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Shox2 is required for the proper development of the facial motor nucleus and the establishment of the facial nerves.

Rosin JM, Kurrasch DM, Cobb J - BMC Neurosci (2015)

Bottom Line: Using a Nestin-Cre driver, we show that elimination of Shox2 throughout the brain results in elevated cell death in the facial motor nucleus at embryonic day 12.5 (E12.5) and E14.5, which correlates with impaired axonal projection properties of vMNs.We also observed changes in the spatial expression of the vMN cell fate factors Isl1 and Phox2b, and concomitant defects in Shh and Ptch1 expression in Shox2 mutants.Furthermore, we demonstrate that elimination of Shox2 results in the loss of dorsomedial and ventromedial subnuclei by postnatal day 0 (P0), which may explain the changes in physical activity and impaired feeding/nursing behavior in Shox2 mutants.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Calgary, 2500 University Drive N.W., BI286D, Calgary, AB, T2N 1N4, Canada. jmrosin2013@gmail.com.

ABSTRACT

Background: Axons from the visceral motor neurons (vMNs) 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 (Phox2b) 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 2 (Shox2) gene in the hindbrain, and since SHOX2 has been shown to regulate transcription of islet 1 (Isl1), an important regulator of vMN development, we sought to determine whether Shox2 is required for the proper development of the facial motor nucleus.

Results: Using a Nestin-Cre driver, we show that elimination of Shox2 throughout the brain results in elevated cell death in the facial motor nucleus at embryonic day 12.5 (E12.5) and E14.5, which correlates with impaired axonal projection properties of vMNs. We also observed changes in the spatial expression of the vMN cell fate factors Isl1 and Phox2b, and concomitant defects in Shh and Ptch1 expression in Shox2 mutants. Furthermore, we demonstrate that elimination of Shox2 results in the loss of dorsomedial and ventromedial subnuclei by postnatal day 0 (P0), which may explain the changes in physical activity and impaired feeding/nursing behavior in Shox2 mutants.

Conclusions: Combined, our data show that Shox2 is required for development of the facial motor nucleus and its associated facial (VII) nerves, and serves as a new molecular tool to probe the genetic programs of this understudied hindbrain region.

No MeSH data available.


Related in: MedlinePlus

LacZ staining highlights truncated facial nerves and impaired axonal projection properties of vMNs in mice lacking Shox2 function. a–j BAC RP23-105B3-lacZ transgenic animals stained with X-gal. a, b Side view of the E11.5 face of control (a) and Nestin-Cre; Shox2flox/− (b) embryos display similar axonal projections of the main branch of the facial nerve (VII) (compare a to b, arrows). c, d Side view of the E12.5 face of control (c) and Nestin-Cre; Shox2flox/− (d) embryos display facial nerve truncation in mice lacking Shox2 function (compare c to d, arrow). e–h Side view of the E13.5 (e, f) and E14.5 (g, h) face of control (e, g) and Nestin-Cre; Shox2flox/− (f, h) embryos highlights the zygomatic (z), temporal (t), superior buccolabial (sbl), inferior buccolabial (ibl) and marginal mandibular (mm) nerve branches in control (e, g) embryos, which are truncated or lost in Nestin-Cre; Shox2flox/− embryos (compare e to f and g to h, arrows). i, j Side view of the P0 face of control (i) and Nestin-Cre; Shox2flox/− (j) pups demonstrate that the facial nerves are absent in Nestin-Cre; Shox2flox/− pups (compare i to j, arrows). ey eye, b buccal nerve, sm superficial masseter nerve. Scale bar 500 μm.
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Fig3: LacZ staining highlights truncated facial nerves and impaired axonal projection properties of vMNs in mice lacking Shox2 function. a–j BAC RP23-105B3-lacZ transgenic animals stained with X-gal. a, b Side view of the E11.5 face of control (a) and Nestin-Cre; Shox2flox/− (b) embryos display similar axonal projections of the main branch of the facial nerve (VII) (compare a to b, arrows). c, d Side view of the E12.5 face of control (c) and Nestin-Cre; Shox2flox/− (d) embryos display facial nerve truncation in mice lacking Shox2 function (compare c to d, arrow). e–h Side view of the E13.5 (e, f) and E14.5 (g, h) face of control (e, g) and Nestin-Cre; Shox2flox/− (f, h) embryos highlights the zygomatic (z), temporal (t), superior buccolabial (sbl), inferior buccolabial (ibl) and marginal mandibular (mm) nerve branches in control (e, g) embryos, which are truncated or lost in Nestin-Cre; Shox2flox/− embryos (compare e to f and g to h, arrows). i, j Side view of the P0 face of control (i) and Nestin-Cre; Shox2flox/− (j) pups demonstrate that the facial nerves are absent in Nestin-Cre; Shox2flox/− pups (compare i to j, arrows). ey eye, b buccal nerve, sm superficial masseter nerve. Scale bar 500 μm.

Mentions: To confirm the persistent lack of proper facial (VII) 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 (BAC) containing the Shox2 gene with a lacZ insertion (previously described BAC RP23-105B3-lacZ transgenic line [37]) to visualize the facial (VII) nerves. While obvious facial (VII) nerve truncations were difficult to visualize at E11.5 (Figure 3a, b), the main branch of the facial (VII) nerve was noticeably truncated, as the smaller ganglionic projections were absent at E12.5 in Nestin-Cre; Shox2flox/− mutant embryos as compared to controls (Figure 3c–d, arrow). Furthermore, consistent with our neurofilament staining, at later embryonic (E13.5 and E14.5) and early postnatal (P0) stages, the zygomatic, temporal, superior buccolabial, inferior buccolabial and marginal mandibular nerve branches of the facial (VII) nerve were observed in controls (Figure 3e, g, i) and appeared truncated or absent in Nestin-Cre; Shox2flox/− mutant animals (Figure 3f, h, j; arrows). For example, by P0 the facial (VII) nerves appeared to be absent in Nestin-Cre; Shox2flox/− mutant animals (Figure 3j, arrows). The buccal and superficial masseter nerves, both sensory components originating from the trigeminal (V) nerve [36], were the only nerves visible in Nestin-Cre; Shox2flox/− mutant animals, leaving the face almost completely devoid of innervation (Figure 3j, arrows). To determine whether the facial (VII) nerve disruptions occurred as a result of the slight reduction in Shox2 expression in the embryonic facial mesenchyme (Additional file 3: Figure S3A–B, arrows), or whether this was related specifically to disruptions in the facial motor nucleus, we conditionally removed Shox2 in all neural crest derivatives using a Wnt1-Cre driver. This resulted in the complete loss of Shox2 expression in the face (Additional file 5: Figure S4A, B), and ultimately did not appear to result in any disruptions to the development of the facial (VII) nerves (Additional file 5: Figure S4C, D). Together these results show impaired axonal projection properties of visceral motor neurons (vMNs) in the facial motor nucleus of the hindbrain in mice lacking Shox2.Figure 3


Shox2 is required for the proper development of the facial motor nucleus and the establishment of the facial nerves.

Rosin JM, Kurrasch DM, Cobb J - BMC Neurosci (2015)

LacZ staining highlights truncated facial nerves and impaired axonal projection properties of vMNs in mice lacking Shox2 function. a–j BAC RP23-105B3-lacZ transgenic animals stained with X-gal. a, b Side view of the E11.5 face of control (a) and Nestin-Cre; Shox2flox/− (b) embryos display similar axonal projections of the main branch of the facial nerve (VII) (compare a to b, arrows). c, d Side view of the E12.5 face of control (c) and Nestin-Cre; Shox2flox/− (d) embryos display facial nerve truncation in mice lacking Shox2 function (compare c to d, arrow). e–h Side view of the E13.5 (e, f) and E14.5 (g, h) face of control (e, g) and Nestin-Cre; Shox2flox/− (f, h) embryos highlights the zygomatic (z), temporal (t), superior buccolabial (sbl), inferior buccolabial (ibl) and marginal mandibular (mm) nerve branches in control (e, g) embryos, which are truncated or lost in Nestin-Cre; Shox2flox/− embryos (compare e to f and g to h, arrows). i, j Side view of the P0 face of control (i) and Nestin-Cre; Shox2flox/− (j) pups demonstrate that the facial nerves are absent in Nestin-Cre; Shox2flox/− pups (compare i to j, arrows). ey eye, b buccal nerve, sm superficial masseter nerve. Scale bar 500 μm.
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Fig3: LacZ staining highlights truncated facial nerves and impaired axonal projection properties of vMNs in mice lacking Shox2 function. a–j BAC RP23-105B3-lacZ transgenic animals stained with X-gal. a, b Side view of the E11.5 face of control (a) and Nestin-Cre; Shox2flox/− (b) embryos display similar axonal projections of the main branch of the facial nerve (VII) (compare a to b, arrows). c, d Side view of the E12.5 face of control (c) and Nestin-Cre; Shox2flox/− (d) embryos display facial nerve truncation in mice lacking Shox2 function (compare c to d, arrow). e–h Side view of the E13.5 (e, f) and E14.5 (g, h) face of control (e, g) and Nestin-Cre; Shox2flox/− (f, h) embryos highlights the zygomatic (z), temporal (t), superior buccolabial (sbl), inferior buccolabial (ibl) and marginal mandibular (mm) nerve branches in control (e, g) embryos, which are truncated or lost in Nestin-Cre; Shox2flox/− embryos (compare e to f and g to h, arrows). i, j Side view of the P0 face of control (i) and Nestin-Cre; Shox2flox/− (j) pups demonstrate that the facial nerves are absent in Nestin-Cre; Shox2flox/− pups (compare i to j, arrows). ey eye, b buccal nerve, sm superficial masseter nerve. Scale bar 500 μm.
Mentions: To confirm the persistent lack of proper facial (VII) 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 (BAC) containing the Shox2 gene with a lacZ insertion (previously described BAC RP23-105B3-lacZ transgenic line [37]) to visualize the facial (VII) nerves. While obvious facial (VII) nerve truncations were difficult to visualize at E11.5 (Figure 3a, b), the main branch of the facial (VII) nerve was noticeably truncated, as the smaller ganglionic projections were absent at E12.5 in Nestin-Cre; Shox2flox/− mutant embryos as compared to controls (Figure 3c–d, arrow). Furthermore, consistent with our neurofilament staining, at later embryonic (E13.5 and E14.5) and early postnatal (P0) stages, the zygomatic, temporal, superior buccolabial, inferior buccolabial and marginal mandibular nerve branches of the facial (VII) nerve were observed in controls (Figure 3e, g, i) and appeared truncated or absent in Nestin-Cre; Shox2flox/− mutant animals (Figure 3f, h, j; arrows). For example, by P0 the facial (VII) nerves appeared to be absent in Nestin-Cre; Shox2flox/− mutant animals (Figure 3j, arrows). The buccal and superficial masseter nerves, both sensory components originating from the trigeminal (V) nerve [36], were the only nerves visible in Nestin-Cre; Shox2flox/− mutant animals, leaving the face almost completely devoid of innervation (Figure 3j, arrows). To determine whether the facial (VII) nerve disruptions occurred as a result of the slight reduction in Shox2 expression in the embryonic facial mesenchyme (Additional file 3: Figure S3A–B, arrows), or whether this was related specifically to disruptions in the facial motor nucleus, we conditionally removed Shox2 in all neural crest derivatives using a Wnt1-Cre driver. This resulted in the complete loss of Shox2 expression in the face (Additional file 5: Figure S4A, B), and ultimately did not appear to result in any disruptions to the development of the facial (VII) nerves (Additional file 5: Figure S4C, D). Together these results show impaired axonal projection properties of visceral motor neurons (vMNs) in the facial motor nucleus of the hindbrain in mice lacking Shox2.Figure 3

Bottom Line: Using a Nestin-Cre driver, we show that elimination of Shox2 throughout the brain results in elevated cell death in the facial motor nucleus at embryonic day 12.5 (E12.5) and E14.5, which correlates with impaired axonal projection properties of vMNs.We also observed changes in the spatial expression of the vMN cell fate factors Isl1 and Phox2b, and concomitant defects in Shh and Ptch1 expression in Shox2 mutants.Furthermore, we demonstrate that elimination of Shox2 results in the loss of dorsomedial and ventromedial subnuclei by postnatal day 0 (P0), which may explain the changes in physical activity and impaired feeding/nursing behavior in Shox2 mutants.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Calgary, 2500 University Drive N.W., BI286D, Calgary, AB, T2N 1N4, Canada. jmrosin2013@gmail.com.

ABSTRACT

Background: Axons from the visceral motor neurons (vMNs) 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 (Phox2b) 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 2 (Shox2) gene in the hindbrain, and since SHOX2 has been shown to regulate transcription of islet 1 (Isl1), an important regulator of vMN development, we sought to determine whether Shox2 is required for the proper development of the facial motor nucleus.

Results: Using a Nestin-Cre driver, we show that elimination of Shox2 throughout the brain results in elevated cell death in the facial motor nucleus at embryonic day 12.5 (E12.5) and E14.5, which correlates with impaired axonal projection properties of vMNs. We also observed changes in the spatial expression of the vMN cell fate factors Isl1 and Phox2b, and concomitant defects in Shh and Ptch1 expression in Shox2 mutants. Furthermore, we demonstrate that elimination of Shox2 results in the loss of dorsomedial and ventromedial subnuclei by postnatal day 0 (P0), which may explain the changes in physical activity and impaired feeding/nursing behavior in Shox2 mutants.

Conclusions: Combined, our data show that Shox2 is required for development of the facial motor nucleus and its associated facial (VII) nerves, and serves as a new molecular tool to probe the genetic programs of this understudied hindbrain region.

No MeSH data available.


Related in: MedlinePlus