Catenin-Dependent Cadherin Function Drives Divisional Segregation of Spinal Motor Neurons

Abstract

Motor neurons that control limb movements are organised as a neuronal nucleus in the developing ventral horn of the spinal cord called the lateral motor column. Neuronal migration segregates motor neurons into distinct lateral and medial divisions within the lateral motor column that project axons to dorsal or ventral limb targets respectively. This migratory phase is followed by an aggregation phase whereby motor neurons within a division that project to the same muscle cluster together. These later phases of motor neuron organisation depend on limb-regulated differential cadherin expression within motor neurons. Initially, all motor neurons display the same cadherin expression profile and this coincides with the migratory phase of motor neuron segregation. Here, we show that in the chicken embryo this early, pan-motor neuron, cadherin function drives the divisional segregation of spinal motor neurons by controlling motor neuron migration. We manipulate pan-motor neuron cadherin function through dissociation of cadherin binding to their intracellular partners. We find that of the major intracellular transducers of cadherin signalling, γ-catenin and α-catenin predominate in the lateral motor column. In vivo manipulations that uncouple cadherin-catenin binding disrupt divisional segregation via deficits in motor neuron migration. Additionally, reduction of the expression of cadherin-7, a cadherin predominantly expressed in motor neurons only during their migration, also perturbs divisional segregation. Our results show that γ-catenin-dependent cadherin function is required for spinal motor neuron migration and divisional segregation and suggest a prolonged role for cadherin expression in all phases of motor neuron organisation.

Introduction

Neuronal nuclei are a recurrent organisational scheme within the central nervous system that clusters functionally related neuronal soma as spatially distinct groups (Cajal., 1995). Despite the critical functions of neuronal nuclei, little is known of the molecular mechanisms that drive their clustering during development, a process termed nucleogenesis (Agarwala and Ragsdale, 2002).

The spinal motor neurons that control limb movement form a neuronal nucleus termed the lateral motor column (LMC) (Jessell, 2000). As with other neuronal nuclei, LMC internal structure is related to the axonal targets of the neurons in the nucleus (Landmesser, 1978). The LMC segregates into lateral (LMCl) and medial (LMCm) divisions related to the dorsal or ventral compartments of the limb to which each division projects. Divisional segregation occurs by inside-out migration of LMCl neurons through the earlier born LMCm (Hollyday and Hamburger, 1977).

Following motor neuron migration, clusters of motor neurons that project axons to an individual muscle emerge (Whitelaw and Hollyday, 1983). Clustering of these so-called motor neuron pools (Romanes, 1964), results from differential expression of members of the type II family of cadherin cell adhesion molecules (Price et al., 2002). Cadherin expression within the LMC is highly dynamic and encompasses a pan-motor neuron phase during divisional segregation with a later motor pool specific phase. For example, expression of cadherin-20, is initiated in all motor neurons soon after their generation and is refined via limb-derived signals to a motor pool specific pattern only after divisional segregation is well underway (Price et al., 2002). Absence of these limb-derived signals perturbs motor pool sorting whilst leaving divisional segregation intact (Haase et al., 2002; Livet et al., 2002). This suggests that divisional segregation and pool sorting are separable and raises the possibility that early, pan motor neuron cadherin expression could drive divisional segregation prior to pool sorting.

The cytoplasmic domain of type II cadherins binds to members of the armadillo family of catenins, β–catenin or γ– catenin (plakoglobin) (Nollet et al., 2000). β–catenin and γ–catenin bind to α–catenin, which anchors cadherin adhesion to the actin cytoskeleton via the linker protein EPLIN (Abe and Takeichi, 2008). Thus, function of all type II cadherins converges on their interaction with either β– or γ–catenin and absence of β– or γ–catenin abrogates cadherin function (Kintner, 1992; Weiss and Nelson, 2006).

We investigated pan-motor neuron cadherin expression in LMC organisation through manipulations of catenin-cadherin binding and reduction of a cadherin predominantly expressed during divisional segregation. Expression of a single amino-acid mutant of γ–catenin, predicted to uncouple interaction with α–catenin, results in a cell autonomous stalling of motor neuron migration with a concomitant disruption of divisional segregation. Dissociation of cadherin from γ-catenin, via expression of an extracellular deleted dominant negative cadherin, also perturbs motor neuron migration and divisional segregation. Finally, knockdown of cadherin-7 perturbs divisional segregation. These data are consistent with a model whereby early pan-motor neuron cadherin function drives the migration of LMC neurons into the ventral horn and suggest a role for cadherin expression throughout nucleogenesis of spinal motor neurons.

Materials and Methods

Chick Embryo Preparation

Fertilised Brown Bovan Gold Hen’s eggs (Henry Stewart Farms, UK) were incubated in a forced draft incubator at 38°C and staged as in Hamburger and Hamilton (1992). All embryos were treated in accordance with the Animals (Scientific Procedures) Act of 1986, UK. Embryos of either sex were used in our experiments.

Labelling of migrating Neurons

HRP (Roche, 50% solution in PBS with 1% Lysolecithin (SIGMA) was pressure injected into the dorsal limb essentially as described in Lin et al., (1998) in order to retrograde label neurons.

BrdU labelling

BrdU (200μl, 1mM (SIGMA)) was injected directly under the embryo and the eggs sealed and returned to the incubator for the desired length of time. Following cryosectioning, embryo sections were incubated for 5 minutes in 2M HCl (Sigma) followed by five washes in PBS each for 5 minutes. Primary antibodies to BrdU and subsequent secondary antibody detection was as described in the section on immunohistochemistry.

In Situ Hybridisation Histochemistry

Digoxigenin (DIG)-labelled anti-sense cRNA probes were used for in situ hybridisation histochemistry on 15 μm thick cryostat sections as in Price et al 2002. Dual in situ hybridisation histochemistry with BrdU labelling was performed by sequential in situ hybridisation followed by a 2M HCl treatment at 20°C for 5 minutes, 3 washes in PBS for 5 minutes each followed by immunohistochemistry for BrdU with secondary antibodies conjugated to Horse Radish Peroxidase (HRP) followed by IMPACT DAB staining according to the manufacturers protocol (Vector Labs).

In Ovo Electroporation

Expression of cDNAs was achieved by in ovo electroporation using an ECM830 electro-squareporator (BTX Inc.). ~0.1μl of DNA constructs (1-10μg/μl in H2O with 0.1% Fast Green (Sigma)) was pressure injected into the lumen of the spinal cord. Five 30 Volt electrical pulses of 50ms duration equally spaced over a 5 second period were applied by placing electrodes adjacent to each side of the spinal cord of the embryo. Embryos were electroporated at HH stages 12-18 and analyzed at HH stages 25-32.

Generation of Constructs

A Full-length cDNA for chick γ–catenin was cloned by screening an E3 chick cDNA library. The sequence of chick γ–catenin has been submitted to GenBank, accession number HM102357. Point mutation (L127A) was generated using the QuikChange kit (Stratagene) following the manufacturers protocol. γ–catenin and γ–catenin (L127A) cDNAs were cloned into a pCAGGS vector containing an internal ribosome entry sequence (IRES) followed by a cDNA encoding nuclear localization sequence tagged β-galactosidase (pCAGGS inlz). Other constructs used in this work include HA-tagged dominant negative GSK, HA-tagged β-catenin ΔARM, HA-tagged β-catenin-1-ins, HA-tagged constitutively active GSK, HA-tagged dominant negative TCF, transposase integrated doxycycline inducible N-cadherinΔ390 (Kawakami and Noda, 2004; Tanabe et al., 2006; Sato et al., 2007; Watanabe et al., 2007), cad-7 shRNA knock-down and control cad-7 shRNA (described, tested and characterised in Barnes et al., 2010, these constructs follow the method described in Das et al., 2006), pCAGGS inlz, CMV eGFP (Invitrogen). In situ hybridisation probes for cad-20 (MN-cad) and cad-12 were described in Price et al., 2002.

Immunohistochemistry

Antibodies used in this study were: Rabbit (R) anti-GFP (Invitrogen, 1/1000), R anti β–catenin (SIGMA, 1/1000), R anti pan-cadherin (SIGMA, 1/1000), R anti HB9 (1/5000), R anti N-cadherin (AbCAM, 1/1000), Dylight 488 conjugated R anti HRP (Jackson), Rat anti HA (Roche, 1/500), Guinea pig (GP) anti-Isl1(2) (1/20000), GP anti FoxP1, Goat (G) anti-HRP (Jackson Labs) (1/2000), G anti β-galactosidase (1/1000), Mouse (M) anti α–E–catenin (Zymed, 1/100), M anti γ-catenin (BD Transduction Laboratories, 1/100), M anti GFP (Invitrogen, 1/100), M anti BrdU (Roche, 1/50), M anti HA (Covance, 1/50). The following mouse monoclonal antibodies were purchased from the Developmental Studies Hybridoma Bank, 745A5 (anti Nkx2.2) PAX6 (anti Pax6), 4F2 (anti Lim1/Lhx1), 2D6 and 4D5 (anti islet-1), 5C10 (anti MNR2/HB9), A2B11 and EAP3 (anti transitin), CCD7-1 (anti cadherin-7). Alkaline phosphatase conjugated Sheep anti-DIG Fab fragments (Roche) (1/5000), Immunocytochemistry was performed essentially as described (Price et al., 2002). Cryostat sections mounted on superfrost plus glass slides were incubated in PBS for 5 minutes followed by incubation in block solution (PBS with 1% Goat serum (Sigma)) for 30 minutes at 20°C. This solution was replaced by antibody diluted in block solution and incubated for 12 to 16 hours at 4°C. Following three washes of 5 minutes in PBS, fluorescent conjugated secondary antibodies were incubated with the sections for 30 minutes at 20°C in block solution, washed and mounted with vectashield fluorescent mounting medium (Vector Labs).

Image Acquisition and data analysis

Images were acquired on a Nikon Eclipse E80i fluorescence microscope equipped with a Nikon DS5M and Hamammatsu ORCA ER digital camera or on a Leica SPE confocal microscope. Quantitation of migration lengths was performed using ImageJ software to trace the pathway from ventricular zone to identified motor neurons using knowledge of the curved nature of the pathway following γ–catenin (L127A) expression.

Divisional Mixing Index

The Divisional Mixing index was calculated by focussing on an individual LMCl cell at a time and counting the number of LMCm that are immediately adjacent to it. The control side of the spinal cord and control electroporations resulted in close to 100% of such cells being surrounded by zero LMCm cells. These results were quantitated for at least 5 different embryos of each phenotype and presented as mean±SEM percentages in each bin of the mixing index.

Results

LMC neurons migrate in an orderly series during divisional segregation

We first characterised the timing of LMC formation as a nucleus in the ventral horn through analysis of the expression of transcription factors that define divisions of the LMC. At lumbar spinal cord levels, expression of the forkhead transcription factor Foxp1 identifies the LMC (Dasen et al., 2008; Rousso et al., 2008). Further, expression of the LIM homeodomain factor islet-1 identifies neurons of the medial division of the lateral motor column (LMCm) (Tsuchida et al., 1994). Neurons of the lateral division of the LMC express Hb9 (William et al., 2003). The expression of these transcription factors persists through the migratory phase of LMC formation (Tsuchida et al., 1994; William et al., 2003; Dasen et al., 2008; Rousso et al., 2008). Neurons of the medial motor column, which project axons to axial or body wall muscles, co-express both islet-1 and HB9 but do not express Foxp1 (Dasen et al., 2008; Rousso et al., 2008).

More than 95% of LMC neurons are born within the 24-hour period before Hamburger and Hamilton (HH) (Hamburger and Hamilton, 1992) stage (st) 23 with the peak generation of LMCm in rostral lumbar regions from st18 to st20 and LMCl occurring at st20 and 21 (Hollyday and Hamburger, 1977; Whitelaw and Hollyday, 1983). We thus assessed the time course of motor neuron migration from st23 to st27. Consistent with previous studies, we find that at HH st23 LMCm neurons dominate in the ventral horn with the majority of LMCl neurons being found medial to this group (Figure 1A-C) (Sockanathan and Jessell, 1998; Lin et al., 1998; William et al., 2003). LMCl neurons are first detected in the lateral ventral horn around st24 (data not shown) and divisional segregation continues until around st27 (Figure 1D-I; Summarised in Figure 1J-L). Therefore, ~24 hours appears to be required for the migration of a given LMCl neuron with LMCl migration extending over a continuous 48-hour period after they are generated. Thus, LMCl neurons initiate their migration in an ordered series during divisional segregation.

Figure 1. Timecourse of LMC divisional segregation.

A-C. Expression in the ventral horn of a stage 23 embryo of Islet1 and HB9 (A), Hb9 and Foxp1 (B) and FoxP1. Foxp1 marks the entire lateral motor column. Hb9 and Islet-1 are LMC divisional markers. Medial is to the left and is illustrated in panel (A). LMCl cells are, at this stage, more medial to the LMCm cells.

D-F Ventral horn expression at stage 25 of the divisional markers Hb9 (D, E) and Islet-1 (D, F) and the LMC marker Foxp1 (E, F). Note that LMCl and LMCm cells are intermingled.

G-I. Columnar segregation is largely complete by stage 27 as assessed by ventral horn expression of Foxp1 (H, I), Islet-1 (G, I) and Hb9 (G, H).

J-L. Summary of the migration of LMCl cells through LMCm cells during motor neuron divisional segregation at stage 23 (J), st25 (K) and st 28 (L).

γ-catenin and α-catenin are expressed by the LMC during divisional segregation

We next characterised the expression of classical cadherin signalling components during the stages of motor neuron migration and divisional segregation (Zhurinsky et al., 2000a; Nollet et al., 2000; Hirano et al., 2003; Yamada et al., 2005; Uemura and Takeichi, 2006). We focussed our attention on the cytoplasmic binding partners for classical cadherins, which play roles critical to cadherin function, α–catenin, β–catenin and γ–catenin. We found that α–catenin is expressed in most neurons in the spinal cord and appeared to be expressed in all spinal motor neurons, including the LMC, at all stages analysed (st20 to st32) (Figure 2A, B; data not shown). γ–catenin is also found in the majority of LMC neurons on both their soma and axons from the time of their first generation to at least HH stage 32 (Figure 2C-J; data not shown).

Figure 2. α-catenin and γ-catenin are expressed in the lateral motor column during motor neuron migration.

A, B. α-catenin immunofluorescence in the lumbar spinal cord at st 25. The whole spinal cord is shown in (A). The ventral horn is shown in (B). Medial is to the left, as shown in B)

C, D. γ-catenin immunofluorescence in the lumbar spinal cord at st 25. The whole spinal cord is shown in (C). The ventral horn is shown in (D). Medial is to the left.

E γ-catenin is expressed within the LMC, as marked by Foxp1 expression.

F. γ-catenin immunofluorescence in motor axons

G. γ–catenin transcript expression in the ventral horn of stage 21 lumbar spinal cord

H-J Lumbar spinal cord expression of γ-catenin transcript at stage 24 (H), st 26 (I) and st29 (J)

β–catenin is expressed by transitin radial glia in the ventral spinal cord

In contrast to α- and γ-catenin, β–catenin expression was found in a radial pattern within the ventral horn (Figure 3A). β–catenin transcript was predominantly found in cells (~14 per section) in a line roughly parallel to the ventricle within the ventral part of the spinal cord, at the ventricular surface and in the floor plate (Figure 3B-E). This expression persisted from st 21 to at least st 29. Outside of this ventricular zone expression, only low levels of β–catenin could be found in only a small number of cells within the ventral horn, this becoming more apparent after st26. Within more dorsal regions of the spinal cord, β–catenin was detected in the majority of cells (Figure 3C-E). Outside the spinal cord, β–catenin expression was also observed at the ventral root exit points (arrow in Figure 3B).

Figure 3. β–catenin is expressed in transitin radial glia.

A β–catenin immunoreactivity in the ventral horn of a stage 25 embryo

B-E. β–catenin transcript expression in stage 21 ventral spinal cord (B), stage 24 (C) stage 26 (D) and stage 29 (E) lumbar spinal cords.

F. BrdU expression (in brown) with β-catenin transcript (in blue) following a one hour pulse application of BrdU to a stage 23 embryo. The right hand panel is a magnification of the area shown by the black lines.

G-I. Double immunofluorescence of γ-catenin (G, I) and β-catenin (H, I) at stage 25. Arrow in I shows β-catenin expression in radial glia, arrowhead shows γ–catenin expression in motor neurons.

J-R. β–catenin (J, K, M, N, P,Q) and transitin (J, L, M, O, P, R) localisation at stage 26 in the ventral horn (J-L), intermediate part of the ventral spinal cord (M-O) and ventral ventricular zone (P-R). Arrowheads show regions of co-localisation in radial glia. Arrows in N and Q show blood vessel expression of β–catenin.

S. Transitin immunofluorescence in the ventral spainal cord of a st 26 embryo. Arrowheads show individual transitin radial glia processes.

T, U. 0.15 micron confocal sections showing HRP labelling of migrating motor neurons (green) and Transitin (red) expression following retrograde tracing of LMCl cells. T. Cell bodies of five different motor neurons are shown. Note the close apposition of the motor neurons to the transitin fibres. U. motor axons could also be observed in close apposition to transitin radial glial processes.

We asked whether the ventral line of cells that express β–catenin are progressing through the cell cycle. A 1-hour pulse of BrdU was applied to embryos at HH st22 and subsequent immunohistochemistry for BrdU and insitu hybridisation for β–catenin was performed. We found that ~30% of BrdU^+ve cells were co-labelled with β–catenin-suggesting that β–catenin expressing cells are synthesising DNA but that only a subset of progenitor cells express detectable levels of β–catenin (Figure 3F). We also confirmed that γ–catenin is predominantly expressed within motor neurons whilst β-catenin is excluded from motor neurons by double immunofluorescence of the two proteins (Figure 3G-I).

The expression of β-catenin within a subset of progenitor cells suggested that the radial staining of β-catenin could be within radial glia. Within the chick spinal cord, radial glia express the intermediate filament protein transitin (Cole and Lee, 1997). Double immunofluorescence staining indicated a co-localisation of transitin expressing radial fibres and β–catenin within the ventral spinal cord (Figure 3J-R). Within the ventral horn, transitin immunofluorescence at HH st26 showed an average of 14 (modal value; range 12 to 15) glial fibres (Figure 3S), similar to the number of β-catenin cells observed in the ventral ventricular zone. Together, this suggests that β–catenin is predominantly expressed in transitin radial glia in the ventral spinal cord and that these radial glia represent a subset of ventral progenitor cells.

Transitin radial glia mark pathways of motor neuron migration

We further characterised the transitin/β-catenin radial glia by asking whether the pathways of migration of LMC neurons coincide with them. We identified subsets of migrating LMCl motor neurons through injection of the retrograde axonal tracer, Horseradish Peroxidase (HRP), into the dorsal limb mesenchyme at st25. Migrating motor neurons were identified by their location within the LMCm domain at the time of analysis. Confocal analysis of 0.15 micron thin optical sections revealed that in all cases (50/50 HRP^+ve neurons analysed within the LMCm) transitin fibres and migrating motor neurons were closely juxtaposed (Figure 3T). Additionally, we found evidence of motor axons following the paths of transitin radial glia (Fig 3U). This suggests that transitin/β-catenin radial glia mark the pathways of LMC migration during LMC divisional segregation.

Overexpression of γ–catenin leaves LMC organisation unperturbed

We focussed our attention on γ–catenin as motor neurons express it predominantly. We cloned a full-length cDNA of chick γ–catenin and found that the transcript is 86% identical to that of human γ–catenin and its amino acid sequence is 89% identical (94% similar) to that of human γ–catenin. Additionally, chick γ–catenin is 69% identical (89% similar) to chick β–catenin-illustrating the high level of conservation between these two armadillo family members (data not shown). We expressed wild-type γ–catenin by in ovo electroporation (Momose et al., 1999) and confirmed expression of the protein, noting in particular its presence at the apical surface of the ventricular zone (data not shown). Overexpression of γ–catenin (marked by nuclear β-galactosidase immunoreactivity in Fig 4A) or the empty DNA vector had no observable effect on the total number of motor neurons or the position of those motor neurons in the ventral horn or the segregation of LMCl and LMCm divisions (Figure 4A, B; data not shown). This suggested that the levels of γ–catenin are saturating with regard to a role in motor neuron migration.

Figure 4. γ–catenin (L127A) expression disrupts LMC migration and divisional segregation.

A, B. Lack of effect of γ-catenin overexpression on divisional segregation at stage 29. Cells that had been electroporated are marked by β-gal immunoreactivity (green in A). Hb9 (A, B) and Islet-1 (B) mark the MMC, LMCl and LMCm in the ventral horn.

C, D. Expression of γ–catenin (L127A) viewed by γ–catenin immunoreactivity. Electroporated cells are marked by β-gal immunofluorescence (red in D). Arrows show apical expression of γ-catenin at the ventricle surface.

E, F. Disruption of β–catenin apical expression following γ–catenin(L127A) expression. Arrows show regions of this disruption.

G, H. Disruption of ZO1 apical expression following γ–catenin(L127A) expression. Left arrow in H shows ZO1 apical expression on the contralateral spinal cord, right arrow shows the disruption on the electroporated side of the spinal cord.

I, J The ventral spinal cord after electroporation with γ–catenin (L127A) at st 18. Foxp1 (red) and BrdU (green) expression after BrdU application from st23 to st28. No-colocalisation of motor nuclei with BrdU is observed, indicating that all motor neurons had been born before st23 following γ–catenin(L127A) expression. J is a blow up of the area shown in I. Dotted lines in I and J illustrate the midline, arrows in I and J show Foxp1 nuclei stalled in their migration that are unlabelled by BrdU.

K-N. Effects of γ–catenin(L127A) expression on divisional segregation in st29 lumbar spinal cords. K, L. The left hand side of the spinal cord is unelectroporated (β-gal immunofluorescence is absent (L). K. LMCm and LMCl segregate normally on the left hand side as viewed by Hb9 (red ) and Islet-1 (green) immunofluorescence. Following γ–catenin(L127A) expression on the right hand side of the spinal cord, LMCl and LMCm segregation is perturbed. The LMC is spread over a much larger area than the control with LMC cells found close to theventricle and LMCl and LMCm cells are intermingled. Arrows in K show some of the LMC cells stalled in their migration. M and N show the ventral horn of a different embryo electroporated by γ–catenin(L127A) showing that both LMCm and LMCl are affected, the midline as at the left side of the panel. Arrows in M illustrate some LMCm and LMCl cells close to the ventricle surface. I, II and III in K show the regions quantitated in O.

O. Quantitation of LMCl neuron position in bins illustrated in K.

P. Quantitation of distance migrated by γ–catenin(L127A) expressing LMCl neurons compared to those on the electroporated side of the spinal cord that did not express γ–catenin(L127A). Error bars in panels O, P indicate SEM.

Exxpression of a point mutation in γ–catenin

Previous work identified a 29 amino acid region of γ–catenin that is both necessary and sufficient for binding to α–catenin (Aberle et al., 1996). This region is highly conserved between different species with only one replacement (to a similar amino acid) between chicken and human γ–catenin . Additionally, single amino-acid mutations in this region can reduce binding to α–catenin to background levels (Aberle et al., 1996). We reasoned that expression of γ–catenin containing such a mutation might uncouple cadherin/ catenin interaction, thus abrogating cadherin function. We generated a single amino acid substitution of chick γ–catenin of L127-A, γ(L127A), which is located in the α–catenin binding domain of γ–catenin and reduces this binding to less than 2% of the wild-type γ–catenin for the human protein (Aberle et al., 1996). We expressed γ(L127A) by in ovo electroporation, confirming its misexpression by immunofluorescence (Figure 4C,D; electroporated cells are marked by nuclear β-gal staining in D). γ(L127A) expression should result in a delocalisation of adherens junctional complexes, which depend on interaction with the actin cytoskeleton via α–catenin. We find that the apical expression of β–catenin in radial glia in the dorsal spinal cord is disrupted following γ(L127A) expression (Figure 4E, F), consistent with its predicted mode of action. Alpha-catenin binds to the tight junction protein ZO1 and disruption of cadherin function follows disruption of ZO1 binding to the cadherin complex (Itoh et al, 1997; Imamura et al, 1999). We thus followed the localisation of ZO1 protein expression following γ(L127A) expression and again found that its localisation to the apical end-feet of radial glia was disrupted (Figure 4G, H). Together, this data suggests that γ(L127A) disrupts cadherin function most likely through uncoupling the cadherin-catenin complex.

Following γ(L127A) expression the total number of LMCm and LMCl motor neurons (56 ± 5; 58 ± 3 motor neurons per section respectively) was not significantly different to the control side of the spinal cord (62 ± 6; 55 ± 5; per section respectively; p>0.05 student’s t-test) indicating that the general differentiation of LMC divisions was not perturbed. We did, however, observe an ~50% decrease in the number of MMC neurons when γ(L127A) was expressed (25 ± 3 versus 15 ± 2; control versus experimental; p<0.05 student’s t-test). Application of BrdU to embryos from st23 to st27, following γ(L127A) expression, revealed that LMC neurons were born before stage 23, as is found in wild-type embryos (Figure 4I, J). Thus, the major program of LMC differentiation in terms of neuron number within each division and the timing of motor neuron generation is not perturbed by γ(L127A) expression. This finding allowed us to investigate the role of γ(L127A) expression on LMC divisional segregation.

LMC neuron position and divisional segregation is perturbed by γ(L127A) cell autonomously

LMC divisional segregation is normally complete before st29, four days after the first generation of LMC neurons. We thus investigated the effect of γ(L127A) expression on divisional segregation at st 29. Following γ(L127A) expression we observed a striking perturbation of LMC neuron positioning. In contrast to the control, non-electroporated side of the spinal cord (Figure 4K-N), the area encompassing the LMC was greatly expanded (over two-fold) with neurons of both LMCl and LMCm found close to the ventricle. Divisional segregation was also severely disrupted with many the Hb9^+ve/Islet-1^−ve LMCl neurons located in a position medial to the Hb9^−ve/Islet-1^+ve LMCm neurons (Arrows in Figure 4K). We also observed mixing of LMCm and LMCl with LMCl neurons found within the domain normally occupied by LMCm neurons.

We quantitated LMCl cell body position by considering the percentage of neurons that were located in three defined medio-lateral bins in the ventral spinal cord, bin I being most medial and bin III the most lateral (Illustrated in Figure 4K). Expression of γ(L127A) results in a mosaic misexpression due to stochastic incorporation of the electroporated construct (indicated by nuclear-localised β–galactosidase). We could thus quantify the cell autonomy of mispositioning of LMC cells in relation to their expression of γ(L127A) (Figure 4O). 54±3% of LMCl neurons and 20% of LMCm neurons that had acquired γ(L127A) were found in bin I, compared to 12% and 3% respectively on the control side of the spinal cord (p<0.001, student’s t-test). In contrast, 85±4% of HB9^+ve/islet-1^−ve/β–gal^−ve LMCl neurons were located in bin III, similar to that found in the control ventral horn (p>0.1, student’s t-test) (Figure 2K). Additionally, we found that ~25% of HB9^+ve/islet-1^−ve/β–gal^+ve LMCl neurons were found within the LMCm, indicating that divisional mixing also occurs following γ(L127A) expression. These data are consistent with a cell autonomous perturbation of LMC divisional segregation and motor neuron positioning in response to γ(L127A) expression.

We next quantitated the distance migrated by either HB9^+ve/islet-1^−ve/β–gal^+ve or HB9^+ve/islet-1^−ve/β–gal^−ve cells. We found that LMCl cells expressing γ(L127-A) had migrated roughly half the distance of controls (Figure 4P). This suggests that LMC cell body positioning defects are due to a perturbation in the distance migrated by the cells.

Stalled LMCl neurons project axons into the limb normally

The perturbation of LMC neuron positioning prompted us to ask whether motor axon trajectory proceeded normally following γ(L127A) expression. To address this, we injected HRP into the dorsal limb mesenchyme at HH st 28 to trace LMCl cell body position following co-expression of γ(L127A) and GFP, the latter to trace motor neuron cell body and axon position. We found that following γ(L127A)/GFP coexpression the region of the spinal cord occupied by HRP^+ve cells was increased compared to controls (Figure 5A-C). Particularly notable was the presence of HRP labelling in neurons located close to the ventricle (arrows in Figure 5B). This indicated that the mispositioned LMCl neurons still project axons to the dorsal limb. In contrast, axonal GFP was not restricted to the dorsal limb mesenchyme suggestive of normal axon trajectories of LMCm and LMCl cells following γ(L127-A) expression (arrows in Figure 5D-F). Additionally, we noted that within the ventral spinal cord GFP fluorescence was enhanced medially compared to control expression of GFP alone (Figure 5G), consistent with the cell autonomous perturbation of LMCl position following γ(L127A) expression. This further suggests that the normal programme of LMC differentiation occurs following expression of γ(L127A) despite aberrant motor neuron positioning.

Figure 5. Motor neuron projections appear normal following γ–catenin (L127A) expression.

GFP and γ–catenin(L127A) were co-electroporated at stage 18 and HRP was injected into the dorsal limb at stage 29.

A, B. GFP (A) and HRP (B) immunofluorescence in one section. Arrows in B show motor neurons close the the ventricular surface that had projected into the dorsal limb. Dotted lines in A and B show the ventricle surface.

C. HRP retrograde labelling in control embryos. Bars in B and C show the medio-lateral extent of retrogradely labelled motor neurons. Note that this is more than twice as long in B compared to C.

D-G. GFP immunofluorescence is more intense medially in the spinal cord than in controls following γ–catenin(L127A)/ GFP co-electroporation.

(D, E) GFP immunofluorescence (E, F) HRP immunofluorescence.

G. Quantitation of average GFP immunofluorescence in the spinal cord verus that found in the lateral LMC following GFP or γ–catenin(L127A)/ GFP co-electroporation. GFP fluorescence is lower in the lateral ventral horn following γ–catenin(L127A) expression, consistent with a defect in motor neuron migration. Arrows in D-F show motor axon tracks to dorsal and ventral limb-note that GFP is present in both whereas HRP immunofluorescence is exclusively in the dorsal limb tracks. Error bars are SEM.

γ–catenin (L127A) expression does not perturb dorsal interneuron positioning

The predominant expression of γ-catenin is within the ventral horn. We thus asked whether the observed migration defect following γ(L127A) expression is specific to the ventral spinal cord. We investigated the effect of γ(L127A) expression on the positioning of both interneurons and motor neurons within the spinal cord at st29 through Lhx1 expression, a LIM homeodomain transcription factor expressed within the LMCl and also within the majority of interneurons within the spinal cord. We also assessed the positioning of the subset of dorsal commissural interneurons that express Isl-1. Consistent with previous results, following γ(L127A) expression, we detected a large number of Lhx1^+ve cells adjacent to the ventral ventricular surface with a concomitant reduction in the number of Lhx1^+ve cells in the lateral ventral horn (Figure 6A-C). However, we saw no defect in positioning of either Lhx1^+ve or Isl-1^+ve dorsal interneurons following γ(L127A) expression (Right facing arrow in figure 6 B,C). This data suggests that the mispositioning of neurons following γ(L127A) expression is specific to the ventral region of the spinal cord and does not result in a general perturbation of cell body positioning in the spinal cord.

Figure 6. Stalled motor neuron migration results in bucking of the ventral Pax6 domain following γ–catenin(L127A) expression.

A-C. Lhx1 expressing ventral interneurons and LMCl neurons (A-C, illustrated by left pointing arrows in B, C)) and islet-1 cells (I) are found close to the venticle (illustrated by dotted lines in A and B). Electroporated neurons are marked by β-gal immunofluorescence (green in A). Right pointing arrows in B, C show absence of effect on dorsal interneuron populations.

D, E. The ventral Pax 6 (green) progenitor domain buckles following γ–catenin(L127A) expression but the Nkx2.2 (red) domain is unperturbed. Electroporation is marked by β-gal immunofluorescence (blue in D). Dotted lines in D show the ventricle surfaces. E is a magnified image of the ventral progenitor domains of the right hand side spainal cord in D. Note that the electroporated cells (stalled LMC neurons) are close to the ventricle whereas the majority of the buckled ventral Pax6 domain is not electroporated.

F. Lhx1 immunofluorescence focussing on the ventricular zone of the adjacent section to that shown in D & E. F’ shows the position of the Lhx1 cells outlined by the dotted lines in relation to the Pax6 domain of E.

G-I. Transitin immunofluorescence (G, H) following electroporation of γ–catenin(L127A). The right hand side of the spinal cord in G was electroporated. H, I are magnified images of the dotted box in G. Dotted lines in H indicate the ventricle surfaces. I. β-gal immunoreactivity showing electroporated cells.

J-L. β-catenin (J, K) immunofluorescence in radial glia in the ventral horn at stage 25 after γ(L127A) expression marked by β-gal immunoreactivity in J and L.

M, N. Islet-1 (green) and Pax6 (red) immunofluorescence at stage 26 following γ–catenin(L127A) expression showing that motor neurons stalled in their migration are found adjacent to the bucked Pax6 domain (as in F, F’). Motor neuron position relative to the Pax6 domain is therefore normal. β-gal immunoreactivity (blue in N) shows electroporated neurons.

O. Summary of the data in this figure showing the relative positions of transitin radial glia, progenitor domains and LMC neurons following γ–catenin(L127A) expression.

Stalled LMC migration causes the motor neuron progenitor domain to buckle

The location of Lhx-1^+ve, Isl-1^+ve/HB9^−ve and Isl-1^−ve/HB9^+ve cells close to the ventricle following γ(L127A) expression prompted us to investigate the integrity of the spinal progenitor cells within the ventricular zone at these later stages of spinal cord development. We analysed the expression at st28 of homeodomain transcription factors expressed within ventral progenitor domains, specifically Pax6 and Nkx2.2, which label adjacent domains (Briscoe et al., 2000). On the γ(L127A) electroporated side of the spinal cord, the v3 (Nkx2.2) progenitor domain was found in a normal ventral position, adjacent to the floor plate. (Figure 6D,E). In contrast, the ventral part of the Pax6 domain ‘folded’ medially and dorsally such that its more ventro-lateral aspect was now closer to the ventricle (Figure 6D,E). We noted that in many embryos it was only the stalled motor neurons and not the ventral Pax6 domain that was electroporated. This suggested that the buckling of the Pax6 domain is a non-cellautonomous effect most likely mediated by γ(L127A) expression in motor neurons. The position of stalled ventral Lhx1 cells fits between the Pax6 domain and the ventricle surface (Figure 6F, F’). The length of the Pax6 domain as a coherent group of cells was, however, similar to the control side of the spinal cord (239.6±8.4μm vs 240.6±7.8μm, p>0.1 student’s t-test). Additionally, the dorsal part of the Pax6 domain resided in a normal position. At this stage, a small number of Pax6 cells are found in the mantle zone of the spinal cord, presumably having migrated from the ventricular zone. We observed that following γ(L127A) expression, the total number of these cells was not significantly different from the control side of the spinal cord (13±1 versus 13±3 cells per section, p>0.1 chi-squared test). This suggests that the general integrity of the Pax6 progenitor domain is not affected by γ(L127A) expression.

Transitin/β-catenin radial glia still traverse to the ventral horn following γ(L127A) expression

Owing to the bucking of the ventral Pax6 progenitor domain, we investigated the morphology of transitin radial glia fibres in the vicinity of stalled motor neurons. We found that in the ventral spinal cord, transitin/ β-catenin fibres still course from the ventricular zone to the pial surface within the ventral horn (Figure 6G-L). However, the folded nature of the ventral Pax-6 domain resulted in these fibres first taking a ventral and then lateral route (Figure 6G, H). Related to the Pax6 domain, the position of stalled LMC neurons located close to the ventricle were, however, normal (Figure 6M, N). This indicates that motor neurons adjacent to the ventricular surface are not within the ventricular zone itself, as defined by the expression of Pax6. Thus, following γ(L127A) expression, the pathway of migration of spinal motor neurons still follows the course outlined by transitin^+ve fibres (Summarised in Figure 6O).

Cadherin dominant negative expression stalls migration of LMC neurons

The effects of γ(L127A) expression suggests a cadherin dependence of γ–catenin function in LMC neuron migration and divisional segregation. We therefore asked whether uncoupling cadherin function from intracellular binding to γ-catenin also perturbed motor neuron divisional segregation. Expression of a truncation of the extracellular domain of N-cadherin, NΔ390, has been shown to act in a cadherin dominant-negative fashion by sequestering endogenous β- or γ-catenin (Fujimori and Takeichi, 1993). Expression of NΔ390 from st18 caused a phenotype similar to γ(L127A) expression, albeit at a reduced level (Figure 7A-F). In contrast to γ(L127A) expression, we did, however, observe a small decrease (~20%) in the total number of motor neurons cells (64±2 Hb9 cells versus 73±2; p<0.05 chi-squared; 39±2 versus 50±2 Islet-1 cells per section; p<0.05 chi-squared). Similar to γ(L127A) expression, N Δ390 expression left the LMC spread over a greater area, approximately double that of controls. Additionally, we found ~5% of both LMCl and LMCm cells adjacent to the ventricular surface (Figure 7G). We quantitated divisional segregation of LMCl and LMCm and found that LMCl cells were found mingled throughout the LMCm division (Figure 7H), consistent with a perturbation of LMC migration.

Figure 7. NΔ390 dominant negative cadherin expression results in a similar phenotype to γ–catenin(L127A) expression in disruption of LMC divisional segregation.

A-C. GFP, Islet-1 and HB9 immunoreactivity following NΔ390 cadherin expression shows stalled motor neurons and perturbed divisional segregation of the LMC.

D-F. Hb9 and Islet-1 immunoreactivity on the contralateral LMC.

Dotted lines in A-F show the ventricular surface.

Lines in C and F show the medio-lateral extent of the LMC-note that following N Δ390 cadherin expression the LMC spans approximately twice the extent of the contralateral LMC.

G. Percentage of Hb9 and Islet-1 cells adjacent to the ventricular zone following N Δ390 cadherin expression. Contralateral spinal cords have zero cells in similar positions.

H. Divisional mixing index following NΔ390 cadherin expression compared to contralateral spinal cord (student’s t-test of bins 0, 1 and 2, p<0.001. χ² p<0.001 of the entire distribution 2df). Error bars in G, H indicate SEM.

I-K. Pax6 expression following NΔ390 cadherin expression, marked by GFP in I. K is a magnified area illustrating the buckled Pax 6 domain (shown by the arrow) in J.

L-N. Transitin immunoreactivity (M, N) following NΔ390 cadherin expression marked by GFP in L. N is a magnified region of M, shown by the black lines. Arrowheads in N show transitin fibres coursing from the ventricle surface to the pial surface of the spinal cord.

Further, similar to γ(L127A) expression, we observed a dorso-medial folding of the ventral Pax6 progenitor domain. However, we noted that the extent of folding of the ventricular zone following NΔ390 expression was smaller than that following γ(L127A) expression, consistent with the reduced effect of NΔ390 compared to γ(L127A) expression (Figure 7I-K). Additionally, similar to γ(L127A) expression, the pathway of transitin^+ve radial glia mirrored the perturbed LMC neuron position and were still associated with the ventricle surface (Figure 7L-N). Thus, the essential features of the phenotype of γ(L127A) expression on LMC migration are captured by expression of a cadherin dominant negative. Taken together, these results suggest that catenin-dependent cadherin function is necessary for the correct radial migration of spinal motor neurons.

Canonical Wnt signalling does not influence LMC migration

The perturbations of cadherin and catenin function described here have the potential to alter canonical Wnt signalling (Nelson and Nusse 2004). We thus asked whether direct perturbations of Wnt signalling alter motor neuron migration and LMC divisional segregation. We expressed constructs in vivo that have been shown to result in the up-regulation or down-regulation of canonical Wnt signalling (Zhurinsky et al., 2000b; Roose et el., 1999; Krylova et al., 2000). Expression of either wild-type β–catenin (Figure 8A-D) or a truncated version of β–catenin (β–cat-1-ins (Zhurinsky et al., 2000b)) (Figure 8E-H) or a dominant negative GSK3β construct (Figure 8I-L) resulted in no observable difference in motor neuron migration compared to the control, unelectroporated side of the spinal cord. Thus, constructs that upregulate Wnt signalling have no effect on LMC divisional segregation.

Figure 8. Upregulation of Canonical Wnt signalling does not disrupt LMC neuron organisation.

A-D. Overexpression of β–catenin. A. Summary of Wnt signalling. B. Hb9 and Islet-1(2) (B,C) immunohistochemistry after β–catenin expression marked by Haemaglutinin immunoreactivity (D).

E-H. Expression of β–catenin-1-ins construct. E. Summary of effect to increase Wnt signalling. F. Lhx1 and islet-1(2) (F, G) immunoreactivity after β–cat-1-ins expression marked by Haemaglutinin immunoreactivity (H).

I-L Lack of effect on LMC organisation of expression of a dominant negative GSK3β construct. I. Schematic of action of the construct. J-L HA-tag (green in K, L), Islet-1 (green in J, red in K) and Lhx1 (red in J and L) immunoreactivity following electroporation of the construct.

Additionally, we sought to down-regulate canonical Wnt signalling pathway through the expression of either wild-type GSK3β (Figure 9A-D) or constitutively active GSK3β (Figure 9E-H) or a dominant negative TCF construct (Ciani et al., 2004) (Figure 9I-L). Again, we observed no differences in spinal motor neuron migration following expression of these constructs. These data suggest that the observed migration and divisional segregation defects following γ(L127A) or NΔ390 expression are not consistent with a perturbation of canonical Wnt signalling.

Figure 9. Downregulation of Canonical Wnt signalling does not disrupt LMC neuron organisation.

A-D. Lack of effect on LMC organisation of expression of wild-type GSK3β. A. Schematic of action of the construct. B-D HA-tag (green in B, C), Islet-1 (red in B, green in D) and Lhx1 (red in C, D) immunoreactivity following electroporation of the construct.

E-L Downregulation of Wnt signalling pathway by expression of constitutively active GSK3β construct (E-H) or dominant negative TCF transcription factor expression (I-L). E, I Summary of action of each construct. Islet-1(2) immunohistochemistry (F, G, J, K) reveals normal motor neuron migration and divisional segregation following expression of the constructs, marked by Haemaglutinin immunoreactivity (F, H, J, L). Islet1(2) immunofluorescence is lower in LMCl than LMCm.

Cadherin-7 is required for LMC migration and divisional segregation

We have shown that uncoupling general cadherin function intracellularly disrupts divisional segregation by neuronal migration. We sought to manipulate the expression of a single cadherin that was predominantly expressed during LMC migration with little or no expression later in development. To begin to address this, we screened expression of type I and type II cadherin family members for such an expression profile. We found that type I cadherins, including N-cadherin, appear excluded from LMC neurons, instead being found in radial fibres in the ventral horn (Figure 10A-D). In contrast, type II cadherins, including cad-7, cad-12 and cad-20 were expressed in the majority of the LMC at early stages of development (Luo et al., 2006; Figure 10E-L). Both cadherin-12 and cadherin-20 also have prominent expression within a number of motor neuron pools after divisional segregation (Figure 10F, H) (Price et al, 2002). In contrast, cad-7 was predominantly expressed early during LMC divisional segregation and is downregulated in the vast majority of LMC neurons after divisional segregation is complete (st28) (Figure 10I-L; Price et al, 2002). Therefore, cad-7 is expressed within the majority of the LMC neurons during their migration and we thus focussed our attention on its function during LMC divisional segregation (Luo et al., 2006).

Figure 10. Pan motor neuron type II cadherin expression and cadherin-7 siRNA restricts LMC neuron migration and divisional segregation.

A. Pan type I cadherin immunolabelling at st28.

B-D. N-cadherin immunoreactivity at st24 (B), st26 (C) and st28 (D)

E, F. Cad-20 in situ hybridisation in the ventral horn at st 20 (E) and at caudal lumbar regions of st 28 (F)

G, H. Cad-12 in situ hybridisation in the ventral horn at st 20 (G) and at caudal lumbar regions of st 28 (H).

I-L. Cad-7 immunohistochemistry in the ventral spinal cord at st20 (I) and in the ventral horn at HH st 24 (J), st 26 (K) and st 28 (L). Cad-7 appears to be expressed in the majority of LMC neurons during their migration (I-K) and is weakly expressed in only a small subset of motor neurons subsequently (L).

M-P. Cad-7 control shRNA expression. Foxp1 (M, N, P) a pan LMC marker and Hb9 immunoreactivity (O, P) show normal segregation and positioning of LMCl, LMCm and MMC following control shRNA expression, marked by dsRed fluorescence in M. Q-U. Cad-7 shRNA perturbs LMC divisional segregation. Q-T. HB9 (Q, S, T) and Islet-1 (R, T) immunohistochemistry at st28 following expression of cad-7 shRNA revealed by dsRed fluorescence (Q).Dotted lines in Q and T indicate the ventricle surface. Note there are motor neurons close to the ventricle surface.

U. Divisional mixing index following cad-7 shRNA expression compared to cells that had not acquired the construct (p<0.001 for the 0 bin, p<0.05 for the 1 and 2 bins, student’s t-test; χ² analysis p<0.001 2df).

V-X. Islet-1 immunohistochemistry (V, W) at st24 reveals a perturbation of LMCm migration following cad-7 shRNA expression (bracket in V and W) revealed by dsRed fluorescence (V, X); note the paucity of dsRed fluorescence in the lateral LMC.

To address a role for cad-7 expression in spinal motor neuron migration we reduced its expression using a previously characterised and successful shRNA approach (Barnes et al., 2010). Following expression of a control cad-7 shRNA construct, visualised by dsRed fluorescence, both migration of LMCm and LMCl cells and their segregation into divisions appeared normal (Figure 10M-P). DsRed fluorescence was also observed laterally as well as medially in the ventral horn (Figure 10M). In contrast, following expression of the cad-7 shRNA construct, cells that had acquired the shRNA were concentrated more medially (Figure 10Q). These data suggest that the cells expressing cad-7 shRNA were perturbed in their migration within the lateral motor column. We next characterised the position of LMCm and LMCl cells following cad-7 shRNA expression. At st27 cad-7 shRNA expression resulted in HB9^+ve isl-1^−ve LMCl cells located medial to the LMCm (Figure 10R-T). Quantitation of the effect of cad-7 shRNA at st 27 showed divisional segregation of LMCm and LMCl was compromised (Figure 10U). Further, at earlier stages (st25) we observed an increase in the number of isl-1^+ve LMCm cells located medially, close to the ventricular zone (Figure 10V-X). Taken together, these data suggest that knockdown of cad-7 stalls the migration of LMC motor neurons and perturbs LMC divisional segregation.

Discussion

Spinal motor neuron migration to the ventral horn is highly organised. Over 95% of motor neurons are born rapidly over ~24 hours, whereas their migration occurs over a substantially longer timecourse (Hollyday and Hamburger, 1977; Whitelaw and Hollyday, 1983; Lin et al., 1998; Sockanathan and Jessell, 1998; William et al., 2003). Thus, owing to their rapid generation, a backlog of motor neurons waiting to migrate is formed. Our data (Schematised in Figure 11) suggest that γ-catenin-dependent cadherin function is required for the migration of spinal motor neurons.

Figure 11. Schematic summary of results.

A. Schematic of motor neuron migration along pathways of transitin radial glia and divisional segregation and schematic of cadherin-catenin interactions.

B. Schematic of the effects of γ(L127A) on migration and divisional segregation. Cad-7 shRNA disrupts divisional segregation and NΔ390 cadherin expression results in migration and divisional segregation effects similar to γ(L127A) and the cad-7 shRNA.

Transitin radial glia as a scaffold for motor neuron migration

Within the ventral spinal cord, coexpression of transitin and β–catenin identifies a subset of progenitor cells with characteristics of radial glia. Expression of β-catenin within radial glia demarcates two domains of ventral progenitor cells; apical cells that do not express it and more basal cells that do express β–catenin. Radial migration of spinal motor neurons has previously been inferred (Barron, 1946; Wentworth, 1984; Leber et al., 1990; Leber and Sanes, 1995; Eide and Glover, 1996) and we suggest that this occurs on transitin radial glia for two reasons. First, retrograde labelling of migrating motor neurons indicated that their migration paths follow transitin radial glia. There are relatively few transitin glia (~14) in the ventral horn and their juxtaposition to migrating motor neurons seems unlikely to result from chance alone. More tellingly, stalled motor neurons were still found along pathways defined by transitin glia following γ(L127A) expression. This would not be predicted by motor neurons taking the most direct route in their migration and strongly suggests that spinal motor neuron migration follows routes labelled by transitin glia.

Motor Neuron Migration Requires γ-catenin Function

Both β– and γ–catenin bind to classical cadherins and link their extracellular interactions to intracellular signalling (Hirano et al., 2003), particularly via α–catenin, which provides a bridge for cadherin function to the actin cytoskeleton (Abe and Takeichi, 2008). Generally, it is believed that β-catenin is the major transducer of cadherin signalling intracellularly. We were thus surprised to find that β-catenin is absent from spinal motor neurons. Instead, α–catenin and γ–catenin are the predominant catenins expressed in the LMC. Thus, within the chick LMC γ–catenin is the major transducer of cadherin function.

Expression of either γ–catenin or γ(L127A) leaves the total number and timing of generation of LMCm and LMCl cells unaffected. Additionally, motor neurons were labelled by retrograde tracing from the limb following γ(L127A) expression, suggesting that the time required for axon growth to the limb (more than 48 hours, Tosney and Landmesser 1985) had occurred despite motor neuron migration being retarded. Thus, γ(L127A) does not perturb the generation of LMC neurons.

Expression of γ(L127A) resulted in a cell autonomous perturbation of LMC motor neuron position; ~60% of γ(L127A)^+ve LMCl MNs were located close to the ventricle with a further ~25% found within the domain occupied by the LMCm. We found that the distances migrated were less than half those compared to control neurons suggesting that migration of LMC neurons was severely compromised by γ(L127A). Importantly, LMC neurons on the experimental side of the spinal cord that had not acquired γ(L127A) behaved similarly to the control side of the spinal cord. Whilst we cannot rule out an additional role for perturbed β–catenin expression in transitin radial glia in contributing to this migration phenotype, the cell autonomous nature of the effect of γ(L127A) strongly suggests that the predominant role for γ(L127A) is within the motor neurons themselves.

Stalled LMC migration causes buckling of the ventral pax6 progenitor domain

The majority of ventral Pax6 cells express neither β-catenin nor γ-catenin. Instead, transitin radial glia represent around 30% of these progenitor cells and express detectable levels of β-catenin. The general integrity of this progenitor domain and position of generation of motor neurons from it appears normal following NΔ390 or γ(L127A) manipulations. The ventral buckling of the Pax6 domain was often seen in embryos where the Pax6 cells were not electroporated but was never observed when motor neurons were not electroporated. Further, motor neurons that had not acquired the γ(L127A) manipulation reached a normal settling position. Thus, the motor neurons stalled in their migration probably contribute most to the buckling of the Pax6 domain. We suggest that the force required for the buckling of the progenitor domain could arise from the continued mitotic activity of the ventral Pax6 domain cells. BrdU labelling following γ(L127A) expression demonstrated that cell division is not halted. Additionally, the number of Lhx1 cells arrested close to the ventricle surface was greater than the number of Hb9+ve/isl1-ve LMCl cells. Thus, the ventral Pax6 domain continues to generate postmitotic Lhx1 interneurons. This cell division might exert a force rearward away from stalled LMC neurons which would result in the progenitor domain buckling at its ventral extent.

Cadherin involvement in motor neuron migration

Cadherin dominant negative expression (Fujimori and Takeichi., 1993) caused a similar phenotype to that of γ(L127A). We found that type I cadherins were not expressed in LMC motor neurons. Of the type II classical cadherins, cad-7 was expressed predominantly in most LMC neurons during divisional segregation and downregulated in the vast majority of the LMC thereafter. Downregulation of cad-7 by shRNA resulted in a similar migration and divisional mixing phenotype to that observed by NΔ390 expression: motor neurons were found close to the ventricle and LMCm and LMCl divisions were mixed. Cad-7 appears not expressed in ventral transitin radial glia. This further suggests that the predominant role for cadherins in motor neuron migration is within the motor neurons themselves. During review of this paper, Demireva et al (2011) reported perturbed LMC organisation following conditional ablation of both β- and γ-catenin from mouse motor neurons. These findings are in broad agreement with those presented here. However, this conditional ablation approach in mouse revealed a much milder migration phenotype than we show for the chicken. Differences in gene expression, rate of motor neuron generation and migration between chicken and mouse may underlie differences in severity of catenin and cadherin manipulations in these different species.

How could cadherin and catenin function facilitate motor neuron migration? A model has recently been proposed whereby α–catenin acts as a molecular clutch that links substrate adhesion to retrograde actin flow during cell migration (Bard et al, 2008). Our results are consistent with a model where both γ(L127A) and NΔ390 constructs perturb γ–catenin function intracellularly within motor neurons, disrupting cell migration, likely mediated at least in part through cad-7-based linkage to the migration machinery. Whether retrograde actin flow is a dominant force in motor neuron migration as it is in other cell types will require further characterisation of the dynamics of cadherin and catenin linkage (Yamada et al, 2005; Drees et al, 2005) to the actin cytoskeleton during motor neuron migration.

Neuronal nucleus formation through regulated cadherin function

The LMC is organised as a neuronal nucleus. Neuronal nuclei are generally found in more evolutionarily ancient regions of the CNS. It seems likely that the mechanisms of nucleogenesis will reveal similarities and differences with organisational mechanisms in evolutionarily newer regions of the CNS, such as the lamination of the cortex (Rakic, 2006; Lui et al, 2011). Radial glia provide a scaffold for excitatory projection neuronal migration in the cortex and are a major progenitor cell-type of the ventricular zone. Additional progenitor cells, generated from radial glia, are found more basally in the sub-ventricular zone (Noctor et al., 2004). Within the spinal cord, radial glia delineate pathways of spinal motor neuron migration, although, in contrast to the cortex, spinal transitin glia represent only a small proportion of the ventral progenitor cells. Additionally, these radial glia are basal to the ventricular progenitor cells. One major difference in the generation of motor neurons from that observed to drive lamination of the cortex is the rate at which LMC neurons are generated. Rapid LMC generation results in a backlog of neurons waiting to migrate. In contrast, neurons in the cortex destined to populate more superficial layers are born sequentially and migrate in a ordered fashion to generate the layers of the laminated cortex (Noctor et al., 2004). It is likely that the availability of spinal radial glia represents a rate limiting step for motor neuron migration.

We have demonstrated that early, pan-motor neuron cadherin function is required for LMC divisional segregation through neuronal migration. Subsequently, differential type II cadherin function is required for motor neuron pool formation (Price et al., 2002; Patel et al., 2006). Our results therefore suggest a prolonged requirement for cadherin function in all phases of motor neuron cell body organisation. How could cadherin-dependent radial LMC migration and later motor pool sorting be coupled? Recent work has suggested that perturbation of reelin signals disrupts later phases of motor neuron organisation (Palmesino et al., 2010). Reelin can regulate cadherin function in the control of neuronal migration in the cortex (Franco et al., 2011). Thus, reelin signalling may provide a mechanism for dissociation of cadherin and catenin mediated radial migration from a later involvement of cadherin expression in motor pool sorting.