Abstract
Rhythmic and patterned locomotion is driven by spinal cord neurons that form neuronal circuits, referred to as central pattern generators (CPGs). Recently, dI6 neurons were suggested to participate in the control of locomotion. The dI6 neurons can be subdivided into three populations, one of which expresses the Wilms tumor suppressor gene Wt1. However, the role that Wt1 exerts on these cells is not understood. Here, we aimed to identify behavioral changes and cellular alterations in the spinal cord associated with Wt1 deletion. Locomotion analyses of mice with neuron-specific Wt1 deletion revealed that these mice ran slower than controls with a decreased stride frequency and an increased stride length. These mice showed changes in their fore-hindlimb coordination, which were accompanied by a loss of contralateral projections in the spinal cord. Neonates with Wt1 deletion displayed an increase in uncoordinated hindlimb movements and their motor neuron output was arrhythmic with a decreased frequency. The population size of dI6, V0 and V2a neurons in the developing spinal cord of conditional Wt1 mutants was significantly altered. These results show that the development of particular dI6 neurons depends on Wt1 expression and loss of Wt1 is associated with alterations in locomotion.
Introduction
In vertebrates, rhythmic activity is generated by a network of neurons, commonly referred to as central pattern generators (CPGs) (Grillner and Zangger 1979; Grillner 1985). CPGs do not require sensory input to produce rhythmical output; however, the latter is crucial for the refinement of CPG activity in response to external cues (Shik and Orlovsky 1976; Rossignol S 1988; Pearson 2003). The locomotor CPGs are located in the spinal cord and consist of distributed networks of interneurons and motor neurons (MN), which generate an organized motor rhythm during repetitive locomotor tasks like walking and swimming (Grillner 1985; McCrea and Rybak 2008).
The spinal cord develops from the caudal region of the neural tube. The interaction of secreted molecules including sonic hedgehog (Shh) and bone morphogenetic proteins (BMPs) provides instructive positional signals to the 12 progenitor cell domains that reside in the neuroepithelium (Alaynick et al., 2011). Each domain is characterized by the expression of specific transcription factor encoding genes that are used to selectively identify these populations. The dI1-dI5 interneurons are derived from dorsal progenitors and primarily contribute to sensory spinal pathways. The dI6, V0-V3 interneurons and MN arise from intermediate or ventral progenitors and are involved in the locomotor circuitry (Goulding 2009).
Whereas the involvement of V0 - V3 neurons in locomotion has been well documented, the role for dI6 neurons in locomotion has only recently been investigated (Andersson et al. 2012; Dyck et al. 2012). In particular, a part of the dI6 population shows rhythmically active neurons (Dyck et al. 2012), and a more defined subpopulation of dI6 neurons expressing the transcription factor Dmrt3, is critical for normal development of coordinated locomotion (Andersson et al. 2012). Another group of dI6 neurons is suggested to express the Wilms tumor suppressor gene Wt1 but has not yet been characterized (Goulding 2009; Andersson et al. 2012).
Wt1 encodes a zinc finger transcription factor that is inactivated in a subset of Wilms tumors, a pediatric kidney cancer (Call et al. 1990; Gessler et al. 1990). Wt1 fulfills a critical role in kidney development; however, the function of Wt1 is not limited to this organ. Phenotypic anomalies of Wt1 knockout mice can be found, among others, in the gonads, heart, spleen, retina and the olfactory system (Kreidberg et al. 1993; Herzer et al. 1999; Moore et al. 1999; Wagner et al. 2002; Wagner et al. 2005). In one of the first reports on Wt1 expression, the spinal cord was described as a prominent Wt1+ tissue (Armstrong et al. 1993; Rackley et al. 1993), however, until now there are no further reports regarding the function of Wt1 in the central nervous system (CNS).
Here, we have examined the role of Wt1 in the developing spinal cord. We performed locomotor analyses of conditional Wt1 knockout mice and used molecular biological and electrophysiological approaches to elucidate the function of Wt1 expressing neurons for locomotion. Our data suggest that Wt1 expressing dI6 neurons contribute to the coordination of locomotion and that Wt1 is needed for proper dI6 neuron specification during development.
Results
Wt1 expressing cells in the spinal cord are dI6 neurons
In order to determine the spatial and temporal pattern of Wt1 expressing cells in the spinal cord, we performed immunohistochemical analyses. Wt1+ cells were detected in the medioventral mantle zone of the developing spinal cord at embryonic day (E) 12.5 (Fig. 1A). Until E15.5, embryonic spinal cords showed a constant amount of Wt1+ cells; thereafter, their number gradually decreased until they could no longer be detected in adult mice (Fig. 1B).
We next wanted to determine the birthdate of Wt1+ cells, defined as the time point when progenitor cells cease to proliferate, leave the ventricular zone and start to differentiate. Using Bromodeoxyuridine (BrdU), the proliferative cells in the ventricular zone were labelled at different embryonic stages (E9.5, E10.5 and E11.5). Immunostaining of these cells for Wt1 at E12.5 revealed that prospective Wt1 expressing cells still proliferate at E9.5 and even at E10.5 (Fig. 1C). At E11.5 Wt1 + cells no longer showed incorporation of BrdU suggesting that they had left the ventricular zone and started their migration and differentiation in the mantel zone at this time-point.
Wt1 has been proposed to label dI6 neurons (Goulding 2009), however, the only available primary data has so far only suggested its presence in a subpopulation of dI6 neurons expressing Dmrt3 (Andersson et al., 2012). In order to closer examine the nature of Wt1+ cells, we performed immunostainings of embryonic spinal cords at E12.5. Cells expressing Wt1 were positive for Pax2 and Lim1/2 labelling dI4, dI5, dI6, V0D and V1 neurons (Tanabe and Jessell 1996; Burrill et al. 1997) while being negative for the post-mitotic V0V marker Evx1 (Moran-Rivard et al. 2001) (Fig. 1D, E). Wt1 expression did not overlap with Lmx1b, a marker specific for dI5 neurons, but did coincide with Lbx1 (Gross et al. 2002) and Bhlhb5 (Skaggs et al. 2011), which commonly occur in the ventral most dI4-dI6 Lbx1+ domain giving rise to dI6 neurons. Thus, these data supports and extends on the previous observations that Wt1 is a marker for a subset of dI6 neurons.
Deletion of Wt1 affects locomotor behavior
To investigate the function of the Wt1+ neurons in the spinal cord, we made use of a Nes-Cre;Wt1fl/fl mouse line (Fig. 2A). At E12.5, no Wt1 mRNA or protein was detected in neurons from this mouse line (Fig. 2A, B). Given the location of the Wt1 + neurons within the ventral dI6 population that has been shown to be involved in regulating locomotion, we performed behavioral tests associated with locomotion to investigate potential phenotypic consequences of deleting Wt1 in spinal cord neurons. Footprints of adult mice walking on a transparent treadmill at fixed speeds (0.15, 0.25, 0.35 m/s) were recorded to analyze different gait parameters (Supplemental Fig. S1A). Nes-Cre;Wt1fl/fl mice revealed a significant reduction in stride frequency for both the fore- and hindlimbs relative to control (Wt1fl/fl) animals at all speeds measured. Heterozygous Wt1 knockout mice (Nes-Cre;Wt1fl/+) did not differ significantly from controls. Stride length, accordingly, was significantly longer in Nes-Cre;Wt1fl/fl animals compared to wild type mice and Nes-Cre;Wt1fl/+. Thus, although Nes-Cre;Wt1fl/fl mice were slightly smaller compared to controls (body mass Wt1fl/fl vs Nes-Cre;Wt1fl/fl males 33 +/− 3.9 vs 25 +/− 3.7 g; females 25 +/− 3.2 g vs 22 +/− 1.4 g; body length males 9.9 +/− 0.4 g vs 9.4 +/− 0.4 cm; females 9.9 +/− 0.4 cm vs 9.8 +/− 0.3 cm), they made longer strides with lower frequency.
To further explore gait alterations, we used X-ray fluoroscopy as a complementary method in a larger cohort of mice (Fig. 2C; Supplemental Fig. S1B; Supplemental Movie 1 and 2). When animals walked voluntarily at their preferred speed, deviations in stride frequency and stride length from the expected value (control baseline) for the given speed were again observed in Nes-Cre;Wt1fl/fl (Fig. 2D), but statistical significance is confirmed only for females. The changes were accompanied by a significant reduction of raw speed and size-corrected speed (= Froude number) in Nes-Cre;Wt1fl/fl mice of both sexes (Supplemental Fig. S1C). While both the duration of stance and swing phase and the distance covered by the trunk and the limbs, respectively, differ between controls and Nes-Cre;Wt1fl/fl by more than 10 percent in males and more than 15 percent in females, the ratio between the two phases, expressed by the Duty factor, remains unaffected (Supplemental Fig. S1D). Thus, the temporal coordination between stance and swing phase in adult Nes-Cre;Wt1fl/fl mice is normal.
We tested whether changes in gait parameters are accompanied by changes in the phase relationships between the limbs (Fig. 2E, F). The footfall pattern of control and Nes-Cre;Wt1fl/fl females did not show significant differences at the same speed of 0.21 m/s (Supplemental Fig 1E). However, the different spread along the X-axis indicates the evenly elongated stance and swing phases.
The symmetry of left and right limb movements expressed as the time-lag between footfalls in percent stride duration of a reference limb was unaffected in the Nes-Cre;Wt1fl/fl mice (Fig. 2E,F - 1 and 2). Also, the timing of forelimb footfalls relative to the hindlimb cycles is very similar between Wt1fl/fl mice and Nes-Cre;Wt1fl/fl mice (Fig. 2E,F – 3 and 4). Significant differences between Wt1fl/fl mice and Nes-Cre;Wt1fl/fl mice were observed in the timing of the hindlimb footfalls relative to the forelimb cycles (Fig. 2E,F – 3 and 4). The touchdown of the ipsilateral and the contralateral hindlimb fall in a later fraction of the forelimb stride cycle in Nes-Cre;Wt1fl/fl mice compared to the Wt1fl/fl mice. The deviation cannot be explained by the differences in animal speed, because the hind-to-forelimb coordination does not show speed-dependent variation.
So far, the limb kinematics of adult Nes-Cre;Wt1fl/fl mice compared to the Wt1fl/fl mice shows subtle differences in gait parameters and interlimb coordination with a high degree of variation. In sum, these differences result in a performance reduction indicated by the overall lower walking velocities.
Deletion of Wt1 results in a disturbed and irregular postnatal locomotor pattern
After having observed altered gait parameters in adult Nes-Cre;Wt1fl/fl animals, we wondered whether gait also would be affected in younger mice. Indeed, Nes-Cre;Wt1fl/fl pups had more difficulty coordinating their fore- and hindlimbs compared to controls when performing air stepping. Although there was no increase in hindlimb synchronous steps, left/right alternating steps were decreased and the number of uncoordinated steps were increased in Nes-Cre;Wt1fl/fl animals (Supplemental Fig 2; Supplemental Movie 3 and 4). We next performed fictive locomotion experiments on isolated spinal cords from control and Nes-Cre;Wt1Ml mice (P0-P3). Fictive locomotor drugs induced a markedly slower, disturbed, more variable pattern of locomotor-like activity in Nes-Cre;Wt1fl/fl spinal cords (n = 6) compared to the stable, rhythmic pattern of locomotor-like activity in control mice (n = 5). Control spinal cords had recorded activity bursts that showed clear left/right (L2 vs L2) and flexor/extensor (L2 vs L5) alternation that persisted throughout activity periods, whereas activity bursts in Nes-Cre;Wt1fl/fl spinal cords were uncoordinated and did not maintain strict left/right or flexor/extensor alternation (Fig 3A-B). The relationship between left/right and flexor/extensor alternation was examined and gave a strong phase preference for alternating bursts in control (Fig. 3C; l/r control, average phase preference: 183.4° R = 0.93; f/e control, 185.2°, R = 0.84). However, spinal cords from mice with a Wt1 deletion showed an irregular locomotor pattern with inconsistent alternation as indicated by the length and direction of the phase vector (Fig. 3C; l/r average phase preference: 165.3°, R = 0.60; f/e 155.2°, R = 0.44). Additionally, the frequency of the ventral root output was decreased (Fig 3D: control; 0.30 ± 0.024 Hz: Nes-Cre;Wt1fl/fl; 0.18 ± 0.08 Hz). This slower rhythm in Nes-Cre;Wt1fl/fl cords could be attributed to altered L2 and L5 activity burst parameters, as Nes-Cre;Wt1fl/fl mice had significantly longer burst, interburst and cycle periods compared to control (Fig. 3E, F). Thus, the deletion of Wt1 results in a disturbed and irregular locomotor pattern, which suggests that there are changes to the neuronal locomotor circuitry that occur following Wt1 deletion.
Wt1+ neurons receive various synaptic inputs and can project commissurally
In order to assess how Wt1+ dI6 neurons are connected within the CPG network, we focused on the innervation pattern of these cells. We used the Wt1-GFP reporter mouse line (Hosen et al. 2007) where Wt1+ neurons are labeled by GFP. In contrast to the restricted localization of Wt1 in the nucleus, GFP is distributed throughout the cytoplasm and labels the soma and major processes (Fig 4A). In combination with antibodies against particular vesicular synaptic transporters, we observed that excitatory (VGLUT2), inhibitory (VGAT) and modulatory (VMaT2) synapses contact the soma of Wt1+ dI6 neurons (Fig. 4B). This shows that Wt1+ dI6 neurons receive excitatory, inhibitory and modulatory inputs suggesting that Wt1 + neurons are positioned to receive a multitude of signals and could act during locomotion to integrate different CPG signals.
Using the Wt1-GFP reporter mouse, we found GFP+ fibers crossing the spinal cord midline beneath the central canal suggesting that Wt1+ neurons project commissural fibers (Fig. 4C). Fluorescent dextran amine retrograde tracing of contralateral projections confirmed that at least part of the Wt1+ dI6 neurons project commissurally (Supplemental Fig 3). We analyzed spinal cord commissural neurons in control (Nes-Cre;Wt1+/+) and homozygous (Nes-Cre;Wt1fl/fl) mice (P1-5) to determine if the deletion of Wt1 alters the total number of commissural neurons and investigated ascending (aCIN), descending (dCIN) and bifurcating (adCIN) subpopulations (Fig. 4 D, E). All traced subpopulations were markedly reduced in Nes-Cre;Wt1fl/fl cords spinal cords compared to controls (Fig. 4F – H).
Loss of Wt1 leads to altered interneuron composition
To assess the possible impact of Wt1 deletion for interneuron development, we analyzed dI6 and non-dI6 populations situated in the embryonic ventral spinal cord. The number of Dmrt3 expressing cells, which constitutes a distinct but partly overlapping dI6 population (Andersson et al. 2012), was significantly decreased in the embryos harboring a loss of Wt1 in the spinal cord already at E12.5 (Fig. 5A) persisting throughout development (E16.5 and P1). At any investigated time point, neurons co-expressing both Wt1 and Dmrt3 were not detected in Nes-Cre;Wt1fl/fl embryos and neonates.
Loss of the transcription factor Dbx1 that is involved in differentiation of the V0 population results in a fate switch of some V0 neurons to become dI6 interneuron-like cells (Lanuza et al., 2004). Thus, we investigated whether populations flanking the dI6 population were affected in Nes-Cre;Wt1fl/fl mice. The Lmx1b+ dI5 population was similar in number when comparing Nes-Cre;Wt1fl/fl with wild type embryos, whereas the number of Evx1+ V0V neurons was significantly increased already at E12.5 (Fig. 5B). This increase was still detectable at E16.5. No differences could be seen in Foxp2+ V1 neurons, Chx10 (V2a) and Gata3 (V2b) neurons and Islet 1/2+ motor neurons between conditional Wt1 knockout and control embryos at E12.5. However, at E16.5 Chx10+ V2a neurons showed a significant decrease in cell number.
To verify the changes of interneuron composition found in the developing Nes-Cre;Wt1fl/fl mice, we made use of a second mouse line, namely Lbx1-Cre;Wt1fl/fl mice. At embryonic stage E16.5, we observed a decrease in the amount of dI6 neurons and an increase in the cell number of Evx1+ neurons similar to Nes-Cre;Wt1fl/fl mice (Fig. 5C). This decline in the number of dI6 neurons and the concomitant increase in the amount of Evx1+ neurons might point to a change in the developmental fate from dI6 neurons into V0 neurons prompted by deletion of Wt1. To test this hypothesis, we ablated the cells destined to express Wt1. We used Lbx1-Cre;Wt1-GFP-DTA mice in which the Diphtheria toxin subunit A (DTA) is expressed from the endogenous Wt1 locus after Cre-mediated excision of a GFP cassette harboring a translational STOP-codon. Cre expression driven by the Lbx1 promoter targets the dI4 to dI6 interneuron populations (Müller et al. 2002). In Lbx1-Cre;Wt1-GFP-DTA embryos, nearly all Wt1+ neurons were ablated at E16.5 (Fig. 5D). The ablation of Wt1+ neurons coincided with a significantly decreased number of Dmrt3+ neurons in Lbx1-Cre;Wt1-GFP-DTA embryos, but did not affect the number of Evx1 + neurons (Fig 5D). Taken together, the results from the Wt1 deletion and the ablation of the Wt1 neurons suggests that the fate switch from dI6 neurons into Evx1+ V0 neurons occurs due to the deletion of Wt1. A postnatal phenotypic behavioral analysis of these mice was not possible because neonates died immediately after birth due to serious respiratory deficits (data not shown).
The analyses of the interneuron composition in developing conditional Wt1 knockout mice and embryos with an ablation of Wt1+ neurons suggest a fate switch within a specific subset of dI6 and V0V neurons that depends on the presence of the cells destined to express Wt1.
The transition of dI6 neurons into Evx1+ V0V neurons upon loss of Wt1 is not direct
In order to further investigate the cellular fate change upon deletion of Wt1 we combined Wt1-GFP and Nes-Cre;Wt1fl/fl animals to generate Nes-Cre;Wt1fl/GFP mice. These mice harbor a constitutive knockout allele of Wt1 due to the insertion of a GFP coding sequence and another conditional Wt1 knockout allele. GFP and Wt1 were co-localized in the ventral spinal cord of Wt1fl/GFP control animals at E13.5, whereas GFP, but not Wt1, was detected in spinal cords of Nes-Cre;Wt1fl/GFP embryos of the same age (Fig 6A). Thus, Nes-Cre;Wt1fl/GFP mice allowed us to inactivate Wt1 while the cells destined to express Wt1 are labelled by GFP.
To investigate whether Wt1 deletion leads to apoptosis in the respective cells, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used. TUNEL+ cells were present in the ventrolateral spinal cords of Wt1fl/GFP control and Nes-Cre;Wt1fl/GFP embryos (Fig 6A). However, TUNEL signals never overlapped with GFP+ dI6 neurons destined to express Wt1, suggesting that Wt1 inactivation in dI6 neurons did not result in cell death.
In order to find out whether cells destined to express Wt1 would directly convert to V0V neurons upon Wt1 inactivation, we performed immunohistochemical analyses. The presence of Dmrt3 and Evx1 in GFP+ dI6 neurons was analyzed in Wt1fl/GFP control and Nes-Cre;Wt1fl/GFP embryos at E12.5 (Fig 6B). The number of GFP+ cells per hemicord was determined and set to 100%. The proportion of Dmrt3+ cells was approximately 13% of all GFP+ cells in spinal cord of E12.5 control embryos. When Wt1 was absent, the amount of Dmrt3+ GFP cells significantly decreased to 4%. In contrast, the proportion of GFP+ dI6 neurons that also showed Evx1 staining was not changed between Wt1fl/GFP control and Nes-Cre;Wt1fl/GFP animals (below 1% for both). Thus, the increase in the amount of Evx1+ V0V neurons observed in mice lacking Wt1, does not seem to result from a direct transition of future Wt1+ dI6 neurons into Evx1+ V0V neurons.
Discussion
In this study, we have examined Wt1, which marks a subset of dI6 neurons. We found that Wt1 is required for proper differentiation of spinal cord neurons and that deletion of Wt1 results in locomotor aberrancies in neonate and adult mice.
Neonates lacking Wt1 in the spinal cord increased the number of uncoordinated steps, which was supported by a markedly slower and variable pattern of locomotor-like activity in isolated Nes-Cre;Wt1fl/fl spinal cords. Adult Nes-Cre;Wt1fl/fl animals showed an increased stride length and a decreased stride frequency resulting in slower absolute locomotor speed, however, these alterations were more subtle compared to locomotion abnormalities seen in neonates. Compensatory adaptation of functional properties during postnatal maturation of neuronal circuits could possibly act to reduce the severity of the deficits seen in neonates. For instance, the corticospinal tract, which allows control of the spinal circuits directly by the motor cortex and overrides the spinal circuits, does not reach the lumbar spinal cord until approximately P7-P9 (Kamiyama et al. 2015).
Wt1+ neurons are unlikely to participate in locomotor rhythm generation per se since a rhythm is established when Wt1 is deleted. We hypothesize that these neurons are involved in the maintenance or modulation of this rhythm. Adult Nes-Cre;Wt1fl/fl animals show a decreased stride frequency. The hindlimb-to-forelimb phase relationship is altered in animals with Wt1 deletion in the spinal cord. This supports a possible role of the Wt1+ dI6 neurons in both timing and limitation of the stride cycle. An involvement in timing of the stride cycle would require an integrative position in the locomotor CPGs, which is compatible with the observed multi-synaptic input to Wt1+ dI6 neurons.
The timing of hindlimb footfalls relative to forelimb footfalls differed between Wt1fl/f and Nes-Cre;Wt1fl/fl mice, particularly at the contralateral limbs, suggesting Wt1 cells to play a role for long range coordination between various spinal cord segments. This phenotype is supported by the observation that at least a fraction of Wt1+ dI6 neurons possess commissural projections and thus are involved in the contralateral communication between the spinal cord halves. If and how the loss of this communication affects the adult locomotor phenotype does not become apparent from our data set of footfall timing but we expect to detect more details by the analysis of intrinsic limb kinematics. Deletion of Wt1 leads to a decline in the number of commissural neurons suggesting an involvement of Wt1 in establishing proper projections of the Wt1+ dI6 neurons. Thus, it will be of future interest to investigate the transcriptome of Wt1+ dI6 neurons and screen for potential target genes involved in axon guidance.
Lack of Wt1 in the spinal cord causes alterations in the differentiation of dI6, V0 and V2a spinal cord neurons (Fig 6C). The inverse alterations in the dI6 and V0 populations suggest a fate change from dI6 to V0-like neurons when Wt1 is inactivated. The putative transition from dI6 to V0-like neurons occurs at the time-point when Wt1 expression would normally start. This instantaneous effect might be due to the derivation of both interneuron populations from neighboring progenitor domains sharing common transcription factors such as Dbx2 (Alaynick et al., 2011). Thus, loss of Wt1 might lead to a switch in developmental programs that are normally repressed; whether this repression occurs cell-autonomously or non-cell-autonomously still has to be determined. In any case, when future Wt1+ cells are ablated an increase of V0-like neurons is no longer observed, suggesting that the fate switch requires the cells about to express Wt1.
The fate change of prospective dI6 to V0-like neurons is complex since dI6 neurons can be subdivided into at least three subsets based on expression of the transcription factor encoding genes Wt1 and Dmrt3 (Fig 6C). Loss of Wt1 not only affects the small number of dI6 neurons that possess Wt1 and Dmrt3 but also the number of neurons that only express Dmrt3. This population is significantly decreased. The presence of Wt1+ neurons therefore is essential to maintain the character of a subset of Dmrt3 neurons. If Wt1 is inactivated, in addition to the cells which are programmed to express Wt1, possibly also Dmrt3+ neurons may differentiate into V0-like neurons.
Two main subpopulations exist within the V0 population (Alaynick et al., 2011): the Evx1+, more ventrally derived V0V and the Evx1 negative, more dorsally derived V0D population, for which no distinct marker has yet been described. The knockout of Dbx1 results in loss of the whole V0 population, whereby Evx1+ V0V neurons acquire a more ventral fate and become V1 neurons, whereas Evx1 negative V0D neurons acquire characteristics of dI6 neurons (Lanuza et al., 2004). This suggests that the V0D, rather than the V0v, neurons closer resemble the dI6 neurons and poses the question whether the fate change from Wt1+ dI6 neurons to Evx1+ V0V-like neurons represents a direct or an indirect transition. The investigations using the Nes-Cre;Wt1fl/GFP mice suggest that the Wt1-deficient dI6 cells do not change their fate directly into Evx1+ V0V-like neurons suggesting an indirect transition. That points to the possibility that the fate change might be achieved by a transition of Wt1+ dI6 neurons into the more closely related Evx1 negative V0D-like neurons, which leads to a putative increase of the V0D population (Fig 6C). The Evx1+ V0V population might, in turn, increase its number to compensate for a higher proportion of V0D-like neurons.
In addition to the changes in the dI6 and V0 population that occur upon Wt1 deletion in the spinal cord, Chx10+ V2a neurons show a slight but significant decrease in their cell number at E16.5 (Fig 6C). This might represent a secondary effect of the alterations in the dI6 and V0 population, which occur already at E12.5. It was reported that V2a neurons directly innervate V0V neurons (Crone et al., 2008). This secondary effect might thus be due to a potential adaptation to the altered interneuron composition in the spinal cord and the necessity to form proper contacts with target cells to build up the neuronal circuits responsible for locomotion.
In sum, the results obtained in this study not only shed light on the so far undescribed necessity for Wt1 in the development of spinal cord neurons and their functional implementation in circuits responsible for locomotion. The data also broadens our view on the complex interplay of the various neuron subpopulations within the spinal cord.
Materials and Methods
Mouse husbandry
All mice were bred and maintained in the Animal Facility of the Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, Germany, according to the rules of the German Animal Welfare Law. Sex- and age-matched mice were used. Animals were housed under specific pathogen-free conditions (SPF), maintained on a 12 hour light/dark cycle, fed with mouse chow and tap water ad libitum. Mice used for analysis of fictive locomotion and projection tracing were kept according to the local guidelines of Swedish law. Wt1fl/fl mice were maintained on a mixed C57B6/J x 129/Sv strain. Wt1-GFP mice (Hosen et al. 2007) were maintained on a C57B6/J strain. Conditional Wt1 knockout mice were generated by breeding Wt1fl/fl females (Gebeshuber et al. 2013) to Nes-Cre;Wt1fl/fl (Tronche et al. 1999) or Lbx1-Cre;Wt1fl/fl mice (Sieber et al. 2007). To generate mice with Wt1 ablated cells, Wt1-GFP-DTA mice were bred with Lbx1-Cre mice. Control mice were sex- and age-matched littermates (wild type or Wt1fl/fl). For plug mating analysis, females of specific genotypes were housed with males of specific genotypes and were checked every morning for the presence of a plug. For embryo analysis, pregnant mice were sacrificed by CO2 inhalation at specific time points during embryo development and embryos were dissected. Typically, female mice between 2 and 6 months were used.
Generation of Wt1-GFP-DTA mice
The Wt1-GFP-DTA mouse line bares an IRES-lox-GFP-lox-DTA cassette that was inserted into intron 3 of the Wt1 locus. This cassette consists of a GFP encoding sequence that ends in a translational STOP-codon and is flanked by loxP sites. Downstream of GFP, the coding sequence for the Diphtheria toxin subunit A (DTA) was incorporated. Before Cre-induction, the IRES cassette ensures the generation of a functional GFP protein. After Cre-mediated excision of the floxed GFP sequence, the DTA is expressed from the endogenous Wt1 promotor.
The Wt1-GFP-DTA model was generated by homologous recombination in embryonic stem (ES) cells. After ES cell screening using PCR and Southern Blot analyses, recombined ES cell clones were injected into C57BL/6J blastocysts. Injected blastocysts were re-implanted into OF1 pseudo-pregnant females and allowed to develop to term. The generation of F1 animals was performed by breeding of chimeras with wild type C57BL/6 mice to generate heterozygous mice carrying the Wt1 knockin allele.
Immunohistochemistry
Embryonic and postnatal spinal cords were dissected. They were either frozen unfixed after 15 min dehydration with 20% sucrose (in 50 % TissueTec/PBS) (post-fix) or fixed for 75 min in 4% paraformaldehyde in PBS (pre-fix). Pre-fixed tissue was cryo-protected in 10%, 20% and 30% sucrose (in PBS) before freezing in cryo-embedding medium (Neg-50 - Thermo Scientific, Kalamazoo, USA). Post- and pre-fix samples were sectioned (12 μm). Post-fixed samples were fixed for 10 min after sectioning and washed with 2% Tween in PBS (PBS-T). For pre-fixed samples, antigen retrieval was performed by incubation in sub boiling 10 mM sodium citrate buffer pH6.0 for 30 min. After blocking with 10% goat serum and 2% BSA in PBS-T (post-fix) or (prefix), sections were incubated with primary antibodies (in blocking solution) using the following dilutions: gBhlhb5 1:50 (Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA), BrdU 1:100 (abcam, Cambridge, UK), shChx10 1:100 (abcam, Cambridge, UK), gpDmrt3 1:5000 (custom made (Andersson et al. 2012)), mEvx1 1:100 (1:3000 pre-fix) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), chGFP 1:1000 pre-fix (abcam, Cambridge, UK), mGFP 1:100 (Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA), rFoxP2 1:800 (abcam, Cambridge, UK), mIslet1/2 1:50 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), gpLbx1 1:20,000 (gift from C. Birchmeier, MDC, Berlin, Germany), Lim1/2 1:50 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), NeuN, 1:500 (Merck, Darmstadt, Germany), rbPax2 1:50 (Thermo Fisher Scientific, Waltham, Massachusetts, USA), rbLmx1b 1:100 (gift from R. Witzgall, University, Regensburg, Germany), rbWt1 1:100 (Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA). Secondary antibodies were applied according to species specificity of primary antibodies. Hoechst was used to stain nuclei. Quantitative analysis of the antibody staining was statistically analyzed using student t-test and two-way ANOVA followed by Tukey’s post hoc test.
Bromodeoxyuridine labeling
To label proliferating cells in the embryonic spinal cord, pregnant mice at E9.5, E10.5 and E11.5 were injected intraperitoneally with 100 μg/g of Bromodeoxyuridine (BrdU) dissolved in 0.9% sodium chloride solution. Embryos were harvested at E12.5 to isolate spinal cords and stain for BrdU and Wt1. Spinal cords were frozen unfixed after 15 min dehydration with 20% sucrose (in 50 % TissueTec/PBS) and sectioned (12 μm). After any of the following treatments, sections were washed with PBS. Antigen retrieval was performed by incubation in 98°C sub boiling 10 mM sodium citrate buffer pH6.0 for 30 min. After treatment with 2N HCl at 37°C for 30 min, sections were incubated with primary antibodies using dilutions mentioned above (Immunohistochemistry). Secondary antibodies were applied according to species specificity of primary antibodies.
RNA isolation and qRT-PCR analysis
Total RNA was isolated from E12.5 embryonic spinal cords using Trizol (Invitrogen) according to manufacturer’s protocol. Subsequently, 0.5 μg of RNA was reverse transcribed with iScript™ cDNA Synthesis Kit (Bio-Rad) and used for qRT-PCR. The primer sequences used for RT-PCR analyses are as follows: TGT TAC CAA CTG GGA CGA CA (Act_foŕ); GGG GTG TGG AAG GTC TCA AA (Act_rev); AGT TCC CCA ACC ATT CCT TC (Wt1_qRT_for); TTC AAG CTG GGA GGT CAT TT (Wt1_qRT_rev). Real time PCR was carried out in triplicates for each sample using Syber®GreenERTM (Thermo Fisher Scientific, USA) and Bio-Rad iCycler™ (Bio-Rad). PCR efficiencies of primer pairs were calculated by linear regression method. Ct values were normalized to the mean of the reference gene, Actin. Relative expression was determined by comparing normalized Ct values of Wt1 conditional knockout and control samples.
Analysis of locomotor behavior
In order to characterize gait parameters, 10 animals per sex and genotype were used. Body masses of the mice varied considerably within the groups and among the groups with significant differences between male Wt1fl/fl and Nes-Cre;Wt1fl/fl mice (Wt1fl/fl: 28 g ± 3 g vs. Nes-Cre;Wt1fl/fl: 23 g ± 3 g; Fs = 31.98; ts = 3.28, P> 0.001) and moderate differences between the female Wt1fl/fl and Nes-Cre;Wt1fl/fl mice (Wt1fl/fl: 25 g ± 5 g vs. Nes-Cre;Wt1fl/fl: 22 g ± 4 g; Fs = 3.80; ts = 1.62, n.s.). We recorded the voluntary walking performance of this larger cohort using high-resolution X-ray fluoroscopy (biplanar C-arm fluoroscope Neurostar, Siemens AG, Erlangen, Germany). Because of body size variation within and among groups, we adjusted treadmill speed dynamically to the individual preferences and abilities of the mice. This method of motion analysis has been described in detail in several recent publications (e.g. Böttger et al., 2011; Andrada et al., 2015; Niederschuh et al., 2015) and will be only briefly summarized here: The X-ray system operates with high-speed cameras and a maximum spatial resolution of 1536 dpi x 1024 dpi. A frame frequency of 500 Hz was used. A normal-light camera operating at the same frequency and synchronized to the X-ray fluoroscope was used to document the entire trial from the lateral perspective. Footfall sequences and spatio-temporal gait parameters were quantified by manual tracking of the paw toe tips and two landmarks on the trunk (occipital condyles, iliosacral joint) using SimiMotion 3D.
Speed, stride length, stride frequency, as well as the durations of stance and swing phases, as well as the distances that trunk or limb covered during these phases were computed from the landmark coordinates collected at touchdown and lift-off of each limb. The phase relationships between the strides of left and right limbs as well as fore- and hindlimbs were determined from footfall sequences as expression of temporal interlimb coordination. As the animals frequently accelerated or decelerated relative to the treadmill speed, the actual animal speed was obtained by offsetting trunk movement against foot movement during the stance phase of the limb. The resulting distance was divided by the duration of the stance phase. Animal speed as well as all temporal and spatial gait parameters were then scaled to body size following the formulas published by Hof (1996): non-dimensional speed =v/gl0, where v is raw speed, g is gravitational acceleration and l0 is the cube root of body mass as characteristic linear dimension, which scales isometrically to body mass; non-dimensional frequency = f/gl0, where f is raw frequency; non-dimensional stride length = l/l0, where l is raw stride length. The scaled spatio-temporal gait parameters change as a function of non-dimensional speed. Therefore, linear regression analyses were computed for each parameter in the male and the female Wt1fl/fl group. The power formulas obtained from regression computation (Y = a + bX) were then used to calculate the expected value for a given non-dimensional speed for each gait parameter (baseline) in each animal of all four groups. The coefficient of determination r2 was computed. The deviations of the measured values of Y from the expected values, the residuals, were determined and are given in percent of deviation. Using these residuals, one-way ANOVA were computed in order to establish the significance of the differences between the means of Wt1fl/fl and Nes-Cre; Wt1fl/fl in males and females. Group means were calculated from the means of 10 animals. Sample size per mouse and limb ranged between 5 and 41 stride cycles with an average sample size of 22 ± 9.
Fictive locomotion
Animals (P0-P3) were euthanized and the spinal cords eviscerated in ice-cold cutting solution containing (in mM): 130 K-Gluconate, 15 KCl, 0.05 EGTA, 20 HEPES, 25 Glucose, pH adjusted to 7.4 by 1M KOH) and then equilibrated in artificial cerebrospinal solution (aCSF) (Perry et al. 2015) for at least 30 minutes before the beginning of experimental procedures. Suction electrodes were attached to left and right lumbar (L) vental roots 2 and 5 (L2 and L5). A combination of NMDA (5 μM) + 5-HT (10 μM) + dopamine (DA) (50 μM) were added to the perfusing aCSF to induce stable locomotor-like output. All chemicals were obtained from Sigma. Recorded signals containing compound action potentials were amplified 10,000 times, and band-pass filtered (100-10 kHz) before being digitized (Digidata 1322A, Axon instruments) and recorded using Axoscope 10.2 (Axon Instruments Inc.) for later off-line analysis. The data was rectified and low-pass filtered using a third-order Butterworth filter with a 5 Hz cut-off frequency before further analysis. Coherence plots between L2 and L2/L5 traces were analysed using a mortlet wavelet transform in SpinalCore (Version 1.1, (Mor and Lev-Tov, 2007)). Preferential phase alignment across channels are shown in the circular plots and burst parameters were analysed for at least 20 sequential bursts, as previously described (Kiehn and Kjaerulff 1996) using an in-house designed program in Matlab (Mathworks R2014b). Ventral root recording preferential phase alignment was assessed by means of circular statistics (Rayleight test) for 20 consecutive cycles as described (Kiehn and Kjaerulff 1996). Burst parameters, including frequency, are presented as the mean ± standard deviation (SD). Burst parameters were compared using the two-tailed Mann-Whitney test or the Kruskal-Wallis analysis of variance test followed by a Dunns post-test comparing all groups.
Tracing of commissural neurons
To examine whether the loss of Wt1 affects spinal cord populations, tracing experiments were conducted as previously described (Rabe et al. 2009; Andersson et al. 2012). Nes-Cre;Wt1fl/fl and Nes-Cre;Wt1++ littermate control mice P0-P5, were prepared as described above (Fictive locomotion). Two horizontal cuts (intersegmental tracing targeting commissural ascending/descending/bifurcating neurons) were made in the ventral spinal cord at lumbar (L) level 1 and between L4 and 5. Fluorescent dextran-amine (FDA, 3000 MW, Invitrogen) was applied at L1 and rhodamine-dextran amine (RDA, 3000 MW, Invitrogen) was applied between the L4/5 ventral roots. Spinal cords were incubated overnight at room temperature, subsequently fixed in 4% formaldehyde (FA) and stored in the dark at 4°C until transverse sectioning (60μm) on a vibratome (Leica, Germany).
Fluorescent images were acquired on a fluorescence microscope (Olympus BX61W1). For quantitative analyses of traced cords, consecutive images were taken between the two tracer application sites using Volocity software (Improvision, Lexington, USA). Captured images were auto-levelled using Adobe Photoshop software. Only cords with an intact midline, as assessed during imaging, were used for analysis.
Traced neurons in Wt1fl/fl control, Nes-Cre;Wt1fl/+ and Nes-Cre;Wt1fl/fl cords were examined for significance using the Kruskal-Wallis analysis of variance test followed by a Dunns post-test comparing all groups. Tracing data are presented as the mean ± standard error of the mean (SEM).
TUNEL-Assay
To detect apoptosis in situ, the TUNEL assay was performed prior to antibody binding. Slides were incubated with TUNEL reaction solution (1x Reaction Buffer TdT and 15 U TdT in ddH2O from Thermo Scientific; 1 mM dUTP-biotin from Roche) at 37 °C for 1 h and washed with PBS afterwards.
Imaging and picture processing
Fluorescent images were viewed in a Zeiss Axio Imager and a Zeiss Observer Z1 equipped with an ApoTome slider for optical sectioning (Zeiss, Germany). Images were analyzed using the ZEISS ZEN2 image analysis software. For quantitative analyses of traced spinal cords, the application sites were identified and consecutive photographs were taken between the two application sites using the OptiGrid Grid Scan Confocal Unit (Qioptiq, Rochester, USA) and Volocity software (Improvision, Lexington, USA). Confocal images were captured on a ZEISS LSM 710 ConfoCor 3 confocal microscope and analyzed using the ZEISS ZEN2 image analysis software. Captured images were adjusted for brightness and contrast using ZEN2 image analysis software and Adobe Photoshop software.
Statistical Analyses
Data are expressed as mean ± SD or as indicated. Groups were compared using two-way ANOVA or two-tailed two-sample equal variance student t-test as determined by group and sample size. If normal distribution of a sample was not confirmed, sample means are compared by using non-parametric Mann-Whitney U test. All statistical analyses were done using GraphPad Prism Software (GraphPad Software inc., San Diego, USA), IBM SPSS Statistics 24 (IMB Corporation, New York, USA), Microsoft Excel (Microsoft Corporation, Redmond, USA) or Matlab (Mathworks, R2014b). Normal distribution was assessed using the D’Agostino-Pearson normality test or Kolmogorov-Smirnov test. Significance was determined as * = P < 0.05, ** = P<0.01, *** = P<0.001.
Acknowledgements
We thank D. Kruspe and R. Peterson for technical assistance, C. Birchmeier (MDC, Berlin, Germany) for providing the Lbx1-Cre mouse line and C. Hübner, H. Heuer and G. Zimmer for critical discussion. This project was supported by grants from German Federal Ministry of Education and Research (Infrafrontier grant 01KX1012) to L.B. and the Swedish Medical Research Council, Hållsten, Ländells, Swedish Brain Foundations to K.K. D.S. received a scholarship from the Leibniz Graduate School on Ageing and Age-Related Diseases (LGSA). F.V.C. was funded by a scholarship from CNPq – Brazil. The FLI is a member of the Leibniz Association and is financially supported by the Federal Government of Germany and the State of Thuringia.