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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 15, 2004 as doi:10.1096/fj.04-2251fje. |
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* Department of Physiology, and
Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina, USA
1 Correspondence: East Carolina University School of Medicine, Brody Bldg. #6N-98, 600 Moye Blvd., Greenville, NC 27858, USA. E-mail: murashoval{at}mail.ecu.edu
SPECIFIC AIMS
During neural development, caudalization and dorsoventral patterning of the neural tube is orchestrated by signaling pathways such as retinoic acid, sonic hedgehog (Shh), bone morphogenetic proteins (BMPs), and Wnt. Interaction between these signaling pathways may be critical for formation of dorsal interneurons. The aim of the current study was to determine whether dorsal interneurons specific for the spinal cord can be generated from mouse embryonic stem (ES) cells using known inductive signals.
PRINCIPAL FINDINGS
1. Wnt3A potentiates neuronal differentiation of ES cells
We examined the effects of Wnt3A applied alone or in combination with other inductive factors including RA, Shh, and BMP2 on the total number of neurons generated from ES cells after 15 days of culture (Fig. 1
). On day 16, cultures were fixed with 4% paraformaldehyde in PBS (pH 7.4) and processed for immunocytochemistry for MAP-2, a neuron-specific marker. Immunocytochemical staining revealed a significant increase in the total number of neurons in group 6 (treated with RA, Shh, BMP2, and Wnt3A) in comparison to all other groups (Fig. 2
A). The treatment in group 5 (RA, Shh, and BMP2) showed a tendency to produce a higher number of neurons relative to groups 1–4 but without reaching a statistical difference. Treatment of ES cells with Wnt3A and RA (group 4) did not affect the total number of neurons compared with RA treatment alone (group 1), nor did treatment with BMP2 and RA (group 3) or with Shh and RA (group 2) affect total counts of neurons in comparison to group 1. Our experiments showed that separate treatment of ES cells with Shh, BMP2, and Wnt3A failed to produce a greater number of neurons whereas the same factors in combination produced a new inductive effect responsible for increased neuronal differentiation. These results indicate that Wnt3A action may depend on Shh and BMP2 signaling.
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2. Wnt3A increases number of Lim2 positive neurons
During spinal cord development, differentiation of neuronal progenitors involves activation of Shh, BMP, and Wnt signaling. Specification of postmitotic motor neurons is directed by transcription factor HB9. In the dorsal portion of spinal cord, postmitotic dorsal interneurons dl2, dl4, and dl6 express the transcription factor Lim1/2. We used antibodies to HB9 and to Lim2 to identify motor neurons and interneurons, respectively. To examine whether Wnt3A treatment of ES cells induces motor or interneurons, we used immunofluorescence with double staining for MAP-2 and HB9 and for MAP-2 and Lim2.
Our experiments showed that all six treatments (groups 1–6) produced mixed populations of HB9 and Lim2 immunopositive cells (Fig. 2B
). The highest number of Lim2 positive cells was observed in group 6. We found that 55.7 ± 5.5% (n=13) of generated neurons were Lim2 positive whereas only 37.7 ± 3.9% (n=10) of generated neurons expressed HB9 (P<0.05). The number of Lim2 immunopositive cells were statistically higher in group 6 (56.0±5.5, n=10) in comparison to either group 5 (32.5± 3.9, n=9) or group 1 (31.4±4.9, n=14) (Fig. 2C
). The number of HB9 positive cells revealed no statistical difference between treatments (Fig. 2D
). We performed an experiment with an antagonist of BMP noggin and inhibitor of Shh SANT-1 showing that a combination of noggin, SANT-1, or noggin+SANT-1 statistically reduced the number of Lim2 positive cells. These results demonstrated that treatment of ES cells with RA, Shh, BMP2, and Wnt3A produced a statistically higher number of Lim2 positive cells. Treatment of ES cells with a combination of RA, Shh, BMP2, and Wnt3A produced a specific effect on neuronal differentiation, suggesting that Wnt3A signaling plays a significant role in specification of interneurons and cross-talks with BMP and Shh signaling pathways.
3. Effect of inductive factors on neurite length and branching
We further evaluated whether selected treatments of ES cells with RA, Shh, BMP2, and Wnt3A may affect neuronal maturation. Comparison of mean neurite length in Lim2 positive cells demonstrated a significant increase of neurite length in group 6 (144.4±18.09 µm, n=10), group 5 (122.0±6.1 µm, n=31), and group 3 (132.8±9.6 µm, n=28) vs. group 1 (100.6±6.9 µm, n=31) (Fig. 2E
). Analysis of mean neurite length in HB9 positive cells revealed a similar increase of neurite growth in groups 6, 5, and group 3 relative to group 1 (see Fig. 2F
). Statistical analysis by ANOVA of mean neurite length between all groups revealed the increase in mean neurite lengths in Lim2 and HB9 positive cells was primarily due to BMP2 treatment.
Quantification of neurite branching for Lim2 positive cells showed that the mean numbers of branches in group 6 (6.3±0.26, n=16) and group 5 (5.9±0.29, n=13) were statistically higher than in group 1 (4.17±0.3, n=21) (Fig. 2G
). Comparison of branching in HB9 positive cells revealed a remarkable similarity (see Fig. 2H
). ANOVA comparison between all groups demonstrated that treatment with Shh+BMP2 was responsible for increased branching in Lim2 and HB9 positive cells. With results on neurite length, these data indicate that the increase observed in maturation reflected by neurite outgrowth and branching was due to BMP2 and Shh signaling in HB9 and Lim2 positive neurons.
4. Generated neurons express synaptic proteins and neurotransmitters
Immunohistochemical staining revealed that Lim2 positive neurons express synapsin, synaptophysin, and postsynaptic protein PSD95. Immunohistochemical analysis of HB9 positive neurons demonstrated that HB9 neurons express the same synaptic proteins (data not shown). Immunofluorescent staining showed that HB9 neurons expressed ChAT, indicating that the generated motor neurons are cholinergic. Further analysis of Lim2 positive neurons revealed that 
50% were immunopositive for GABA-producing glutamate decarboxylase, GAD67; the rest were immunopositive for glutamate. Neurotransmitter release (glutamate and GABA) after stimulation with K+ was confirmed by HPLC.
5. Neuronal progenitors express MAP-2 and synapsin
We examined mitotic figures using immunostaining with anti-phospho-histone H3 (ser10) antibodies. Phosphorylation at Ser10 of histone H3 is tightly correlated with chromosome condensation during mitosis and serves as a marker of dividing cells. At day 16, a few dividing (2–3%) were still identified in cell populations by staining cultures for histone H3. These cells were immunopositive for MAP-2 and synapsin. Triple staining of the cells for DAPI, histone H3, and synapsin revealed neuronal cells possibly undergoing the last mitotic division. The results indicate the majority of cells were synchronized during the differentiation procedure, with only a few cells not having completed specialization.
CONLUSIONS AND SIGNIFICANCE
The present study examined whether application of inductive signals involved in neural development of the spinal cord may direct mouse ES cell differentiation into spinal interneurons. RA was used in some studies to induce neuralization of ES cells; Shh was used to produce motor neurons from ES cells. Interaction between Shh and BMP was shown to regulate interneuron development from dorsal telencephalic progenitors. Recently, Wnts have been implicated in promotion of neuronal differentiation in neural stem cell culture and specification of dorsal character.
Recent observations of the action of inductive factors have revealed their novel properties and effects in vivo and in vitro. Shh, BMPs, and Wnt have emerged as factors that control axonal guidance and growth. BMP7 has been implicated in the regulation of axonal repulsion and BMP6 stimulated neurite outgrowth in culture. In our experiments we observed that BMP2 stimulated neurite outgrowth, reflected by significant increases of mean neurite length in all groups treated with BMP2. Neurite outgrowth was increased in Lim2 and HB9 cell populations. These results indicate that BMP2 has a neurotrophic effect on differentiating progenitors, thereby affecting neurite growth. Here we provide the first evidence that the Shh+BMP2 combination significantly increased the mean number of branches. This treatment increased neurite branching in Lim2 as well as in HB9 positive cells, indicating that cross-talk between Shh and BMP2 signaling may be an important regulator of neurite outgrowth in different neuronal cell types.
The Wnt/ß-catenin pathway has been identified as a critical mediator of dendritic morphogenesis and formation of functional neuronal networks. Another report showed that Wnt3A protein added to neural stem cells promoted differentiation into MAP-2 positive neuronal cells. In our experiments, addition of Wnt3A to Shh/BMP2 pretreated cells significantly increased total number of MAP-2 positive neurons. In MAP-2 positive neurons, we observed a dramatic increase in the quantity of Lim2 positive neurons. These results demonstrate that Wnt signaling promotes neuronal differentiation of stem cells along specific lineages. Application of Shh, BMP2, and Wnt3A stimulated differentiation of ES cells into MAP-2 positive neurons possessing neuronal marker for dorsal interneurons (Lim2), synaptic proteins (synapsin, synaptophysin, PSD95), and functional neurotransmitter machineries (GAD67 and glutamate) characteristic for spinal interneurons. Shh, BMP2, and Wnt3A applied separately failed to produce significant morphogenic effects, but the same factors in combination and sequence produced a new, inductive action responsible for neuronal differentiation toward dorsal interneurons. Experiments with antagonist of BMP noggin and inhibitor of Shh SANT-1 revealed that a noggin, SANT-1, or noggin+SANT-1 combination abolished Wnt3A inductive action. These data showed that differentiation into dorsal interneurons depends on BMP and Shh signaling, leading us to hypothesize that Wnt3A inductive action relies on cross-talk with Shh and BMP2 signaling pathways (Fig. 3
).
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Future studies should yield important insight into mechanisms that control development of dorsal horn neuronal populations. Identification of these mechanisms may provide the means to generate specific classes of interneurons from ES cells for therapeutic applications.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2251fje;
This article has been cited by other articles:
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  S. LEV, I. KEHAT, and L. GEPSTEIN Differentiation Pathways in Human Embryonic Stem Cell-Derived Cardiomyocytes Ann. N.Y. Acad. Sci., June 1, 2005; 1047(1): 50 - 65. [Abstract] [Full Text] [PDF]
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