BACKGROUND
in the hindbrain, motor neuron progenitors are born in a region-specific manner, neighboring the floor plate, in response to their level of exposure to different morphogenetic gradients  <cit> .
these neuronal progenitors differentiate in a rhombomere-specific pattern, which requires the correct spatiotemporal expression of the homeotic  gene clusters .
motor neuron progenitors in the hindbrain can differentiate into somatomotor neurons , which innervate the skeletal muscles of the body, or visceromotor neurons , which innervate autonomic ganglia  and branchial arch-derived muscles   <cit> .
facial  motor neurons, which originate from rhombomeres 4/ <dig>  belong to the vmn class of motor neurons.
at embryonic day  <dig>  , facial motor neurons begin to migrate caudally to the pial side of the hindbrain to form the facial motor nucleus, which subsequently segregates into two lobes  <cit> .
despite the elucidation of the genetic mechanisms that specify smn identity, the corresponding pathways responsible for the establishment of vmns are less well understood.
furthermore, even though sonic hedgehog  signaling is known to be required for the development of all motor neurons , the upstream regulators of shh expression and function during late embryonic and early postnatal development, especially within the vmns of the facial motor nucleus, remain largely unknown  <cit> .
the paired-like homeobox 2b  gene is expressed in both proliferating progenitors and post-mitotic motor neurons and is required for the development of vmns  <cit> .
similarly, the homeodomain transcription factor islet <dig> , which is expressed in both post-mitotic smns and vmns, plays a broad role in motor neuron identity and specification  <cit> .
in the absence of isl <dig>  mice die during embryogenesis around e <dig> .
this embryonic lethality is associated with an increase in cell death in the hindbrain and neural tube, suggesting that loss of isl <dig> function results in the death of cells that were fated to differentiate into motor neurons  <cit> .
in the trigeminal and dorsal root ganglia , isl <dig> appears to play distinct roles during early  versus late  development  <cit> .
in cranial ganglia, isl <dig> is required for cell survival and isl <dig> mutants show thinner blunted facial  ganglia  <cit> .
however, the precise role of isl <dig> in these cell types and in their respective hindbrain nuclei, such as the facial motor nucleus, remains poorly documented.
the adult facial motor nucleus contains seven subnuclei; the lateral, dorsolateral, dorsal intermediate, ventral intermediate, dorsomedial and ventromedial nuclei are located within the main nucleus, while the seventh, the dorsal accessory nuclei, is located above the main nucleus  <cit> .
neurons innervating the nasolabial musculature are located in the lateral facial motor nucleus, while neurons supplying the auricular musculature are located in the medial facial motor nucleus  <cit> .
together the lateral, intermediate and medial subnuclei comprise  <dig>   <dig> , and  <dig> %, respectively, of the motor neurons in the adult facial motor nucleus, with the dorsal accessory nuclei contributing the final  <dig> %  <cit> .
studies with mouse knockout models have shown that shox <dig> is required for normal development of the humerus and femur , the anterior palate  <cit> , the temporomandibular joint of the jaws  <cit> , the sinoatrial valves and pacemaker region of the heart  <cit> , and trkb-positive mechanosensory neurons of the dorsal root ganglia  <cit> .
most recently, shox <dig> has been shown to play an important role in development of the inferior colliculus and cerebellum  <cit> .
we suspected a function for shox <dig> during facial motor nucleus development given our previous study demonstrating a novel role for shox <dig> in the hindbrain  <cit> , and since shox <dig> has been shown to regulate transcription of isl <dig>  <cit> , an important regulator of vmn development  <cit> .
in the current study we show that conditional inactivation of shox <dig> in the brain results in impaired axonal projections of vmns and a concomitant loss of medially located neurons in the facial motor nucleus postnatally.
furthermore, we demonstrate that shox2-mutant neonates display impaired feeding behavior, perhaps due to facial paralysis as a result of improper development and function of the facial nerves.
RESULTS
shox <dig> expression in the face and facial motor nucleus during development
whole-mount in situ hybridization  at e <dig>  showed shox <dig> expression in the trigeminal  and facial  ganglia, in addition to the developing maxillary process and mandibular arch .
shox <dig> continued to be expressed in the trigeminal  ganglia, maxillary process and mandibular arch at e <dig>  .
we also visualized the shox <dig> expression pattern with a novel shox2lacz allele .
x-gal staining of embryos carrying the shox2lacz allele accurately reproduced the endogenous shox <dig> expression pattern with increased sensitivity and less background compared to wish .
at e <dig> , shox2lacz/+ embryos showed lacz staining in the trigeminal  and facial  ganglia of the embryo .
similarly at e <dig> , lacz staining was visible in the trigeminal  and facial  nerves of the embryo, in addition to the maxillary process and mandibular arch .
lacz continued to be expressed in the trigeminal  ganglion of the embryonic face from e <dig>  to e <dig> , and could be seen in the developing pharyngeal arch .
at e <dig>  lacz expression was present in the facial mesenchyme that contributes to the maxilla and mandible .
however, from e <dig>  onward lacz staining was no longer observed in the facial  ganglia .
postnatally  lacz expression was maintained in the facial mesenchyme surrounding the facial  nerves, but not within the axons themselves .
examination of shox <dig> expression early embryonically in the developing brain showed shox <dig> staining in a region of post-mitotic neurons adjacent to the floor plate , a region where phox2b+/isl1+ are localized   <cit> .
later in the p <dig> brain, shox <dig> was expressed in the facial motor nucleus .
similarly, in shox2lacz/+ animals, lacz staining was strongly expressed in both the lateral and medial lobes of the facial motor nucleus ; however, staining was not visible within the trigeminal nucleus .figure  <dig>
shox <dig> mrna and lacz expression in the developing embryonic face and postnatal facial motor nucleus.
a, b whole-mount in situ hybridization  at e <dig>   and e <dig>   show shox <dig> expression in the trigeminal  and facial  ganglia, in addition to the developing maxillary process  and mandibular arch .
c–e in situ hybridization  on whole p <dig> brains  and sagittal  or coronal  sections through the brain show shox <dig> expression in the facial motor nucleus .
f–j x-gal staining of shox2
lacz/+ embryos at e <dig>  , e <dig>  , e <dig>  , e <dig>  , and e <dig>   shows that shox <dig> is expressed in the trigeminal and facial ganglion , in addition to the developing maxillary process and mandibular arch.
starting at e <dig>  and continuing to e <dig> , shox <dig> expression becomes restricted to the trigeminal ganglion and the facial mesenchyme that will contribute to the maxilla and mandible, however it is absent from the facial ganglia.
k–o x-gal staining of shox2
lacz/+ pups in the p <dig> face , brain  and sagittal  or coronal  sections through the brain show shox <dig> expression in the tissue surrounding the facial nerves , with the exception of the nerves themselves, and shox <dig> expression in the facial motor nucleus .
ey eye, nv trigeminal nucleus, sbl superior buccolabial nerve, ibl inferior buccolabial nerve, mm marginal mandibular nerve, cb cerebellum, mb midbrain.
scale bar 500 μm.
nestin-creshox2-mutant pups exhibit defects in feeding behavior
to determine the functional role of shox <dig> in the facial motor nucleus, we used nestin-cre  <cit>  to delete the shox <dig> gene conditionally in neural tissue throughout the central nervous system .
we crossed nestin-cre; shox2+/− mice with shox2flox/flox mice  <cit>  to obtain conditional mutant nestin-cre; shox2flox/− embryos and neonates.
shox <dig> expression was eliminated in the brain of nestin-creshox2-mutant embryos, including the facial motor nucleus .
all nestin-cre shox2-mutant pups died between  <dig> to 26 h following birth.
this limited our characterization of shox <dig> function to embryonic and neonatal stages.
p <dig> nestin-cre; shox2flox/− mutant pups displayed reduced activity and impaired feeding behavior as early as 6 h following birth , which correlated with a lack of milk in the stomachs of nestin-cre; shox2flox/− mutant pups .
since shox <dig> is required for the proper development of the palate  <cit> , we analyzed the anterior and posterior palate of nestin-cre; shox2flox/− mutant pups.
we did not observe any obvious defects or malformations in the palate of nestin-cre; shox2flox/− mutant pups , suggesting that the absence of milk in shox2-mutant neonatal stomachs was not due to cleft palate malformations.
nestin-cre; shox2flox/− mutant pups also displayed tremors 16–26 h following birth , which could be related to defects in neural circuitry or result from a loss of shox <dig> function in other regions of the hindbrain.
facial  nerve branching defects in nestin-cre shox2-mutant animals
since shox2-mutant neonates displayed impaired feeding but an apparently normal palate, we hypothesized that shox <dig> mutants fail to nurse properly due to facial paralysis.
to test this hypothesis, we first sought to determine if the abnormal feeding behavior resulted from the loss of a subset of axons in the nerves of the face.
we stained control and nestin-cre shox2-mutant embryos and p <dig> heads with the 2h <dig> antibody that visualizes nerves by detecting neurofilaments .
in e <dig>  nestin-cre; shox2flox/− conditional knockout embryos the trigeminal nerves  appeared normal .
to confirm that the trigeminal nerves  were intact even in the complete absence of shox <dig>  we performed neurofilament staining on e <dig>  shox2-null embryos   <cit> .
while the trigeminal nerves  remained intact, the facial nerves  were truncated and failed to branch properly in both nestin-cre; shox2flox/− conditional knockout and shox2-null embryos .
at e <dig>  the facial  nerves continued to be truncated as compared to controls .
neurofilament staining at e <dig>  displayed a range in severity of facial  nerve truncation, with figure 2f showing the least severe truncation and figure 2h having the most severe truncation we observed among seven embryos.
later at e <dig>  , e <dig>  , and early postnatal  stages, the zygomatic, superior buccolabial, inferior buccolabial and marginal mandibular nerve branches of the facial  nerve could be seen in controls   <cit> , however these distinct facial  nerve branches were truncated or absent in nestin-cre; shox2flox/− mutant animals .
similar to e <dig> , facial  nerve truncation ranged in severity at the later embryonic and early postnatal time-points analyzed .
the severity in facial  nerve truncation observed in nestin-cre; shox2flox/− mutant animals, displayed in figure 2j, l, n, are representative of the most common truncations observed.figure  <dig> facial nerve truncations in shox2-mutant animals.
a–d side view of the e <dig>   or e <dig>   face of control , nestin-cre; shox2
flox/−  and shox2
−/−  embryos stained with the 2h <dig> anti-neurofilament antibody.
at e <dig>  the main branch of the facial nerve  begins to separate into smaller projections in control embryos , while a single branch is visible in nestin-cre; shox2
flox/− conditional knockout embryos .
at e <dig>  the facial nerve is noticeably truncated in the shox2
−/− embryos , while the trigeminal nerve  remains intact.
e–h side view of the e <dig>  face of control  and nestin-cre; shox2
flox/−  embryos stained with the 2h <dig> antibody display the range in severity of facial nerve truncation, with f being the least severe truncation and h being the most severe truncation observed in nestin-cre; shox2
flox/− embryos .
side view of the e <dig>  , e <dig>   and p <dig>  face of control  and nestin-cre; shox2
flox/−  animals stained with the 2h <dig> antibody.
at e <dig>  and e <dig>  the zygomatic , superior buccolabial , inferior buccolabial  and marginal mandibular  nerve branches can be seen in control  embryos but are not visible in nestin-cre; shox2
flox/−  embryos .
at p <dig> the truncated remnants of what appears to be the superior buccolabial nerve were observable in nestin-cre; shox2
flox/− pups .
ey eye, b buccal nerve, sm superficial masseter nerve.
scale bar 500 μm.
to confirm the persistent lack of proper facial  nerve branching and development, particularly at late embryonic and early postnatal time-points when the larger head size led to confounding background staining, we used a second transgenic line, this one carrying a bacterial artificial chromosome  containing the shox <dig> gene with a lacz insertion  to visualize the facial  nerves.
while obvious facial  nerve truncations were difficult to visualize at e <dig>  , the main branch of the facial  nerve was noticeably truncated, as the smaller ganglionic projections were absent at e <dig>  in nestin-cre; shox2flox/− mutant embryos as compared to controls .
furthermore, consistent with our neurofilament staining, at later embryonic  and early postnatal  stages, the zygomatic, temporal, superior buccolabial, inferior buccolabial and marginal mandibular nerve branches of the facial  nerve were observed in controls  and appeared truncated or absent in nestin-cre; shox2flox/− mutant animals .
for example, by p <dig> the facial  nerves appeared to be absent in nestin-cre; shox2flox/− mutant animals .
the buccal and superficial masseter nerves, both sensory components originating from the trigeminal  nerve  <cit> , were the only nerves visible in nestin-cre; shox2flox/− mutant animals, leaving the face almost completely devoid of innervation .
to determine whether the facial  nerve disruptions occurred as a result of the slight reduction in shox <dig> expression in the embryonic facial mesenchyme , or whether this was related specifically to disruptions in the facial motor nucleus, we conditionally removed shox <dig> in all neural crest derivatives using a wnt1-cre driver.
this resulted in the complete loss of shox <dig> expression in the face , and ultimately did not appear to result in any disruptions to the development of the facial  nerves .
together these results show impaired axonal projection properties of visceral motor neurons  in the facial motor nucleus of the hindbrain in mice lacking shox <dig> figure  <dig>
lacz staining highlights truncated facial nerves and impaired axonal projection properties of vmns in mice lacking shox <dig> function.
a–j bac rp23-105b3-lacz transgenic animals stained with x-gal.
a, b side view of the e <dig>  face of control  and nestin-cre; shox2
flox/−  embryos display similar axonal projections of the main branch of the facial nerve  .
c, d side view of the e <dig>  face of control  and nestin-cre; shox2
flox/−  embryos display facial nerve truncation in mice lacking shox <dig> function .
e–h side view of the e <dig>   and e <dig>   face of control  and nestin-cre; shox2
flox/−  embryos highlights the zygomatic , temporal , superior buccolabial , inferior buccolabial  and marginal mandibular  nerve branches in control  embryos, which are truncated or lost in nestin-cre; shox2
flox/− embryos .
i, j side view of the p <dig> face of control  and nestin-cre; shox2
flox/−  pups demonstrate that the facial nerves are absent in nestin-cre; shox2
flox/− pups .
ey eye, b buccal nerve, sm superficial masseter nerve.
scale bar 500 μm.
loss of shox <dig> in the facial motor nucleus results in elevated apoptosis
since shox2-mutant embryos and neonates display impaired axonal projection properties and were unable to nurse properly, we next wanted to determine if development of the facial motor nucleus was disrupted in the absence of shox <dig>  both neurofilament and lacz staining of p <dig> control and nestin-cre; shox2flox/− or nestin-cre; shox2lacz/flox pup brains demonstrated that the facial motor nucleus was severely reduced in size, with disruptions in neuronal spatial organization in mice lacking shox <dig> function .
while the symmetry between the left and right facial motor nuclei appears to be relatively maintained , the two lobes that normally comprise the facial motor nucleus are no longer present .
we next investigated whether an increase in apoptosis could account for the changes observed in the facial motor nucleus of shox2-mutant brains.
we found an increase in the number of apoptotic cells present at e <dig>  and e <dig>  in the facial motor nucleus of shox2-mutants ; however, no significant increase in apoptosis was detected in the e <dig>  or p <dig> facial motor nucleus of shox2-mutant animals .
we observed a ~ <dig> -fold increase in the number of cleaved active-casp3 + cells in the e <dig>  facial motor nucleus of nestin-cre; shox2flox/− animals as compared to controls .
we also observed a striking ~ <dig> -fold increase in the number of cleaved active-casp3 + cells in the e <dig>  facial motor nucleus of nestin-cre; shox2flox/− animals as compared to controls .
while the slight elevation in apoptosis detected in the e <dig>  and p <dig> facial motor nucleus of shox2-mutant animals was not significantly different from controls , we did observe a ~ <dig> -fold increase in the number of cleaved active-casp3 + cells at p <dig> when we included the tissue immediately surrounding the facial motor nucleus of nestin-cre; shox2flox/− animals  in our cell count .
moreover, when we calculated the area of shox2lacz + cells remaining in sagittal sections through the center of the facial motor nucleus of nestin-cre; shox2lacz/flox animals as compared to controls at e <dig>   and p <dig> , we observed a ~44% decrease in the area of shox2lacz + cells in the e <dig>  facial motor nucleus  and a ~53% decrease in the area of shox2lacz + cells in the p <dig> facial motor nucleus .
therefore, the higher levels of apoptosis are correlated with a decrease in the size of the facial motor nucleus, and taken together, the results demonstrate that apoptosis contributes to the disruptions observed in the facial motor nucleus of nestin-cre; shox2flox/− animals.figure  <dig>
nestin-cre driven conditional shox2-deletion results in disruptions in the facial motor nucleus and changes in cell death.
a, b ventral view of the p <dig> brain of control  and nestin-cre; shox2
flox/−  animals stained with the 2h <dig> antibody demonstrate that the facial motor nucleus  is severely reduced in nestin-cre; shox2
flox/− pups .
c–f x-gal staining of p <dig> control  and nestin-cre; shox2
lacz/flox  pup brains  show shox <dig> expression in the facial motor nucleus and demonstrate that the nucleus is severely reduced in size  in mice lacking shox <dig> function.
cleaved active-casp <dig> immunostaining on e <dig>  , e <dig>  , e <dig>   and p <dig>  control  and nestin-cre; shox2
flox/− mutant  sagittal sections through the facial motor nucleus .
i quantification of the number of cleaved active-casp3 + cells present in the e <dig>  facial motor nucleus  of control and nestin-cre; shox2
flox/− embryos .
l quantification of the number of cleaved active-casp3 + cells present in the e <dig>  facial motor nucleus  of control and nestin-cre; shox2
flox/− embryos .
o quantification of the number of cleaved active-casp3 + cells present in the e <dig>  facial motor nucleus  of control and nestin-cre; shox2
flox/− embryos .
r quantification of the number of cleaved active-casp3 + cells present in the p <dig> facial motor nucleus  of control and nestin-cre; shox2
flox/− embryos .
s quantification of the number of cleaved active-casp3 + cells present in and around the p <dig> facial motor nucleus  of control and nestin-cre; shox2
flox/− embryos .
t, u representative binary particle area diagrams from imagej displaying shox2lacz + cell staining at e <dig>   and p <dig>  in controls and nestincre; shox2
lacz/flox facial motor nuclei.
v quantification of the area of shox2lacz + cells present in sagittal sections through the center of the e <dig>  facial motor nucleus  of control and nestin-cre; shox2
lacz/flox embryos .
w quantification of the area of shox2lacz + cells present in sagittal sections through the center of the p <dig> facial motor nucleus  of control and nestin-cre; shox2
lacz/flox animals .
cb cerebellum.
a–f
scale bar 500 μm.
g–q
scale bar 250 μm.
shox <dig> is required for normal expression of markers of vmn identity in the facial motor nucleus
given the marked reduction in size of the facial motor nucleus in shox <dig> mutants , we next sought to investigate whether factors other than facial  nerve truncation and cell death could be changing in the facial motor nucleus following the loss of shox <dig>  prior to e <dig> , we did not observe any obvious changes in gene expression or disruptions to the migratory patterns of vmns .
at e <dig> , however, sagittal sections of the facial motor nucleus showed reductions in the overall size of the nucleus in nestin-cre; shox2lacz/flox embryos , which was consistent with the regional increase in the number of cleaved active-casp3 + cells at this time-point .
since shox <dig> has been previously shown to regulate isl <dig>  <cit> , an important regulator of vmn development  <cit> , we next examined the expression of isl <dig> in shox2-mutant animals.
we observed a regionally restricted loss of isl <dig> and the hindbrain vmn determinant phox2b in the facial motor nucleus as early as e <dig>  in nestin-cre; shox2flox/− mutant embryos as compared to controls .
since loss of isl <dig> function in the trigeminal and drg have been shown to result in changes in the expression of contactin  <dig> , tubulin β- <dig> chain  and peripherin  <dig>   <cit> , and given that phox2b-deficient mice have been used to identify the facial motor nucleus as a source of slit homolog  <dig>  signaling  <cit> , we next examined the expression patterns of these genes.
although subtle changes in the spatial patterns and expression levels of cntn <dig> and tubb <dig> were observed, periph <dig>  solute carrier family  <dig>  and slit <dig> phenocopied the regionally specific absence of isl <dig> and phox2b in the facial motor nucleus at e <dig>  in nestin-cre; shox2flox/− mutant embryos .
the changes in expression for these genes continued to be observed between nestin-cre; shox2flox/− mutant embryos and controls at e <dig>  .
together, these results show that shox <dig> is required for normal expression of markers of vmn identity at e <dig> .figure  <dig> loss of shox <dig> in the brain results in subtle changes in the expression pattern of isl <dig> and phox2b in the e <dig>  facial motor nucleus.
a diagram of a sagittal section through the e <dig>  brain highlights the regions represented in b–r.
b x-gal stained sagittal sections through e <dig>  embryos shows decreased staining in the facial motor nucleus  of nestin-cre; shox2
lacz/flox  embryos that lack shox <dig> function, as compared to controls .
c–r ish on e <dig>  control  and nestin-cre; shox2
flox/−  sagittal sections shows loss of shox <dig> expression , and subtle changes in the spatial expression patterns of isl <dig> , phox2b , cntn <dig> , tubb <dig> , periph , slc18a <dig>  and slit <dig>  in the developing facial motor nucleus.
the dashed-circles in paired panels being compared  are the same size, and this applies from b′ to r. scale bar 250 μm.
figure  <dig>
shox <dig> is required for the proper development of the facial motor nucleus.
a diagram of a sagittal section through the p <dig> brain highlights the regions represented in b–v.
b x-gal stained sagittal sections through p <dig> brains show a decrease in staining levels in the facial motor nucleus  of nestin-cre; shox2
lacz/flox  animals as compared to controls .
c–v ish on p <dig> control  and nestin-cre; shox2
flox/−  sagittal sections shows loss of shox <dig> expression , and decreases in isl <dig> , phox2b , cntn <dig> , tubb <dig> , periph , slc18a <dig> , slit <dig> , shh  and ptch <dig>  expression in the facial motor nucleus.
nv trigeminal nucleus, rtn retrotrapezoid nucleus.
scale bar 250 μm.
loss of shox <dig> results in severe developmental disruptions to the facial motor nucleus
in contrast to the subtle changes at e <dig> , postnatally we observed a drastic reduction in the size of the facial motor nucleus and changes to the overall organization of motor neurons in nestin-cre; shox2flox/− mutant animals , consistent with the phenotype displayed in figure  <dig>  specifically, lacz staining of p <dig> control and nestin-cre; shox2lacz/flox sagittal sections of the facial motor nucleus demonstrate that the nucleus is reduced in size in mice lacking shox <dig> .
the expression levels of isl <dig>  phox2b, cntn <dig>  tubb <dig>  periph, slc18a <dig> and slit <dig> were all strikingly reduced in the p <dig> facial motor nucleus of nestin-cre; shox2flox/− mutant animals as compared to controls .
since conditional inactivation of shox <dig> in the brain was previously shown to result in the down-regulation of shh expression in dorsal-residing purkinje cells of the cerebellum  <cit> , we next investigated whether loss of shox <dig> function in the facial motor nucleus interfered with the expression of shh.
the expression of both shh and patched  <dig>  were absent in the facial motor nucleus of nestin-cre; shox2flox/− mutant animals .
although the loss of shox <dig> in the brain resulted in drastic reductions in the size of the facial motor nucleus and the expression of the above-mentioned genes, the spared vmns remaining in the facial motor nucleus of shox <dig> mutants appeared to be mostly localized to the dorsal regions of the nucleus .
this suggested that there could be a region-specific loss of distinct subnuclei in the facial motor nucleus in shox2-mutant animals.
therefore we examined the expression patterns of these same genes in coronal sections of the facial motor nucleus, as this would allow us to more clearly observe the six subnuclei that comprise the main facial motor nucleus; specifically the lateral, dorsolateral, dorsal intermediate, ventral intermediate, dorsomedial and ventromedial nuclei  <cit> .
lacz staining of p <dig> control and nestin-cre; shox2lacz/flox coronal sections of the facial motor nucleus demonstrated that elimination of shox <dig> resulted in the loss of medially-localized subnuclei, specifically the dorsomedial and ventromedial nuclei, in addition to severe disruptions to the development of the intermediate nuclei, especially the dorsal intermediate .
similar to what we observed at p <dig> in sagittal sections of the facial motor nucleus , the expression of isl <dig>  phox2b, cntn <dig>  tubb <dig>  periph, slc18a <dig>  slit <dig>  shh and ptch <dig> were restricted to the lateral or intermediate regions in coronal sections of the facial motor nucleus, or completely lost in nestin-cre; shox2flox/− mutant animals as compared to controls .
while the regional changes in expression were variable between the genes examined, in general the dorsomedial, ventromedial and dorsal intermediate subnuclei of the facial motor nucleus appeared to be most strongly affected in shox2-mutant animals , which may reflect a regional specific requirement for shox <dig> in the facial motor nucleus or may indicate that there are populations of vmns within the facial motor nucleus that do not express shox <dig> during development.
together, the results suggest that shox <dig> is required for the development of specific subcompartments within the broader facial motor nucleus.figure  <dig> loss of shox <dig> function in the facial motor nucleus interferes with the proper development of the medial subnuclei.
a diagram of a coronal section through the p <dig> brain highlights the region represented in panels b–v, particularly the  <dig> neuronal clusters that form the facial motor nucleus  .
b x-gal stained coronal sections through p <dig> brains shows a loss of medial neuronal clusters in the facial motor nucleus of nestin-cre; shox2
lacz/flox  brains as compared to controls .
c–v ish on p <dig> control  and nestin-cre; shox2
flox/−  coronal sections shows loss of shox <dig> expression , and decreases in isl <dig> , phox2b , cntn <dig> , tubb <dig> , periph , slc18a <dig> , slit <dig> , shh  and ptch <dig>  expression in the facial motor nucleus.
da dorsal accessory, dm dorsomedial, vm ventromedial, di dorsal intermediate, vi ventral intermediate, dl dorsolateral, lat lateral.
scale bar 250 μm.
discussion
in this study, we used a nestin-cre driver and a conditional knockout strategy to determine the contribution of shox <dig> to the development of the facial motor nucleus.
we found that mice lacking shox <dig> function failed to develop proper vmns in the medial subnuclei normally present in the facial motor nucleus, which was accompanied by truncated or absent facial  nerve projections.
our results have uncovered an unexpected and novel role for shox <dig> in the development of the facial motor nucleus and facial  nerves.
specifically, shox <dig> is required for cell survival and perhaps plays a region-specific role in the development and/or maintenance of normal transcriptional programs of vmns of the facial motor nucleus.
ultimately, in the absence of shox <dig>  pups lack facial  nerves and display abnormal feeding behavior.
shox <dig> is required for cell survival in the facial motor nucleus
nestin-cre-mediated deletion of shox <dig> in the brain resulted in a significant increase in the number of apoptotic cells present at e <dig>  and e <dig>  in the facial motor nucleus of shox2-mutants.
given that truncated and abnormal facial  nerve projections could be observed in nestin-cre; shox2flox/− mutants from e <dig>  onward, apoptosis could:  contribute to the disruptions in the axonal projection properties of vmns in the facial motor nucleus, or  result from a lack of peripheral trophic support for the facial  nerves in shox <dig> mutants.
similarly, the changes observed in the size of the p <dig> facial motor nucleus also likely results from elevated cell death embryonically in shox2-mutant animals.
however, given that there are ~ <dig>  neurons that comprise the facial motor nucleus  <cit> , future analysis is needed to quantify cell death accurately over a developmental time course to determine if the levels of cell death we observe in shox2-mutant animals is sufficient to account for the disruptions observed in the facial motor nucleus in the absence of shox <dig>  this is similar to what has been documented in isl <dig> mutants, as loss of isl <dig> is associated with increased cell death in the trigeminal, drg, hindbrain and neural tube prior to e <dig> , while later developmentally isl <dig> functions during subtype specification  <cit> .
accordingly, if shox <dig> is required to maintain proper isl <dig> expression in vmns of the facial motor nucleus, similar to what has been observed during heart development  <cit> , and isl <dig> is required to induce the subsequent differentiation of adjacent neurons, similar to what has been observed in the neural tube  <cit> , this could explain the elevated cell death observed in neurons adjacent to the facial motor nucleus at p <dig>  pattyn et al.
<cit>  have shown that unlike phox2b and isl <dig>  nkx <dig> proteins  are not required for the establishment of vmn identity, yet nkx <dig> proteins are necessary to prevent vmns from trans-differentiating into improper neuronal subtypes.
similar to shox <dig> mutants, mice lacking nkx <dig> function display impaired axonal projection properties of vmns and facial nerve disruptions  <cit> .
together, the shox2-mutant model could provide an additional example to support the argument of pattyn et al.
that vmn differentiation requires multiple inputs rather than one dominant determinant  <cit> .
alternatively, necrosis in the facial motor nucleus region could be responsible for the observed ectopic increase in the number of apoptotic cells in tissue immediately surrounding the facial motor nucleus.
shox <dig> may play a regional specific role in the transcriptional programs of vmns during development of the facial motor nucleus
in this study we have shown that shox <dig> is expressed in the facial motor nucleus and provide evidence that shox <dig> plays an important role during facial motor nucleus development.
we have demonstrated that loss of shox <dig> function results in the down-regulation or loss of isl <dig>  phox2b, cntn <dig>  tubb <dig>  periph, slc18a <dig> and slit <dig> in the facial motor nucleus.
since loss of isl <dig> function in the trigeminal and drg have been shown to result in changes in the expression of cntn <dig>  tubb <dig> and periph  <cit> , and given that phox2b-deficiency influences slit <dig> expression  <cit> , we propose that the observed changes in gene expression in shox <dig> mutants could result from direct or indirect regulation of isl <dig> and/or phox2b by shox <dig>  since isl <dig> can rescue shox2-mediated bradycardia, and isl <dig> has been shown to be a transcriptional target of shox <dig> in vitro  <cit> , it would not be surprising if shox <dig> regulates isl <dig> in the facial motor nucleus.
as conditional inactivation of shox <dig> in the brain was previously shown to result in the down-regulation of shh expression in dorsal-residing purkinje cells of the cerebellum  <cit> , we also investigated whether loss of shox <dig> function in the facial motor nucleus interfered with expression of shh.
similar to what was observed in the cerebellum, the expression of both shh and ptch <dig> were reduced or lost in the facial motor nucleus of shox2-mutant animals, suggesting that shox <dig> acts upstream of shh signaling, or that the neurons that would normally express shh underwent apoptosis earlier embryonically in shox2-mutant animals.
despite the known critical function of shh in motor neuron generation , the role of shh in the later development of the vmns of the facial motor nucleus remains largely unknown.
in rats, shh appears to play a role in the regeneration of the facial motor nucleus and facial  nerves following nerve injury  <cit> .
therefore, as the facial motor nucleus normally undergoes massive cell death from e <dig> to p <dig>  <cit> , the late-onset of shh expression in the facial motor nucleus could be localized to specific vmns for cell survival.
this hypothesis will be tested in future studies.
together, it appears that shox <dig> is necessary for proper isl <dig> and phox2b expression levels in the developing facial motor nucleus, and is subsequently required for late-onset shh expression; whether this results from facial  nerve disruption and embryonic apoptosis in shox <dig> mutants or occurs via direct or indirect regulation of isl <dig>  phox2b and/or shh has yet to be determined.
since the phenotype observed in the facial motor nucleus of shox <dig> mutants, including the changes in gene expression, were most prominent in the medial and intermediate subnuclei, factors other than shox <dig> must contribute to the development and survival of the lateral subnuclei of the facial motor nucleus.
the observed phenotype could reflect a regional specific requirement for shox <dig> in the facial motor nucleus or indicate that there are populations of vmns within the facial motor nucleus that do not express shox <dig>  the latter hypothesis is supported by results depicted in figure s2f, h, j, as the shox <dig> expression domain appears more restricted then those of isl <dig> and phox2b; therefore, future analysis will be required to examine whether shox2−/isl1+ and/or shox2−/phox2b+ cell populations exist in the facial motor nucleus.
alternatively, if vmns destined for the lateral subnuclei of the facial motor nucleus are specified first, this could explain why they are the least disrupted group of nuclei apparent in shox <dig> mutants.
however, considering the severe disruptions to facial  nerve development observed in shox <dig> mutants, including those that innervate the nasolabial musculature that are supplied by neurons located in the lateral subnuclei of the facial motor nucleus  <cit> , it is also possible that shox <dig> is required for the proper development of the lateral facial motor nuclei.
this is further supported by changes in the expression of cntn <dig>  tubb <dig>  shh and ptch <dig> in the lateral facial motor nucleus.
taken together, it appears that shox <dig> may play a role in the transcriptional programs of vmns during development of the facial motor nucleus; whether this occurs via a regional requirement for shox <dig> in the facial motor nucleus or influences the development of the entire facial motor nucleus in an equal manner has yet to be determined.
disruptions in the development of the facial motor nucleus influences the axonal projections of vmns of the facial  nerves in shox2-mutant animals
as shox2-mutant neonates display impaired feeding behavior, we wondered if this was due to a loss of a subset of axons in the nerves present in the face or whether it reflected a more substantial innervation deficit.
our findings demonstrate that the loss of shox <dig> causes severe truncations and disruptions to the development of the facial  nerves, which could result from:  a lack of peripheral trophic support for the facial  nerves,  vmn cell death,  facial motor nucleus defects that influence the axonal projections of vmns and target acquisition of the facial  nerves, or a combination thereof.
it should be mentioned, however, that disruptions in peripheral trophic support for the facial  nerves would have to result from shox2’s autonomous function in the facial  nerves themselves, and not as a result of shox <dig> playing a role as a peripheral target, since loss of shox <dig> expression in the face in wnt1-cre; shox2flox/− mutant animals does not disrupt facial  nerve development.
given that the face of shox2-mutant neonates are severely devoid of innervation, the behavioral abnormalities exhibited by shox2-mutant animals could be attributed to facial paralysis.
disruption of hoxb <dig> function has been shown to result in facial nerve defects in mice, where the phenotype is similar to that of human patients with bell’s palsy or moebius syndrome  <cit> .
similarly, other hox-mutant mouse models have displayed disruptions in facial  nerve development, which correlate with facial impairments  <cit> .
moreover, isl <dig> mutants display disruptions in the development of the facial  nerves, however similar to shox <dig> mutants, early postnatal lethality has limited the behavioral analysis of these animals  <cit> .
to determine if disruption to the facial motor nucleus is the cause of the early postnatal lethality of nestin-cre; shox2flox/− animals, future studies could employ more restricted hindbrain cre lines to specifically remove shox <dig> expression from the facial motor nucleus.
if regionally restricted loss of shox <dig> in facial motor nucleus does not result in postnatal lethality, future studies could examine shox2-mutant behavior later postnatally and in the adult to test whether shox2-mutant animals have control over their face or are experiencing facial paralysis.
CONCLUSIONS
taken together, these results demonstrate that elimination of shox <dig> in the brain results in disruptions in the development of the facial  nerves and the facial motor nucleus.
the present study demonstrates that in the absence of shox <dig> the murine face develops with severe innervation deficits, ultimately highlighting the importance of shox <dig> to proper facial motor nucleus development.
the shox2-mutant model can now be exploited to further our understanding of normal neuronal development and circuit formation; processes that are likely dysregulated in human facial paralysis disorders.

 in the hindbrain, motor neuron progenitors are born in a region-specific manner, neighboring the floor plate, in response to their level of exposure to different morphogenetic gradients. these neuronal progenitors differentiate in a rhombomere-specific pattern, which requires the correct spatiotemporal expression of the homeotic gene clusters. motor neuron progenitors in the hindbrain can differentiate into somatomotor neurons, which innervate the skeletal muscles of the body, or visceromotor neurons, which innervate autonomic ganglia and branchial arch-derived muscles. facial motor neurons, which originate from rhombomeres 4/ belong to the vmn class of motor neurons. at embryonic day, facial motor neurons begin to migrate caudally to the pial side of the hindbrain to form the facial motor nucleus, which subsequently segregates into two lobes. despite the elucidation of the genetic mechanisms that specify smn identity, the corresponding pathways responsible for the establishment of vmns are less well understood. furthermore, even though sonic hedgehog signaling is known to be required for the development of all motor neurons, the upstream regulators of shh expression and function during late embryonic and early postnatal development, especially within the vmns of the facial motor nucleus, remain largely unknown. the paired-like homeobox 2b gene is expressed in both proliferating progenitors and post-mitotic motor neurons and is required for the development of vmns. similarly, the homeodomain transcription factor islet, which is expressed in both post-mitotic smns and vmns, plays a broad role in motor neuron identity and specification. in the absence of isl mice die during embryogenesis around e. this embryonic lethality is associated with an increase in cell death in the hindbrain and neural tube, suggesting that loss of isl function results in the death of cells that were fated to differentiate into motor neurons. in the trigeminal and dorsal root ganglia, isl appears to play distinct roles during early versus late development. in cranial ganglia, isl is required for cell survival and isl mutants show thinner blunted facial ganglia. however, the precise role of isl in these cell types and in their respective hindbrain nuclei, such as the facial motor nucleus, remains poorly documented. the adult facial motor nucleus contains seven subnuclei; the lateral, dorsolateral, dorsal intermediate, ventral intermediate, dorsomedial and ventromedial nuclei are located within the main nucleus, while the seventh, the dorsal accessory nuclei, is located above the main nucleus. neurons innervating the nasolabial musculature are located in the lateral facial motor nucleus, while neurons supplying the auricular musculature are located in the medial facial motor nucleus. together the lateral, intermediate and medial subnuclei comprise, and %, respectively, of the motor neurons in the adult facial motor nucleus, with the dorsal accessory nuclei contributing the final %. studies with mouse knockout models have shown that shox is required for normal development of the humerus and femur, the anterior palate, the temporomandibular joint of the jaws, the sinoatrial valves and pacemaker region of the heart, and trkb-positive mechanosensory neurons of the dorsal root ganglia. most recently, shox has been shown to play an important role in development of the inferior colliculus and cerebellum. we suspected a function for shox during facial motor nucleus development given our previous study demonstrating a novel role for shox in the hindbrain, and since shox has been shown to regulate transcription of isl, an important regulator of vmn development. in the current study we show that conditional inactivation of shox in the brain results in impaired axonal projections of vmns and a concomitant loss of medially located neurons in the facial motor nucleus postnatally. furthermore, we demonstrate that shox2-mutant neonates display impaired feeding behavior, perhaps due to facial paralysis as a result of improper development and function of the facial nerves.  shox expression in the face and facial motor nucleus during development whole-mount in situ hybridization at e showed shox expression in the trigeminal and facial ganglia, in addition to the developing maxillary process and mandibular arch. shox continued to be expressed in the trigeminal ganglia, maxillary process and mandibular arch at e. we also visualized the shox expression pattern with a novel shox2lacz allele. x-gal staining of embryos carrying the shox2lacz allele accurately reproduced the endogenous shox expression pattern with increased sensitivity and less background compared to wish. at e, shox2lacz/+ embryos showed lacz staining in the trigeminal and facial ganglia of the embryo. similarly at e, lacz staining was visible in the trigeminal and facial nerves of the embryo, in addition to the maxillary process and mandibular arch. lacz continued to be expressed in the trigeminal ganglion of the embryonic face from e to e, and could be seen in the developing pharyngeal arch. at e lacz expression was present in the facial mesenchyme that contributes to the maxilla and mandible. however, from e onward lacz staining was no longer observed in the facial ganglia. postnatally lacz expression was maintained in the facial mesenchyme surrounding the facial nerves, but not within the axons themselves. examination of shox expression early embryonically in the developing brain showed shox staining in a region of post-mitotic neurons adjacent to the floor plate, a region where phox2b+/isl1+ are localized. later in the p brain, shox was expressed in the facial motor nucleus. similarly, in shox2lacz/+ animals, lacz staining was strongly expressed in both the lateral and medial lobes of the facial motor nucleus; however, staining was not visible within the trigeminal nucleus.figure  shox mrna and lacz expression in the developing embryonic face and postnatal facial motor nucleus. a, b whole-mount in situ hybridization at e and e show shox expression in the trigeminal and facial ganglia, in addition to the developing maxillary process and mandibular arch. c–e in situ hybridization on whole p brains and sagittal or coronal sections through the brain show shox expression in the facial motor nucleus. f–j x-gal staining of shox2 lacz/+ embryos at e, e, e, e, and e shows that shox is expressed in the trigeminal and facial ganglion, in addition to the developing maxillary process and mandibular arch. starting at e and continuing to e, shox expression becomes restricted to the trigeminal ganglion and the facial mesenchyme that will contribute to the maxilla and mandible, however it is absent from the facial ganglia. k–o x-gal staining of shox2 lacz/+ pups in the p face, brain and sagittal or coronal sections through the brain show shox expression in the tissue surrounding the facial nerves, with the exception of the nerves themselves, and shox expression in the facial motor nucleus. ey eye, nv trigeminal nucleus, sbl superior buccolabial nerve, ibl inferior buccolabial nerve, mm marginal mandibular nerve, cb cerebellum, mb midbrain. scale bar 500 μm. nestin-creshox2-mutant pups exhibit defects in feeding behavior to determine the functional role of shox in the facial motor nucleus, we used nestin-cre to delete the shox gene conditionally in neural tissue throughout the central nervous system. we crossed nestin-cre; shox2+/− mice with shox2flox/flox mice to obtain conditional mutant nestin-cre; shox2flox/− embryos and neonates. shox expression was eliminated in the brain of nestin-creshox2-mutant embryos, including the facial motor nucleus. all nestin-cre shox2-mutant pups died between to 26 h following birth. this limited our characterization of shox function to embryonic and neonatal stages. p nestin-cre; shox2flox/− mutant pups displayed reduced activity and impaired feeding behavior as early as 6 h following birth, which correlated with a lack of milk in the stomachs of nestin-cre; shox2flox/− mutant pups. since shox is required for the proper development of the palate, we analyzed the anterior and posterior palate of nestin-cre; shox2flox/− mutant pups. we did not observe any obvious defects or malformations in the palate of nestin-cre; shox2flox/− mutant pups, suggesting that the absence of milk in shox2-mutant neonatal stomachs was not due to cleft palate malformations. nestin-cre; shox2flox/− mutant pups also displayed tremors 16–26 h following birth, which could be related to defects in neural circuitry or result from a loss of shox function in other regions of the hindbrain. facial nerve branching defects in nestin-cre shox2-mutant animals since shox2-mutant neonates displayed impaired feeding but an apparently normal palate, we hypothesized that shox mutants fail to nurse properly due to facial paralysis. to test this hypothesis, we first sought to determine if the abnormal feeding behavior resulted from the loss of a subset of axons in the nerves of the face. we stained control and nestin-cre shox2-mutant embryos and p heads with the 2h antibody that visualizes nerves by detecting neurofilaments. in e nestin-cre; shox2flox/− conditional knockout embryos the trigeminal nerves appeared normal. to confirm that the trigeminal nerves were intact even in the complete absence of shox we performed neurofilament staining on e shox2-null embryos. while the trigeminal nerves remained intact, the facial nerves were truncated and failed to branch properly in both nestin-cre; shox2flox/− conditional knockout and shox2-null embryos. at e the facial nerves continued to be truncated as compared to controls. neurofilament staining at e displayed a range in severity of facial nerve truncation, with figure 2f showing the least severe truncation and figure 2h having the most severe truncation we observed among seven embryos. later at e, e, and early postnatal stages, the zygomatic, superior buccolabial, inferior buccolabial and marginal mandibular nerve branches of the facial nerve could be seen in controls, however these distinct facial nerve branches were truncated or absent in nestin-cre; shox2flox/− mutant animals. similar to e, facial nerve truncation ranged in severity at the later embryonic and early postnatal time-points analyzed. the severity in facial nerve truncation observed in nestin-cre; shox2flox/− mutant animals, displayed in figure 2j, l, n, are representative of the most common truncations observed.figure  facial nerve truncations in shox2-mutant animals. a–d side view of the e or e face of control, nestin-cre; shox2 flox/− and shox2 −/− embryos stained with the 2h anti-neurofilament antibody. at e the main branch of the facial nerve begins to separate into smaller projections in control embryos, while a single branch is visible in nestin-cre; shox2 flox/− conditional knockout embryos. at e the facial nerve is noticeably truncated in the shox2 −/− embryos, while the trigeminal nerve remains intact. e–h side view of the e face of control and nestin-cre; shox2 flox/− embryos stained with the 2h antibody display the range in severity of facial nerve truncation, with f being the least severe truncation and h being the most severe truncation observed in nestin-cre; shox2 flox/− embryos. side view of the e, e and p face of control and nestin-cre; shox2 flox/− animals stained with the 2h antibody. at e and e the zygomatic, superior buccolabial, inferior buccolabial and marginal mandibular nerve branches can be seen in control embryos but are not visible in nestin-cre; shox2 flox/− embryos. at p the truncated remnants of what appears to be the superior buccolabial nerve were observable in nestin-cre; shox2 flox/− pups. ey eye, b buccal nerve, sm superficial masseter nerve. scale bar 500 μm. to confirm the persistent lack of proper facial nerve branching and development, particularly at late embryonic and early postnatal time-points when the larger head size led to confounding background staining, we used a second transgenic line, this one carrying a bacterial artificial chromosome containing the shox gene with a lacz insertion to visualize the facial nerves. while obvious facial nerve truncations were difficult to visualize at e, the main branch of the facial nerve was noticeably truncated, as the smaller ganglionic projections were absent at e in nestin-cre; shox2flox/− mutant embryos as compared to controls. furthermore, consistent with our neurofilament staining, at later embryonic and early postnatal stages, the zygomatic, temporal, superior buccolabial, inferior buccolabial and marginal mandibular nerve branches of the facial nerve were observed in controls and appeared truncated or absent in nestin-cre; shox2flox/− mutant animals. for example, by p the facial nerves appeared to be absent in nestin-cre; shox2flox/− mutant animals. the buccal and superficial masseter nerves, both sensory components originating from the trigeminal nerve, were the only nerves visible in nestin-cre; shox2flox/− mutant animals, leaving the face almost completely devoid of innervation. to determine whether the facial nerve disruptions occurred as a result of the slight reduction in shox expression in the embryonic facial mesenchyme, or whether this was related specifically to disruptions in the facial motor nucleus, we conditionally removed shox in all neural crest derivatives using a wnt1-cre driver. this resulted in the complete loss of shox expression in the face, and ultimately did not appear to result in any disruptions to the development of the facial nerves. together these results show impaired axonal projection properties of visceral motor neurons in the facial motor nucleus of the hindbrain in mice lacking shox figure  lacz staining highlights truncated facial nerves and impaired axonal projection properties of vmns in mice lacking shox function. a–j bac rp23-105b3-lacz transgenic animals stained with x-gal. a, b side view of the e face of control and nestin-cre; shox2 flox/− embryos display similar axonal projections of the main branch of the facial nerve. c, d side view of the e face of control and nestin-cre; shox2 flox/− embryos display facial nerve truncation in mice lacking shox function. e–h side view of the e and e face of control and nestin-cre; shox2 flox/− embryos highlights the zygomatic, temporal, superior buccolabial, inferior buccolabial and marginal mandibular nerve branches in control embryos, which are truncated or lost in nestin-cre; shox2 flox/− embryos. i, j side view of the p face of control and nestin-cre; shox2 flox/− pups demonstrate that the facial nerves are absent in nestin-cre; shox2 flox/− pups. ey eye, b buccal nerve, sm superficial masseter nerve. scale bar 500 μm. loss of shox in the facial motor nucleus results in elevated apoptosis since shox2-mutant embryos and neonates display impaired axonal projection properties and were unable to nurse properly, we next wanted to determine if development of the facial motor nucleus was disrupted in the absence of shox both neurofilament and lacz staining of p control and nestin-cre; shox2flox/− or nestin-cre; shox2lacz/flox pup brains demonstrated that the facial motor nucleus was severely reduced in size, with disruptions in neuronal spatial organization in mice lacking shox function. while the symmetry between the left and right facial motor nuclei appears to be relatively maintained, the two lobes that normally comprise the facial motor nucleus are no longer present. we next investigated whether an increase in apoptosis could account for the changes observed in the facial motor nucleus of shox2-mutant brains. we found an increase in the number of apoptotic cells present at e and e in the facial motor nucleus of shox2-mutants; however, no significant increase in apoptosis was detected in the e or p facial motor nucleus of shox2-mutant animals. we observed a ~ -fold increase in the number of cleaved active-casp3 + cells in the e facial motor nucleus of nestin-cre; shox2flox/− animals as compared to controls. we also observed a striking ~ -fold increase in the number of cleaved active-casp3 + cells in the e facial motor nucleus of nestin-cre; shox2flox/− animals as compared to controls. while the slight elevation in apoptosis detected in the e and p facial motor nucleus of shox2-mutant animals was not significantly different from controls, we did observe a ~ -fold increase in the number of cleaved active-casp3 + cells at p when we included the tissue immediately surrounding the facial motor nucleus of nestin-cre; shox2flox/− animals in our cell count. moreover, when we calculated the area of shox2lacz + cells remaining in sagittal sections through the center of the facial motor nucleus of nestin-cre; shox2lacz/flox animals as compared to controls at e and p, we observed a ~44% decrease in the area of shox2lacz + cells in the e facial motor nucleus and a ~53% decrease in the area of shox2lacz + cells in the p facial motor nucleus. therefore, the higher levels of apoptosis are correlated with a decrease in the size of the facial motor nucleus, and taken together, the results demonstrate that apoptosis contributes to the disruptions observed in the facial motor nucleus of nestin-cre; shox2flox/− animals.figure  nestin-cre driven conditional shox2-deletion results in disruptions in the facial motor nucleus and changes in cell death. a, b ventral view of the p brain of control and nestin-cre; shox2 flox/− animals stained with the 2h antibody demonstrate that the facial motor nucleus is severely reduced in nestin-cre; shox2 flox/− pups. c–f x-gal staining of p control and nestin-cre; shox2 lacz/flox pup brains show shox expression in the facial motor nucleus and demonstrate that the nucleus is severely reduced in size in mice lacking shox function. cleaved active-casp immunostaining on e, e, e and p control and nestin-cre; shox2 flox/− mutant sagittal sections through the facial motor nucleus. i quantification of the number of cleaved active-casp3 + cells present in the e facial motor nucleus of control and nestin-cre; shox2 flox/− embryos. l quantification of the number of cleaved active-casp3 + cells present in the e facial motor nucleus of control and nestin-cre; shox2 flox/− embryos. o quantification of the number of cleaved active-casp3 + cells present in the e facial motor nucleus of control and nestin-cre; shox2 flox/− embryos. r quantification of the number of cleaved active-casp3 + cells present in the p facial motor nucleus of control and nestin-cre; shox2 flox/− embryos. s quantification of the number of cleaved active-casp3 + cells present in and around the p facial motor nucleus of control and nestin-cre; shox2 flox/− embryos. t, u representative binary particle area diagrams from imagej displaying shox2lacz + cell staining at e and p in controls and nestincre; shox2 lacz/flox facial motor nuclei. v quantification of the area of shox2lacz + cells present in sagittal sections through the center of the e facial motor nucleus of control and nestin-cre; shox2 lacz/flox embryos. w quantification of the area of shox2lacz + cells present in sagittal sections through the center of the p facial motor nucleus of control and nestin-cre; shox2 lacz/flox animals. cb cerebellum. a–f scale bar 500 μm. g–q scale bar 250 μm. shox is required for normal expression of markers of vmn identity in the facial motor nucleus given the marked reduction in size of the facial motor nucleus in shox mutants, we next sought to investigate whether factors other than facial nerve truncation and cell death could be changing in the facial motor nucleus following the loss of shox prior to e, we did not observe any obvious changes in gene expression or disruptions to the migratory patterns of vmns. at e, however, sagittal sections of the facial motor nucleus showed reductions in the overall size of the nucleus in nestin-cre; shox2lacz/flox embryos, which was consistent with the regional increase in the number of cleaved active-casp3 + cells at this time-point. since shox has been previously shown to regulate isl, an important regulator of vmn development, we next examined the expression of isl in shox2-mutant animals. we observed a regionally restricted loss of isl and the hindbrain vmn determinant phox2b in the facial motor nucleus as early as e in nestin-cre; shox2flox/− mutant embryos as compared to controls. since loss of isl function in the trigeminal and drg have been shown to result in changes in the expression of contactin, tubulin β- chain and peripherin, and given that phox2b-deficient mice have been used to identify the facial motor nucleus as a source of slit homolog signaling, we next examined the expression patterns of these genes. although subtle changes in the spatial patterns and expression levels of cntn and tubb were observed, periph solute carrier family and slit phenocopied the regionally specific absence of isl and phox2b in the facial motor nucleus at e in nestin-cre; shox2flox/− mutant embryos. the changes in expression for these genes continued to be observed between nestin-cre; shox2flox/− mutant embryos and controls at e. together, these results show that shox is required for normal expression of markers of vmn identity at e.figure  loss of shox in the brain results in subtle changes in the expression pattern of isl and phox2b in the e facial motor nucleus. a diagram of a sagittal section through the e brain highlights the regions represented in b–r. b x-gal stained sagittal sections through e embryos shows decreased staining in the facial motor nucleus of nestin-cre; shox2 lacz/flox embryos that lack shox function, as compared to controls. c–r ish on e control and nestin-cre; shox2 flox/− sagittal sections shows loss of shox expression, and subtle changes in the spatial expression patterns of isl, phox2b, cntn, tubb, periph, slc18a and slit in the developing facial motor nucleus. the dashed-circles in paired panels being compared are the same size, and this applies from b′ to r. scale bar 250 μm. figure  shox is required for the proper development of the facial motor nucleus. a diagram of a sagittal section through the p brain highlights the regions represented in b–v. b x-gal stained sagittal sections through p brains show a decrease in staining levels in the facial motor nucleus of nestin-cre; shox2 lacz/flox animals as compared to controls. c–v ish on p control and nestin-cre; shox2 flox/− sagittal sections shows loss of shox expression, and decreases in isl, phox2b, cntn, tubb, periph, slc18a, slit, shh and ptch expression in the facial motor nucleus. nv trigeminal nucleus, rtn retrotrapezoid nucleus. scale bar 250 μm. loss of shox results in severe developmental disruptions to the facial motor nucleus in contrast to the subtle changes at e, postnatally we observed a drastic reduction in the size of the facial motor nucleus and changes to the overall organization of motor neurons in nestin-cre; shox2flox/− mutant animals, consistent with the phenotype displayed in figure  specifically, lacz staining of p control and nestin-cre; shox2lacz/flox sagittal sections of the facial motor nucleus demonstrate that the nucleus is reduced in size in mice lacking shox. the expression levels of isl phox2b, cntn tubb periph, slc18a and slit were all strikingly reduced in the p facial motor nucleus of nestin-cre; shox2flox/− mutant animals as compared to controls. since conditional inactivation of shox in the brain was previously shown to result in the down-regulation of shh expression in dorsal-residing purkinje cells of the cerebellum, we next investigated whether loss of shox function in the facial motor nucleus interfered with the expression of shh. the expression of both shh and patched were absent in the facial motor nucleus of nestin-cre; shox2flox/− mutant animals. although the loss of shox in the brain resulted in drastic reductions in the size of the facial motor nucleus and the expression of the above-mentioned genes, the spared vmns remaining in the facial motor nucleus of shox mutants appeared to be mostly localized to the dorsal regions of the nucleus. this suggested that there could be a region-specific loss of distinct subnuclei in the facial motor nucleus in shox2-mutant animals. therefore we examined the expression patterns of these same genes in coronal sections of the facial motor nucleus, as this would allow us to more clearly observe the six subnuclei that comprise the main facial motor nucleus; specifically the lateral, dorsolateral, dorsal intermediate, ventral intermediate, dorsomedial and ventromedial nuclei. lacz staining of p control and nestin-cre; shox2lacz/flox coronal sections of the facial motor nucleus demonstrated that elimination of shox resulted in the loss of medially-localized subnuclei, specifically the dorsomedial and ventromedial nuclei, in addition to severe disruptions to the development of the intermediate nuclei, especially the dorsal intermediate. similar to what we observed at p in sagittal sections of the facial motor nucleus, the expression of isl phox2b, cntn tubb periph, slc18a slit shh and ptch were restricted to the lateral or intermediate regions in coronal sections of the facial motor nucleus, or completely lost in nestin-cre; shox2flox/− mutant animals as compared to controls. while the regional changes in expression were variable between the genes examined, in general the dorsomedial, ventromedial and dorsal intermediate subnuclei of the facial motor nucleus appeared to be most strongly affected in shox2-mutant animals, which may reflect a regional specific requirement for shox in the facial motor nucleus or may indicate that there are populations of vmns within the facial motor nucleus that do not express shox during development. together, the results suggest that shox is required for the development of specific subcompartments within the broader facial motor nucleus.figure  loss of shox function in the facial motor nucleus interferes with the proper development of the medial subnuclei. a diagram of a coronal section through the p brain highlights the region represented in panels b–v, particularly the neuronal clusters that form the facial motor nucleus. b x-gal stained coronal sections through p brains shows a loss of medial neuronal clusters in the facial motor nucleus of nestin-cre; shox2 lacz/flox brains as compared to controls. c–v ish on p control and nestin-cre; shox2 flox/− coronal sections shows loss of shox expression, and decreases in isl, phox2b, cntn, tubb, periph, slc18a, slit, shh and ptch expression in the facial motor nucleus. da dorsal accessory, dm dorsomedial, vm ventromedial, di dorsal intermediate, vi ventral intermediate, dl dorsolateral, lat lateral. scale bar 250 μm. discussion in this study, we used a nestin-cre driver and a conditional knockout strategy to determine the contribution of shox to the development of the facial motor nucleus. we found that mice lacking shox function failed to develop proper vmns in the medial subnuclei normally present in the facial motor nucleus, which was accompanied by truncated or absent facial nerve projections. our results have uncovered an unexpected and novel role for shox in the development of the facial motor nucleus and facial nerves. specifically, shox is required for cell survival and perhaps plays a region-specific role in the development and/or maintenance of normal transcriptional programs of vmns of the facial motor nucleus. ultimately, in the absence of shox pups lack facial nerves and display abnormal feeding behavior. shox is required for cell survival in the facial motor nucleus nestin-cre-mediated deletion of shox in the brain resulted in a significant increase in the number of apoptotic cells present at e and e in the facial motor nucleus of shox2-mutants. given that truncated and abnormal facial nerve projections could be observed in nestin-cre; shox2flox/− mutants from e onward, apoptosis could: contribute to the disruptions in the axonal projection properties of vmns in the facial motor nucleus, or result from a lack of peripheral trophic support for the facial nerves in shox mutants. similarly, the changes observed in the size of the p facial motor nucleus also likely results from elevated cell death embryonically in shox2-mutant animals. however, given that there are ~ neurons that comprise the facial motor nucleus, future analysis is needed to quantify cell death accurately over a developmental time course to determine if the levels of cell death we observe in shox2-mutant animals is sufficient to account for the disruptions observed in the facial motor nucleus in the absence of shox this is similar to what has been documented in isl mutants, as loss of isl is associated with increased cell death in the trigeminal, drg, hindbrain and neural tube prior to e, while later developmentally isl functions during subtype specification. accordingly, if shox is required to maintain proper isl expression in vmns of the facial motor nucleus, similar to what has been observed during heart development, and isl is required to induce the subsequent differentiation of adjacent neurons, similar to what has been observed in the neural tube, this could explain the elevated cell death observed in neurons adjacent to the facial motor nucleus at p pattyn et al. have shown that unlike phox2b and isl nkx proteins are not required for the establishment of vmn identity, yet nkx proteins are necessary to prevent vmns from trans-differentiating into improper neuronal subtypes. similar to shox mutants, mice lacking nkx function display impaired axonal projection properties of vmns and facial nerve disruptions. together, the shox2-mutant model could provide an additional example to support the argument of pattyn et al. that vmn differentiation requires multiple inputs rather than one dominant determinant. alternatively, necrosis in the facial motor nucleus region could be responsible for the observed ectopic increase in the number of apoptotic cells in tissue immediately surrounding the facial motor nucleus. shox may play a regional specific role in the transcriptional programs of vmns during development of the facial motor nucleus in this study we have shown that shox is expressed in the facial motor nucleus and provide evidence that shox plays an important role during facial motor nucleus development. we have demonstrated that loss of shox function results in the down-regulation or loss of isl phox2b, cntn tubb periph, slc18a and slit in the facial motor nucleus. since loss of isl function in the trigeminal and drg have been shown to result in changes in the expression of cntn tubb and periph, and given that phox2b-deficiency influences slit expression, we propose that the observed changes in gene expression in shox mutants could result from direct or indirect regulation of isl and/or phox2b by shox since isl can rescue shox2-mediated bradycardia, and isl has been shown to be a transcriptional target of shox in vitro, it would not be surprising if shox regulates isl in the facial motor nucleus. as conditional inactivation of shox in the brain was previously shown to result in the down-regulation of shh expression in dorsal-residing purkinje cells of the cerebellum, we also investigated whether loss of shox function in the facial motor nucleus interfered with expression of shh. similar to what was observed in the cerebellum, the expression of both shh and ptch were reduced or lost in the facial motor nucleus of shox2-mutant animals, suggesting that shox acts upstream of shh signaling, or that the neurons that would normally express shh underwent apoptosis earlier embryonically in shox2-mutant animals. despite the known critical function of shh in motor neuron generation, the role of shh in the later development of the vmns of the facial motor nucleus remains largely unknown. in rats, shh appears to play a role in the regeneration of the facial motor nucleus and facial nerves following nerve injury. therefore, as the facial motor nucleus normally undergoes massive cell death from e to p, the late-onset of shh expression in the facial motor nucleus could be localized to specific vmns for cell survival. this hypothesis will be tested in future studies. together, it appears that shox is necessary for proper isl and phox2b expression levels in the developing facial motor nucleus, and is subsequently required for late-onset shh expression; whether this results from facial nerve disruption and embryonic apoptosis in shox mutants or occurs via direct or indirect regulation of isl phox2b and/or shh has yet to be determined. since the phenotype observed in the facial motor nucleus of shox mutants, including the changes in gene expression, were most prominent in the medial and intermediate subnuclei, factors other than shox must contribute to the development and survival of the lateral subnuclei of the facial motor nucleus. the observed phenotype could reflect a regional specific requirement for shox in the facial motor nucleus or indicate that there are populations of vmns within the facial motor nucleus that do not express shox the latter hypothesis is supported by results depicted in figure s2f, h, j, as the shox expression domain appears more restricted then those of isl and phox2b; therefore, future analysis will be required to examine whether shox2−/isl1+ and/or shox2−/phox2b+ cell populations exist in the facial motor nucleus. alternatively, if vmns destined for the lateral subnuclei of the facial motor nucleus are specified first, this could explain why they are the least disrupted group of nuclei apparent in shox mutants. however, considering the severe disruptions to facial nerve development observed in shox mutants, including those that innervate the nasolabial musculature that are supplied by neurons located in the lateral subnuclei of the facial motor nucleus, it is also possible that shox is required for the proper development of the lateral facial motor nuclei. this is further supported by changes in the expression of cntn tubb shh and ptch in the lateral facial motor nucleus. taken together, it appears that shox may play a role in the transcriptional programs of vmns during development of the facial motor nucleus; whether this occurs via a regional requirement for shox in the facial motor nucleus or influences the development of the entire facial motor nucleus in an equal manner has yet to be determined. disruptions in the development of the facial motor nucleus influences the axonal projections of vmns of the facial nerves in shox2-mutant animals as shox2-mutant neonates display impaired feeding behavior, we wondered if this was due to a loss of a subset of axons in the nerves present in the face or whether it reflected a more substantial innervation deficit. our findings demonstrate that the loss of shox causes severe truncations and disruptions to the development of the facial nerves, which could result from: a lack of peripheral trophic support for the facial nerves, vmn cell death, facial motor nucleus defects that influence the axonal projections of vmns and target acquisition of the facial nerves, or a combination thereof. it should be mentioned, however, that disruptions in peripheral trophic support for the facial nerves would have to result from shox2’s autonomous function in the facial nerves themselves, and not as a result of shox playing a role as a peripheral target, since loss of shox expression in the face in wnt1-cre; shox2flox/− mutant animals does not disrupt facial nerve development. given that the face of shox2-mutant neonates are severely devoid of innervation, the behavioral abnormalities exhibited by shox2-mutant animals could be attributed to facial paralysis. disruption of hoxb function has been shown to result in facial nerve defects in mice, where the phenotype is similar to that of human patients with bell’s palsy or moebius syndrome. similarly, other hox-mutant mouse models have displayed disruptions in facial nerve development, which correlate with facial impairments. moreover, isl mutants display disruptions in the development of the facial nerves, however similar to shox mutants, early postnatal lethality has limited the behavioral analysis of these animals. to determine if disruption to the facial motor nucleus is the cause of the early postnatal lethality of nestin-cre; shox2flox/− animals, future studies could employ more restricted hindbrain cre lines to specifically remove shox expression from the facial motor nucleus. if regionally restricted loss of shox in facial motor nucleus does not result in postnatal lethality, future studies could examine shox2-mutant behavior later postnatally and in the adult to test whether shox2-mutant animals have control over their face or are experiencing facial paralysis.  taken together, these results demonstrate that elimination of shox in the brain results in disruptions in the development of the facial nerves and the facial motor nucleus. the present study demonstrates that in the absence of shox the murine face develops with severe innervation deficits, ultimately highlighting the importance of shox to proper facial motor nucleus development. the shox2-mutant model can now be exploited to further our understanding of normal neuronal development and circuit formation; processes that are likely dysregulated in human facial paralysis disorders. 
using a nestin-cre driver, we show that elimination of shox throughout the brain results in elevated cell death in the facial motor nucleus at embryonic day and e, which correlates with impaired axonal projection properties of vmns. furthermore, we demonstrate that elimination of shox results in the loss of dorsomedial and ventromedial subnuclei by postnatal day, which may explain the changes in physical activity and impaired feeding/nursing behavior in shox mutants. given that our previous study identified a novel role for the short stature homeobox gene in the hindbrain, and since shox has been shown to regulate transcription of islet, an important regulator of vmn development, we sought to determine whether shox is required for the proper development of the facial motor nucleus. we know that vmns, like all motor neurons, depend on sonic hedgehog signaling for their generation. short stature homeobox geneislet sonic hedgehog visceral motor neurons facial motor nucleusfacial nervesissue-copyright-statement© the author 2015 axons from the visceral motor neurons project from nuclei in the hindbrain to innervate autonomic ganglia and branchial arch-derived muscles. although much is known about the events that govern specification of somatic motor neurons, the genetic pathways responsible for the development of vmns are less well characterized. similarly, the paired-like homeobox 2b gene, which is expressed in both proliferating progenitors and post-mitotic motor neurons, is essential for the development of vmns. combined, our data show that shox is required for development of the facial motor nucleus and its associated facial nerves, and serves as a new molecular tool to probe the genetic programs of this understudied hindbrain region.
 axons from the visceral motor neurons project from nuclei in the hindbrain to innervate autonomic ganglia and branchial arch-derived muscles. although much is known about the events that govern specification of somatic motor neurons, the genetic pathways responsible for the development of vmns are less well characterized. we know that vmns, like all motor neurons, depend on sonic hedgehog signaling for their generation. similarly, the paired-like homeobox 2b gene, which is expressed in both proliferating progenitors and post-mitotic motor neurons, is essential for the development of vmns. given that our previous study identified a novel role for the short stature homeobox gene in the hindbrain, and since shox has been shown to regulate transcription of islet, an important regulator of vmn development, we sought to determine whether shox is required for the proper development of the facial motor nucleus.  using a nestin-cre driver, we show that elimination of shox throughout the brain results in elevated cell death in the facial motor nucleus at embryonic day and e, which correlates with impaired axonal projection properties of vmns. we also observed changes in the spatial expression of the vmn cell fate factors isl and phox2b, and concomitant defects in shh and ptch expression in shox mutants. furthermore, we demonstrate that elimination of shox results in the loss of dorsomedial and ventromedial subnuclei by postnatal day, which may explain the changes in physical activity and impaired feeding/nursing behavior in shox mutants.  combined, our data show that shox is required for development of the facial motor nucleus and its associated facial nerves, and serves as a new molecular tool to probe the genetic programs of this understudied hindbrain region. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords short stature homeobox geneislet sonic hedgehog visceral motor neurons facial motor nucleusfacial nervesissue-copyright-statement© the author 2015 