Multiple roles of Activin/Nodal, BMP, FGF and Wnt/ β-catenin signaling in the Neural Patterning of adherent Human Embryonic Stem Cell Culture

newfileneuro

Time Lapse of neural stem cells (University of Victoria Medical Sciences)

Introduction

9-Sep_#23

hESC line was differentiated toward neuronal lineage (Ganat and Studer)

Can you imagine regenerating any cell type from skin cells to major neural cells through just one cell? Human embryonic stem cells (hESC) are known for their self-renewal ability through the action of signaling molecules and can differentiate along the three germ layers (Alexander et al. 2013). They have great therapeutic potential for regenerating tissue and for understanding developmental processes such as neural induction. The ability for hESC to undergo neural induction can lead to great breakthroughs in neuroscience and understanding the development in the human brain. (Dottori et al. 2011).

In this paper, several important genes have been looked at and categorize to their domain. The pathways of Activin/Nodal, BMP, FGF and Wnt/ β-catenin have been analyzed to understand the neural induction, AP neural patterning and eye field specification in hESCs. The authors tested the specification of the neuroectoderm by manipulating the pathways and seeing if the manipulation can influence forebrain patterning, and eye field gene expression.

Activin/ Nodal Activin/Nodal pathway is part of the TGFβ growth factor family. It induces a variety of developmental pathways such as in mesodermal and endodermal cell types, ventral-dorsal patterning, and neural patterning.
BMP Bone morphogenetic proteins, a group of growth factors that induce many different morphologies throughout the body. BMPs are very important for neural induction as their inhibition leads to specific neural fates.
FGF Fibroblast Growth Factors are also a family of growth factors. In development, FGF is responsible for mesoderm induction, anterior-posterior patterning, limb development, and neural induction.
Wnt/β-catenin Once Wnt binds to the receptor, β-catenin moves into the nucleus and binds to a transcription factor. Wnt/β-catenin regulates body axis patterning, cell proliferation and cell fate specification, especially neuronal differentiation.

Table 1: Signals and some of their functions

Experimentation and Results

There has been many studies on the induction of the neuroectoderm. Some of these include, inhibition of Activin/nodal, and BMP antagonism by Noggin/SB or FGF. The authors decided to compare the effects of Noggin/SB and FGF with inhibition of Activin/Nodal (Figure 1).  An immunofluorescence analysis was conducted of cells differentiated for 14 days with Activin/Nodal Signaling (SB), SB+Noggin and SB+FGF2. In Figure 1, majority of the cells were shown to be positive for the neural marker, NESTIN. SB treatments resulted in low SOX2, and βIII-tubulin expression (used in neuroectodermal identity).

However after 12-16 days of differentiation, SB exhibited slightly lower amounts of the neuroectodermal markers than the other two categories. This indicates that neural development can occur in the absence of Noggin and FGF2. Noggin and FGF2 still exhibited higher amounts of expression than SB alone indicating that both of these genes are required by hESC in neuroectoderm. Further experimentation indicated that FGF2 was slightly stronger and downregulated pluripotency. Therefore, Activin/Nodal down-regulation plays a role, but the key actor is FGF2 and its inhibition on BMP signaling to enhance neural induction.

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Figure 1 (From Figure 1A) : Treatments with SB + Noggin or SB + FGF2 promote neuroectoderm specification in hESC cultures, but have opposite effects on the expression of PAX6 and CDX2. Immunofluorescence analysis with NESTIN, SOX2 and βIII-tubulin antibodies in hESCs cultured for 14 days. Cells treated with SB expressed NESTIN, but very few cells were positive for SOX2 and βIII-tubulin.

After looking at the interaction between SB, SB+Noggin and SB+FGF2, the authors decided to look at the positional identity of the neuroectoderm by these signals. They conducted a real-time PCR of AP neural patterning markers. Previous research shows that FGF2 and Noggin are BMP antagonists, (with FGF2 acting later) and they induced neuroectoderm formation as well as being major factors involved in the self-renewal of hESCs (Arau´zo-Bravo et al 2011; Dottori et al 2011). SB+FGF2 showed up-regulation in the hindbrain/spinal cord (Figure 2c).  SB+Noggin showed up-regulation in the forebrain markers (Figure 2b). Both showed up-regulation in the midbrain (Figure 2c). This concluded that the SB-FGF2 specifies the intermediate and the posterior of the neuroectoderm and SB+Noggin specifies for anterior regions. The results are consisted with previous research that the hESC acquire anterior neuroectrodermal fates in the presence of BMP and Activin/ Nodal inhibitor’s.

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Figure 2 (From Figure 2): Treatment of hESC cultures with SB + Noggin or SB + FGF2 induces neuroectoderm with different Anteroposterior positional identities using Real-time PCR. Undifferentiated hESCs cultured with Activin + FGF2 (A + F) were used as control while testing SB, SB and Noggin (SB + N) or SB and FGF2 (SB + F), (a,b) Compared to SB treatments, SB + N or SB + F treatment enhances transcription of some neuroectoderm markers. However, those cells show specific upregulation of forebrain (SB+N) or hindbrain/spinal cord genes (SB+F). Upregulation of midbrain genes is detected in both conditions.

As for Wnt/β-catenin signaling, previous research has shown that they work together with FGF and Retinoic acid to signal posterior neural patterning (Kudoh Et al, 2004). Due to SB+Noggin exhibiting some expression in the midbrain region, the authors observed the gene markers interaction with Fzd 8Δ (Frizzled8, prevents activation of endogenous receptors). After experimentations, it is shown that Fzd 8Δ rescues the forebrain expression genes and down regulates hindbrain genes (Figure 3).

figure 3

Figure 3 (from Figure 3B): Increased concentration of Fzd 8Δ leads to increased expression of Forebrain genes and reversed expression of hindbrain genes

fig 4

Figure 4 (From Figure 5B) : SB+Noggin hESC were treated and not treated with XAV, a molecule that causes β-catenin degradation. . XAV treatment results in improved expression of forebrain genes and downregulation of midbrain genes.

The authors also discovered that β-catenin degradation leads to forebrain specification concluding that midbrain specification is mediated by the activation of β-catenin in Wnt signals. Since Wnt/β-catenin is involved in the midbrain specification, it is concluded that Wnt/ β-catenin signaling promotes neural fates from posterior to forebrain. Inhibition of this pathway will lead to efficient forebrain specification (figure 4)

For eye field specification, the writers tested a previous hypothesis that constitutive inhibition of Activin/Nodal and BMP signaling hinders eye field specification in hESCs. They found that SB+Noggin repressed eye field genes and specifies another set of cephalic genes. Further experimentation shows that Activin/Nodal inhibition interferes with eye field genes approximately in the first four days of differentiation. Noggin (BMP inhibitor) repressed eye field genes after 6-8 days of differentiation.

The results from these experiments are very significant because they show that there are key mechanisms in controlling neural patterning. By using hESC, they proved that these patternings exhibited in model organisms can happen in a stem cell line. With the use of hESC, there is more insights into the signaling of neural induction and eye field specification.

Conclusion

The study shows the inhibition of BMP signaling or activation of FGF signaling are required for effective neural induction in this system. The signals have different outcomes in the positions of the AP axis in the induced neuroectoderm. They also show the specification of positional fates posterior to forebrain is dependent on Wnt/β-catenin. There is also a difference in FGF2 and Noggin signaling and their outcomes, despite both being BMP antagonists. Lastly they found that the eye field fate is inhibited by Activin/Nodal and Bmp signaling. All of these results indicate that BMP, FGF, on Wnt/β-catenin work together to specify the neuroectoderms.

Signals Position
BMP Forebrain and midbrain positional identities (anterior)
Activin/Nodal All (especially forebrain) and eye field
Wnt/β-Catenin Midbrain and Forebrain (effects posterior to forebrain)
FGF Midbrain/hindbrain/spinal cord (posterior)

Table 2: Signals and positions of the brain they specify

They also conclude with a proposed pathway controlling neural induction using the neuroectodermal markers (Figure 5).

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Figure 5 (From Figure 7): Proposed models of the signaling pathways controlling neural induction in adherent, chemically defined hESC cultures (a) In the embryo, Activin/Nodal and BMP inhibition and FGF regulate hESCs transformation into the neuroectoderm. Β-Catenin signaling determines posterior or anterior specification. In the anterior neuroectoderm, Activin/Nodal signaling determines eye field or forebrain expression. (b). Neural induction is promoted by BMP antagonists (Chordin, Noggin) and FGFs and is inhibited by high levels of Activin/Nodal signalling. AP patterning is controlled by RA, Wnts and FGFs and their antagonist, Dkk1. Eye field specification is marked by upregulation of eye field-specific transcription factors (EFTFs) and involves higher levels of Activin/Nodal and BMP signaling compared with forebrain specification.

Significance

This can allow us to have more insight to signals that regulate different areas of the neural system such as forebrain patterning, control of Activin/Nodal and BMP for eye field specification, and hESC differentiation. There are great strides in using neural stem cells to create brain tumors and other defects.

Strengths and Weaknesses

The paper goes into a lot on the signals and their pathways. Sometimes it was confusing when there was a chunk of text filled with acronyms because it was difficult to differentiate signals and neuroectoderm markers. They should have done fluorescent indicators on the embryo so that the readers would have a better understanding where the signals took place in each domain. They also spoke about a lot of data that was not represented by a figure.

References

Alexander M., Biagioni S., Lupo G., Harris W.A., Miranda E., Novorol C., Pedersen R., Smith J.R., Vallier L. (2013). Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signaling in the anterior neural patterning of adherent human embryonic stem cell cultures. Open Biology.

Arau´zo-Bravo M.J., Coulon P., Frank S., Greber B., Han D.W. Moritz S., Muller-Molina A.J., Pape H., Scholer H., Zhang M. (2011). FGF signalling inhibits neural induction in human embryonic stem cells. The EMBO Journal 30, 4874-4884.

City of Hope’s Dr. Margarita Gutova talks about using neural stem cells to treat brain tumors. (2014, January 16).YouTube. Retrieved April 27, 2014, from https://www.youtube.com/watch?v=ac8ABya0f00

Dottori M., Hughes S.H., Tay C. (2011). Neural development in human embryonic stem cells—applications of lentiviral vectors. Journal of Cellular Biochemistry1 1955-1962.

Kudoh T, Concha ML, Houart C, Dawid IB, Wilson SW. 2004 Combinatorial Fgf and Bmp signalling patterns the gastrula ectoderm into prospective neural and epidermal domains. Development 131, 3581–3592.

Neurons growing in a cell culture [gif]. University of Victoria Medical Sciences 4/19/2014. http://the-science-llama.tumblr.com/post/44631342440/infinity-imagined-neurons-growing-in-a-cell

Transcription factor SOX-2 – SOX2 – Homo sapiens (Human). (n.d.).Transcription factor SOX-2 – SOX2 – Homo sapiens (Human). Retrieved April 24, 2014, from http://www.uniprot.org/uniprot/P48431

Yosif Ganat and Lorenz Studer, Memorial Sloan-Kettering Cancer Center, New York, NY

800.656.7625. (n.d.).Immunofluorescence Microscopy. Retrieved April 27, 2014, from http://www.rockland-inc.com/fluorescence_microscopy.aspx

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