ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2012

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 825

Crossing the Midline

Locomotor Neuronal Circuitry Formation

FATIMA MEMIC

ISSN 1651-6206ISBN 978-91-554-8500-9urn:nbn:se:uu:diva-182692

Dissertation presented at Uppsala University to be publicly examined in B22 BMC,Husargatan 3, Uppsala, Saturday, December 1, 2012 at 09:15 for the degree of Doctor ofPhilosophy (Faculty of Medicine). The examination will be conducted in English.

AbstractMemic, F. 2012. Crossing the Midline: Locomotor Neuronal Circuitry Formation. ActaUniversitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations fromthe Faculty of Medicine 825. 43 pp. Uppsala. ISBN 978-91-554-8500-9.

Networks at various levels of the nervous system coordinate different motor patterns such asrespiration, eye or hand movements and locomotion. Intrinsic rhythm-generating networks thatare located in the spinal cord generate motor behaviors that underlie locomotion in vertebrates.These networks give a continuous and measurable coordinated rhythmic motor output and arereferred to as locomotor central pattern generators (CPGs). Characterization of the mammalianlocomotor CPG and its molecular control is depending on the identification of participatingneurons and neuronal populations. In this thesis I present work where we have studied thesignificance of subpopulations of neurons in the formation and function of the left-right circuitry.In summary, we show that the axon guidance receptor DCC has a central role in the formation ofspinal neuronal circuitry underlying left-right coordination, and that both Netrin-1 and DCC arerequired for normal function of the locomotor CPG. Commissural interneurons (CINs), whichsend their axons across the ventral midline in the spinal cord, play a critical role in left–rightcoordination during locomotion. A complete loss of commissural axons in the spinal cord, asseen in the Robo3 null mutant mouse, resulted in uncoordinated fictional locomotor activity.Removing CIN connections from either dorsal or ventral neuronal populations led to a shift fromalternation to strict synchronous locomotor activity. Inhibitory dI6 CIN have been suggested aspromising candidate neurons in coordinating bilateral alternation circuitry. We have identifiedthat Dmrt3, expressed in inhibitory dI6 CINs, is a crucial component for the normal developmentof coordinated locomotor movements in both horses and mice. We have also concluded thatthe prominent hopping phenotype seen in hop/hop mice is a result of abnormal developmentalprocesses including induction from the notochord and Shh signaling. Together, these findingsincrease our knowledge about the flexibility in neuronal circuit development and further confirmthe role of dI6 neurons in locomotor circuits.

Keywords: CPG, CIN, neuronal network, locomotion, left-right alternation

Fatima Memic, Uppsala University, Department of Neuroscience, Box 593,SE-751 24 Uppsala, Sweden.

© Fatima Memic 2012

ISSN 1651-6206ISBN 978-91-554-8500-9urn:nbn:se:uu:diva-182692 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-182692)

Somewhere, something incredible is waiting to be known -Dr Carl Sagan

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Rabe, N., Gezelius, H.,# Vallstedt, A.,# Memic, F., Kullander,

K., (2009) Netrin-1-dependent spinal interneuron subtypes are required for the formation of left-right alternating locomotor circuitry . Journal of Neuroscience, Dec 16;29(50):15642-9

II Rabe Bernhardt, N.,# Memic, F.,# Gezelius, H., Thiebes, A.L., Vallstedt, A. and Kullander, K., (2012) DCC mediated axon guidance of spinal interneurons is essential for normal locomo-tor central pattern generator function. Dev Biol. Jun 15;366(2):279-89

III Memic, F., Wootz, H., Renier, N., Lapshyna, K., Chédotal, A. and Kullander, K., The role of Robo3 subpopulations in the spi-nal locomotor CPG (Manuscript)

IV Andersson, L.S., # Larhammar, M., # Memic, F., # Wootz, H., # Schwochow, D., Rubin, C.J., Patra, K., Arnason, T., Wellbring, L., Hjälm, G., Imsland, F., Petersen, J., McCue, M., Mickelson, J., Cothran, G., Ahituv, N., Roepstorff, L., Mikko, S., Vallstedt, A., Lindgren, G., Andersson, L and Kullander, K., (2012) # Mu-tations in DMRT3 affect locomotion in horses and spinal circuit function in mice. Nature Aug 30;488:642-646

V Memic, F., # Bernhardt, N.,# Tran, M., Chersa, T., Wootz, H.,

Strömstedt, L., Andersson, L., Whelan, P. and Kullander K., The hop mutation causes a restricted merge of the ventral lum-bar spinal cord resulting in fused CPG half-centers and syn-chronous hind-limb gait (Manuscript)

# Equal contribution

Contents

Introduction ..................................................................................................... 9  Neuronal networks ...................................................................................... 9  Neuronal networks that coordinate locomotion .......................................... 9  

Spinal cord neurons involved in the CPG ............................................ 10  Commissural interneurons and CPG function ..................................... 11  

Development of neuronal networks .......................................................... 11  Patterning and cell specification .......................................................... 11  Axon guidance ..................................................................................... 14  Downstream signalling ........................................................................ 18  

Aims .............................................................................................................. 19  

Methodological considerations ..................................................................... 20  Animals ..................................................................................................... 20  Retrograde tracing of commissural interneurons ...................................... 21  In situ hybridization .................................................................................. 22  Immunohistochemistry ............................................................................. 24  Imaging and picture processing ................................................................ 24  Ventral root recordings ............................................................................. 24  Trans-synaptic tracing of interneurons ..................................................... 25  

Results and conclusions ................................................................................ 26  Paper I and II ............................................................................................ 26  Paper III .................................................................................................... 27  Paper IV .................................................................................................... 28  Paper V ..................................................................................................... 29  

Future prospects ............................................................................................ 31  

Acknowledgements ....................................................................................... 33  

References ..................................................................................................... 35  

Abbreviations

BMP Bone morphogenic protein CIN Commissural interneuron CNS Central nervous system CPG Central Pattern Generator Cre Cyclic recombinase DCC Deleted in colorectal cancer Dmrt3 Doublesex and mab-3 related transcription factor 3 HD Homeodomain MNs Motor neurons Ntn1 Netrin-1 Robo3 Roundabout receptor 3 Shh Sonic Hedgehog VIAAT Vesicular Inhibitory Amino Acid Transporter Vglut2 Vesicular glutamate transporter 2 Wnt Wingless type

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Introduction

Neuronal networks Networks at various levels of the nervous system coordinate different motor patterns such as respiration, eye or hand movements and locomotion. These neuronal networks form functional units that can be used by the nervous system to generate necessary reaction to external stimuli and control of the body. Some networks are present at birth, whereas others mature during development to be modified and perfected through learning. Knowledge about the basic components of the neuronal networks on the cellular and molecular level as well as their connectivity is essential to understand nor-mal brain function and diseases of the nervous system.

Neuronal networks have been well studied and characterized in simpler vertebrates like lamprey and Xenopus tadpole (Roberts et al., 1998, McLean et al., 2000), but the complexity of the mammalian nervous system limits our current knowledge about neuronal circuits in mammals (Butt and Kiehn, 2003). A useful model system for studying the organization of neuronal net-works and the participating neurons are the neuronal networks that control rhythmic movements, like breathing, feeding, swimming, and walking. The possibility to relate the network activity with an actual behavior combined with mouse genetics provides a good model system to study the components of a functional neuronal circuit in higher vertebrates (Kiehn and Kullander, 2004).

Neuronal networks that coordinate locomotion Intrinsic rhythm-generating networks that are located in the spinal cord gen-erate the motor behaviors that underlie locomotion in vertebrates. These networks give a continuous and measurable coordinated rhythmic motor output and are referred to as locomotor central pattern generators (CPGs) (Grillner, 2003, Kiehn and Kullander, 2004, Goulding and Pfaff, 2005, Grillner, 2006). Locomotor CPGs are inactive at rest but are activated by tonic signals usually originating from higher brain centers.

The CPG controlling hind limb muscle activity during locomotion is lo-calized in the ventral part of the lumbar spinal cord. Each half of the spinal cord contains a CPG capable of rhythm generation. Cross-inhibitory actions

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between the two sides ensure left/right coordination of walking while cross-inhibitory actions between CPGs on different rostrocaudal levels of the spi-nal cord control coordination of ipsilateral flexors and extensors muscles (Bracci et al. 1996; Kjaerulff et al., 1994; Kjaerulff and Kiehn, 1996).

Spinal cord neurons involved in the CPG Characterization of the mammalian locomotor CPG and its molecular control depends on the identification of its components, the participating neurons and neuronal populations. Spinal cord neurons likely to participate in loco-motor CPG are shown in Figure 1.

There are five subtypes of cholinergic neurons present in the vertebrate spinal cord, including somatic motor neurons, preganglionic autonomic neu-rons, partition cells (lamina VII), central canal cluster cells (lamina X), and small dorsal horn cells scattered in lamina III-V (Houser et al., 1983, Barber et al., 1984, Phelps et al., 1984, Borges and Iversen, 1986, Sherriff and Henderson, 1994). The somatic motor neurons are the largest and most abundant cholinergic neurons located in the ventral spinal cord. They direct-ly innervate limb muscles and are therefore considered to be the functional output of the locomotor CPG. The vesicular acetylcholine transporter (VAChT) and the choline acetyl transferase (ChAT) can be used as markers for these populations of neurons. Partition cells and central canal cells are also believed to be involved in the control of movement as the spinal motor neurons receive cholinergic input (Nagy et al., 1993, Li et al., 1995, Arvidsson et al., 1997).

Ipsilateral projecting glutamatergic excitatory interneurons (EIs) are thought to excite other ipsilaterally located CPG neurons and motor neurons. In the lamprey these neurons have been shown to be the most likely source for intrinsic rhythm generation in locomotor networks (Grillner, 2003). However, the exact identity and precise function of mammalian EIs during locomotion has not been established.

Renshaw cells are inhibitory interneurons that are excited by axon collat-erals from motor neurons (MNs) and in turn mediate recurrent inhibition to motor neurons and Ia interneurons. This negative feedback system is likely to contribute to stabilize and “fine-tune” motor output (Pratt and Jordan, 1987).

Ia interneurons are part of the spinal reflex pathway. Ia interneurons pro-vide inhibitory feedback onto ipsilateral motor neurons both during the stretch reflexes and locomotion suggesting that this neuronal population is involved in a variety of locomotor behavior.

Commissural interneurons (CINs), which send their axons across the ven-tral midline in the spinal cord, play a critical role in left–right coordination during locomotion. (Butt et al., 2002, Butt and Kiehn, 2003, Lanuza et al., 2004).

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Figure 1. Schematic illustration of spinal locomotor CPG and its components that likely participate in locomotor networks. (CPG) central pattern generator, (CIN) commissural interneuron, (eIN) excitatory interneuron, (Ia) Ia inhibitory interneuron, (MN) motor neuron and (R) Renshaw cell.

Commissural interneurons and CPG function Spinal CINs have been anatomically classified into four different types in rodents (Eide et al., 1999, Stokke et al., 2002, Nissen et al., 2005) based on the direction of their axonal projections. Ascending (aCIN) axons travel rostrally after crossing the midline and descending (dCIN) axons travel cau-dally. Bifurcating (adCIN) axons diverge after crossing the midline and trav-el both rostrally and caudally. And finally short-range intrasegmental CINs axons remain locally within 1.5 segments of their somata. CINs make inhibi-tory or excitatory synapses onto motor neurons and other interneurons on the opposite side of the spinal cord (Bannatyne et al., 2003, Birinyi et al., 2003, Butt and Kiehn, 2003, Lanuza et al., 2004, Quinlan and Kiehn, 2007). In the neonatal rat, dCINs have been demonstrated to be involved in left–right al-ternation during fictive locomotion (Butt et al., 2002, Butt and Kiehn, 2003).

Development of neuronal networks Patterning and cell specification All neural functions depend on the precise assembly of neuronal circuits during embryonic development. A fundamental step in this process is the generation of distinct classes of neurons at precise locations with the help of positional cues.

In vertebrate embryos, the notochord, located ventral to the neural tube in the midline of the embryo is essential for dorsoventral patterning of the par-axial mesoderm and neural tube (Fig. 2). Mutations disrupting notochord development, or developmental experiments involving both transplantation

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and removal of the notochord, showed that a notochord-derived signal can induce the formation of the floor plate, the ventral-most fate of the spinal cord (Placzek et al., 1991, Yamada et al., 1991). Thus, the notochord is nec-essary for the induction of ventral cell fates in the paraxial mesoderm and neural tube. Among the signals secreted by the notochord are the Hedgehog (Hh) proteins and in particular, sonic hedgehog (Shh) that is expressed both in the notochord and floor plate (Echelard et al., 1993). Shh is crucial for determination of ventral cell fates. It induces a range of ventral spinal cord fates by graded signaling while simultaneously suppressing the expression of characteristic dorsal genes (Yamada et al., 1991, Placzek, 1995). Bone mor-phogenic proteins (BMPs) and Wingless –type (Wnts) proteins are secreted from the roof plate of the vertebrate neural tube, and form a gradient that is important for the determination of dorsal neural cell fates (Lee et al., 2000, Liem et al., 2000).

The establishment of progenitor domains Shh morphogen signaling is regulating the expression of a set of transcrip-tion factors, including members of the homeodomain (HD) protein and basic helix-loop-helix (bHLH) families (Goulding et al., 1993, Pierani et al., 1999, Briscoe et al., 2000, Novitch et al., 2001). An integrative transcriptional code defines spatially specific progenitor domains along the dorso-ventral (DV) axis of the neural tube. The dorsal progenitor domains are named dI1-dI6 and the ventral domains p0-p3 and pMN. From each domain one or more distinct neuronal subpopulations are generated. The identity of the subpopu-lations is determined by the combination of progenitor domain expressed transcription factors. Dorsal domains give rise to six early classes of neu-rons, dI1 to dI6, and ventral domains give rise to four classes of interneu-rons, V0 to V3 and motor neurons (Fig. 2)(Gross et al., 2000, Bermingham et al., 2001, Moran-Rivard et al., 2001, Pierani et al., 2001, Lanuza et al., 2004).

CINs are considered to originate from both the dorsal and ventral inter-neuron populations and have been studied in left-right locomotor coordina-tion. dI3 interneurons are unlikely to contribute to CPG coordination, since they have been suggested as part of ascending pathways including the spino-cerebellar and the spinothalamic tract (Gross et al., 2000, Bermingham et al., 2001). The inhibitory dI6 neurons, which settle in lamina VII and VIII, have been suggested to participate in left-right alternating circuitry. This is sup-ported by the idea that inhibitory commissural connections are the major pathways responsible for coordinating the left-right phasing during locomo-tion (Grillner and Wallén, 1980, Cohen and Harris-Warrick, 1984, Soffe et al., 1984, Jankowska and Noga, 1990, Cowley and Schmidt, 1995, Lanuza et al., 2004, Kiehn, 2006). V0 interneurons arise from P0 progenitor cells ex-pressing the Dbx1 homeodomain (HD) protein and consist of two popula-tions of cells, V0V and V0D neurons (Moran-Rivard et al., 2001, Pierani et

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al., 2001, Lanuza et al., 2004). Dbx1 is expressed in progenitor cells giving rise to both V0V and V0D neurons, whereas Evx1 is found in postmitotic V0V interneurons only. Deletion of Dbx1 led to a loss of both V0v and V0D re-sulting in irregular episodes of synchrony and alternation in left and right coordination during in vitro locomotion. However in Evx1 null mice loss of V0V neurons gave a normal pattern. V3 neurons arise from Nkx2.2 positive p3 progenitor domain, are predominantly excitatory and settle in lamina VIII. Genetic manipulations of V3 neurons indicate that they contribute to a regular and balanced motor rhythm distributing excitatory drive to other neurons (Briscoe et al., 1999, Nissen et al., 2005, Zhang et al., 2008).

Several other populations of neurons are considered to be involved in var-ious aspects of locomotion. V1 interneurons expressing Engrailed-1 (En1) migrate to lamina VII and develop local projections (Burrill et al., 1997, Saueressig et al., 1999). A subset of V1 interneurons, the Renshaw cells, mediate recurrent inhibition to motor neurons and Ia inhibitory interneurons that receive input from Ia sensory afferent neurons of the spinal reflex path-way (Sapir et al., 2004, Alvarez et al., 2005). V2 interneurons developing from p2 progenitor also migrate laterally to lamina VII. Deletion and silenc-ing of V2a interneurons with a Chx10-DTA, results in greater variability in cycle period and amplitude of locomotor bursts during fictive locomotion (Crone et al., 2008). Also, the adult mice show a speed dependent loss of left-right alternation, defined by a transition to synchronous gait at high speed (Crone et al., 2009).

Figure 2. Patterning and cell specification in the spinal cord. Gradients of Shh and BMP/Wnts regulate the expression of different HD and bHLH transcription factors in a dorso-ventral manner. Cross-repressive interactions between pairs of these pro-teins generate six dorsal and five ventral distinct progenitor domains that each give rise to a specific cell type. The neuronal cell types can be recognized by their specif-ic expression profiles of transcription factors.

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Axon guidance A critical stage during neuronal circuit development is the formation of specific connections between neurons and their target cells. The ability of axons and dendrites to navigate and connect to their appropriate target de-pends on the attractive and repulsive cues from the embryonic environment (Tessier-Lavigne and Goodman, 1996). These cues exist in either diffusible or cell surface-associated forms and can either attract or repel growth cones. The midline of the developing spinal cord is an important choice point for growing axons. Many axons grow towards the midline and must then decide whether or not to cross over to the opposite side. After reaching the midline, non-crossing and crossing axons turn on the ipsilateral (same) or contrala-teral (opposite) side of the midline. Subsequently, these growth cones/axons must further decide whether to extend in the rostral or caudal direction paral-lel to the midline. Both classes of axons are expelled from the midline once they begin to extend in the longitudinal direction. Specialized midline cells function as intermediate targets that provide growth cones with long- and short-range guidance cues which positively or negatively influence axonal growth (Bovolenta and Dodd, 1990). Many molecules have been implicated in the guidance of these processes including growth-promoting factors, cell adhesion molecules (CAMs) and extracellular matrix molecules (ECMs). In addition four conserved families of guidance signals have been described: Netrins, Ephrins, Slits and Sema-phorines (Culotti and Merz, 1998, Frisen et al., 1999, Brose and Tessier-Lavigne, 2000, Nakamura et al., 2000)

Figure 3. Guidance molecules and their receptors

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The Netrin/DCC system Netrins were the first family of secreted guidance cues to be found in both invertebrate and vertebrate nervous systems (Kennedy, 2000). Netrins func-tion through two classes of transmembrane receptors: UNC-40 (Hedgecock et al., 1990, Chan et al., 1996) and UNC-5 (Leung-Hagesteijn et al., 1992) in C. elegans, and their respective mammalian homologues named Deleted in colorectal cancer (DCC), neogenin (Keino-Masu et al. (1996), (Fazeli et al., 1997) and UNC-5H-1, -2, -3 and -4 (Leonardo et al., 1997). Studies in mammals indicate bidirectional function of Netrins; DCC/UNC-40 is re-quired for both the attractive and repulsive responses, whereas UNC-5 is required only for the repulsion of some axons (Leung-Hagesteijn et al., 1992, Chan et al., 1996, Keino-Masu et al., 1996, Fazeli et al., 1997).

The most studied member of the netrins is netrin-1. During the develop-ment, it is found at the floor plate and the neuroephithelial cells in the ventral spinal cord as well as at the midline and the ventral ventricular zone in the brain. At the midline, secreted netrin-1 attracts distinct neuronal populations (Kennedy et al., 1994, Keino-Masu et al., 1996, Serafini et al., 1996) as well as repels those axons that should not cross the midline (Leonardo et al., 1997). In the spinal cord netrin-1, attracts and stimulates the outgrowth of dorsal commissural axons. These are axons destined to cross the midline at the floor plate in order to connect to neurons on the contralateral side. In netrin-1 mutant mice the majority of commissural axons extend ventrally but do not reach and cross the midline. DCC mutant mice show similar defects in axon projection, suggesting that DCC is the receptor that mediates netrin-1 signaling on commissural neurons in the developing spinal cord. Several intracellular responses of netrin-1-mediated signaling have been identified. Netrin-1 interaction with its receptors DCC or UNC-5 activates downstream signaling molecules, thereby reorganizing the actin cytoskeleton to generate distinct guidance responses (Figure 3). Netrin-1 binding to DCC initiates attraction by clustering of multiple DCC molecules through their intracellu-lar P3 domains (Serafini et al., 1996). Interaction between the DCC P1 do-main and UNC-5 DB domain is leading to long-range repulsion while bind-ing of netrin-1 to UNC-5 alone leads to short-range repulsion (Colamarino and Tessier-Lavigne, 1995, Hong et al., 1999).

The Slit/Robo system One of the most essential ligand-receptor pairs among the axon guidance molecules is constituted by the Roundabout receptors (Robo) and their Slit ligands.

Robos were identified in Drosophila in a mutant screen for genes that control the midline crossing of commissural axons (Seeger et al., 1993, Kidd et al., 1998) while Slit was discovered in Drosophila as a protein secreted by midline glia (Rothberg et al., 1988, Rothberg et al., 1990). There are homo-

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logues of both proteins in many species and the three mammalian Slit genes (Slit1–Slit3), secreted by the floor plate, are acting through four Robo genes (Robo1-Robo4) expressed by growing CNS neurons (Rothberg et al., 1988, Battye et al., 1999, Brose et al., 1999, Kidd et al., 1999, Li et al., 1999, Long et al., 2004, Sabatier et al., 2004). The mammalian Robo3 has only been detected in the nervous system and its function differs from the roles of the other mammalian Robos (Jen et al., 2004, Marillat et al., 2004, Sabatier et al., 2004, Barber et al., 2009). In the developing spinal cord and hindbrain, Robo3 is expressed at high levels on commissural axons until they have crossed the floor plate (Marillat et al., 2004, Sabatier et al., 2004, Tamada et al., 2008). These findings have led to a model in which, before midline crossing, the presence of Robo3 in commissural axons interferes with Slit/Robo repulsion. This allows commissural axons and neurons to progress towards the ventral midline under the action of chemoattractants, such as netrin 1. After crossing, Robo3 expression is downregulated, allowing the activation of Robo1 and Robo2 by Slit, which triggers axon repulsion (Fig. 4).

All Robo receptors can be alternatively spliced to generate various isoforms. In the case of Robo3, the two splice variants, Robo3.1 and Ro-bo3.2, have opposite effects on commissural axons (Chen et al., 2008). Ro-bo3.1 is expressed before crossing and seems to suppress premature respon-siveness to Slits, whereas Robo3.2 is expressed after midline crossing and appears to cooperate with Robo1 and Robo2 to repel commissural axons away from the floor plate thus preventing recrossing. In mice, mutations in all three Slit genes lead to axons aberrantly crossing or stalling at the midline (Rothberg et al., 1990, Kidd et al., 1999, Long et al., 2004). Effects on motor and sensory motor behaviors have been reported in Robo3 deficient humans, zebrafish and mice (Jen et al., 2004, Amoiridis et al., 2006, Burgess et al., 2009, Renier et al., 2010). Mutations in human Robo3 were discovered in patients with a rare autosomal recessive disease named horizontal gaze palsy with progressive scoliosis (HGPPS). Characteristic for HGPPS patients is that both the descending corticospinal tract motor projections and the as-cending lemniscal sensory projections are abnormally uncrossed (Haller et al., 2008, Jen, 2008). In addition, patients are unable to perform conjugate lateral eye movements (Jen, 2008).

Robo3 knockout mice have a complete loss of ventral commissure in spi-nal cord and hindbrain as most commissural axons fail to cross the midline resulting in ipsilateral rather than contralateral projections giving rise to mo-tor and sensory deficit (Marillat et al., 2004, Sabatier et al., 2004, Tamada et al., 2008). However behavioral studies on Robo3 null mice are precluded as they die shortly after birth. However, recently, a unique genetic tool de-signed to probe the function of selected hindbrain commissures was generat-ed by crossing a novel Robo3 conditional knockout line with transgenic mice expressing Cre recombinase in specific subsets of hindbrain commissural

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neurons (Renier et al., 2010). There is increasing evidence that the signaling pathways of Slit/Robo and netrin 1/Dcc are linked. In the presence of Slit, Robo1 binds to DCC to silence netrin 1 attraction (Stein and Tessier-Lavigne, 2001). Whether DCC can interact directly with the other Robo receptors to modulate attraction or repulsion is currently unknown though.

Figure 4. Roundabout (Robo) receptors mediate midline repulsion in response to Slit in ipsilateral, precrossing and postcrossing contralateral axons. Schematics showing commissural axons before, during, and after crossing the CNS midline. DCC-Net attracts the axons to the ventral midline. In precrossing axons the sensitivity to Slit is antagonized by the Robo3.1 isoform. After crossing, Robo3.2 prevents recrossing.

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Downstream signaling Neuronal development through cell activation, repulsion, growth cone col-lapse or modulation of the cytoskeleton is possible due to activation of dif-ferent signaling pathways. Netrins, slits, ephrins and semas, have all been implicated to activate the Rho-family GTPases (Li et al., 2002b, Hu et al., 2005). A number of intracellular adapters and mediators transmit DCC sig-naling upon netrin-1 binding including the cAMP-protein kinase A pathway (Bouchard et al., 2004), GTPases Rho pathway, activated by GEFs (Guanine nucleotide exchange factors)(Li et al., 2002a, Luo, 2002), MAP-Kinases pathway (Campbell and Holt, 2001), MAP1B: Microtubule Associated Pro-tein 1B (Del Rio et al., 2004) and cGMP-Protein Kinase G pathway (Song and Poo, 1999). Slits, acting through Robo receptors, lead to decreased lev-els of active Cdc42 and increased RhoA and Rac activity (Wong et al., 2001, Fan et al., 2003). Ephrins, through Eph receptors, result in increased RhoA activity as well, but they also cause transient, decreased Rac activity in RGCs (Wahl et al., 2000, Jurney et al., 2002). Sema treatment via plexin-B1 activates RhoA (Swiercz et al., 2002) and sequesters active Rac (Vikis et al., 2000, Hu, 2001).

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Aims

The overall aim of my doctorial work was to investigate organization, for-mation and function of spinal cord neuronal networks. Transgenic mice with abnormal left-right coordination have been a fundamental research tool and have enabled studies of specific neuronal population functions in the loco-motor CPG. Specific aims of this work are presented below:

Paper I and II: To identify subpopulations of neurons involved in left-right coordination and study the role of axon guidance molecules netrin-1 and Dcc in the development of a functional locomotor CPG Paper III: To dissect the role of Robo3 expressing subsets of spinal cord commissural neurons in CPG coordination using a Robo3lox/lox allele. Paper IV: To explore the Dmrt3 influence on the development of a coordi-nated locomotor network in the spinal cord Paper V: To identify the mechanism responsible for the aberrant locomotion phenotype in the functionally validated mouse mutant hop

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Methodological considerations

Animals All experiments involving animals have been approved by the appropriate local Swedish ethical committee (C147/7, C79/9 or C248/11).

Ntn1 (Ntn1Gt(pGT1.8TM)629Wcs ) and Dcc (DCCtm1Wbg ) mice were obtained from Marc Tessier-Lavigne. Mice were maintained on C57BL/6J and 129x1/SvJ backgrounds, respectively (Serafini et al., Fazeli et al.). Dcckanga were imported from The Jackson Laboratory and maintained in a C.AKR background.

Dmrt3 null mice were generated by deleting a sequence covering exon 1 of Dmrt3. All animals used for experiments were from Dmrt3 heterozygous breedings.

CByJ.Cg-hop/J mice were imported from Jackson Laboratory (N12 on a backcross-intercross to BALB/cByJ). The colony was kept on the BALB/cByJ background for analysis of the locomotor phenotype and out-crossed to the C57BL/6J background for positional cloning and morphology studies.

For free floating in-situ hybridization, C57/Bl6 mice were anesthetized with an intraperitoneal injection of a 50/50 (volume) mixture of Dormitor (70 µg/g bodyweight, Orion) and Ketalar (7 µg/g bodyweight, Pfitzer). Mice were perfused by intracardial infusion of phosphate buffered saline (PBS) followed by 4% formaldehyde in PBS. Spinal cords were dissected and im-mersed in 4% formaldehyde in PBS over night. Lumbar spinal cord tissue was embedded in 4% agarose and 60 µm sections were cut using a vi-bratome (Leica, Germany). Sections were dehydrated in graded series of methanol washes before storage in 100% methanol in –20°C.

For developmental studies animals were mated overnight and females checked for vaginal plug the next morning. In the morning of plug, embryos were staged as E0.5. Embryos were collected at different developmental stages from E10.5 to E18.5. For dissection of embryos, pregnant females were killed by cervical dislocation and embryos were removed. Embryos were used immediately for tracings or fixed in 4% formaldehyde for 2 h and then transferred to 30% sucrose in PBS at 4°C over night. Embryos were

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embedded in OCT and 12 µm sections were cut using a cryostat (CM1800, Leica, Germany), collected onto Superfrost slides (Menzel-Gläser, Germa-ny) and stored at -80°C until usage.

Retrograde tracing of commissural interneurons Spinal cords from adult and neonatal mice at P0–P3 were prepared according to following protocol. Mice were anesthetized with Isoflurane (Baxter, Swe-den) and decapitated. During dissection mice were kept in ice-cold oxygen-ated (95% O2/5% CO2) artificial cerebrospinal fluid (aCSF mM: 128 NaCl, 4,69 KCl, 2.5 CaCl2, 1.25 MgSO4, 1.18 KH2PO4, 25 NaHCO3, 22 glucose). Mice were pinned down in a dissection tray and eviscerated. The vertebral column was cleaned of adherent tissue and opened ventrally. After the spinal roots were cut, the spinal cord was removed and pinned down. Fluorescent dextran-amines 3,000 Da rhodamine-dextran-amine (RDA) and 3,000 Da fluorescein-dextran-amine (FDA) (Invitrogen, Sweden) were used for retro-grade tracing of commissural interneurons (CINs) as described previously (Glover, 1995). The tracers were dissolved in a small drop of distilled water to allow collection of minute quantities of tracer on the tip of a fine tungsten needle. Two different application protocols were used for labeling of intra-segmental and intersegmental projection neurons. Intrasegmental tracing with FDA was used to show local projecting commissural interneurons. A sagittal cut was made parallel to the ventral commissure along the entire L2 segment and FDA tracer was immediately applied. Intersegmental tracing with FDA and RDA was used to locate ascending (aCINs), descending (dCINs) and bifurcating commissural interneurons (adCINs). Therefore tho-racic root 13 (T13) and lumbar root 3 (L3) were identified. With a 20-minute interval, a small cut was made across the ventral and ventrolateral funiculi immediately caudal to each of these roots. A different tracer was applied into each cut. For both application protocols excess dye that diffused away from the cut was removed by using a small pipette to avoid contamination of adja-cent areas. Preparations were then incubated in a dark box in oxygenated aCSF pH 7.4 at room temperature for 12–16 hours. Samples were fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS), pH 7.4 and stored dark at 4°C for 1 week. Spinal cords were washed in PBS and embedded in 4% Agarose in 1xTAE. 60 µm thick transverse sections were cut on a vibratome (Leica), collected on glass slides, and mounted with 2.5% DABCO (Sigma, Sweden) in glycerol containing 0.1M Tris pH 8.6. Slides were stored in the dark at –20°C.

Tracings on E12.5 embryos were performed essentially the same way, but the spinal cord remained within the vertebral column during tracer applica-tion and incubation (Nissen et al., 2005). Preparations were incubated for 12–16 h and then fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate-

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buffered saline (PBS), pH 7.4 and stored in dark at 4°C for 1 week. Samples for in situ hybridization and immunohistochemistry studies were fixed for 2 hours and then transferred to 30% sucrose in PBS at 4°C over night. The samples were embedded in OCT and 12 µm sections were cut using a cryo-stat (CM1800, Leica, Germany), collected onto Superfrost slides (Menzel-Gläser, Germany) and stored in the dark at -80°C until usage for additional staining.

All Sections were viewed in an Olympus BX61WI microscope (Olympus) with separate filters to view RDA and FDA. The application sites were iden-tified and photographs with a 4x objective were taken of all sections of the L2 level (intrasegmental tracing) or between the two application sites (in-tersegmental tracing). Higher magnification photographs were taken with a 10x objective using the OptiGrid Grid Scan Confocal Unit (Qioptiq, Roches-ter USA) and Volocity software (Improvision, Lexington USA).

In situ hybridization Linearization of cDNA clones was performed using appropriate restriction enzymes (Fermentas, Germany). Probes were in vitro transcribed using Sp6, T3 or T7 RNA polymerase in the presence of digoxigenin (DIG)- or fluores-cein (FITC) -UTP (Roche, Sweden) followed by removal of DNA template with DnaseI (Fermentas, Germany). Probes were quality controlled on aga-rose gels, quantified using the Nanodrop® ND-1000 Spectrophotometer (NanoDrop Technologies, Delaware, USA) and subsequently aliquoted be-fore storage in -80 °C.

For in situ hybridization on freefloating spinal cord, sections were rehy-drated in consecutive washes for 10 min in 75%, 50% and 25% methanol in PBT, bleached in 6% hydrogen peroxide in PBT for 15 min and treated with 0.5% Triton X-100 for 5 min. The sections were then digested with protein-ase K (10 µg/ml) in PBT for 15 min. The digestion was stopped with a wash in glycine (Scharlau Chimie, Spain; 2mg/ml) in PBT for 5 min, and sections were then postfixed in 4% formaldehyde for 20 min. The sections were pre-hybridized at 65°C in hybridization buffer (50% formamide, 5xSSC pH 4.5, 1% SDS, 50 µg/ml tRNA (Sigma) and 50 µg/ml heparin (Sigma)) for two hours prior to addition of probe. 1 µg/ml probe was added to the hybridiza-tion buffer and sections were hybridized over night at 65°C. Excess probe was removed by washes in wash buffers (50% formamide, 5xSSC pH 4.5 and 1% SDS; 50% formamide, 2xSSC pH 4.5 and 0.1% tween-20) at 65°C for 3 times 30 min each. The sections were then transferred to blocking solu-tion (1% blocking reagent in TBST) for 2 h before addition of anti-DIG or anti-FITC AP(1:5000) diluted in blocking solution and incubated over night at 4°C. The sections were treated with levamisole (0.5 mg/ml) in TBST and levamisole (0.5mg/ml) in NTMT (100 mM NaCl, 10 mM Tris-HCl pH 9.5,

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50 mM MgCl2 and 0.1% Tween-20) before developing in BM purple AP substrate (Roche) at 37°C 1h – 4 days. Between additions of new chemicals, sections were washed with either PBT (prior to addition of probe) or TBST (after addition of probe).

Two-color in situ hybridization experiments used the same conditions, with the following modifications. Sections were hybridized with both FITC-labeled and DIG-labeled probe simultaneously. The FITC probe was devel-oped first with either INT/BCIP (Roche) or SIGMAFAST™ Fast Red TR/Naphthol AS-MX (Sigma). When the first signal was developed the AP was inactivated by heating to 65°C for1 h and washing with 0.1 M Glycine-HCl (pH 2.2) for 30 min. After the inactivation step the sections were blocked, incubated with anti-Digoxigenin antibody and developed with BM purple.

For in situ hybridization on cryosections, these were thawed and fixed for 10 minutes in 4% formaldehyde, washed in PBS and treated with proteinase K (Sigma, 1 µg/ml diluted in 50mM Tris-HCl / 5mM EDTA pH7.5) for 5 minutes. Following refixation and washes in PBS, the slides were acetylated for 10 minutes in a mixture of 1.3% triethanolamine (Sigma), 0.2% acetic anhydride (Fluka, Germany) and 0.06% HCl in water. Slides were then washed in PBS and prehybridized for 6 hours in hybridization solution with-out probe (50% formamide (Fluka), 5 x SSC, 5 x Denhardt´s, 250 mg/ml yeast transfer RNA (Sigma), 500 mg/ml sheared salmon sperm DNA (Am-bion) and 2% blocking reagent (Roche) in water). Probes were diluted to 1 µg/ml in hybridization solution and heated to 80°C. Sections were then hy-bridized with 100 ml hybridization solution for 16 hours at 70°C. The next day, slides were dipped in prewarmed 5 x SSC, transferred to 0.2 x SSC and incubated for 2 hours at 70°C. Following one wash in 0.2 x SSC at room temperature and one wash in B1-solution (0.1 M Tris-HCl pH7.5 and 0.15 M NaCl), sections were immunoblocked with 10% fetal calf serum in B1 and then incubated over night at 4ºC with alkaline phosphatase-conjugated anti-DIG (Roche, Germany) diluted 1:5000 in B1 containing 1% fetal calf serum. The following day, slides were washed in B1, equilibrated in B3 (0.1 M Tris-HCl, pH9.5, 0.1 M NaCl, 50 mM MgCl2) and color developed in a 10% poly vinyl alcohol (Sigma) solution also containing 100 mM Tris-HCl pH 9.5, 100 mM NaCl, 5 mM MgCl2, 0.17% NBT (Roche), 0.17% BCIP (Roche) and 1 mM levamisole (Sigma-Aldrich, Germany).

The VIAAT probe covers nucleotides 588-2072. Dcc probe covering nu-cleotides 654-3077 (Cooper et al.,1995) VAChT and Vglut2 (Wallen-Mackenzie et al., 2006) probes have been described earlier.

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Immunohistochemistry For immunohistochemistry sections were incubated with, in PBS, 0.3% Tri-ton X-100 (Sigma, Sweden) at 4°C over night. The following day, slides were washed in PBS and incubated with DAPI (Sigma, Sweden) and Alexa fluorescent secondary antibodies (Molecular Probes, USA) diluted 1:200 in PBS with 0.3% Triton X-100 and 3% BSA for more than 2 hours at room temperature. Slides were mounted with 2.5% DABCO (Sigma, Sweden) in glycerol containing 0.1M Tris pH 8.6.

Antibodies on E12.5 embryo spinal cords were used in 5% goat serum, 0.3% BSA in PBS. Antibodies were used in the following dilutions; mNkx2.2 1:100 (Hybridoma bank, USA), mEvx1 1:50 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), gpLbx1 1:10000 (kind gift from C. Birchmeier MDC, Berlin, Germany), rPax2 1:1000 (Covance, UK) mBrn3a 1:500 (Chemicon, Sweden) 1:100 mIsl1 (Hybridoma bank), 1:500 mLhx1/5 (Hybridoma bank), 1:8000 Lhx2/9 (kind gift from T.Jessell), mShh 1:100 (Hybridoma bank), mMap2 1:500(Chemicon, Sweden), rbNeuN 1:1000 (Chemicon) and MBP 1:500 (AbCam), mPax7 1:100 (Developmental Studies Hybridoma Bank), rbLmx1b 1:2000 (gift from Yu-Qiang Ding), rJagged 1:20 (Developmental Studies Hybridoma Bank), rbWt1 1:500 (Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA), chGFP 1:1000 (Abcam). The anti-Dmrt3 an-tibody was custom made in guinea pig using the immunizing peptide CKQSIYTEDDYDERS-amide (Innovagen, Lund, Sweden). Quantitative analysis of the antibody staining was statistically analysed using one-way ANOVA followed by Bonferroni’s post hoc test.

Imaging and picture processing Brightfield stainings were captured on a MZ16F dissection microscope with DFC300FX camera and FireCam software (Leica, Germany). Fluorescent stainings were captured on an Olympus microscope with an Optigrid system (Thales, USA) and Volocity software (Improvision, UK).

Ventral root recordings P0-P2 mice were anesthetized with isoflurane and decapitated, eviscerated and submerged in ice-cold dissection buffer containing the following (in nM: 128 NaCL, 4.69 KCl, 25 NaHCO3, 1.18 KH2PO4, 3.5 MgSO4, 0.25 CaCl2 and 22 D-Glucose; equilibrated with 95% O2 and 5% CO2). The spinal cord was carefully dissected out of the spinal column and placed in a Sylgard-coated recording chamber. The spinal cord was then left for 30 minutes to

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recover continuously perfused with aCSF containing the following (in nM: 128 NaCL, 4.69 KCl, 25 NaHCO3, 1.18 KH2PO4, 1.25 MgSO4, 2.5 CaCl2 and 22 D-Glucose; equilibrated with 95% O2 and 5% CO2). Suction elec-trodes were attached to left and right L2 and L5 roots and locomotion was induced using 6µM NMDA and 6µM 5HT or 5µM NMDA, 10µM 5HT and 50µM Dopa. Three mice were also treated with 500µM Nipecotic acid and 500µM Sarcosine. Fictive locomotion was analyzed using the Matlab-based programs Spinalcore and Neurodata (Zhang et al., 2008). For analysis in Spinalcore the data was downsampled to 100 Hz and rectified. A Morlet wavelet transform was used to extract the phase and frequency from 400 seconds of recorded fictive locomotion. For analysis in Neurodata, 600 se-conds of fictive locomotion was low-pass filtered at 5 Hz, rectified and high-pass filtered at 0.01 Hz. The resulting trace was analyzed for burst, interburst and cycle period duration as previously described (Zhang et al., 2008). The Neurodata analysis was also used to create the circular plots.

Trans-synaptic tracing of interneurons Attenuated pseudorabies virus strain Bartha (PRV Bartha) expressing en-hanced green fluorescent protein (PRV152) was generously provided by Dr. Lynn Enquist (Princeton University, Princeton, NJ). PRV152 has been wide-ly used for transneuronal tracing (Card et al., 1999, Jovanovic et al., 2010) as the cell body, nucleus and processes of the infected neurons are filled with EGFP. The virus was grown in pig kidney cells and the titers were set to a final concentration of 4.86x108 plaque forming units (pfu/µl). The experi-ments were performed on P2 mice. All procedures were carried out accord-ing to Bio Safety level 2 conditions in accordance with the Work Environ-ment Authority in Sweden. 24 h prior to the viral injection the mice were injected (IP, 50 µg/g in 0.9% physiological saline) with a retrograde label Fluorogold (FG; Fluorochrome, Denver, CO) to produce global labeling of somatic motor neurons. The animals were anesthetized on ice and 3-4 µl injections of PRV152 were made in multiple sites of the tibialis anterior (TA) or gastrocnemius (GC) muscle using a 10µl Hamilton syringe with a 34 gauge needle (NanoFil, World Precision Instruments). 38-40 h post-injection the mice were decapitated, the spinal cords dissected and immersed in 4% paraformaldehyde overnight. The following day the tissue was cryo-protected in 30% sucrose, cast frozen in O.C.T (Tissue Tek, Histolab, Gothenburg, Sweden) sectioned at 14 µm on a cryostat and processed using immunohistochemistry as described above.

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Results and conclusions

Paper I and II In paper I and II, the role of netrin-1 and Dcc in the development of a func-tional locomotor CPG was studied. Our studies of artificial locomotor behavior of netrin-1 (Ntn1Gt/Gt) and Dcc (Dcc-/-) mutant mice show that Ntn1Gt/Gt mice display synchrony in left-right coordination while Dcc-/- mice exhibit a variety of locomotor output includ-ing left-right alternation, synchrony and uncoordinated periods. With retro-grade tracing of CINs, we could observe a severe reduction of projections over the midline in both Ntn1Gt/Gt and Dcc-/- although a small number of CINs (around 20%) can still be found in both Dcc-/- and Ntn1Gt/Gt mice at P0. Based on the neurotransmitter profiles of the remaining CINs we show that in Ntn1Gt/Gt mice, more excitatory CINs remain, which results in a relative shift towards more excitatory signals being transmitted over the midline. In contrast, in Dcc-/- mice both excitatory and inhibitory CINs were similarly affected. However, the severe reduction of CINs in combination with the resulting shift in the neurotransmitter balance over the midline could explain the observed locomotor pattern and the difference between the phenotype in netrin-1 and Dcc mutant mice. Considering that not all commissural neurons seem to depend on Netrin-DCC signaling we investigated the developmental origin of the remaining CINs in DCC-/- and Ntn1-/- mice by using embryonic tracings in combination with antibody detection of neuronal subpopulations. In absence of netrin-1 we found a severe reduction of dI1– dI3, dI5, dI6 and V0d neurons, a minor reduction of V0v and no loss of the most ventral V3 CINs (Paper I). In contrast, the loss of DCC affected the CINs from all la-beled developmental subpopulations to the same severe extent (Paper II).

These results show that CINs originating from different dorsal and ventral progenitor domains are dependent on DCC signaling to properly cross the midline. In Paper II we propose that V3 derived CINs, could be an important component of the left-right synchrony circuit based on the fact that the re-maining V3 subpopulation in Ntn1Gt/Gt mice is sufficient to maintain a coor-dinated strictly synchronous left-right activity.

In summary our data shows a crucial role of DCC for proper formation of spinal neuronal circuitry underlying left-right coordination, and that both Netrin-1 and DCC are required for normal development and function of the locomotor CPG.

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Paper III Here we studied the significance of neurons originating from dorsal or ven-tral subpopulations in the formation and function of the left-right circuitry. To inhibit midline axon crossing from selected subpopulations of CINs we used a Robo3lox/lox allele crossed to transgenic lines expressing Cre recom-binase in specific/restricted subsets of spinal cord CINs.

By crossing the Robo3lox/lox mice with a cre line under the phosphoglycer-ate kinase (PGK) promotor, we prevented CINs originating from all progeni-tor domains to cross the midline as PGK is expressed in all somatic cells. Thereby we could mimic a full Robo3 knockout. The spinal cords from Ro-bo3:PGKcre mice showed an uncoordinated fictive locomotion with contin-uous shifting between alternation and synchronous rhythms between the left and right roots of the same lumbar level. The ipsilateral flexion/extension coordination remained alternating.

Three subpopulations of CINs are believed to originate in the dorsal spi-nal cord, more precisely from the dI2, dI5 and dI6 progenitor domains. The transcription factor Pax 7 is expressed in dI2-dI6 dorsal progenitor domains. Using Pax7cre we could prevent dI2-dI6 originating CINs from crossing the midline, allowing us to study the effect of remaining ventral CIN on the lo-comotor CGP. The opposing situation was enabled through Nkx6.2cre (NK6 homeobox 2) as it is expressed in p1-p3ventral derived populations of neu-rons. This permitted us to study the remaining dorsal and the ventrally de-rived V0 CIN. Both Robo3:Pax7cre and Robo3:Nkx6.2cre mice showed a strictly synchronous left-right fictive locomotion. While the ipsilateral coor-dination remained alternating in Robo3:Nkx6.2cre mice, the flexion-extension pattern in Robo3:Pax7cre mice was affected. Thus, neurons coor-dinating flexion-extension may have a dorsal origin. The burst duration time was significantly longer for the Robo3:Pax7cre compared to the control while the interburst duration time increased for both Robo3:PGKcre and Pax7:cre. Further, the synchronous left-right coordination in Robo3:Nkx6.2 and Robo3:Pax7cre could not be pushed to alternation by applying GABA and Glycine blockers to the bath although a more robust flexion-extansion was observed in Robo3:Pax7cre.

Using retrograde intrasegmental tracings, we investigated of locally short and long projecting CINs. Both kinds of CINs were completely absent in Robo3:PGKcre and severely reduced in both Robo3:Pax7 and Ro-bo3:Nkx6.2 mice compared to controls. The remaining CINs in Robo3:Pax7 (42%) and Robo3:Nkx6.2 (32%) were consistently distributed in the ven-tromedial area of the spinal cord.

By removing Robo3, the mice had a complete loss of commissural axons in the spinal cord resulting in uncoordinated fictional locomotor activity. Removing Robo3 in Pax7 or Nkx6.2 neuronal populations led to a shift from alternation to strict synchronous locomotor activity. This indicates that CINs

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responsible for left-right alternation originate from both dorsal and ventral domains. Furthermore, neurons originating from dorsal progenitors could be involved in flexion-extension coordination, emphasizing the importance of investigating these populations in relation to locomotor network coordina-tion. Paper IV Inhibitory dI6 neurons, which settle in the ventral lamina VII and VIII (Kiehn, 2006, Brownstone and Wilson, 2008) have in previous studies been suggested as promising candidate neurons in coordinating bilateral alterna-tion circuitry (Kjaerulff and Kiehn, 1996). In this paper we show that Dmrt3, expressed in inhibitory dI6 CINs, is important for the normal development of coordinated locomotor movements in both horses and mice. Dmrt3 was found in a screen for genes expressed predominantly in cholinergic cells located in the ventral spinal cord (Enjin et al., 2010). In order to characterize the origin of the Dmrt3 cells, we used markers for dorsal and ventral progen-itors which give rise to subclasses of interneurons. We found that Dmrt3 immunopositive cells are generated near the ventral extent of the Pax7+ do-main, and overlap with the Pax2 domain marking dI4, dI5 and V0d progeni-tors. However the Dmrt3 expression did not coincide with the dI5 markers Lmx1b or Brn3a. Labeling with the dI4-6 postmitotic marker Lbx1 indicated that the Dmrt3+ population arise from the most ventral Lbx1 domain. Fur-thermore, a partial overlap was observed between Dmrt3 and WT1, a marker previously suggested to label the dI6 population (Goulding, 2009). With retrograde tracing in spinal cords it was possible to show that Dmrt3+ inter-neurons extend projections both ipsi- and contralaterally. Trans-synaptic pseudorabies virus based tracing revealed direct connections to motor neu-rons both ipsi- and contralateral to the injected muscle. Finally the Dmrt3+ dI6 interneurons were co-labeled with VIAAT demonstrating that they are inhibitory. In Dmrt3-/- mice the dI6 population, with the flanking dI5 and V0d populations, remained of normal sizes. In contrast, the number of WT1+ neurons increased while the number of commissural interneurons decreased. Taken together these results suggest that Dmrt3 is expressed in a population of inhibitory dI6 interneurons projecting ipsi- and contralaterally with direct connections to motor neurons.

To explore the functional effect of Dmrt3 in coordinated locomotor net-works, we used Dmrt3-/- mice. The CPG output of the lumbar spinal cord controlling the hind limbs was studied with drug-induced fictive locomotion. A stable rhythm was observed in the isolated spinal cords from both wild type and heterozygous mice, while the spinal cords from Dmrt3-/- mice dis-played an uncoordinated and irregular firing rhythm in both left-right and flexion-extension coordination, as well as increased burst and interburst du-

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ration. Limb coordination in neonatal mice was tested in an air-stepping setup, showed increased uncoordinated step movements and absent alternat-ing hind limbs movement in Dmrt3-/- mice. Motor coordination in adult mice was tested by having the mice walking and running on a treadmill. Of the five speeds tested, Dmrt3-/- mice, but not wild type mice, failed at the two highest speeds. Also, at the highest speed analyzed (20 cm/s), a significant increase in stance and swing times was observed in Dmrt3-/- mice, indicating that Dmrt3-/- have a longer stride.

In a parallel study Leif Anderssons group identified a region associated to the ability to pace in Icelandic horses. There are three naturally occurring gaits; walk, trot and canter/gallop. However, some horses have alternate gaits; pace, regular rhythm ambling, lateral ambling and diagonal ambling. Toelt is a regular ambling gait characteristic for the Icelandic horse. Se-quencing of the Dmrt3 gene in a pace and a non-pace Icelandic horse identi-fied a nonsense mutation in the Dmrt3 gene. The mutation, a single nucleo-tide substitution, leads to a premature stop codon and expression of a trun-cated version of Dmrt3 affecting the pattern of locomotion in horses. Do-mestic horses with the ability to perform alternate gaits are all homozygous for the Dmrt3 mutation while non-gaited horses are homozygous for the wild type allele. The Dmrt3 mutation has accumulated in harness race horses in which the transition from trot to gallop leads to disqualification from the race.

In conclusion, Dmrt3 is critical for the development of a coordinated lo-comotor network in mice, showing that spinal circuits controlling stride and is of great importance for gait control in horse locomotion. These findings have increased our knowledge about the flexibility in neuronal circuit devel-opment and further confirm the role of dI6 neurons in locomotor circuits. Paper V In this paper we set out to functionally validate a mouse model called hop. Hop-sterile (hop) mutation arose in a stock in 1967 in the C57BL/10J strain at The Jackson Laboratory. The homozygous mutants have several distinct phenotypes such as preaxial polydactyly of all feet, sperm tail deficiency resulting in male sterility, dilated lateral ventricles, hydrocephalus and the characteristic hopping gate using the hind limbs simultaneously.

Our analysis of spinal cord morphology revealed a phenotype with de-fects in the ventral spinal cord, including an absent midline, a poorly defined white/grey matter border, misplaced central canal and missing artery of Adamkiewicz. Further histological studies showed that these defects are exclusively found in the lumbar part, and not in thoracic, cervical or sacral levels of the spinal cord. Gait analysis revealed that during locomotion the hop/hop mice alternate their forelimbs while they move their hind limbs

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synchronously. Ventral root recordings of neonatal mice confirmed the syn-chronous gait observed in adult mice.

Studies on E12.5 spinal cord revealed a missing floor plate and Nkx2.2 progenitor domain together with migration defect of neurons from all ana-lyzed progenitor domains in hop/hop mice. Additionally we observed re-duced amounts of the axon guidance molecules Shh and netrin-1as well as ventrally absent ephrinB3. Taken together, these results indicate that the prominent phenotype seen in hop/hop mice is a result of abnormal develop-mental processes including induction from the notochord and Shh signaling.

In the early 20th century Thomas Graham-Brown proposed that two sys-tems of neurons, termed half-centers are underlying coordinated movements. Our data indicate that in hop mutant mice the two CPG half-centers are fused to form one CPG center, with a synchronous gait as a functional conse-quence.

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Future prospects

We propose in paper I that CINs from ventral V3 domain are a part of left-right synchrony network and in paper II an alternative mechanism underly-ing midline guidance for ventral originating CINs, were DCC might act in-dependent of netrin-1. Studies in C.elegans have reported netrin-1-independent function of DCC/UNC-40 in cell and axon migration, however no example of netrin-1-independent DCC signaling in vertebrates has been found (Hedgecock et al., 1990, Yu et al., 2002). Therefore, in order to evalu-ate V3 interneuron axon responses in vitro one could use a reporter line such as TomatoAi14 together with a Cre line with a V3 specific promoter. This experimental approach would enable us to visualize V3 neurons and the axonal growth cone in tissue culture using fluorescence microscopy. Axon outgrowth on E11.5 mouse spinal cord explants could be cultured for out-growth assays, either alone or with a mouse floor plate and notochord, or COS cell aggregates transfected with control, netrin-1, Shh, or other candi-date-protein expression plasmids. By this approach, the axonal response of developing V3 neurons to a set of different tissues and guidance cues within these different environments could be assessed. In paper III we have used a genetic tool to effectively prevent contralateral projections from several populations of neurons. To further narrow down and possibly pinpoint the neuronal populations involved in left-right coordi-nation we could use selectively expressing Cre recombinase, e.g. Sim1cre, Nkx2.2cre, WT1cre, Dbx1cre. We could also combine the findings in paper III and IV. Generating a Dmrt3cre and thereafter combining it with the con-ditional Robo3, we would prevent the contralateral projections in the Dmrt3 population of neurons though still allowing the ipsilateral connections to form. Studying the functional output of these mice in both fictive locomotion and adult locomotion would further increase our knowledge about the left-right coordination.

An interesting follow up study to the characterization of Dmrt3 positive neurons would be to combine the Dmrt3cre with a fluorescent reporter line thus allowing us to follow the Dmrt3 cells from when they are born to their location in the adult spinal cord. Moreover we could analyze these mice together with the markers for different neuronal populations, to reinforce our findings from paper IV stating that Dmrt3 neurons originate from the dI6 population. To broaden the functional output studies in paper IV we could

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combine the Dmrt3cre with Vesicular inhibitory amino acid transporter (VI-AATlox/lox) thereby taking away the inhibitory signaling from these neurons. Another interesting supplement would be to make a Dmrt3 conditional knockout. The experimental possibilities with this tool are numerous. Acute silencing in adult mice can be done by incorporating the allatostatin receptor that upon delivery of allatostatin, silences neurotransmission. An alternative is to use an inducible version of the Cre protein that is modified to translo-cate to the nucleus upon addition of estrogen analogs, thus allowing control of onset. An additional possibility is Cre recombinase–dependent opsin-expressing viruses allowing optogenetic (light) on/off control.

With transsynaptic pseudovirus tracing, we have concluded that the Dmrt3 neurons make synaptic connections to motor neurons. Monosynaptic rabies virus tracing (Stepien et al., 2010) that has higher efficiency, together with Dmrt3cre driving the Tomato reporter expression, could provide a more detailed study of the spatial distribution and connectivity of Dmrt3 neurons. This analysis would also visualize the segmental distribution as well as syn-aptic contacts to spinal motor neurons. The hop gene is believed to be functionally important in neuronal circuits because mutations in this gene give rise to the characteristic rabbit like gait also displayed by other mouse mutants with defects in the central pattern generator output. In paper V, we have focused identifying the mutation/gene responsible for the synchronous hopping gait. Analysis of two-paired SOLiD sequencing data has not shown any major changes in the hop genome. Using positional cloning technique with known SNP’s we have narrowed down the area where the hop gene is located, from 50Mbp to 7Mbp on chromosome 6. We have considered several candidate genes located in the interesting area. One of them is an intraflagellar transport (IFT) protein named Ttc26. Studies conducted on other IFT mutants have shown similar phenotypic defects as we have found in the hop mutant with ventral spinal cord patterning defects, loss of ventral cell types and polydactyly (Huangfu et al., 2003, Liu et al., 2005, Haycraft et al., 2007). Mutations that inactivate the process of in-traflagellar transport block all responses to Sonic and Indian hedgehog (Hh) in the mouse embryo (Scholey, 2003). This is in accordance with our find-ings that the defects seen in the hop mutant originate from deficient Shh signaling. Therefore a possible approach forward is extending the embryo studies to early stages of development including notochord formation and floor plate induction as well as RT-PCR studies to examine the levels of Ttc26 in the hop mutant.

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Acknowledgements

At the end of this journey I am grateful to all the people who have supported and encouraged me on the way. First, I would like to thank my supervisor Klas Kullander for giving me the opportunity to work in his group. Thank you for your support, for challeng-ing me by giving me a lot of freedom and for your office door always being open, even for the simplest matters. I would also like to thank my second supervisor, Hanna Wootz, for opening the world of electrophysiology to me. Your magic ephys-fingers have saved me many times. I am also grateful for all discussions and help with writing this thesis. A big thank you to Nadine, for not giving up on me when I was your stu-dent, for teaching me the research foundation on which I stand today, for motivation, guidance and friendship. I also wish to thank present and past colleagues in Kullander, MacKenzie, Leao and Lagerström groups and everybody at the Department of Neuro-science for creating a stimulating work environment. I would also like to thank my former and present office colleagues. A special thanks to: Christiane, the most thorough researcher I know, for always answering my questions, for your friendship and the outdoor trips you took me on. Kia, for your interest and advice concerning research and life. Kasia, the hardest working person I know, thank you for always helping me when I needed it the most. Åsa M, for being an inspiration. Emma, for your bubbling nature and for changing my life one year ago. Hermany, for your genuine warmth and big heart. Chetan, for always offering a helping hand. Martin, for good collaborations and sharing hours of teaching. Malin, for your support. Hanna P, for talks about life and dogs. My students, Jo-han B, Kateryna and Anders Eriksson for a job well done. Bejan, Sharn, Martina, Emil for pleasant company in the lunchroom. Anders E, Karin, Carolina, Casey, Nicole, Greta this place hasn’t been the same since you left. Smitha for being such a sweet person and for cooking best Indian food.

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Thanks to the former and present heads of the department, Håkan Aldsko-gius and Ted Ebendal. Ulla, Emma, Lena, Cecilia, Maria for doing the administration work. Birgitta, for taking care of us. Susanne, Sussie, Eva, Jonna, Jenny for taking good care of the animals. I am also grateful for collaborations with prominent researchers who have contributed with their knowledge, mice and equipment, specially Patrick Whelan from University of Calgary, Canada, Alain Chédotal from Univer-site Pierre et Marie Curie, Paris, France and Leif Andersson from Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsa-la and their respective group members.

I am very thankful for my family and friends, this thesis would not have been possible without you. Lina, my rock to lean on, and Wilma, the sweetest girl I know, thank you for making my everyday life full of joy. Anders N for enriching my Satur-day mornings. Kerstin, Janne and Agneta for opening your arms and homes to me. Sanja, my dearest friend, thank you for all the emotional support, for always listening and encouraging me onwards. My sister Selma, my cousins Sanja and Jasmina, thank you for always being by my side, for your unconditional love and all the laughters we con-tinuously have. Thank you Huso, Dina, Emil for your support. The biggest appreciation I owe to my parents. They bore me, raised me, sup-ported me, taught me, and loved me. Mama i tata, riječi nemogu opisati koliko sam vam dužna, hvala za sve što činite za mene.

35

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