs in response to acetylcholine (ACh) acting on muscarinic ACh receptor 4. GAP formation in the small intestine was regulated at the level of ACh production, as GCs rapidly formed GAPs in response to ACh analogs. In contrast, colonic GAP formation was regulated at the level of GC responsiveness to ACh. Myd88-dependent microbial sensing by colonic GCs inhibited the ability of colonic GCs to respond to Ach to form GAPs and deliver luminal antigens to colonic LP-antigen-presenting cells (APCs). Disruption of GC microbial sensing in the setting of an intact gut microbiota opened colonic GAPs, and resulted in recruitment of neutrophils and APCs and production of inflammatory cytokines. Thus GC intrinsic sensing of the microbiota has a critical role regulating the exposure of the colonic immune system to luminal substances.Mucosal Immunology advance online publication, 9 July 2014; doi:10.1038/mi.2014.58.Mucosal Immunology 07/2014; · 7.54 Impact Factor
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ABSTRACT: Background information: The vertebrate basic helix-loop-helix transcription factor Atoh1 is essential for maturation and survival of mechanosensory hair cells of the inner ear, neurogenesis, differentiation of the intestine, homeostasis of the colon and is implicated in cancer progression. Given that mutations in Atoh1 are detected in malignant tumors, study of functionally different Atoh1 alleles and homologues might yield useful avenues for investigation. The predicted sequence of chicken Atoh1 (cAtoh1) has large regions of dissimilarity to that of mammalian Atoh1 homologues. We hypothesize that cAtoh1 might have intrinsic functional differences to mammalian Atoh1.Results: In this study we cloned and sequenced the full open reading frame of cAtoh1. In overexpression experiments we show that this sequence is sufficient to generate a cAtoh1 protein capable of inducing hair cell markers when expressed in nonsensory regions of the developing inner ear, and that morpholino-mediated knock-down using a section of the sequence 5’ to the start codon inhibits differentiation of hair cells in the chicken basilar papilla. Furthermore, we compare the behavior of cAtoh1 and human Atoh1 (hAtoh1) in embryonic mouse cochlear explants, showing that cAtoh1 is a potent inducer of hair cell differentiation and that it can overcome Sox2 mediated repression of hair cell differentiation more effectively than human Atoh1.Conclusions: cAtoh1 is both necessary and sufficient for avian mechanosensory hair cell differentiation. The non-conserved regions of the cAtoh1 coding region have functional consequences on its behavior.This article is protected by copyright. All rights reservedBiology of the Cell 11/2014; · 3.87 Impact Factor
Excitatory neurons of the proprioceptive,
interoceptive, and arousal hindbrain networks
share a developmental requirement for Math1
Matthew F. Rosea, Kaashif A. Ahmadb, Christina Thallerc, and Huda Y. Zoghbia,b,d,e,1
aProgram in Developmental Biology, Departments ofbPediatrics,cBiochemistry and Molecular Biology, anddMolecular and Human Genetics, andeHoward
Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030
Contributed by Huda Y. Zoghbi, October 7, 2009 (sent for review September 3, 2009)
Hindbrain networks important for sensation and arousal contain
diverse neuronal populations with distinct projections, yet share
specific characteristics such as neurotransmitter expression. The re-
tal origin, and the timing of their migration remains unclear. Mice
essential for hearing, balance, and unconscious proprioception. By
projection neurons of the dorsal column nuclei and for vital compo-
nents 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, includ-
ing glutamate, acetylcholine, somatostatin, corticotropin releasing
hormone, and nitric oxide. These findings identify twenty novel
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
auditory ? dorsal columns ? medial lemniscus ? proneural ? rhombic lip
hindbrain, including the auditory, vestibular, and proprioceptive
systems. It also requires regulation of an organism’s arousal state,
of the reticular activating system.
Interestingly, many components of these various systems share a
developmental requirement for the proneural transcription factor
mouse atonal homolog 1 (Math1, Atoh1) (1–6). Math1 expression
begins around embryonic day E9.5 in the rhombic lip (RL), the
dorsal-most neuroepithelium of the developing hindbrain, and
spans the length of the pons, cerebellum, and medulla (7, 8). Early
Math1-dependent neuronal populations have been identified pri-
marily in the rostral pons and cerebellum by using Math1 lacZ
knock-in and Math1-creERT2 transgenic mice (1, 3, 4), whereas the
caudal pons and medulla have remained comparatively uncharac-
the full extent of Math1’s contribution to various hindbrain net-
works has yet to be revealed.
Proprioception has been divided anatomically into unconscious
and conscious pathways. In the unconscious pathway, sensory
inputs synapse with precerebellar neurons in the spinal cord and in
to the cerebellum to coordinate movement ‘‘unconsciously’’ (9).
The conscious proprioceptive network, by contrast, sends input to
the cortex via the cuneate and gracile dorsal column nuclei in the
medulla (10), which relay it to the thalamus via excitatory gluta-
matergic fibers in the medial lemniscus (11). Math1 is required for
glutamatergic neurons in the ECu and other precerebellar nuclei
(unconscious proprioception), but no reports have linked Math1
ovement requires an accurate representation of body posi-
tion in space that utilizes multiple sensory inputs to the
with conscious proprioception. Indeed, the origin of the dorsal
column nuclei as well as their genetic relationship to ECu neurons
has remained unclear.
Similarly, auditory information projects along two distinct hind-
brain pathways, from the ventral cochlear nucleus (VC) to either
the adjacent dorsal cochlear nucleus (DC) or the superior olive
nucleus (SON) in the ventral medulla. The DC analyzes frequency
differences whereas the SON determines the source of sounds
relative to the position of the body (12, 13). Both pathways send
excitatory glutamatergic projections via the lateral lemniscus to the
inferior colliculus (12, 14, 15). However, although RL-derived
origin of the SON has yet to be reported.
In this study, we generated a targeted hormone-inducible
Math1Cre*PRallele to ensure a native Math1 expression pattern. We
labeled temporally distinct subsets of Math1-expressing lineages
and traced their projections. In addition, we characterized changes
null mice. These experiments revealed a novel Math1-dependent
caudal RL migratory stream in the medulla, doubled the number
of reported Math1 hindbrain lineages, and identified new Math1-
dependent neurotransmitters of the conscious proprioceptive, in-
teroceptive (‘‘visceral proprioceptive’’), vestibular, auditory, and
Novel Math1 Lineages in the Medulla and Pons. Known Math1-
dependent caudal rhombic lip (cRL) lineages migrate in the
posterior precerebellar extramural migratory stream (PES) over
several days to form the ECu and lateral reticular (LRt) nuclei in
the medulla (3, 6, 17). The peak migration occurs around E12 in
mice (18). By using Math1LacZ/?knock-in mice (1), we uncovered
an earlier migration from the cRL at E10.5 (Fig. 1A, yellow arrow)
that was contiguous with the later-forming PES (black arrowhead).
This early migration, which we term the caudal rhombic-lip migra-
tory stream (CLS), appeared to form several unreported Math1
sections through an E16.5 Math1LacZ/?hindbrain identified many
nuclei containing new Math1 lineages in both the medulla and
to known lineages (light blue), as best approximated from multiple
brain atlases (SI Text).
Temporal Classification of Math1 Hindbrain Lineages.Tobetterassess
the fate of these new Math1 lineages and characterize the time of
their formation, we targeted a hormone-inducible Cre*PR con-
Author contributions: M.F.R. and H.Y.Z. designed research; M.F.R., K.A.A., and C.T. per-
formed research; M.F.R. analyzed data; and M.F.R., K.A.A., and H.Y.Z. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
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single doses of RU486 on E9.5, E10.5, E12.5, or E13.5. Analysis at
E18.5 revealed distinct and reproducible patterns of labeled hind-
brain cells for each induction time, indicating a temporal resolution
of at least 24 h for this Cre. The labeling included new (yellow
arrowheads) and known (black arrowheads) Math1 lineages (Fig.
1C–F?), and persisted in the adult (Fig. S3). In contrast, Cre
induction in mice lacking Math1 (Math1Cre*PR/?;ROSALacZ/?) pri-
marily labeled cells near the RL (Fig. 1 G and G?). For higher
resolution imaging, we crossed Math1Cre*PRmice to the ROSAEYFP
Cre-reporter line (20). Math1-null brains (E18.5) induced at E10.5
had increased labeling of the choroid plexus (CP), with a concor-
dant decrease in Math1 medullary lineages (compare Fig. 1 I and
to the RL (21), this observation parallels that in the spinal cord
where Math1-dependent lineages contribute to the roof plate in
Math1-null mice (22).
The schematics in Fig. 2A summarize nuclei containing Math1
populations labeled at E10.5 (yellow) versus E12.5/E14.5 (black),
with boxed regions shown to the right (Fig. 2 B and C). The
included the cuneate (Cu) and gracilis (Gr) dorsal column nuclei
(Fig. 2B, level 6a), and the principal sensory trigeminal (Pr5) and
medial portion of the spinal trigeminal–interpolar division (Sp5I)
nuclei (Fig. 2B, level 5a, 2F). In contrast, most unconscious
proprioceptive lineages were labeled at E12.5–E14.5, including the
external granule layer (EGL), ECu, and LRt (Fig. 2C, levels 2, 6a,
6b), as well as the lateral portion of Sp5I (Fig. 2C, level 5a), and the
intertrigeminal region (ITR), prepositus (Pr), and roller precer-
Time course (E10.5–16.5) of LacZ expression in the medulla of Math1LacZ/?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 Math1Cre*PR/
?;ROSALacZ/?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 arrow-
heads) lineages. (G) A Math1Cre*PR/?;ROSALacZ/?(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 Math1Cre*PR/?;
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 Math1Cre*PR/?and CP cells in Math1Cre*PR/?
60 ?m; C–G, 1,000 ?m; C?–G?, 900 ?m; H and I, 400 ?m; H? and I?, 100 ?m.)
(A) Schematics of hindbrain nuclei containing early- (yellow) and late-forming
(black) Math1 lineages (listed to right in corresponding colors) on coronal he-
misections, rostral to caudal (levels 1–6, Fig. 1B). (B and C) Magnified boxed
regions in A from corresponding sections of E18.5 Math1Cre*PR/?;ROSAEYFP/?and
at level 2 marks the midbrain/hindbrain junction.) The regions shown contain
(D and E) Corresponding regions to B and C from E18.5 WT and Math1-null
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
neurons. Abbreviations: Please see Table 1 for list of nuclei. (Scale bar: B–E, 500
?m; F and G, 83 ?m; F and G Insets, 28 ?m.)
Rose et al.PNAS ?
December 29, 2009 ?
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ebellar nuclei (Fig. 2G). Similarly, neurons of the medial (MVe)
and spinal (SpVe) vestibular nuclei were labeled at E10.5 (Fig. 2F),
whereas the more lateral vestibular nucleus X (X) was labeled at
and DC were labeled at E10.5 (Fig. 2B, level 3b, and 2F), whereas
the more lateral VC and cochlear granule neurons were labeled at
E12.5 (Fig. 2G).
Induction at E10.5 also labeled nuclei vital for interoception,
including Barrington’s (BN) and the parabrachial/Ko ¨lliker-Fuse
(PB/KF) nuclei (Fig. 2B, levels 2, 3a), as well as those critical for
arousal: the pedunculopontine tegmental (PPTg), lateral dorsal
tegmental (LDT), and medullary reticular (MdD) nuclei (Fig. 2B,
levels 1, 3a, 6b). Moreover, induction at E10.5 labeled cells near
multiple medullary nuclei associated with respiration: the ros-
troventrolateral reticular nucleus (RVL), pre-Bo ¨tzinger complex
(preBo ¨tC), and rostral ventral respiratory group (rVRG) (Fig. 2B,
levels 4, 5b, 6b), recently described in further detail (23). Table 1
lists all new and known Math1 hindbrain populations.
We also evaluated several transcription factors and neuro-
as Math1 autoregulation (24). Expression of Lhx9 and Barhl1 at
E18.5, known Math1-dependent transcription factors (3, 6, 25),
segregated with Math1 lineages induced at E10.5 and E12.5,
respectively (Fig. 2 D–G), and were lost in Math1-null hindbrains
(Fig. 2 D and E). Lhx2 and Barhl2, also Math1-dependent (6, 26),
appeared similar to Lhx9 [Movies S1–S48 (AVI)]. [These lin-
eages did not coexpress Phox2b, distinguishing them from
recently identified neurons with intraparenchymal Math1 ex-
pression (23).] Complete sets of serial sections labeled for each
transcription factor in both WT and Math1-null hindbrains are
available online [Movies S1–S48 (AVI)].
Subsets of Hindbrain Glutamatergic, Somatostatin, CRH, Nitric Oxide,
Cholinergic, and Levodopa Neurons Require Math1.LossofMath1led
to loss/reduction in markers for multiple hindbrain neurotransmit-
ters: glutamate (Vglut1, Vglut2, and Vglut3), somatostatin (Sst),
corticotropin releasing hormone (Crh), nitric oxide (Nos1), acetyl-
choline (Vacht), and levodopa (Th) [Fig. 3 B–G, Table 1, and
Movies S1–S48 (AVI)]. Vglut2 was largely absent from the Math1-
pons (Fig. 3B, levels 1, 2, 3b), and the Cu/Gr dorsal column nuclei
in the medulla (Fig. 3B, level 6a). Other nuclei primarily lost
neurons with high Vglut2 expression, whereas low-expressing ones
remained, presumably from non-Math1-dependent lineages (27):
the microcellular tegmental (MiTg), PPTg, LDT/BN, Sp5I, RVL,
MdD, rVRG, and near the preBo ¨tC (Fig. 3B, levels 1, 3a, 4, 5a, 5b,
6b). Several other regions likewise lost many Vglut2 neurons [Mov-
ies S1–S48 (AVI), Table 1]. We quantified the Vglut2 expression
by the VC at P0, was entirely lost. Most other neurons that express
Vglut1 later (e.g., granule and precerebellar neurons) also require
Math1 (1–3). By comparison, Vglut3 was only lost from the DC
[Movies S1–S48 (AVI)]. The Dll and PB in the pons and the Cu/Gr
in the medulla also lost much of their Sst expression (Fig. 3C, levels
1, 2, 6a).
Math1-null mice also lost most Crh expression from the hind-
brain, including the PPTg, PB, and BN in the pons, and the RVL,
Sp5I, ECu, preBo ¨tC, Cu, and LRt in the medulla (Fig. 3D, levels
pons, components of the reticular activating system (Fig. 3 E and F,
levels 1, 3a), and from the preBo ¨tC, Cu, and rVRG in the medulla
and LDT, indicating these cholinergic neurons require Math1 in
also expressed Math1-dependent tyrosine hydroxylase (Th) (Fig.
3G, levels 1–2) but lacked subsequent enzymes required for nor-
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
somatostatin (SST: Sst), corticotropin releasing hormone (CRH: Crh), nitric oxide
(NO: Nos1), acetylcholine (ACh: Vacht), and levodopa (L-Dopa: tyrosine hydrox-
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 ?m.)
Subsets of glutamatergic, somatostatin, CRH, nitric oxide, cholinergic,
tive, and conscious proprioceptive systems arise from the RL. (A) Schematic of
C) Dotted outlines mark approximate boundaries of these three nuclei on adja-
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 colabeling of EYFP (somas) with Sst (cytoplasmic ? extracellular). Abbre-
C?, and E?, 80 ?m; Lower frames of B, C, and E, 40 ?m.)
Nitric oxide, CRH, and somatostatin neurons of the arousal, interocep-
www.pnas.org?cgi?doi?10.1073?pnas.0911579106 Rose et al.
epinephrine/dopamine synthesis [Fig. S5 and Movies S1–S48
(AVI)]. These neurons may produce levodopa, similar to more
rostral neurons that are reportedly involved in interneuronal shut-
tling for catecholamine production (28). Also, some enkephalin
(Penk1) expression was lost from the PB and DN, serotonin receptor
1a (Htr1a) disappeared from the Dll, and somatostatin receptor 2
(Sstr2) was lost from the EGL and DC [Movies S1–S48 (AVI)]. In
contrast, other neurotransmitter markers for GABA, glycine, nor-
epinephrine, dopamine, serotonin, substance P, and thyrotropin
releasing hormone showed only slight rearrangements due to loss
of surrounding Math1 lineages [Fig. S5 and Movies S1–S48 (AVI)].
To assess the cell autonomy of these new Math1-dependent
CRH, and SST in select nuclei from Math1Cre*PR/?;ROSAEYFP/?hind-
brains induced at E10.5. We found colabeling of early Math1
lineages with Nos1 in the LDT and CRH in the adjacent BN in the
in the medulla (Fig. 4 D and E).
Math1 Lineages Form the Excitatory Hindbrain Output Tracts for
Conscious and Unconscious Proprioception and Hearing. The primary
excitatory output tracks for the conscious and unconscious propri-
within the Cu/Gr, DN, and DC/SON, respectively (9, 11, 12). To
assess whether the Math1 lineages in these nuclei correspond to
these excitatory projection neurons, we labeled their processes
with membrane-targeted GFP by crossing the Math1Cre*PRto
TaumGFP_NLacZCre-reporter mice (29).
from the Cu/Gr nuclei to the medial lemniscus (ml) in the ventral
medulla (Fig. 5A?, and Fig. 5 B and B?, level 5). The ml was labeled
along its entire length (Fig. 5B, levels 2–5) up to the VPL nucleus
of the thalamus (Fig. 5 A? and B?, level 1), where the labeled fibers
thalamic Sst expression is known to arise from the cuneothalamic
projections of the dorsal column nuclei, which coexpress Sst and
Vglut2 (11). Induction at E10.5 also labeled the lateral lemniscus
(ll) that contains axons of the auditory system from the SON and
DLL to the inferior colliculi (Fig. 5 A and B?, level 3). Likewise,
projections of the deep cerebellar nuclei extended through the
superior cerebellar peduncle (scp) (Fig. 5 A? and B?, level 4) and
decussated in the pons (xscp) (Fig. 5B, level 3) to contralateral red
nuclei (R), key targets of the cerebellar nuclei (Fig. 5 A? and B?,
In contrast, induction at E12.5 labeled fibers of the inferior
Table 1. Summary of the Math1 hindbrain rhombic lip lineages
superior cerebellar peduncle. Math1Cre*PR/?; ROSAEYFP/?; TaumGFP_nLacZ/?hind-
brains (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.
in the VPL thalamic nucleus. (Although somas also expressed EYFP, primarily
mGFP processes were visible at this magnification.) (B) Coronal sections from
somas (red) (from the TaumGFP_nLacZ/?), with selected projections (yellow arrow-
heads) and nuclei (white arrowheads) indicated. (C–D?) Lateral (C and D) and
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
?m; B, 800 ?m; B?, 215 ?m.)
Early Math1 lineages project in the medial and lateral lemnisci and the
Rose et al.PNAS ?
December 29, 2009 ?
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cerebellar peduncle (Fig. 5C) through which the ECu and LRt
project (Fig. 5C?), and induction at E14.5 labeled fibers of the
middle cerebellar peduncle (Fig. 5D) known to contain the PN
projections (Fig. 5D?). Likewise, projections to the SON (likely
from the VC) were seen with induction at E12.5 (Fig. 5C?).
In this study, we combined histological analysis, in situ hybrid-
ization, and fate-mapping to identify unreported Math1-
dependent lineages in the perinatal hindbrain. We find that
throughout the hindbrain, distinct subsets of Math1-dependent
rhombic lip (RL) lineages express the same transcription factors
and neurotransmitters and contribute to nuclei of the same
networks. Within the conscious and unconscious proprioceptive,
interoceptive, auditory, and arousal networks, Math1 lineages
appear to serve similar functions, such as forming the primary
excitatory output tracts. It is remarkable that distinct hindbrain
networks which process divergent types of sensory information,
all rely on the contribution of Math1-dependent rhombic lip
The hindbrain is arranged as a series of anterior–posterior
that similar Math1-dependent lineages arise from the RL at com-
parable times in various rhombomeres. The early migration (E9.5–
10.5) of the CLS of the medulla occurs in parallel to the early
portion of the rostral RL migratory stream (RLS) that populates
the cerebellum and pons (3). Both of these early migrations
generate Vglut2-positive neurons and express Lhx9. In contrast, the
external granule layer and posterior precerebellar extramural mi-
gratory stream (PES), which exit the RL at E12.5–14.5 as later
portions of the RLS and CLS, respectively (3, 17), form Vglut1-
positive neurons that express Barhl1. Thus, distinct Math1 lineages
arising in the RL at similar times in different rhombomeres share
parallel developmental trajectories and gene expression.
Proprioception, originally defined as an organism’s awareness of
its own movement and position of its body parts, includes both a
conscious network that transmits sensory information to the cortex
(10) and an unconscious cerebellar network that coordinates loco-
motion (9, 33). Although many glutamatergic neurons of the
unconscious network, including the ECu in the dorsal medulla, are
known to arise from the RL and require Math1 (1–6, 17), little is
known about the origins of conscious proprioceptive neurons. The
lie adjacent to the ECu (10). The majority of Cu/Gr neurons form
two days before the ECu (18, 34), and many express Vglut2 and Sst
and project to the thalamus via well-defined tracks, including the
medial lemniscus (11). We show that the Cu/Gr contain Math1
lineages arising mostly at E10.5, matching the peak time of forma-
tion for Cu/Gr neurons (18), and express Lhx9 and Sst. In addition,
they project via the medial lemniscus to the thalamus where their
projections overlap with Sst processes. In the absence of Math1, the
Cu/Gr nuclei lose most all Vglut2 and Sst expression. Hence, the
excitatory neurons of conscious and unconscious proprioception in
the medulla appear to arise from the RL as early and late portions
gene-expression differences correspond to this temporal distinc-
tion, with Math1 serving a central role in the development of both
This developmental pattern parallels that in the vestibular and
auditory systems, where we now show that Lhx9-expressing gluta-
matergic projection neurons of the SON arise early from the RL
and require Math1 just like those of the dorsal cochlear nucleus.
Thus, we find that many glutamatergic neurons of the auditory,
vestibular, and proprioceptive systems throughout the hindbrain
require Math1, arise from the rhombic lip at similar times, share
expression of specific neurotransmitters and transcription factors,
and appear to serve similar roles in each system.
Many NO and CRH neurons critical for interoception and
arousal also require Math1. The Math1-dependent NO lineages
include those in the pedunculopontine tegmental (PPTg) and
lateral dorasal tegmental (LDT) nuclei in the pons. Together with
the adjacent norepinephrine (NE) neurons of the locus coeruleus
constitute an important component of the reticular activating
system (RAS) vital for arousal. Likewise, CRH neurons in the
PPTg and in Barrington’s nucleus (BN), located immediately
adjacent to the LDT and LC (36), are also Math1-dependent. The
BN contains the largest group of CRH neurons in the hindbrain,
responds to the interoceptive inputs of bladder and colon disten-
sion, and projects to the LC to increase the activity of NE neurons
and stimulate arousal (37–39).
This LDT/BN/LC complex lies at a functional intersection of
arousal and interoception. Although these three nuclei have been
described as anatomically separate, in some species their respective
NO, CRH, and NE neurons are intermingled (40). This pattern of
associated NO, CRH, and catecholaminergic neurons repeats
A1/C1, and A2/C2 nuclei. Although the NE neurons in each of
these regions require the proneural gene Mash1 (31), we now
share a similar dependence on Math1. These results uncover a
previously unsuspected genetic and developmental relationship
between CRH and NO neurons of the interoceptive and arousal
systems, and suggests a model of these neurons forming together
throughout the hindbrain similar to the proprioceptive system.
each other within specific networks. In addition, some Math1-
dependent neurons form connections between the networks. For
instance, the PPTg neurons of the RAS receive extensive auditory
inputs that have been proposed to mediate the auditory startle
response that provides arousal from sleep (41). Similarly, the CRH
neurons of the BN connect with the RAS to stimulate arousal in
response to interoceptive input (38, 39). The NO/acetylcholine
neurons of the RAS connect back to proprioceptive nuclei such as
the spinal trigeminal nucleus in the medulla, where cholinergic
stimulation increases the excitability of glutamatergic projection
neurons (42). Some of these neurons then connect to the thalamus
as part of conscious proprioception and others contribute to the
cerebellar unconscious proprioceptive network (34). This associa-
tion between developmental origin and subsequent functional
connectivity forms a leitmotif throughout Math1-dependent hind-
Math1-dependent hindbrain lineages and demonstrated that dif-
ferences in marker expression correlate with temporal origin.
Patterns of RL lineage development are conserved throughout the
fates (glutamate, somatostatin, CRH, nitric oxide, acetylcholine,
and levodopa). This study provides evidence for the association
between Math1 and conscious proprioception and identifies new
Math1-dependent components of the unconscious proprioceptive,
auditory, vestibular, interoceptive, and arousal hindbrain networks,
demonstrating a genetic, developmental, and functional link be-
tween these diverse sensory systems.
Materials and Methods
Generation of an Inducible Math1Cre*PRLine. We used a second-generation
Cre-progesterone receptor fusion (Cre*PR), amplifying the sequence from pNN-
hCre19V336A-PR650–914 (43). We ligated the Cre*PR with Math1 5? and 3?
targeting arms such that the Math1 transcription start site and first five Math1
?g of RU486 (Mifepristone, Sigma) was administered to pregnant dams by
interperitoneal injection at E9.5, E10.5, E11.5, E12.5, E13.5, E14.5, or E18.5. To
prevent abortion, progesterone (Sigma) was coadministered (see SI Text).
www.pnas.org?cgi?doi?10.1073?pnas.0911579106Rose et al.
Mouse Strains, Staging, and Genotyping. We used two Math1-null alleles which
contain either LacZ (Math1LacZ) or HPRT (Math1?) in place of the Math1 coding
region (1, 2). The null embryos carried one Math1Cre*PRallele and one Math1?
allele. Three Cre reporter lines were used: ROSALacZ, ROSAEYFP, and TaumGFP_nLacZ
(19, 20, 29) (see SI Text).
Immunohistochemistry and X-gal Staining. E18.5/P0 brains were fixed 5–10 h in
found online in the SI Text.
RNA in Situ Hybridization (ISH) Screen. The 24 probes were amplified from
using primers from the Allen Brain Atlas (www.brain-map.org). From 34 E18.5
serial fresh frozen sections were cut. ISH for each probe was performed on
complete sets from multiple littermate-matched WT and Math1-null hindbrains
at 1.2 ?m/pixel [see Movies S1–S48 (AVI) and SI Text for list of probes). Image
brightness and contrast were normalized by using Adobe Photoshop.
ACKNOWLEDGMENTS. We thank R. Atkinson, A. Liang, B. Antalffy, N. Ao, and
Y. Liu for technical assistance, J. Dodd (Columbia University, New York, NY) for
kindly providing the Lhx2/9 antibody, and T. Klisch, A. Flora, and B. Jusiak for
comments on the manuscript. This work was supported by Gene Expression and
and Stroke and a Baylor Research Advocates for Student Scientists Scholarship;
K. A. A. was supported by Pediatric Scientist Development Program Award
ment. H. Y. Z. is an investigator of the Howard Hughes Medical Institute.
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