Hoxd10 overexpression in the chick LS neural tube shifts the segmental complement of motoneuron subtypes toward LMCl
Prior studies suggest that Hoxd10 plays an instructive role in defining broad LMC character and position (Carpenter et al., 1997
; Shah et al., 2004
). However, recent analyses of mutants with the loss of both Hoxc10 and Hoxd10 function (Wu et al., 2008
) raise the possibility that Hoxd10 also specifically promotes LMCl development. In a normal embryo, most LMCl motoneurons can be defined by their expression of Lim1 and their projections to dorsal limb musculature (Tsuchida et al., 1994
). We noted that rostral LS segments, which express high levels of Hoxd10, contain a larger complement of Lim1+ motoneurons than caudal segments at stage 29. This difference is likely to persist because rostral segments contain substantially more dorsally projecting motoneurons than caudal segments at stage 36 (). These observations raise the possibility that Hoxd10 is instrumental in defining this distinguishing feature of rostral LS segments.
To test this hypothesis, we asked if overexpression in chick LS segments would influence LS motoneuron subtype complement in a manner predicted by normal expression. Full-length hoxd10
, along with the ires-egfp
sequence from pIRES2-EGFP (Clontech), was cloned into vectors that drive gene expression under the postmitotic motoneuron-specific Hb9 promoter (Arber et al., 1999
; Thaler et al., 1999
). A construct expressing EGFP alone under the same promoter was used as a control. Constructs were transfected into the neural tube via in ovo electroporation before motoneurons are born (stages 14-16, Hollyday and Hamburger, 1977
) and most embryos were sacrified either at early stages of motoneuron differentiation (stages 22-early 25) or after motor column formation (stages 29-30). While evidence of transfection was often present in multiple LS segments, we focused mainly on LS2 for assessments of motoneuron subtype complement.
Endogenous Hb9 is expressed by all motoneurons immediately following their exit from the ventricular zone (Tanabe et al., 1998
; Thaler et al., 1999
, Arber et al., 1999
). It is widely expressed at stages 22-24 but by stage 29, maintained only in a subset of motoneurons (William et al., 2003
). At stages 24, evidence of Hoxd10 overexpression and colocalization with EGFP was readily detected, although a decreasing medial to lateral gradient of Hoxd10 expression suggests that the earliest born (more lateral) motoneurons may have begun to lose Hoxd10 expression (). To assess the effect of this early increase in Hoxd10, LS2 sections from stage 23-early 25 Hb9
d10 embryos were immunolabeled with antibodies against Isl1(2), a panmotoneuron marker, and Lim1, a marker for LMCl motoneurons (Tsuchida et al., 2004), as well as EGFP (). In this and all subsequent experiments, counts of motoneurons were made on three non-adjacent sections per embryo with 4-6 embryos making up each experimental group (). In stage 23-early 25 Hb9
d10 embryos, we found no difference in the mean total number of motoneurons per section between transfected and non-transfected sides (). However, a significant increase in the mean number of Lim1+ motoneurons was present (). Further, while Lim1+ motoneurons made up only 29% of the total motoneuron population on the transfected side, they made up 42% of the transfected (EGFP+) population (). These data are compatible with the hypothesis that Hoxd10 overexpression initiated an early fate switch from medial (LMCm or MMC) to lateral (LMCl) motoneuron differentiation pathways. To discriminate between an LMCm-to-LMCl vs. an MMC-to-LMCl switch, transfected LS2 sections were immunolabeled with Foxp1, a Forkhead domain transcription factor that is normally expressed by both LMCl and LMCm motoneurons, but not by MMC motoneurons (Rousso et al., 2008
; Dasen et al., 2008
). Total numbers of Foxp1+, Isl1(2)+ cells were similar on transfected and non-transfected sides, suggesting an LMCm-to-LMCl switch (, ).
Figure 2 Transfection with an Hb9 promoter-driven Hoxd10 construct (Hb9d10) yields transient overexpression of Hoxd10 and a transient increase in Lim1+ MNs. A-B. Increased Hoxd10 expression and co-localization with EGFP in an LS2 section from a stage (more ...)
Quantification of motoneuron transcription factor expression in control and Hox-electroporated chick LS segments
Transfected LS2 sections were also examined in stage 29 Hb9
d10 embryos. Unlike in stage 23-25 embryos, visible signs of Hoxd10 overexpression were absent () and counts of both total motoneuron numbers and Lim1+ populations were similar on transfected and non-transfected sides (, ). These observations suggest that Hoxd10 overexpression under the Hb9 promoter is downregulated at early stages of motoneuron differentiation and that fate changes initiated by Hoxd10 overexpression are transient. The latter implies, in turn, that the early specification of Lim1+ motor neurons is labile. Normally, many LMCl motoneurons in LS2 retain high levels of Hoxd10 through stage 29 (see ) and it is possible that these cells require sustained expression of Hoxd10 to maintain a Lim1+ phenotype.
To determine if sustained Hoxd10 overexpression effects long-term changes in subtype complement, Hoxd10 was cloned into the pMES vector. This vector utilizes a β-actin promoter to drive expression in all neural cells and includes an ires-egfp
to report protein expression (see Eberhart et al., 2002
). In LS2 sections from β-actin
d10 embryos, transfected (EGFP+) cells co-express high levels of Hoxd10 through stage 29 (). A construct expressing EGFP alone under the β-actin promoter was used as a control.
LS2 sections from stage 29 β-actin
d10 embryos were initially stained with anti-Lim1 and anti-Isl1(2) to identify and quantify Lim1+ LMCl motoneurons on transfected and non-transfected sides of the cord (). In a normal embryo, LS2 contains a population of LMCl motoneurons that are Lim1-, but can be distinguished from LMCm and MMC motoneurons by their lack of Isl1 expression (Lin et al., 1998
). Staining with a digoxigenin-tagged in situ probe against Isl1
in combination with anti-Isl1(2) was thus used to specifically identify Isl1+ LMCm + MMC populations (). Counts revealed two notable effects of Hoxd10 overexpression with the β-actin
d10 construct (, ). First, total motoneuron numbers were reduced by 26% on transfected sides of the cord, with no comparable reduction in β-actin
control embryos. Second, this reduction disproportionately affected the Isl1
+, LMCm+MMC population such that subtype proportions in the motoneuron population as a whole were shifted in the LMCl direction. Isl1
+ proportions decrease from 51% to 40% on non-transfected vs. transfected sides while Lim1+ proportions increased from 35% to 43% (, ). Counts of Lim1+ proportions made in LS5 of Hb9
d10 embryos showed a similar effect (see ).
To examine subtype proportions within transfected populations alone, LS2 sections from β-actin
control and β-actin
d10 embryos were triple labeled with anti-Lim1, -Isl1(2), and -EGFP antibodies (). As above, anti-Lim1 staining allowed the identification of most LMCl motoneurons (). To roughly identify and isolate LMCm + MMC motoneurons from the Lim1- LMCl population, we capitalized on the distribution of fluorescence intensity normally seen in the Isl1(2)+ population. “Brightly” stained Isl1(2)+ cells (Isl1(2)high
) appear in medial portions of the motor columns and correspond spatially to the position of Isl1
+ motoneurons (see , compare to ). “Lightly” stained populations (Isl1(2) low)
) are located laterally, corresponding to the positions of Lim1+ and Lim1- LMCl populations. We utilized a fluorescence intensity threshold function (see Materials and Methods) to isolate and count Isl1(2)high
motoneurons, assuming the number of Isl1(2)high
cells to be an approximation of LMCm and MMC, the two Isl1+ populations. As can be seen in , EGFP+ motoneurons in β-actin
d10 embryos showed an increase in the LMCl:LMCm+MMC ratio that paralleled the increase found in the motor columns as a whole (see ).
Many EGFP+ motoneurons in β-actin
d10 embryos showed two additional LMCl features: lateral position and dorsal axonal projections. We quantified the position of EGFP+ motoneurons by superimposing a tripartite grid over individual LS2 sections (). In β-actin
d10 embryos, most transfected motoneurons were located laterally in accord with an LMCl identity, while transfected motoneurons in β-actin
control embryos showed a more widespread distribution. If the segmental complement of motoneurons has shifted towards an increase in LMCl proportions, then one might expect an increase in the proportion of EGFP+ axons that project to dorsal limb regions in β-actin
d10 embryos. To address this possibility, the paths of EGFP+ and neurofilament+ axons were examined in the anterior (crural) plexus region at stage 29 and at an early stage of muscle nerve formation (stage 26-27). In β-actin
control embryos (n=3, stage 27, n=3, stage 29), EGFP+ axons contributed substantially to both femoral and obturator nerve trunks that project to anterior dorsal and ventral limb regions, respectively (). While the EGFP+ axonal population is likely to have included some sensory axons originated from transfected neural crest, these observations suggest that our protocols generally resulted in the transfection of both dorsally and ventrally projecting neurons. In half (n=3/6) of the stage 26-27 β-actin
d10 embryos examined, a pattern similar to control was found; however, in the remaining stage 26-27 embryos and in all stage 29 embryos (n=6), most EGFP+ axons appeared to diverge at the crural plexus to project along dorsal pathways ().
In sum, these data clearly indicate a shift in motoneuron complement in the LMCl direction following transfection with the β-actin
d10 construct. The interpretation of these changes however, is confounded by the substantial motoneuron cell death observed. One possible explanation is that the early and high levels of Hox produced by transfection with the β-actin
d10 construct had a toxic effect on all motoneurons. This effect, in turn, may have partially masked a promotion of LMCl subtype differentiation, an influence in line with the observed increase in Lim1+ cells in stage 23-25 Hb9
d10 embryos. It is equally possible, however, that high Hox levels were particularly toxic to those motoneurons that withdrew from the cell cycle early and contained the least diluted foreign DNA. Since LMCm motoneurons are normally born before LMCl motoneurons (Hollyday and Hamburger, 1977
), LMCm motoneurons may have been preferentially lost due to this toxicity. Given this possibility, we sought to complement our overexpression analyses with an assessment of motoneuron subtype development using a Hoxd10 loss-of-function paradigm.
LMCl motoneuron numbers are reduced in rostral lumbar segments in a Hoxd10 loss of function mouse mutant
In the mouse embryonic spinal cord, Hoxd10 is expressed in lumbar (L) segments (Dolle and Duboule, 1989
) and inactivation of Hoxd10 alone or in combination with other Hox10 genes leads to shifts in the position of the thoracic-lumbar boundary (Carpenter et al., 1997
; Lin and Carpenter, 2003
; Tarchini et al., 2005
; Wu et al., 2008
). To assess the specific effects of Hoxd10 loss on motoneuron subtypes, we chose to examine subtype distribution in a Hoxd10 loss-of-function mutant in which disruption of the Hoxd10 locus was accomplished via insertion of a neo cassette in exon 2. (Carpenter et al., 1997
). Previous descriptions of this mutant indicated alterations in the axial and appendicular skeleton, and peripheral nerve changes, with no apparent changes in either Hoxd9 or Hoxd11 expression (Carpenter et al., 1997
). Our central aims were to determine if LMCl motoneuron numbers were reduced with inactivation of Hoxd10 alone, mirroring our Hoxd10 overexpression data, and to determine if such a reduction was restricted to a specific rostrocaudal LS subdomain.
Homozygous mutant embryos and wildtype littermates were collected at E12.5-E13.5, just after lumbar motoneurons are normally born but before the peak period of cell death (Lance-Jones, 1982
). To distinguish subtypes, spinal cord sections were double-stained with anti-Hb9 and Isl1(2) (Arber, 1999
) or with anti-nNOS and Isl1(2) (Wu et al., 2008
). In both mutant and wildtype embryos, the spatial arrangement of subtypes was similar (). Visceral motoneurons were identified as nNOS+, Isl1(2)+ cells in the posterior thoracic (T) and rostral lumbar (L) cord (VM, ). LMCl motoneurons were uniquely identified as Hb9+, Isl1(2)- cells located in the ventral horn cluster. (It should be noted that the Isl1(2) antibody appears to stain only Isl1+ cells in the mouse embryo. See also Wu et al., 2008
.) Hb9+, Isl1(2)- cells occupied a lateral position corresponding to the position of motor pools projecting to dorsal limb muscles (). LMCm motoneurons were Isl1(2)+, ± low-level expression of Hb9+, and occupied dorsal and medial positions corresponding to the position of many ventrally projecting motor pools (). MMC motoneurons showed high expression of both Isl1(2) and Hb9 and occupied a ventromedial position (). The border between LMCm and MMC motoneurons was identified as a visible space between the two clusters and/or a sharp increase in the level of HB9 staining (see ).
Figure 4 Lumbar LMCl MNs are reduced in number in a loss-of-function Hoxd10 mutant mouse embryo at E12.5-E13.5. A-H. Representative sections through lumbar motor columns from Hoxd10 +/+ and Hoxd10 -/- embryos. A-B. Sections stained with anti-nNOS and anti-Isl1(2) (more ...)
Counts of individual subtypes were made on one side of the cord in sections taken at the mid level of spinal nerve exit for T12 through L6, sections corresponding roughly to the T12/T13 through LS6/S1 segment borders. The most marked differences between mutant and wildtype littermates were significant decreases in LMCl numbers at rostral levels (T12/13-L2/3; ). At T12/13-LS1/2, small increases in VM numbers accompanied these LMCl decreases (). Although VM increases were not significant, these observations hint at a possible conversion from LMCl to VM. Since a prominent VM is characteristic of thoracic segments, these data suggest a shift from a rostral lumbar to a posterior thoracic identity. In mutants, caudal increases in LMCm motoneuron numbers and decreases in MMC numbers were occasionally found (), an observation also compatible with the idea of a shift in LMC position.
The above observations match prior morphological evidence of a 1/2 segment shift in segment identity in mutant neonates (Carpenter et al., 1997
). However, a shift in the position of the LMC as a whole cannot fully account for observed differences between mutant and wildtype numbers. In mutants, there was a significant decline in LMCl numbers when compared to wildtype littermates (+/+ = 281 ± 26 cells, -/- = 245 ± 15 cells, p=0.046). There was, however, no decline in total LMC numbers (+/+ = 645 ± 55 cells, -/- = 639 ± 42 cells), suggesting that an increase in LMCm accompanied the LMCl loss. As they stand, our histograms indicate that changes in LMCl and LMCm numbers occur at different segmental levels (). However, if one assumes a 1/2 segment shift in the position of the mutant LMC and corrects for it by shifting the mutant curves to the left, declines in LMCl numbers at rostral levels (approximately LS2-4) appear to be accompanied by small increases in LMCm numbers, suggestive of conversion from an LMCl to an LMCm identity. In sum, Hoxd10 mutants show evidence of both a shift in the positioning of the T/L boundary and a decrease in the size of the rostral LMCl. The latter observation favors the hypothesis that Hoxd10 biases subtype development towards an LMCl phenotype in rostral LS or lumbar segments.
In the chick embryo, ectopic Hoxd11 expression leads to a reduction in LMCl motoneuron numbers
In the normal chick embryo, Hoxd11 expression is restricted to caudal LS segments. To address Hoxd11 function, we asked if ectopic expression of Hoxd11 in rostral LS segments would lead to the appearance of features normally characteristic of caudal LS segments. Since motor columns within caudal LS segments differ from those of rostral LS segments in that they contain fewer LMCl motoneurons, we asked first if ectopic Hoxd11 expression in rostral LS segments would lead to a decrease in the numbers and/or proportion of LMCl motoneurons. Electroporations were carried out as for Hoxd10 overexpression studies using Hb9- or β-actin-driven constructs. Analyses of subtype complement were made at stage 29 in both Hb9
d11 and β-actin
d11 embryos. Ectopic expression of Hoxd11 was evident in the former () as well as the latter (data not shown).
Figure 5 Ectopic expression of Hoxd11 via transfection with an Hb9 promoter construct (Hb9d11) leads to a shift in MN subtype complement in favor of medial subtypes. A-C. Ectopic Hoxd11 expression at rostral LS levels at stages 24 and 29 and co-localization (more ...)
d11 embryos, transfected sides of the LS2 cord showed a >40% decrease in Lim1+ (LMCl) motoneurons (). This decrease exceeded a small overall decrease in the total Isl1(2) motoneuron population (see ; ). Further, few if any transfected motoneurons were located laterally, in the normal domain of Lim1+ motoneurons (). Concomitant with a decrease in Lim1+ motoneurons, LS2 sections from Hb9
d11 embryos showed significant increases in the numbers and proportions of Isl1(2)high
cells on transfected vs. non-transfected sides (). This increase was also observed in sections probed for Isl1+ mRNA ().
When transfected (EGFP+) populations were examined in isolation, we found decreases in Lim1+ motoneuron proportions that paralleled those in the motor columns as a whole but were more extreme (). For example, the ratio of transfected Lim1+ motoneurons in Hb9
d11 embryos vs. Hb9
control embryos was 12%/34%, whereas the equivalent ratio for total Lim1+ motoneurons neurons on transfected vs. non-transfected sides of Hb9:d11 embryos was 25%/37%. Similarly, larger proportionate increases in Isl1(2)high
motoneurons were evident in Hb9
d11 embryos when transfected populations were assessed in isolation. These findings suggest that the changes initiated by ectopic Hoxd11 expression arose in part by a cell-autonomous mechanism.
d11 embryo, the decrease in the size of the transfected motor column was substantial, as with β-actin
d10 embryos (). Nevertheless, in β-actin
d11 embryos, as in Hb9:d11 embryos, the Lim1+ population was disproportionately reduced in both LS2 and LS5. LS2 sections from β-actin
d11 embryos showed significant increases in the proportion of Isl1
+ motoneurons on transfected vs. non-transfected sides (). These findings stand in contrast to findings obtained in β-actin
d10 embryos, in which early born, Isl1
+ motoneurons were disproportionately reduced and late-born Lim1+ motoneurons were preserved. We suggested earlier that the preferential loss of Isl1
+ motoneurons might be due to a toxic effect on early born cells that underwent few cell cycles after electroporation. The finding of a substantial reduction in late born, Lim1+ motoneurons in β-actin
d11 embryo suggests that any toxic effects of early and high levels of Hox may be unrelated to time of cell cycle withdrawal and that Hoxd10 and Hoxd11 influence progenitors and/or postmitotic cells in quite different ways.
Our data suggest that ectopic Hoxd11 has shifted the subtype complement of rostral LS segments to resemble that of more caudal segments: Lim1+ (LMCl) motoneurons become less prominent, Isl1+ (LMCm+MMC) motoneurons, more prominent. Given that the Isl1+ molecular profile is shared by multiple motoneuron populations, especially early in the differentiation process (Pfaff et al., 1996
), we next sought to examine more distinctive markers of caudal segment identity.
Ectopic Hoxd11 expression leads to the appearance of novel axonal projections from rostral LS motoneurons to a caudal thigh muscle
The caudilioflexorius is a thigh muscle normally innervated by LMCm motoneurons located exclusively within the Hoxd11 domain (LS6-8, Landmesser, 1978
; Hollyday, 1980
). To determine if motoneurons in rostral LS segments would project to the caudilioflexorius after transfection with Hoxd11, we mapped the positions of this motor pool on transfected and non-transfected sides of Hb9
d11 embryos. Muscle injections were performed at stages 29-30 using rhodamine-conjugated dextran as a retrograde tracer. Caudilioflexorius pools on transfected and non-transfected sides were similar in size (n=7, mean pool size on transfected side=210 ± 36 cells, mean pool size on non-transfected side=197 ± 51 cells), and the vast majority of dextran+ motoneurons were located in a normal rostrocaudal position. However, the number of dextran+ cells located in segments rostral to LS6 was increased on transfected sides (). When expressed as mean percentage of total, dextran+ cells in LS3-5 made up 2±1% of the caudilioflexorius pool on non-transfected sides, but 15.4±6% on transfected sides (p=0.051). It is important to point out that EGFP+, dextran+ motoneurons were few in number, most EGFP+ motoneurons being located medial to the caudilioflexorius pool. However, rostrally positioned dextran+ cells on transfected sides appeared to be EGFP+ (). These observations suggest that a small number of transfected motoneurons in rostral segments may have acquired a novel caudal LS identity and been able to reach the caudilioflexorius.
Figure 6 MNs in rostral LS segments appear to demonstrate a caudal (caudilioflexorius) identity after transfection with Hb9d11 but also show abnormalities in cell positioning and axon outgrowth. A. Horizontal section showing caudilioflexorius pools (dextran+ (more ...)
To examine the possibility that Hoxd11 transfection had a global effect on motor pool organization, we mapped the position of motoneurons projecting to the ventral shank complex. In a normal embryo, this group of muscles is innervated by LMCm motoneurons in segments located both within and outside the Hoxd11 expression domain (LS3-7, Landmesser, 1978
; Hollyday, 1980
). In the transfected sides of Hoxd11-electroporated embryos, ventral shank pools were normally positioned on the rostrocaudal axis with no indication of a rostral extension (, n=7). These pools contained a few EGFP+, dextran+ motoneurons at the medial edge of the dextran+ pool but, as seen above, most EGFP+ motoneurons occupied a more medial position (). In contrast, in control embryos (transfected with HB9 driven EGFP alone), EGFP+ motoneurons were often more laterally positioned and contributed in greater numbers to ventral shank pools (n=3, ).
In sum, the above data implicate Hoxd11 in specifying characteristics unique to a caudal LS subdomain. However, we were surprised to find so few EGFP+ motoneurons projecting to the ventral shank or caudilioflexorius muscle. We think it unlikely that most EGFP+ axons were projecting to other limb muscles because dextran injections at other sites also yielded low numbers or a complete absence of EGFP+, dextran+ cells (n=4 injections of full dorsal + ventral thigh and shank musculature; n=3 injections of the adductors of the ventral thigh; n=4 injections of the iliofibularis of the dorsal thigh; n=4 injections of the ischioflexorius of the ventral thigh).
To address this issue further, we examined the peripheral course of EGFP+ axons in a subset of Hb9
d11 embryos at stages 26-27 (n=6) and stage 29 (n=6). EGFP distribution was examined either in whole mount at the time of sacrifice or in sections stained additionally with anti-neurofilament. EGFP+ axons made substantial contributions to major limb nerve trunks () and to axial nerves. However, the distal extent of EGFP+ axons was often less than that of non-transfected, neurofilament+ axons (). Further, despite the fact that assessments of LIM profiles indicated a reduction in Lim1+ (LMCl) motoneurons, no qualitative difference was evident in the distribution of EGFP+ axons to dorsal vs. ventral nerve trunks (see ). These observations suggest that axon outgrowth from many transfected motoneurons was delayed and/or that these axons were unable to detect and respond appropriately to peripheral guidance cues.
Two additional observations support the notions of abnormal axon-target interactions and a potential developmental delay. The ETS transcription factor, Pea3, is normally expressed in caudilioflexorius motoneurons in response to peripheral signals (Lin et al., 1998
). While Pea3+, EGFP+ motoneurons were occasionally found in Hb9
d11 embryos (), they were very rare (approximately 1-3 cells per embryo, n=6 embryos). Thus, despite our observations of novel projections to the caudilioflexorius, it would appear that, in most cases, peripheral interactions were not sufficient to induce Pea3 expression. The guidance molecule, Slit2, is normally expressed widely by early differentiating motoneurons but becomes restricted to motoneuron subsets by stage 29 (Holmes and Niswander, 2001
; Holmes et al., 1998
; Lance-Jones, personal observations). We chose to examine Slit2 expression in LS sections from a subset of stage 29 Hb9
d11 embryos because of studies implicating Slit-robo signaling in both neuronal migration and motoneuron pathfinding (Geisen et al., 2008
; Hammond et al., 2005
). In these embryos (n=7), Slit2 expression was noticeably higher than normal in regions corresponding to the position of most transfected cells (), suggesting an arrest in maturation and a possible molecular correlate to the medial bias of transfected motoneurons.
In Hoxd11 transfected segments, motoneurons demonstrate a molecular profile suggestive of a suppression of LMC differentiation
Our finding of a marked decrease in LMCl motoneurons with ectopic Hoxd11 expression in rostral LS segments, coupled with the rostral extension of the caudilioflexorius pool, suggest a caudalization of segment identity. However, the abnormalities in motoneuron position and projections described above prompted us to characterize the molecular profiles of motoneurons within transfected segments in greater detail.
We show herein that misexpression of Hoxd11 in rostral LS segments increases the proportion of motoneurons expressing the LIM transcription factor Isl1. This marker is normally expressed by all newly generated motoneurons but maintained only in mature LMCm and MMC motoneurons (Tsuchida et al., 1994
; Pfaff et al., 1996
). To differentiate between LMCm and MMC, we examined expression of Foxp1 and the LIM transcription factor Lim3, which have recently been shown to act in opposition to one another to direct motoneurons towards an LMC or MMCm fate, respectively (Rousso et al., 2008
; Dasen et al., 2008
). In stage 29 Hb9
d11 embryos, we noted a reduction in the number of Foxp1+, LMC motoneurons on the transfected side of the spinal cord (), though some transfected cells did express Foxp1 (arrows in ). In contrast, Lim3+ cells appeared to be present in increased numbers and in a less clustered pattern than normal (). Numerous EGFP+, Lim3+ cells were evident in individual sections () and counts of Lim3+ motoneurons (Lim3+, Isl1/2+ cells) indicated a small but significant increase on transfected vs. non-transfected sides (). In sum, the data presented here suggest that (1) ectopic Hoxd11 increased the proportion of MMCm motoneurons at the expense of the LMC, and (2) settling patterns may have been altered. However, the size of the Lim3+ population increase was considerably smaller than the decrease in the Foxp1+ population, suggesting that some motoneurons took on an alternate fate.
Figure 7 Segments transfected with Hoxd11 show decreases in cells with an LMC molecular profile and increases in cells with profiles characteristic of MMC motoneurons and V2a interneurons. A-C. Distribution and numbers of Foxp1+, Isl1(2)+, LMC MNs on non-transfected (more ...)
Several investigators have recently discussed the existence of lateral MMC (MMCl) cells at limb-innervating levels (Luria and Laufer, 2007
; Rousso et al., 2008
; Dasen et al., 2008
). These motoneurons express neither Foxp1 nor Lim3, but do express high levels of Isl1, and the POU transcription factor, Scip. In order to include this population in our analyses, we examined expression of Scip in a subset of stage 29 Hb9
d11 embryos. Prior studies (see Rousso et al., 2008
) suggest that Scip is highly expressed by MMCl motoneurons, although it is not an exclusive marker. It may additionally be expressed at low levels by MMCm motoneurons, by a small, dispersed population of Foxp1+ LMCm motoneurons at all LS levels, and by a discrete dorsolateral pool of Foxp1+ LMCm motoneurons at caudal LS levels (Luria and Laufer, 2007
; Rousso et al, 2008
). Following electroporation with Hb9
d11, we observed an increase in the total number of Scip+ motoneurons in LS2 (). We found no increase in Scip+/Foxp1+ motoneurons (), implying that the increase in Scip+ expression affected the MMC exclusively. Two observations suggest that this increase impacts MMCl motoneurons. Many Scip+ motoneurons expressed high levels of Isl1, as assessed through fluorescence intensity measurements following anti-Isl1(2) staining (, bottom half of stacked graph). Furthermore, while most Scip+ transfected motoneurons coexpressed Lim3, some did not (arrows in ). Taken together, these observations suggest a specific increase in the MMCl. Interestingly, we also noted that caudal segments (LS7-8) normally possess an expanded population of MMC motoneurons ().
In sum, these data suggest that ectopic Hoxd11 caudalizes the rostral LS cord in two ways: by instructing motoneurons to project to a caudal target (the caudilioflexorius), and by promoting the development of the MMC at the expense of the LMC. The observed decrease in the size of the LMC (Foxp1+ population), however, continues to exceed increases in both MMC cell types (Scip+ and/or Lim3+), implying that some motoneurons failed to differentiate into a recognized, mature phenotype.
Finally, it should be noted that the transfected sides of stage 29 Hb9
d11 embryos appeared to show an increased number of Lim3+, Isl1(2)- cells within or just dorsal to the motor columns (see ). In normal embryos, V2a interneurons occupy a similar position, are Lim3+, Isl1(2)-and Chx10+, and arise from a progenitor domain neighboring that of motoneurons (see Ericson et al., 1997
; Briscoe et al., 2000
). To identify this population, we stained sections from stage 29 Hb9
d11 embryos with anti-Chx10 as well as anti-EGFP. A few Chx10+ cells were EGFP+, but the vast majority of Chx10+ cells were EGFP- (). Counts of the latter revealed a small but significant increase on transfected vs. non-transfected sides of the cord (mean number of Chx10+, EGFP- cells per section=70±5 on transfected side, 54±2 on non-transfected side, n=4 embryos, 3 sections per embryo, p=0.003, ). These data raise the possibility of a non-cell autonomous effect of ectopic Hoxd11 on V2a interneurons; however, a detailed characterization of interneuron profiles at different stages will be needed to address this possibility further.
Ectopic Hoxd11 downregulates the expression of RALDH2
We next sought a mechanistic explanation for observed shifts in motoneuron subtype distribution in Hoxd10- and Hoxd11-electroporated embryos. Prior studies have suggested that motoneuron-derived retinoic acid (RA) plays a critical role in the establishment of the LMC and later, the LMCl (Solomin et al., 1998
; Sockanathan and Jessell, 1998
; Sockanathan et al., 2003
; Ji et al., 2009
), and have linked expression of the RA synthetic enzyme, retinaldehyde dehydrogenase 2 (RALDH2), with Hox function in brachial spinal regions (Dasen et al., 2003
; Vermot et al., 2005
). We therefore hypothesized that Hoxd10 and Hoxd11 may regulate subtype distribution through modulation of RALDH2 expression.
Because prior studies of motoneuron-derived RA focused primarily on brachial levels, we first assessed RALDH2 patterns in the normal LS cord. At stage 23-24, RALDH2 is expressed at all LS levels, but only by Isl1/2+ motoneurons that have reached definitive motor column regions (). By stage 29, RALDH2 expression is limited to particular motoneuron groups and varies by segment (). In LS2, RALDH2 expression is restricted to a lateral crescent-shaped cluster, corresponding positionally to the LMCl. In LS4, the domain of RALDH2 expression has shifted to medial regions and overlaps with the area of Isl1(2)high cells (LMCm). Expression levels gradually taper in more caudal segments – by LS6 motoneuron RALDH2 is barely detectable.
Figure 8 RALDH2 expression is reduced in chick LS segments with ectopic expression of Hoxd11. A-F. Normal patterns of RALDH2 expression among MNs (Isl1(2)+ cells) within LS2, LS4, and LS6 sections at stage 24 (A-C) and stage 29 (D-F). G-I. RALDH2 and Isl1(2) expression (more ...)
To examine the effects of Hox misexpression on RALDH2, sections from mid-LS (LS3-4) segments of stage 23-24 Hb9
Hox and β-actin
Hox and stage 29 β-actin
Hox embryos were stained with antibodies targeting RALDH2 and Isl1(2). The mean pixel intensity of RALDH2 staining within motor regions was determined using NIH ImageJ. In order to correct for any differences in motor column size on transfected and non-transfected sides, regions containing high Isl1(2) expression were manually circumscribed and mean pixel intensity of RALDH2 staining determined for that region alone. The circumscribed area comprised the entire motor column at stage 23-24. In stage 29 sections, it corresponded to medial motor column regions (see ). We observed no significant change in RALDH2 expression following transfection with Hoxd10 constructs (). In embryos transfected with Hoxd11 constructs, however, we noted a significant (p<0.01) decline in mean pixel intensity of RALDH2 staining on the transfected side of the cord (). Furthermore, while Hoxd10-transfected motoneurons were often RALDH2+ in rostral LS segments, there was little if any overlap between EGFP and RALDH2 expression in Hoxd11-transfected segments (). These data reveal that ectopic Hoxd11 leads to a downregulation of the expression of RALDH2, and raise the possibility that endogenous Hoxd11 prevents or arrests formation of the LMC and LMCl in caudal segments by decreasing RALDH2, and as a consequence, the local concentration of RA.