Rosin et al. BMC Neurosci (2015) 16:39 DOI 10.1186/s12868-015-0176-0 RESEARCH ARTICLE Open Access Shox2 is required for the proper development of the facial motor nucleus and the establishment of the facial nerves Jessica M Rosin1, Deborah M Kurrasch2† and John Cobb1*† 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. Keywords: Short stature homeobox 2 (Shox2) gene, Islet 1 (Isl1), Sonic hedgehog (Shh), Visceral motor neurons (vMNs), Facial motor nucleus, Facial (VII) nerves Background homeotic (Hox) gene clusters [3–6]. Motor neuron pro- In the hindbrain, motor neuron progenitors are born in genitors in the hindbrain can differentiate into somato- a region-specific manner, neighboring the floor plate, motor neurons (sMNs), which innervate the skeletal in response to their level of exposure to different mor- muscles of the body, or visceromotor neurons (vMNs), phogenetic gradients [1, 2]. These neuronal progenitors which innervate autonomic ganglia (general vMNs) and differentiate in a rhombomere-specific pattern, which branchial arch-derived muscles (special vMNs) [3, 7, 8]. requires the correct spatiotemporal expression of the Facial (VII) motor neurons, which originate from rhom- bomeres 4/5, belong to the vMN class of motor neurons. At embryonic day 11.25 (E11.25), facial motor neurons *Correspondence: [email protected] begin to migrate caudally to the pial side of the hindbrain †Deborah M Kurrasch and John Cobb contributed equally to this work 1 Department of Biological Sciences, University of Calgary, 2500 University to form the facial motor nucleus, which subsequently Drive N.W., BI286D, Calgary, AB T2N 1N4, Canada segregates into two lobes [6, 9, 10]. Despite the elucida- Full list of author information is available at the end of the article tion of the genetic mechanisms that specify sMN identity, © 2015 Rosin et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rosin et al. BMC Neurosci (2015) 16:39 Page 2 of 17 the corresponding pathways responsible for the estab- to regulate transcription of Isl1 [34], an important regu- lishment of vMNs are less well understood. Furthermore, lator of vMN development [7, 8, 19, 22]. In the current even though sonic hedgehog (SHH) signaling is known to study we show that conditional inactivation of Shox2 in be required for the development of all motor neurons [7, the brain results in impaired axonal projections of vMNs 11–15], the upstream regulators of Shh expression and and a concomitant loss of medially located neurons in the function during late embryonic and early postnatal devel- facial motor nucleus postnatally. Furthermore, we dem- opment, especially within the vMNs of the facial motor onstrate that Shox2-mutant neonates display impaired nucleus, remain largely unknown [16]. feeding behavior, perhaps due to facial paralysis as a The paired-like homeobox 2b (Phox2b) gene is result of improper development and function of the facial expressed in both proliferating progenitors and post- nerves. mitotic motor neurons and is required for the develop- ment of vMNs [17, 18]. Similarly, the homeodomain Results transcription factor islet1 (ISL1), which is expressed in Shox2 expression in the face and facial motor nucleus both post-mitotic sMNs and vMNs, plays a broad role during development in motor neuron identity and specification [7, 8, 17, 19, Whole-mount in situ hybridization (WISH) at E11.5 20]. In the absence of Isl1, mice die during embryogenesis showed Shox2 expression in the trigeminal (V) and facial around E11.5. This embryonic lethality is associated with (VII) ganglia, in addition to the developing maxillary an increase in cell death in the hindbrain and neural tube, process and mandibular arch (Figure 1a). Shox2 contin- suggesting that loss of Isl1 function results in the death of ued to be expressed in the trigeminal (V) ganglia, max- cells that were fated to differentiate into motor neurons illary process and mandibular arch at E12.5 (Figure 1b). [19]. In the trigeminal and dorsal root ganglia (DRG), Isl1 We also visualized the Shox2 expression pattern with appears to play distinct roles during early (i.e. neurogen- a novel Shox2lacz allele (described in “Methods”). X-gal esis) versus late (i.e. subtype specification) development staining of embryos carrying the Shox2lacz allele accu- [21]. In cranial ganglia, Isl1 is required for cell survival rately reproduced the endogenous Shox2 expression and Isl1 mutants show thinner blunted facial (VII) gan- pattern with increased sensitivity and less background glia [22]. However, the precise role of Isl1 in these cell compared to WISH (Additional file 1: Figure S1). At types and in their respective hindbrain nuclei, such as the E10.5, Shox2lacZ/+ embryos showed lacZ staining in the facial motor nucleus, remains poorly documented. trigeminal (V) and facial (VII) ganglia of the embryo The adult facial motor nucleus contains seven subnu- (Figure 1f). Similarly at E11.5, lacZ staining was vis- clei; the lateral, dorsolateral, dorsal intermediate, ventral ible in the trigeminal (V) and facial (VII) nerves of the intermediate, dorsomedial and ventromedial nuclei are embryo, in addition to the maxillary process and man- located within the main nucleus, while the seventh, the dibular arch (Figure 1g). LacZ continued to be expressed dorsal accessory nuclei, is located above the main nucleus in the trigeminal (V) ganglion of the embryonic face [23, 24]. Neurons innervating the nasolabial musculature from E12.5 to E13.5, and could be seen in the developing are located in the lateral facial motor nucleus, while neu- pharyngeal arch (Figure 1h, i). At E14.5 lacZ expression rons supplying the auricular musculature are located in was present in the facial mesenchyme that contributes the medial facial motor nucleus [23, 24]. Together the to the maxilla and mandible (Figure 1j). However, from lateral, intermediate and medial subnuclei comprise 43, E12.5 onward lacZ staining was no longer observed 27.1, and 28.7%, respectively, of the motor neurons in in the facial (VII) ganglia (Figure 1h–j). Postnatally the adult facial motor nucleus, with the dorsal accessory (P0) lacZ expression was maintained in the facial mes- nuclei contributing the final 1.2% [23]. enchyme surrounding the facial (VII) nerves, but not Studies with mouse knockout models have shown within the axons themselves (Figure 1k). Examination of that Shox2 is required for normal development of the Shox2 expression early embryonically in the developing humerus and femur [25–27], the anterior palate [28], the brain showed Shox2 staining in a region of post-mitotic temporomandibular joint of the jaws [29], the sinoatrial neurons adjacent to the floor plate (Additional file 2: Fig- valves and pacemaker region of the heart [30, 31], and ure S2A–B), a region where Phox2b+/Isl1+ are localized TrkB-positive mechanosensory neurons of the dorsal (Additional file 2: Figure S2C–E) [18]. Later in the P0 root ganglia [32]. Most recently, Shox2 has been shown brain, Shox2 was expressed in the facial motor nucleus to play an important role in development of the inferior (Figure 1c–e). Similarly, in Shox2lacZ/+ animals, lacZ colliculus and cerebellum [33]. We suspected a function staining was strongly expressed in both the lateral and for Shox2 during facial motor nucleus development given medial lobes of the facial motor nucleus (Figure 1l–o); our previous study demonstrating a novel role for Shox2 however, staining was not visible within the trigeminal in the hindbrain [33], and since SHOX2 has been shown nucleus (Figure 1n, arrowhead). Rosin et al. BMC Neurosci (2015) 16:39 Page 3 of 17 Figure 1 Shox2 mRNA and lacZ expression in the developing embryonic face and postnatal facial motor nucleus.