Written by: Nabhojit Banerjee

Though the developmental process of the nervous system is an extraordinarily complicated one, there have been a few mechanisms that appear to be of particular importance. Specifically, concentration gradients of particular proteins called morphogens have been known to implement control over cell differentiation. Though other mechanisms of action are known, it is often anticipated, due to circumstantial suggestions such as location of secretion and in vitro assays, that concentration gradients of particular proteins implicated in nervous system development are the most likely parameter through which those proteins elicit their effects.

Recently, however, the work done by Varadarajan et al 2017 and Andrews et al 2017 has suggested that such assumptions might be misguided more often than once thought. Varadarajan et al found that the protein netrin-1 acts through localized accumulation on cell surfaces through short-range secretion by neural progenitor cells rather than through a concentration gradient established by long-range secretion from the floor plate as was previously thought.

Andrews et al found that Bone Morphogenetic Protein (BMP) can signal interneuron specificity through a mechanism whereby the identities of multiple versions of BMP are used in a “mix and match” manner solely through ligand-receptor binding. Taken together, these results indicate that there might be a number of ways in which proteins can influence the developmental process, rather than in just one stereotypical way, even for those proteins once thought to be likely candidates for being morphogenic actors.

This is a fundamental advance because concentrations gradients have been shown to be but one tool used in the developmental process rather than the only (or even primary) one for molecules once thought to operate solely through morphogenetic activity—the use of the proteins is what seems to be important, not the specific manner in which they are used.

Concentration gradients of protein molecules were first demonstrated to control neural patterning during development with the discovery of the Shh (sonic hedgehog) gene by Krauss et al 1993, Echelard et al 1993, and Riddle et al 1993. Shh was identified as a homolog of hh (hedgehog), a gene discovered by Nusslein-Volhard et al 1980 found to control the segmentation of the Drosophila embryo. Shh is first secreted by the embryonic notochord in order to induce formation of the floor plate (FP) on the ventral side of the neural tube.

Subsequently, the floor plate itself secretes Shh. A concentration gradient of Shh is established form the ventral side of the embryo to the dorsal side. Particular concentrations of Shh along the gradient correspond to the activation of different combinations of transcription factors, including those of the Pax, Olig2, Nkx, and Dbx families (Shh also interacts with other molecules to elicit effects, such as hedgehog-interacting protein and vitronectin).

It is thought that Shh acts through the intermediary Gli proteins (primarily Gli2 and Gli3 in vertebrates, with Gli2 being a promoter of gene expression and Gli3 being an inhibitor). The graded presence of Shh is converted into graded activation profiles of Gli2 and Gli3, ultimately resulting in clean boundaries between cell-fate endpoints. As found by Charron et al 2003, Shh also seems to influence axon migration behaviour, acting as a guidance cue.

Again, this is done in a graded manner, with low concentrations of Shh acting as an attractor of axons and higher concentrations acting as a repellent. Thus, both in the morphogenic and migratory aspects of the developmental process of the nervous system, concentration gradients of Shh are clearly important mediators of the extremely complex and precise processes that must occur for development to be executed correctly. Similar mechanisms were found for molecules such as retinoic acid and bone morphogenetic protein (Casci 2008 and Gamez et al 2013).

Until the past decade more short-range local effects and mechanisms (or other effects and mechanisms altogether) of certain proteins circumstantially seemingly similar to Shh were, for various reasons, not as readily considered as the possibility that they were morphogens. However, Varadarajan et al found that a compound once thought to follow the usual model of operating through a long-range diffusion gradient in fact appears to achieve its effects through another mechanism.

Varadarajan et al studied the protein netrin-1, a molecule known to direct the migration of axons toward the ventral midline in the developing embryo. Based on its presence at the floor plate (Kennedy et al 1994) and in vitro studies (de la Torre et al 1997, Ming et al 1997, and Sloan et al 2015), it had long been hypothesized that a netrin-1 concentration gradient secreted by the floor plate was likely responsible for directing axons in a very similar manner as Shh.

Based on the fact that netrin-1 mRNA is also present in neural progenitor cells in the ventricular zone, however, Varadarajan et al decided to study whether these cells could also be involved in eliciting axon-directing effects. In order to do this, Varadarajan et al used a number of genetically engineered reporter mice to selectively remove sources of netrin-1 in the developing embryos. First, the effects of removing the floor plate (and thus netrin-1 produced by the floor plate) were examined by producing Gli2-/- mice, in which, due to disruption of the activity of the Gli2 trascriptional regulator (which transduces Shh signaling), the floor plate (FP) is ablated, thus eliminating FP-derived netrin1 (but, importantly, ventricular zone (VZ) cells are unaffected, and so VZ-derived netrin1 Is still present). In tracking the migration of spinal neurofilaments in E11.5 mice, Varadarajan et al found that the neurofilaments in Gli2-/- migrate in the same manner as control littermates.

By contrast, Gli2/netrin1 double mutants, in which ventricular zone neurons are also ablated (thus also removing VZ neurons), exhibit very aberrant neurofilmaent migrations. Next, using the Shh::Cre and Pax::Cre transgenic lines in combination with the netrinflox/netrinflox allele, netrin-1 loss-of-function was induced in both the floor plate and in ventricular zone neurons. Again, it was found that absence of netrin-1 derived from the floor plate did not seem to have an effect on neurofilament migration while absence of netrin-1 derived from ventricular zone neural progenitors caused aberrant migration (100% increase in neurons incorrectly innervating the ventricular zone rather than avoiding it).

Finally, it was found that netrin-1 could not be detected in the dorsal-most spinal cord, but is deposited on the pial surface of the developing embryo by spinal progenitor cells. Furthermore, netrin1 is absent from the pial surface in netrin1(lacZ/lacZ) mutants (in which netrin1-guided axon guidance is extremely aberrant)). This leads to the likelihood that VZ-derived netrin1, through its interaction with the basement membrane protein laminin, achieves its axon-guiding effects through hapotaxis (the directed growth of cells along an adhesive surface) instead of through a diffusible concentration gradient as once thought.

Another case that was once held to be emblematic of the concentration gradient via diffusion model has been that of the molecule bone morphogenic protein (BMP). BMP is important throughout the developing body, and has profound effects on the development of the nervous system. BMP gradients are found in contraposition to Shh gradients. Where Shh concentration is high, BMP concentration is low and vice versa. Partly because BMP is secreted from the roof plate of the dorsal midline, a seemingly corresponding structure to the floor plate of the ventral midline (from where Shh is secreted), it had been thought that BMP worked exclusively as a morphogenic gradient in the same way as Shh (Lee et al 1999).

Studies in Drosophila (Kwan et al 2016; and Bier et al 2015), zebrafish (Tribulo et al 2003 and Tucker et al 2008) and the developing telencephalon (Watanabe et al 2016) have provided corroborating support that BMPs can act as morphogens. However, while it was known that BMP was required for directing dorsal spinal cord patterning, there was no evidence for the assumption that BMP patterns the dorsal spinal cord in the same manner that Shh patterns the ventral spinal cord, especially since multiple BMPs were found to be present (a concentration gradient should only require one BMP).

Andrews et al investigated the role of BMP’s in neural development with this fact in mind and ultimately found that BMP does, in fact, seem to operate in a manner quite distinct from the morphogenic activity of Shh. Andrews et al used the developing chicken embryo and cultured mouse stem cells as model systems for their study. First, they determined that Bmp4 and Bmp 7 were the only BMPs expressed at early stages in the developing chicken embryo.

Therefore, they hypothesized the Bmp4 and Bmp7 were the main two BMPs responsible for directing interneuron specification in the dorsal spinal cord. Subsequently, they used in ovo electroporation to misexpress Bmp4 and Bmp7 to see whether they could affect specification of distinct populations of interneurons (this was done by tracking phosphorylated (activated) states of the known transcription factor complements of specific interneuron types using immunohistochemistry).

Both Bmp4 and Bmp7 up-regulate the phosphorylation of Smad1/5/8 protein, albeit at different levels (65% and 30% for Bmp 4 and Bmp7 respectively). Moreover, Bmp4 and Bmp7 seem to have differential effects for a number of different interneuron types, with Bmp7 consistently up-regulating Mafb+ RP cells and decreasing the number of Pax2+ dI4 cells, and Bmp4 consistently increasing the number of dI1, dI2, and dI3 cells. These results suggest the BMP activity is highly signal-specific, and depends most on BMP ligands binding to receptors, instead of acting through concentration gradients.

In order to further support this model, Andrew et al established an in vitro protocol to assess whether BMPs operate in a similar fashion in mouse ESCs. They found that a different complement of BMPs was present in the mouse dorsal spinal cord, including BMP5, BMP6, BMP7, and GDF7. It was found that, as in vivo, different BMPs direct different dorsal spinal fates, with mouse BMP6 acting as the functional equivalent of chicken BMP7.

Finally, morphogenic activity of BMPs was assessed in the dorsal spinal cord. Andrews et al altered the level of induced BMP4 signaling by, through i in ovo electroporating, establishing a concentration series consisting of different ratios of the CAG::gfp and CAG::Bmp4 vectors into chicken embryos, keeping the total concentration of DNA constant at 500 ng/μl. Ratios used included 99:1, 19:1, and 9:1 (all of CAG::Gfp: CAG::Bmp4). As the concentration of CAG::Bmp4 increased, pSmad1/5/8 also increased between 20% to 130% compared to control electroporations.

Most importantly, different concentrations of Bmp4 were able to direct the same range of cellular fates. All conditions resulted in increased numbers of dI1s, dI2s and dI3s—the effects became progressively larger as the concentration of BMP4 increased. Thus, lower levels of BMP4 did not promote a more ventral-dorsal identity as predicted by the morphogen models; only efficiency was improved. These results were corroborated in vitro. BMP4, BMP5, BMP6 or BMP7 were tested to see whether they can direct EBs towards different dorsal spinal fates.

They observed that the concentration of a given BMP controls the efficiency by which it drives its specific range of cellular identities, just as in in vivo. Specifically, BMP4 can direct EBs to express Lhx2 (dI1) and Isl1 (dI3), and increasing the concentration of BMP4 drives EBs to express progressively higher levels of both Lhx2 and Isl1. Similarly, increasing BMP6 levels results in progressively higher levels of Msx1 and Isl1. In no case did they observe that low concentrations of BMPs can direct ventral-dorsal patterning and that high concentrations can direct dorsal-most cellular identities as predicted by the morphogen model—only efficiency of expression was changed.

The findings of Varadarajan et al and Andrews et al clearly demonstrate that even though morphogenic (concentration-mediated) activity of proteins is certainly an important component of the developmental process as a whole, it is not the only way in which proteins can orchestrate the patterning of the nervous system and should not be over-anticipated even when circumstance or previous data makes such a mechanisms “likely.” This fundamentally advances our understanding of nervous system development because it both expands and refines our expectations when searching for new information about the involvement of proteins in future experimental studies.

 

References:

1. Riddle RD, Johnson RL, Laufer E, Tabin C (1993). Sonic hedgehog mediates the polarizing activity of the ZPA. Cell. 75 (7): 1401–16.
2. Krauss S, Concordet JP, Ingham PW (1993). A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell. 75 (7): 1431–44.
3. Echelard Y, Epstein DJ, St-Jacques B, Shen L, Mohler J, McMahon JA, McMahon AP (1993). Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell. 75 (7): 1417–30.
4. Nüsslein-Volhard C, Wieschaus E (1980). Mutations affecting segment number and polarity in Drosophila. Nature. 287 (5785): 795–801.
5. Charron F, Stein E, Jeong J, McMahon AP, Tessier-Lavigne M (2003). The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance. Cell. 113 (1): 11–23.
6. Casci R (2008). Retinoic acid passes the morphogen test. Nature Reviews Genetics. 9 (7): 45-48.
7. Gamez B, Rodriguez-Carballo E, Ventura F (2013). BMP signaling in telencephalic neural cell specification and maturation. Frontiers in Cellular Neuroscience. 7(87): 156-78.
8. Kennedy TE, Serafini T, de la Torre JR, Tessier-Lavigne M (1994). Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal chord.Cell. 78(3):425-35.
9. de la Torre JR, Höpker VH, Ming GL, Poo MM, Tessier-Lavigne M, Hemmati-Brivanlou A, Holt CE (1997). Neuron. 19(6): 1211-24
10. Ming GL, Song HJ, Berninger B, Holt CE, Tessier-Lavigne M, Poo MM (1997). c-AMP dependent growth cone guidance by netrin-1. Neuron. 19(6): 1225-35
11. Sloan TF, Qasaimeh MA, Juncker D, Yam PT, Charron F (2015). Integration of shallow gradients of Shh and netrin-1 guides commissural axons. PLoS Biol. 13(3):e1002119.
12. Lee KJ, Jessel Tm (1999). The specification of dorsal cell fates in the vertebrate central nervous system. Annual Review of Neuroscience. 22 (261): 261-94
13. Kwan CW, Gavin-Smyth J, Ferguson EL, Schmidtt-Ott U (2016). Functional evolution of a morphogenetic gradient. eLife 5: e20894.
14. Bier E, De Robertis EM (2015). Embryo development. BMP gradients: A paradigm for morphogen-mediated developmental patterning. Science. 348:aaa5838.
15. Tribulo C, Aybar MJ, Nguyen VH, Mullins MC, Mayor R (2003).
Regulation of Msx genes by a Bmp gradient is essential for neural crest specification. Development. 130:6441–6452.
16. Tucker JA, Mintzer KA, Mullins MC (2008). The BMP signaling gradient patterns dorsoventral tissues in a temporally progressive manner along the anteroposterior axis. Developmental Cell. 14:108–119.
17. Watanabe M, Fung ES, Chan FB, Wong JS, Coutts M, Monuki ES (2016).
BMP4 acts as a dorsal telencephalic morphogen in a mouse embryonic stem cell culture system. Biology Open. 5:bio.012021–1843.
18. Varadarajan SG, Kong JH, Phan KD, Kao T, Panaitof C, Cardin J, Eltzschig H, Kania A, Butler SJ (2017). Netrin1 produced by neural progenitors, not floor plate cells, is required for axon guidance in the spinal cord. Neuron. 94(4): 790–799.
19. Andrews MG, del Castillo LM, Ochoa-Bolton E, Yamauchi K, Smogorzewski J, Butler SJ (2017). BMPs direct sensory interneuron identity in the developing spinal cord using signal-specific not morphogenic activities. eLife. 6:e30647