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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

Loss of Shox2 in the brain results in subtle changes in the expression pattern of Isl1 and Phox2b in the E14.5 facial motor nucleus. a Diagram of a sagittal section through the E14.5 brain highlights the regions represented in b–r. b X-gal stained sagittal sections through E14.5 embryos shows decreased staining in the facial motor nucleus (nVII) of Nestin-Cre; Shox2lacZ/flox (B″) embryos that lack Shox2 function, as compared to controls (B′). c–r ISH on E14.5 control (c, e, g, i, k, m, o, q) and Nestin-Cre; Shox2flox/− (d, f, h, j, l, n, p, r) sagittal sections shows loss of Shox2 expression (compare c to d, dashed-circle), and subtle changes in the spatial expression patterns of Isl1 (compare e to f, arrow), Phox2b (compare g to h, arrow), Cntn2 (compare i to j, dashed-circle), Tubb3 (compare k to l, dashed-circle), Periph (compare m to n, arrow), Slc18a3 (compare o to p, arrow) and Slit2 (compare q to r, arrow) in the developing facial motor nucleus. The dashed-circles in paired panels being compared (e.g. B′ and B″) are the same size, and this applies from B′ to r. Scale bar 250 μm.
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Fig5: Loss of Shox2 in the brain results in subtle changes in the expression pattern of Isl1 and Phox2b in the E14.5 facial motor nucleus. a Diagram of a sagittal section through the E14.5 brain highlights the regions represented in b–r. b X-gal stained sagittal sections through E14.5 embryos shows decreased staining in the facial motor nucleus (nVII) of Nestin-Cre; Shox2lacZ/flox (B″) embryos that lack Shox2 function, as compared to controls (B′). c–r ISH on E14.5 control (c, e, g, i, k, m, o, q) and Nestin-Cre; Shox2flox/− (d, f, h, j, l, n, p, r) sagittal sections shows loss of Shox2 expression (compare c to d, dashed-circle), and subtle changes in the spatial expression patterns of Isl1 (compare e to f, arrow), Phox2b (compare g to h, arrow), Cntn2 (compare i to j, dashed-circle), Tubb3 (compare k to l, dashed-circle), Periph (compare m to n, arrow), Slc18a3 (compare o to p, arrow) and Slit2 (compare q to r, arrow) in the developing facial motor nucleus. The dashed-circles in paired panels being compared (e.g. B′ and B″) are the same size, and this applies from B′ to r. Scale bar 250 μm.

Mentions: Given the marked reduction in size of the facial motor nucleus in Shox2 mutants (Figure 4), we next sought to investigate whether factors other than facial (VII) nerve truncation and cell death could be changing in the facial motor nucleus following the loss of Shox2. Prior to E14.5, we did not observe any obvious changes in gene expression or disruptions to the migratory patterns of vMNs (Additional file 2: Figure S2F-K, M-R). At E14.5, however, sagittal sections of the facial motor nucleus showed reductions in the overall size of the nucleus in Nestin-Cre; Shox2lacZ/flox embryos (Figure 5b), which was consistent with the regional increase in the number of cleaved active-CASP3 + cells at this time-point (Figure 4k). Since SHOX2 has been previously shown to regulate Isl1 [34], an important regulator of vMN development [7, 19, 22], we next examined the expression of Isl1 in Shox2-mutant animals. We observed a regionally restricted loss of Isl1 and the hindbrain vMN determinant Phox2b in the facial motor nucleus as early as E14.5 in Nestin-Cre; Shox2flox/− mutant embryos as compared to controls (Figure 5e–h, arrows). Since loss of Isl1 function in the trigeminal and DRG have been shown to result in changes in the expression of contactin 2 (Cntn2), tubulin β-3 chain (Tubb3) and peripherin 1 (Periph) [38], and given that Phox2b-deficient mice have been used to identify the facial motor nucleus as a source of Slit homolog 2 (Slit2) signaling [39], we next examined the expression patterns of these genes. Although subtle changes in the spatial patterns and expression levels of Cntn2 and Tubb3 were observed, Periph1, solute carrier family 18 (Slc18a3) and Slit2 phenocopied the regionally specific absence of Isl1 and Phox2b in the facial motor nucleus at E14.5 in Nestin-Cre; Shox2flox/− mutant embryos (Figure 6i–r, dashed-circle and arrows). The changes in expression for these genes continued to be observed between Nestin-Cre; Shox2flox/− mutant embryos and controls at E16.5 (data not shown). Together, these results show that Shox2 is required for normal expression of markers of vMN identity at E14.5.Figure 5


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)

Loss of Shox2 in the brain results in subtle changes in the expression pattern of Isl1 and Phox2b in the E14.5 facial motor nucleus. a Diagram of a sagittal section through the E14.5 brain highlights the regions represented in b–r. b X-gal stained sagittal sections through E14.5 embryos shows decreased staining in the facial motor nucleus (nVII) of Nestin-Cre; Shox2lacZ/flox (B″) embryos that lack Shox2 function, as compared to controls (B′). c–r ISH on E14.5 control (c, e, g, i, k, m, o, q) and Nestin-Cre; Shox2flox/− (d, f, h, j, l, n, p, r) sagittal sections shows loss of Shox2 expression (compare c to d, dashed-circle), and subtle changes in the spatial expression patterns of Isl1 (compare e to f, arrow), Phox2b (compare g to h, arrow), Cntn2 (compare i to j, dashed-circle), Tubb3 (compare k to l, dashed-circle), Periph (compare m to n, arrow), Slc18a3 (compare o to p, arrow) and Slit2 (compare q to r, arrow) in the developing facial motor nucleus. The dashed-circles in paired panels being compared (e.g. B′ and B″) are the same size, and this applies from B′ to r. Scale bar 250 μm.
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Fig5: Loss of Shox2 in the brain results in subtle changes in the expression pattern of Isl1 and Phox2b in the E14.5 facial motor nucleus. a Diagram of a sagittal section through the E14.5 brain highlights the regions represented in b–r. b X-gal stained sagittal sections through E14.5 embryos shows decreased staining in the facial motor nucleus (nVII) of Nestin-Cre; Shox2lacZ/flox (B″) embryos that lack Shox2 function, as compared to controls (B′). c–r ISH on E14.5 control (c, e, g, i, k, m, o, q) and Nestin-Cre; Shox2flox/− (d, f, h, j, l, n, p, r) sagittal sections shows loss of Shox2 expression (compare c to d, dashed-circle), and subtle changes in the spatial expression patterns of Isl1 (compare e to f, arrow), Phox2b (compare g to h, arrow), Cntn2 (compare i to j, dashed-circle), Tubb3 (compare k to l, dashed-circle), Periph (compare m to n, arrow), Slc18a3 (compare o to p, arrow) and Slit2 (compare q to r, arrow) in the developing facial motor nucleus. The dashed-circles in paired panels being compared (e.g. B′ and B″) are the same size, and this applies from B′ to r. Scale bar 250 μm.
Mentions: Given the marked reduction in size of the facial motor nucleus in Shox2 mutants (Figure 4), we next sought to investigate whether factors other than facial (VII) nerve truncation and cell death could be changing in the facial motor nucleus following the loss of Shox2. Prior to E14.5, we did not observe any obvious changes in gene expression or disruptions to the migratory patterns of vMNs (Additional file 2: Figure S2F-K, M-R). At E14.5, however, sagittal sections of the facial motor nucleus showed reductions in the overall size of the nucleus in Nestin-Cre; Shox2lacZ/flox embryos (Figure 5b), which was consistent with the regional increase in the number of cleaved active-CASP3 + cells at this time-point (Figure 4k). Since SHOX2 has been previously shown to regulate Isl1 [34], an important regulator of vMN development [7, 19, 22], we next examined the expression of Isl1 in Shox2-mutant animals. We observed a regionally restricted loss of Isl1 and the hindbrain vMN determinant Phox2b in the facial motor nucleus as early as E14.5 in Nestin-Cre; Shox2flox/− mutant embryos as compared to controls (Figure 5e–h, arrows). Since loss of Isl1 function in the trigeminal and DRG have been shown to result in changes in the expression of contactin 2 (Cntn2), tubulin β-3 chain (Tubb3) and peripherin 1 (Periph) [38], and given that Phox2b-deficient mice have been used to identify the facial motor nucleus as a source of Slit homolog 2 (Slit2) signaling [39], we next examined the expression patterns of these genes. Although subtle changes in the spatial patterns and expression levels of Cntn2 and Tubb3 were observed, Periph1, solute carrier family 18 (Slc18a3) and Slit2 phenocopied the regionally specific absence of Isl1 and Phox2b in the facial motor nucleus at E14.5 in Nestin-Cre; Shox2flox/− mutant embryos (Figure 6i–r, dashed-circle and arrows). The changes in expression for these genes continued to be observed between Nestin-Cre; Shox2flox/− mutant embryos and controls at E16.5 (data not shown). Together, these results show that Shox2 is required for normal expression of markers of vMN identity at E14.5.Figure 5

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