© 2019. Published by The Company of Biologists Ltd | Development (2019) 146, dev174045. doi:10.1242/dev.174045 RESEARCH ARTICLE Teashirt 1 (Tshz1) is essential for the development, survival and function of hypoglossal and phrenic motor neurons in mouse Charlotte Chaimowicz1, Pierre-Louis Ruffault1, Cyril Chéret1,*, Andrew Woehler2, NiccolòZampieri3, Gilles Fortin4, Alistair N. Garratt5 and Carmen Birchmeier1,‡ ABSTRACT the phrenic motor column located in the mid-cervical spinal cord. Feeding and breathing are essential motor functions and rely on the The tongue has crucial functions in feeding and is innervated by activity of hypoglossal and phrenic motor neurons that innervate the the hypoglossal motor neurons in the lower brainstem. The activity tongue and diaphragm, respectively. Little is known about the genetic of phrenic and hypoglossal motor neurons has to be tightly programs that control the development of these neuronal subtypes. coordinated to avoid maladaptive outcomes such as the swallowing The transcription factor Tshz1 is strongly and persistently expressed of air or the blockage of airways (Moore et al., 2014). Hence, in developing hypoglossal and phrenic motor neurons. We used hypoglossal and phrenic motor neurons and the circuitry that conditional mutation of Tshz1 in the progenitor zone of motor neurons coordinates their activity are essential for animal survival. (Tshz1MNΔ) to show that Tshz1 is essential for survival and function of Nevertheless, relatively little is known about the formation of the hypoglossal and phrenic motor neurons. Hypoglossal and phrenic motor neurons that relay breathing and feeding commands. motor neurons are born in correct numbers, but many die between All motor neurons that innervate skeletal muscle, i.e. somatic embryonic day 13.5 and 14.5 in Tshz1MNΔ mutant mice. In addition, motor neurons, derive from ventral progenitor cells expressing the innervation and electrophysiological properties of phrenic and transcription factor Olig2 and share a common transcriptional + + hypoglossal motor neurons are altered. Severe feeding and program (Isl1/2 , Hb9 ; Hb9 is also known as Mnx1) during their breathing problems accompany this developmental deficit. Although early initial development that distinguishes them from other motor neuron survival can be rescued by elimination of the pro- neuronal types (Jessell, 2000). However, at later developmental apoptotic factor Bax, innervation, feeding and breathing defects stages they acquire subtype-specific identities that correlate with the persist in Bax−/−; Tshz1MNΔ mutants. We conclude that Tshz1 is an muscle groups they innervate (Dasen et al., 2008; De Marco Garcia essential transcription factor for the development and physiological and Jessell, 2008; Livet et al., 2002). According to their subtype function of phrenic and hypoglossal motor neurons. identity, the ventrally derived motor neurons initiate expression of specific sets of transcription factors and either maintain or KEY WORDS: Tshz1, Motor neurons, Phrenic motor column, downregulate Isl1/2 and Hb9 (Arber et al., 1999; Briscoe et al., Hypoglossal nucleus, Aerophagia, Breathing, Mouse 1999; Pfaff et al., 1996). In addition, their cell bodies cluster, forming motor pools in specific positions along the rostro-caudal INTRODUCTION and medio-lateral axes of the hindbrain and spinal cord (Bonanomi Breathing and feeding are motor behaviors that are essential for the and Pfaff, 2010; Dasen and Jessell, 2009). Compared with motor survival of all terrestrial vertebrates. They rely on complex neuronal neuronal subtypes that innervate different limb muscles, little is networks, including motor neurons that are the ‘executive’ known about the genetic programs important for the development of components controlling the activity of specific muscle groups hypoglossal and/or phrenic motor pools. (Moore et al., 2014). The diaphragm muscle is essential for Genes that specifically affect hypoglossal motor neuron inspiration during breathing and is innervated by motor neurons of development have not yet been identified. Hypoglossal motor neurons locate near the midline of rhombomeres 7/8 of the hindbrain and express Isl1/2 and Hb9. They are organized 1Developmental Biology/Signal Transduction, Max Delbrück Center for Molecular topographically according to which tongue muscle they innervate Medicine (MDC) in the Helmholtz Society, 13125 Berlin, Germany. 2Systems Biology Imaging, Max Delbrück Center for Molecular Medicine (MDC) in the (Aldes, 1990, 1995; Krammer et al., 1979; Lewis et al., 1971; Helmholtz Society, 13125 Berlin, Germany. 3Development and Function of Neural Odutola, 1976). Dorsal hypoglossal motor neurons innervate the Circuits, Max Delbrück Center for Molecular Medicine (MDC) in the Helmholtz retrusor muscles (i.e. extrinsic hyoglossus, extrinsic palatoglossus, Society, 13125 Berlin, Germany. 4UMR9197, CNRS/UniversitéParis-Sud, Paris- Saclay Institute of Neuroscience, 1 Avenue de la Terrasse, 91190 Gif-sur-Yvette, intrinsic inferior longitudinalis and intrinsic superior longitudinalis) France. 5Institute of Cell Biology and Neurobiology, Charité-Universitätsmedizin and express Foxp1 (Chen et al., 2016). In contrast, motor neurons Berlin, Virchowweg 6, 10117 Berlin, Germany. *Present address: Institute for Integrative Neuroanatomy, Charité- located in the ventral half of the hypoglossal nucleus innervate the Universitätsmedizin Berlin, Charitéplatz 1, 10115 Berlin, Germany. protrusor muscles (i.e. extrinsic genioglossus, intrinsic verticalis ‡ and intrinsic transversus) and express Pou3f1 (Chen et al., 2016). Author for correspondence ([email protected]) Finally, motor neurons innervating extrinsic and intrinsic muscles C.Chaimowicz, 0000-0002-5998-7482; P.-L.R., 0000-0002-3378-3828; A.W., segregate in the lateral and medial hypoglossal nucleus, respectively 0000-0001-5157-9313; N.Z., 0000-0002-2228-9453; G.F., 0000-0002-2123-8603; (Aldes, 1995; Chen et al., 2016; Dobbins and Feldman, 1995; A.N.G., 0000-0001-9631-3041; C.B., 0000-0002-2041-8872 McClung and Goldberg, 2002). This is an Open Access article distributed under the terms of the Creative Commons Attribution More information is available for phrenic motor neurons that License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. locate to the cervical spinal cord (C3-C5). Previous studies showed that mutations causing respiratory distress often affect the function Received 27 November 2018; Accepted 9 August 2019 and/or survival of phrenic motor neurons (Turgeon and Meloche, DEVELOPMENT 1 RESEARCH ARTICLE Development (2019) 146, dev174045. doi:10.1242/dev.174045 2009). These mutations can either affect the survival of all motor of GFP in several neuronal types, among them motor neurons. neurons, for instance Hb9, ErbB3, CLAC-P (Col25a1)orFzd3 (Hua Detailed immunohistochemical analysis using anti-GFP and pan- et al., 2013; Riethmacher et al., 1997; Tanaka et al., 2014; Yang motor neuron antibodies, such as anti-Isl1/2 or anti-choline et al., 2001), or specifically phrenic motor neurons, e.g. Hoxa5/c5 acetyltransferase (ChAT), demonstrated the presence of GFP in genes and Pou3f1 (Bermingham et al., 1996; Machado et al., 2014; hypoglossal motor neurons (nXII) from embryonic day (E) 12.5 to Philippidou et al., 2012). Indeed, Pou3f1 (also known as Scip or postnatal day (P) 0.5 (Fig. 1A). GFP was detected in 85% of the Oct6) is strongly expressed in phrenic neurons, and its ablation in Isl1/2+ hypoglossal motor neurons at E12.5, but not in Olig2+ mice leads to disorganization of the phrenic motor column and early progenitors (Fig. 1A,B). GFP expression persisted and was detected postnatal lethality due to breathing failure within the first hours of in around two-thirds of hypoglossal motor neurons at E15.5 and life (Bermingham et al., 1996). The Hoxa5/c5 null mutation in P0.5. Subsets of GFP+ hypoglossal motor neurons co-expressed motor neurons impairs the expression of phrenic specific markers Foxp1 and Pou3f1 (Fig. S1A,B). The persistent expression of and the correct localization and survival of phrenic motor Tshz1 in hypoglossal motor neurons was confirmed by in situ neurons, resulting in a complete absence of diaphragm hybridization at P0.5 (Fig. S1C). Tshz1 expression was spatially innervation (Philippidou et al., 2012). When phrenic motor restricted to subdomains of the hypoglossal nucleus at P0.5, a neurons are generated from embryonic stem cells, the combined finding that became apparent in a 3D reconstruction of the action of Pou3f1 and Hoxa5 is necessary for the acquisition of hypoglossal nucleus (Fig. 1C; see Fig. S5 for examples of their identity (Machado et al., 2014). sections used for the reconstruction). The position of hypoglossal The transcription factor teashirt zinc finger homeobox 1 (Tshz1) motor neurons was digitally reconstructed in three dimensions by belongs to the evolutionarily conserved family of Teashirt genes that assigning Cartesian coordinates to each motor neuron identified in are characterized by the presence of three atypical zinc finger motifs, consecutive histological sections (see also Materials and Methods). an N-terminal acidic domain and a binding motif for the co- The position of neurons of the adjacent dorsal nucleus of the vagus repressor CtBP (C-terminal binding protein). In addition, nerve (nX) was used as a landmark. In the rostral hypoglossal mammalian Tshz