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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

Shox2 mRNA and lacZ expression in the developing embryonic face and postnatal facial motor nucleus. a, b Whole-mount in situ hybridization (WISH) at E11.5 (a) and E12.5 (b) show Shox2 expression in the trigeminal (V) and facial (VII, arrow) ganglia, in addition to the developing maxillary process (mp) and mandibular arch (ma). c–e In situ hybridization (ISH) on whole P0 brains (c, ventral viewpoint) and sagittal (d) or coronal (e) sections through the brain show Shox2 expression in the facial motor nucleus (nVII, arrows). f–j X-gal staining of Shox2lacZ/+ embryos at E10.5 (f), E11.5 (g), E12.5 (h), E13.5 (i), and E14.5 (j) shows that Shox2 is expressed in the trigeminal and facial ganglion (f, g, arrow), in addition to the developing maxillary process and mandibular arch. Starting at E12.5 and continuing to E14.5, Shox2 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 Shox2lacZ/+ pups in the P0 face (k), brain (l, side viewpoint; m, ventral viewpoint) and sagittal (n) or coronal (o) sections through the brain show Shox2 expression in the tissue surrounding the facial nerves (k), with the exception of the nerves themselves, and Shox2 expression in the facial motor nucleus (l–o, arrows). 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.
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Fig1: Shox2 mRNA and lacZ expression in the developing embryonic face and postnatal facial motor nucleus. a, b Whole-mount in situ hybridization (WISH) at E11.5 (a) and E12.5 (b) show Shox2 expression in the trigeminal (V) and facial (VII, arrow) ganglia, in addition to the developing maxillary process (mp) and mandibular arch (ma). c–e In situ hybridization (ISH) on whole P0 brains (c, ventral viewpoint) and sagittal (d) or coronal (e) sections through the brain show Shox2 expression in the facial motor nucleus (nVII, arrows). f–j X-gal staining of Shox2lacZ/+ embryos at E10.5 (f), E11.5 (g), E12.5 (h), E13.5 (i), and E14.5 (j) shows that Shox2 is expressed in the trigeminal and facial ganglion (f, g, arrow), in addition to the developing maxillary process and mandibular arch. Starting at E12.5 and continuing to E14.5, Shox2 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 Shox2lacZ/+ pups in the P0 face (k), brain (l, side viewpoint; m, ventral viewpoint) and sagittal (n) or coronal (o) sections through the brain show Shox2 expression in the tissue surrounding the facial nerves (k), with the exception of the nerves themselves, and Shox2 expression in the facial motor nucleus (l–o, arrows). 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.

Mentions: Whole-mount in situ hybridization (WISH) at E11.5 showed Shox2 expression in the trigeminal (V) and facial (VII) ganglia, in addition to the developing maxillary process and mandibular arch (Figure 1a). Shox2 continued to be expressed in the trigeminal (V) ganglia, maxillary process and mandibular arch at E12.5 (Figure 1b). We also visualized the Shox2 expression pattern with a novel Shox2lacz allele (described in “Methods”). X-gal staining of embryos carrying the Shox2lacz allele accurately reproduced the endogenous Shox2 expression pattern with increased sensitivity and less background compared to WISH (Additional file 1: Figure S1). At E10.5, Shox2lacZ/+ embryos showed lacZ staining in the trigeminal (V) and facial (VII) ganglia of the embryo (Figure 1f). Similarly at E11.5, lacZ staining was visible in the trigeminal (V) and facial (VII) nerves of the embryo, in addition to the maxillary process and mandibular arch (Figure 1g). LacZ continued to be expressed in the trigeminal (V) ganglion of the embryonic face from E12.5 to E13.5, and could be seen in the developing pharyngeal arch (Figure 1h, i). At E14.5 lacZ expression was present in the facial mesenchyme that contributes to the maxilla and mandible (Figure 1j). However, from E12.5 onward lacZ staining was no longer observed in the facial (VII) ganglia (Figure 1h–j). Postnatally (P0) lacZ expression was maintained in the facial mesenchyme surrounding the facial (VII) nerves, but not within the axons themselves (Figure 1k). Examination of Shox2 expression early embryonically in the developing brain showed Shox2 staining in a region of post-mitotic neurons adjacent to the floor plate (Additional file 2: Figure S2A–B), a region where Phox2b+/Isl1+ are localized (Additional file 2: Figure S2C–E) [18]. Later in the P0 brain, Shox2 was expressed in the facial motor nucleus (Figure 1c–e). Similarly, in Shox2lacZ/+ animals, lacZ staining was strongly expressed in both the lateral and medial lobes of the facial motor nucleus (Figure 1l–o); however, staining was not visible within the trigeminal nucleus (Figure 1n, arrowhead).Figure 1


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)

Shox2 mRNA and lacZ expression in the developing embryonic face and postnatal facial motor nucleus. a, b Whole-mount in situ hybridization (WISH) at E11.5 (a) and E12.5 (b) show Shox2 expression in the trigeminal (V) and facial (VII, arrow) ganglia, in addition to the developing maxillary process (mp) and mandibular arch (ma). c–e In situ hybridization (ISH) on whole P0 brains (c, ventral viewpoint) and sagittal (d) or coronal (e) sections through the brain show Shox2 expression in the facial motor nucleus (nVII, arrows). f–j X-gal staining of Shox2lacZ/+ embryos at E10.5 (f), E11.5 (g), E12.5 (h), E13.5 (i), and E14.5 (j) shows that Shox2 is expressed in the trigeminal and facial ganglion (f, g, arrow), in addition to the developing maxillary process and mandibular arch. Starting at E12.5 and continuing to E14.5, Shox2 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 Shox2lacZ/+ pups in the P0 face (k), brain (l, side viewpoint; m, ventral viewpoint) and sagittal (n) or coronal (o) sections through the brain show Shox2 expression in the tissue surrounding the facial nerves (k), with the exception of the nerves themselves, and Shox2 expression in the facial motor nucleus (l–o, arrows). 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.
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Fig1: Shox2 mRNA and lacZ expression in the developing embryonic face and postnatal facial motor nucleus. a, b Whole-mount in situ hybridization (WISH) at E11.5 (a) and E12.5 (b) show Shox2 expression in the trigeminal (V) and facial (VII, arrow) ganglia, in addition to the developing maxillary process (mp) and mandibular arch (ma). c–e In situ hybridization (ISH) on whole P0 brains (c, ventral viewpoint) and sagittal (d) or coronal (e) sections through the brain show Shox2 expression in the facial motor nucleus (nVII, arrows). f–j X-gal staining of Shox2lacZ/+ embryos at E10.5 (f), E11.5 (g), E12.5 (h), E13.5 (i), and E14.5 (j) shows that Shox2 is expressed in the trigeminal and facial ganglion (f, g, arrow), in addition to the developing maxillary process and mandibular arch. Starting at E12.5 and continuing to E14.5, Shox2 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 Shox2lacZ/+ pups in the P0 face (k), brain (l, side viewpoint; m, ventral viewpoint) and sagittal (n) or coronal (o) sections through the brain show Shox2 expression in the tissue surrounding the facial nerves (k), with the exception of the nerves themselves, and Shox2 expression in the facial motor nucleus (l–o, arrows). 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.
Mentions: Whole-mount in situ hybridization (WISH) at E11.5 showed Shox2 expression in the trigeminal (V) and facial (VII) ganglia, in addition to the developing maxillary process and mandibular arch (Figure 1a). Shox2 continued to be expressed in the trigeminal (V) ganglia, maxillary process and mandibular arch at E12.5 (Figure 1b). We also visualized the Shox2 expression pattern with a novel Shox2lacz allele (described in “Methods”). X-gal staining of embryos carrying the Shox2lacz allele accurately reproduced the endogenous Shox2 expression pattern with increased sensitivity and less background compared to WISH (Additional file 1: Figure S1). At E10.5, Shox2lacZ/+ embryos showed lacZ staining in the trigeminal (V) and facial (VII) ganglia of the embryo (Figure 1f). Similarly at E11.5, lacZ staining was visible in the trigeminal (V) and facial (VII) nerves of the embryo, in addition to the maxillary process and mandibular arch (Figure 1g). LacZ continued to be expressed in the trigeminal (V) ganglion of the embryonic face from E12.5 to E13.5, and could be seen in the developing pharyngeal arch (Figure 1h, i). At E14.5 lacZ expression was present in the facial mesenchyme that contributes to the maxilla and mandible (Figure 1j). However, from E12.5 onward lacZ staining was no longer observed in the facial (VII) ganglia (Figure 1h–j). Postnatally (P0) lacZ expression was maintained in the facial mesenchyme surrounding the facial (VII) nerves, but not within the axons themselves (Figure 1k). Examination of Shox2 expression early embryonically in the developing brain showed Shox2 staining in a region of post-mitotic neurons adjacent to the floor plate (Additional file 2: Figure S2A–B), a region where Phox2b+/Isl1+ are localized (Additional file 2: Figure S2C–E) [18]. Later in the P0 brain, Shox2 was expressed in the facial motor nucleus (Figure 1c–e). Similarly, in Shox2lacZ/+ animals, lacZ staining was strongly expressed in both the lateral and medial lobes of the facial motor nucleus (Figure 1l–o); however, staining was not visible within the trigeminal nucleus (Figure 1n, arrowhead).Figure 1

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