We have shown that NSCs derived from mouse embryonic cortex and spinal cord have similar proliferative abilities, but have significant differences in gene expression that are maintained in vitro and thus are likely to be cell intrinsic. We found that several genes previously implicated in NSC regulation were not differentially expressed by cortical and spinal cord derived neurospheres, suggesting that the overall genetic regulatory mechanisms of regionally distinct NSC populations is similar. Additionally, we have identified genes that are enriched in spinal cord neurospheres. Further studies can determine which of these genes are expressed specifically by multipotent NSC and which are expressed by neuronally and/or glially restricted progenitors as well as which genes are expressed by ventrally and dorsally derived neural stem and progenitors.
Surprisingly, we found two populations of spinal cord derived NSCs: one that expresses the cell surface antigen LeX and markers that are differentially expressed between spinal cord and cortical derived neurospheres; and one that does not express LeX, nor markers of regional identity. We provide evidence that these populations are lineage-related with the LeX negative NSCs giving rise to LeX positive NSCs, but not vice-versa.
NSC behaviors are similar for cortical and spinal cord derived NSC
In this study we found that the overall stem cell characteristics of self-renewal and tripotency are similar amongst cortical and spinal cord derived NSCs. Spinal cord neural stem cells are not responsive to EGF at embryonic day 11. This inability to generate neurospheres in response to EGF alone at early developmental time points is consistent with the rostral causal gradient of EGF-R expression as shown by Rao and colleagues 
. Previous studies of brain derived NSCs have also found that bFGF responsive NSCs are present prior to EGF responsive NSCs 
. In addition to comparable mitogen responsiveness, clonal neurosphere forming ability is similar between cortical and spinal cord derived NSC. Furthermore, the expression of selected genes shown to regulate or be expressed by NSCs is not different by RT-PCR or by microarray. This suggests that the overall genetic mechanisms regulating NSC behaviors are similar between cortical and spinal cord derived NSCs. The largest group of differentially expressed genes are involved in patterning, including many homeobox genes, demonstrating that patterning and NSC are closely tied together. Additionally, several differentially expressed genes are involved in sensing and responding to external environments, including genes involved in cell migration and cell adhesion suggesting that regionally distinct NSC may be apt to respond to region specific niches.
Neural precursors maintain anterior-posterior patterning
Our studies demonstrate that some patterning persists in spinal cord-derived NSCs and suggest that there is a fundamental difference between brain and spinal cord NSCs. These findings are in seeming contradiction to some previous studies that demonstrated a lack of putative spinal cord markers in cultured progenitors. For example, one study cited a lack of detectable Hoxd1 or Hoxb9 in cultured spinal cord progenitors as evidence of the lack of regional identity 
. However, Hoxd1 has not previously been described as expressed by spinal cord derived precursors, and Hoxb9 is expressed by 0.3% of spinal cord derived precursors, which is potentially below the limit of detection for traditional RT-PCR 
. Furthermore hoxb9 is expressed by committed motor neuron progenitors that may not be present in undifferentiated neurospheres 
. Therefore, selecting appropriate markers of spinal cord identity is necessary to determine whether regional identity is maintained in vitro. Here, we have presented a set of Hox genes that are enriched in spinal cord derived neurospheres that can be used in future studies as markers of regional identity.
The development of the CNS from a single sheet of neuroepithelium requires tight temporal and spatial regulation of cell type generation. Previous work by Temple and colleagues demonstrated that the temporal pattern of neurogenesis preceding gliogenesis is maintained by NSCs in vitro 
. It may therefore not be surprising that regional identity is also cell intrinsic and maintained in vitro. The data presented here, indicate that there is a persistence of the spinal cord specific genes, Hoxc10 and Hoxd10 in serial, clonal cultures. Validating our results using clonal derived, multiply passaged neurospheres, ensures that the gene expression differences we observed were due to either direct differences in NSC gene expression or in genes expressed by NSC progeny rather than other cells that are contaminating the neurosphere cultures. These data support the hypothesis that at least some aspects of spinal cord identity are encoded in the NSC at the times examined, and point to the importance of discovering mechanisms that mediate this identity.
Not all aspects of regional identity, however, are maintained in culture, indicating that there is some plasticity in regionalization. Gabay et al. (2003) demonstrated that cultures of NSCs from either the dorsal or ventral spinal cord rapidly lose their identity in vitro, gaining markers of the other region 
. Although we did not perform our dissections in such a way as to separate dorsal from ventral cord, when we examined gene expression in clonally derived neurospheres, we found expression of several dorsal- ventral markers, consistent with the notion that dorsoventral identity is not maintained. A lack of retention of molecular regionalization has also been described when others have examined characteristics of NSC derived from different brain regions. For example, Emx2, a forebrain-expressed homeodomain factor, is expressed ectopically in neurospheres derived from non-forebrain regions 
. In addition, dorsal brain derived progenitors begin to express genes associated with ventral identity and clonal brain derived neurospheres express markers of multiple dorsoventral precursor domains 
. In addition, Hack et al. (2004) demonstrated a down-regulation of dorsal and ventral specifying transcription factors in neurospheres derived from different brain regions 
The maintenance of rostrocaudal, but not dorsoventral, patterning has been demonstrated in transplantation studies where lateral ganglionic eminenece derived precursors differentiated into host region (dorsoventral) specific neurons in the diencephalon and mesencephalon but continued to express Bf1, a telencephalic marker 
. However, there is a limit to this seeming plasticity. When brain-derived progenitors are placed in culture, the homeodomain genes that they express are indicative of being brain, rather than spinal cord-derived as shown here and by others 
. Furthermore, a lack of molecular regionalization, does not necessarily translate into a loss of regional identity. Horiguchi et al., (2004) showed that neurospheres derived from different brain regions, that expressed the same region specific transcription factors, had distinct proliferation rates and differentiated into neurons specific to the region from which the progenitors were isolated 
. Thus while not all aspects of regional identity are immutable, some aspects of rostrocaudal identity are maintained by NSCs.
One potential criticism in assigning a particular gene as being cortical or spinal cord enriched is that different regions have somewhat different developmental timing sequence. One might propose that a gene that is expressed at one point in the development of an early developing region may be expressed at later times in a later developing region, and not represent true region-specificity. Therefore, in the current study, we examined gene expression in neurospheres derived from multiple developmental stages. Our data indicate that several markers of spinal cord and cortical neurospheres maintained their expression at all stages and times in culture examined. These observations suggest that the differences we observed were in fact based on the region of origin rather than different developmental process that were occurring within the brain and spinal cord at the time NSCs were isolated (or at the same embryological age).
The differences between brain and spinal cord NSCs also seem to be carried through to the tumors that they could potentially give rise to. We, and others, have isolated stem cell-like cells from CNS tumors, consistent with the hypothesis that mutations in NSCs or progenitors derived from them cause tumors 
. In an elegant study, Taylor et al., described these cancer stem cells in ependymomas 
. Their gene expression studies demonstrated strong differences in genes, including homeodomain proteins, expressed by brain and spinal cord derived tumors. We found significant overlap between our lists of differentially expressed genes (Supplemental Table 5
). Of note, we did not find any overlap between genes enriched in cortical ependymomas and spinal cord derived neurospheres nor overlap between spinal cord ependymomas and cortical derived neurospheres. Analysis of our gene expression data in normal murine NSC revealed similar sets of differentially-expressed genes, indicating both that ependymomas do likely arise from a regionally specified stem or progenitor cell and that the regional gene expression differences we observe here are likely to be of relevance to human spinal cord neural stem and progenitor cells.
Heterogeneity amongst NSC populations
We were able to generate clonally passagable, tripotent neurospheres from both LeX positive and LeX negative cells. The discrimination of cells based on their expression of LeX resulted in two different cell populations. LeX positive cells express many markers of spinal cord identity, which are largely absent from LeX negative cells. We found that there is a lineage relationship between LeX negative and positive NSCs (). LeX negative NSCs were able to self-renew as well as generate the LeX expressing NSCs. Since most differentiated cells do not express LeX, it would be expected that a LeX positive cell would give rise to both LeX negative (differentiated) and LeX positive (progenitor) cells, which is indeed the case. However, LeX negative spinal cord NSCs not only self-renew, but also give rise to LeX positive NSCs. Furthermore, although LeX negative NSCs do not express the typical spinal cord homeodomain genes, the clonal neurospheres that arise from these cells do. These data demonstrate that a LeX negative cell, one with an as yet identified spinal cord patterning program, gives rise to the LeX positive NSC population, one with explicit spinal cord properties (). It will be important to define what mechanisms underlie this regional identity and trigger the expression of region-specific genes.
Lineage relationship of spinal cord derived NSCs.
We were not able to definitively isolate the LeX negative NSC population directly from developing spinal cord tissue. We believe that this is because the majority of LeX negative cells in primary tissue are differentiated cells and the percent of LeX negative neural stem cells in primary tissue is below the limit of detection for cell sorting. The exceedingly small percentage of LeX negative NSCs likely present in primary tissue, coupled with the harshness of cell sorting has made the isolation of LeX negative NSCs in vivo problematic. The identification of additional markers to markers to enrich for this population will potentially enable verification of this population in vivo. We believe that the existence of a LeX negative NSC population is not an artifact of tissue culture as there are significant differences in the gene expression of LeX positive and negative NSC. The differences in gene expression between LeX expressing and non-expressing NSCs demonstrate that these different populations are not simply the result of instability of LeX expression. Additionally, the lineal relationship we have shown here provides further evidence of an additional stem cell population that is present in the embryonic mouse spinal cord that is not present in the brain.
Implications for neural repair
Our observations demonstrate that there are fundamental differences between spinal cord and brain-derived NSCs and identify some of the characteristics that are specific to spinal cord NSCs. This raises the possibility that spinal cord NSCs possess information that would make them more likely to produce spinal cord appropriate cell types and therefore be more likely to successfully replace cells and/or integrate into damaged host spinal cord. Furthermore, we do not yet understand the implications of the discovery of the different, but related LeX negative and LeX positive NSCs. It is possible that the LeX negative NSCs will be less restricted and differentiate into a broader range of cell types or be more expandable in culture, thus making them more useful for repair. Our studies also will serve as a springboard to identify genes and pathways that regulate spinal cord NSC proliferation and differentiation—pathways that may be different from those utilized by brain NSCs. This understanding may allow for enhanced production of spinal cord NSCs from pluripotent cells, such as embryonic stem cells, as well as an improved ability to stimulate repair from endogenous NSCs following spinal cord injury.