A role for Nogo-A as an inhibitor of CNS regeneration is well known, where expression is restricted to the axon ensheathing oligodendrocytes [7–10]. Nogo-A found within the plasma membrane of oligodendrocytes inhibits neurite outgrowth by promoting growth cone collapse. It was unexpected, therefore, when we and others found this inhibitory protein expressed during CNS development, and in particular, within developing neurons [15–17, 19, 25, 26]. Our study profiles the expression of NOGO-A as it relates to key phases of CNS development: induction, proliferation, migration/differentiation, and network establishment. While the embryonic expression of NOGO-A has been reported [15, 25, 26], most fetal studies have focused on its role as an inhibitor of CNS regeneration [17, 18, 13, 27]. Our results imply that during development Nogo-A has a function that is different from its "adult" role of inhibiting CNS regeneration.
Conservation of the Nogo-A Sequence
When aligned with human, rat, mouse and xenopus amino acid sequences, the chicken NOGO-A sequence has a high degree of identity (>75%) in the carboxyl-terminal, reticulon-specific portion that is present in all three major isoforms (A, B and C). Multiple alignment of widely different species also revealed eight conserved regions (CR1-8) within the Nogo-A specific coding sequence (Fig 2). This conservation implies preservation of domains that are functionally significant. Previous functional domains have been identified in regions common to all Nogo isoforms [Nogo66 in the C-terminus, NiRΔ2 (rat-aa 59–172) in the shared Nogo-A/B regions], and in the Nogo-A specific region [NiGΔ20 (rat-aa 544–725)], but have been linked to inhibitory roles in neurite outgrowth and cell spreading . The NiGΔ20 inhibitory domain overlaps three highly conserved regions identified in our analysis (CR4-6) suggesting a conserved function across taxa. Interestingly, NiRΔ2 does not correspond to one of the highly conserved regions and previous reports have demonstrated poor preservation of this region in amphibians . Collectively, these data may indicate a mammalian specific role for NiRΔ2. Several highly conserved regions remain in this long protein without known functional roles. It is likely that one or more of these regions are important for Nogo-A's participation in development, however, further studies are needed to validate this prospect.
Differential Regulation of NOGOduring Development
Expression of the NOGO-C isoform was broad and persistent in the developing brain by northern analysis and was in accord with adult expression profiles, where NOGO-C can be found in neurons as well as skeletal muscle . The larger (NOGO-A and -B) isoforms showed progressive expression with development (Fig 1C). Interestingly, we identified two NOGO-A transcripts in the chick, which confirms an earlier report identifying two NOGO-A bands by immunoblot . Sequence analysis against the published chicken genome verified the two bands as alternative splice products, with the smaller band resulting from a secondary splice site in exon 1, similar to what is observed in Xenopus . It is unclear what functional advantage these two splice variants offer, however, there appears to be differential stage-specific expression by northern analysis which suggests stage-specific roles.
A Role for Nogo-A in CNS induction
The onset of neurulation is defined by formation of the neural plate. NOGO-A is induced in the neural plate during its formation and persists in structures derived from neuroectoderm. Furthermore, our data demonstrate that transformation of non-neural ectoderm into presumptive neuroectoderm by ectopic FGF is characterized by rapid induction of SOX3 (a "pre-neural" marker) and NOGO-A (Fig 8B,E). Recent microarray analysis supports this finding with the identification of NOGO-A in human and porcine neural precursor cells . These findings suggest that Nogo-A participates in specifying neuronal identity and/or differentiation.
However, non-neuronal NOGO-A expression was also present in the primitive streak and node, structures which precede neural plate formation (HH5, Fig 3). NOGO-A expression persists within these structures until their regression. This differential induction indicates tissue specific and temporal-spatial regulation of NOGO-A. Interestingly, there was asymmetrical right-sided NOGO-A expression within the primitive node (Hensen's node) and scant expression in the notochord and presumptive neural groove immediately overlying the notochord. This unique expression pattern is complimentary to the expression of Sonic Hedgehog (SHH), a morphogen critical to patterning of the developing neural tube (See additional file 1). NOGO-A also exhibits focal expression associated with somite formation at what may be the sites of future dorsal root ganglia and spinal nerves. We and others have shown that dorsal root ganglia have robust NOGO-A expression (See additional file 2) [15, 13].
The cells of the neural plate, primitive streak/node and somites all demonstrate rapid growth and patterned structural transformations. It is possible that Nogo-A may have neural-specific roles and additional broader roles related to morphogenesis. Changes in cell shape and migration are orchestrated by cytoskeletal reorganization. Nogo-A signaling has been linked to the downstream activation of the small GTPase, RhoA . In development, the Rho family, through Rho kinases, regulates cytoskeletal reorganization associated with changes in cell shape such as the formation of axons and dendrites [32–34]. Moreover, the published expression of Rho Kinase α  overlaps the expression of NOGO-A shown here. Inhibition of Rho kinases results in disrupted formation of the brain/neural tube, reduced caudal extension of the primitive streak and loss of left-right asymmetry. The asymmetric expression of NOGO-A within the primitive node, and its expression within the primitive streak may indicate an additional role for this protein in organogenesis and tissue identity.
Additional roles in later CNS development
Many molecules critical to CNS development have multiple functions. Molecules such as FGFs, SHH, BMPS and Wnts, once thought to play isolated roles in development, have now also been linked with several steps in neurulation and CNS development including formation of the neuronal circuitry through axon guidance and synaptogenesis . The expression of NOGO-A at key phases of CNS development from neural induction to definitive network establishment suggests that this factor may also have multiple functions.
To examine potential roles of Nogo-A during specific phases of CNS development we utilized the chick optic tectum. This highly structured region serves as an ideal model for understanding formation of the vertebrate brain, attaining a high level of complexity while completing most of its maturation prior to hatching. NOGO-A was present in the tectum at all stages of embryonic development observed, nevertheless, clear patterns emerged when looking at embryonic stages correlating to the specific phases of CNS development.
Nogo-A during Proliferation and Migration
Proliferation is a critical part of early development of the expanding neural tube. We saw no correlation between NOGO-A expression and Proliferative Nuclear Cell Antigen (PCNA) positivity when comparing neuroepithelium during peak proliferative and relative quiescent stages (E5 vs E10, respectively) . Thus, it is unlikely that Nogo-A has a role in proliferation.
From E6 onward a migratory zone of post-mitotic neurons can be visualized in the tectum . NOGO-A expression in the E7 tectum was homogenously expressed across the pre-migratory generative zone, the expanding migratory layer, and the post-migratory, first neuronal lamina. Immunostaining for neuronal and glial cells linked NOGO-A expression to the soma of neurons, however, NOGO-A expression was not limited to the neuronal population actively undergoing migration. Thus, our data does not support a role for Nogo-A limited to directing the migration of neurons. Rather, the continued expression of NOGO-A within all populations of developing neurons is further support of a role marking early neural identity and may be related to the state of maturity/differentiation. This concept is in agreement with previous studies that reported a high neuronal NOGO-A mRNA expression during differentiation of the spinal cord , and a down regulation of NOGO-A mRNA following migration and terminal differentiation of rat olfactory neurons  and cerebellar granule cells .
Nogo-A during Neuronal Differentiation
Neuronal differentiation is also characterized by the outgrowth of neurites and the formation of synapses. Neuritogenesis and synaptogenesis coupled with later refinement of connections by cell death and neurite pruning comprise the process of "network establishment." The Stratum Griseum Centrale (SGC) of the chick optic tectum is sparsely populated by large, multi-polar principal efferent neurons with extensively branched dendrites and robust projecting axons . By E10, extension of axons and dendrites is abundant in the SGC and intense expression of NOGO-A can be seen within these large neurons. Heightened NOGO-A expression can also be seen in nearby tectal associated nuclei, which are conspicuous by their large, projecting neurons.
By E14, the first synaptic junctions of the tectum are observed. However, their numbers rise drastically between E18 and the first hours after hatching with more intense retinal input . Loss of retinal input to the tectum can be disturbed by embryonic enucleation of the chick eye optic anlagen. Deafferentation causes neuronal degeneration of the SGC through loss of synaptic input and prevention of forming synaptic connections . Correspondingly, there is an overall decrease in NOGO-A expression within the SGC and the tectal associated nuclei suggesting NOGO-A expression is dependent upon synaptic activity (Caltharp, unpublished data).
Increased Nogo-A immunoreactivity has been demonstrated at the onset of axon growth in developing rat olfactory neurons  and also within sprouting dendrites of cerebellar Purkinje cells . Interestingly, over expression of Nogo-A within COS cells leads to the formation of long processes that resemble neurites . As mentioned earlier, Nogo-A can regulate RhoA dependent cytoskeletal reorganization which is intensely active during neuronal differentiation and neuritogenesis. Collectively, these data combined with our findings provide strong support for a Nogo-A specific role in neuronal differentiation and neuritogenesis.
Nogo-A during the Transition to Adulthood
At E10, NOGO-A expression within the tectum is strictly neuronal. Oligodendrocyte related myelination in the chick tectum takes place between E12–E17  and accordingly, unmyelinated fiber tracts within the tectum are distinctive for an absence of NOGO-A. By E20, these same fiber tracts have begun to acquire myelination by oligodendrocytes and are now positive for NOGO-A expression. Studies marking glial differentiation found Nogo-A to be expressed at all stages of oligodendrocyte development [18, 13] and our data found NOGO-A expression within cells of tectal associated nuclei that were neither positive for neuron, astrocyte or glial progenitor specific stains. This later NOGO-A expression within smaller cells of the tectum is a likely sign of increased oligodendrocyte formation and may reflect the beginning transition of expression from neuronal differentiation and network establishment to myelin derived maintenance of the circuitry.