Nikita Chaudhri

Faculty Mentor: Dr. Vidya Chandrasekaran

The Role of Sonic Hedgehog on Dendritic Growth in Sympathetic Neurons

Abstract

Dendrites are the parts of a neuron that receive signals from other neurons and transmit

these signals down the cell body. Previous studies have observed that dendrites retract in cases

of neurodegenerative disease, neuronal injury, stress, and aging. Therefore, it is necessary to

identify and understand the molecules that could potentially rebuild the dendritic arbor of a

neuron in order to restore its function. In this study, we explored a new player, Sonic Hedgehog

(Shh), for its importance in the dendritic growth pathway of sympathetic neurons. Shh has been

shown to have many neuronal effects in many different systems, such as setting up the neural

crest and the identity of spinal neurons early in embryonic development. This study shows that

Shh is also needed for dendritic growth at later stages of development. Our data indicates that

cells treated with Shh had an increase in the number of dendrites and growth of dendritic arbor of

neuronal cells compared to untreated cells. This effect was specific to dendrites, did not affect

cell survival, and was mediated by the Ptc and Smo signaling pathway.

1. Introduction

Dendrites are important parts of the neuronal cell that are responsible for receiving

signals from other neurons. Across the nervous system, there is a huge diversity in the

branching of dendrites, or dendritic trees. The extent of these dendritic trees depends on the

number of neurons a given neuronal cell communicates with (1). For example, sensory neurons

communicate with a couple of cells and have one dendrite. On the other hand, Purkinje neurons

in the cerebellum communicate with over 200,000 cells and have an elaborate dendritic arbor

(2). The diversity of neurons and the establishment of their arbor take place during embryonic

development (3). An understanding of the molecules controlling dendritic growth and

retraction will provide a better understanding of how this diversity is generated.

In addition to embryonic development, dendritic growth and retraction play an important

role in neuronal injury when dendrites pull back and can no longer receive information from

other neurons in the body. Dendritic retraction associated with neuronal damage is observed in

many neurodegenerative diseases such as Multiple Sclerosis, Alzheimer’s disease, and

Parkinson’s disease (4). Thus, knowing all the molecules that control dendritic growth will

provide a better understanding of how to induce this growth and design therapies for

neurodegeneration and neuronal injury. This study focuses on one family of molecules, the Shh

family, to explore its effects on dendritic growth.

Shh has been shown to be important in many aspects of neuronal development in

embryos. Early in development, Shh is needed for establishing the identity of spinal neurons (5).

Later during development, Shh seems to be needed for maintaining the identity of interneurons

in the ventral telencephalon and setting up the nigrostriatal circuit (6, 7). Furthermore, Shh is

needed in axon guidance, neurite outgrowth, and synapse formation in dopaminergic, retinal, and

hippocampal neurons in the central nervous system (8, 9). These effects of Shh seem to be

mediated through its normal pathway, which involve Patched (Ptc) and Smoothened (Smo)

receptors and Gli proteins (9). Shh has a role throughout embryonic development and well into

postnatal development in many cell types. However, in the peripheral nervous system, Shh is

known to be important for early patterning of the ganglia and neural crest induction, which form

the precursors of sympathetic neurons (10, 11) with no studies showing the role of Shh at later

stages of embryonic development. One of the key processes that occur late in embryonic

development is the growth of dendrites in these sympathetic neurons (12). Therefore, the

purpose of this research is to look for the effects of Shh on dendritic growth in sympathetic

neurons at later stages of development.

2. Materials and Methods

2.1 Materials

E21 rats and BMP-7 were gifted by Dr. Pamela Lein’s Lab at UC Davis Department of

Molecular Biosciences. Recombinant Shh-N was obtained from R&D Systems (Minneapolis,

MN). Ptc Antibody and Smo Antibody were obtained from Abcam (Cambridge, MA). NGF

(125mg/ml) was obtained from Harlan Bioproducts. Prionex (10% solution) was obtained from

Millipore (Billerica, MA). Cyclopamine and SMI-32 antibody were obtained from Calbiochem

(Billerica, MA). All other supplies were obtained from Invitrogen (Grand Island, NY).

2.2 Tissue Culture

Sympathetic neurons were dissociated from the superior cervical ganglia (SCG) of E21

perinatal rats according to previously described methods (13). Cells were plated on 24-well

plates containing glass cover slips coated with poly-D-lysine (BD Biosciences, 100 µg/ml).

Cultures were maintained in serum free medium (DMEM and F12, 1:1) containing 0.5 mg/ml

bovine serum albumin (BSA), 1.4 mM L-glutamine, 10 µg/ml insulin, 5.5 µg/ml transferin, 38.7

nM selenium and 0.1 µg/ml β-nerve growth factor (NGF). After 24 hours, the cells were treated

with cytosine-β-D arabinoside (Ara-C) at 1 µM for 48 hours. Then, cells were treated with the

treatment conditions.

2.3 Morphological Analyses

Dendritic growth was analyzed by immunostaining cells with SMI-32 antibody against

dendritic proteins (1). In this process, neuronal cultures were fixed in 4% paraformaldehyde for

20 min, permeablized with 0.5% Triton X – 100 in 1X PBS for 5 min, blocked with 5% BSA for

20 min, and then incubated with SMI-32 as the primary antibody (1:5000 dilution in 5% BSA)

overnight at 4ºC. A fluorescent secondary antibody (1:1000 dilution in 5% BSA) was added to

the cells for one hour and the cover slips were mounted onto slides in Prolong Gold with DAPI.

Dendritic length and arbor were quantified using the Image J image analysis system. The

dendritic arbor of a cell was specifically quantified by summing the lengths of all the dendrites

and dendrite branches of the cell. Dendrites were counted using fluorescent microscopy, and the

data was statistically analyzed using Sigma Plot.

Cellular distribution of Ptc, Smo, and Smad-1 were visualized by microscopy after

cultures had been immunostained according to the process described above except with Phospho-

Smad-1 (1:100 dilution in 5% BSA), Ptc (1:500 dilution in 5% BSA), and Smo (1:250 dilution in

5% BSA), rabbit polyclonal antibodies directed against Smad, Ptc, and Smo as the primary

antibody.

2.4 Western Blot Analysis

The Western Blot Analysis of Ptc and Smo protein was carried out according to the

Invitrogen Manuel for the NuPAGE® electrophoresis system protocol (2010) under reducing

conditions. The 4-12% Bis-Tris Gel was loaded with a ladder, control lysate (3.51 µg), BMP-7

lysate (3.51 µg) and ran for one hour at 200 V in MES running buffer. The gel was then blotted

and transferred onto nitrocellulose membranes at 60 V for one hour. The blots were then

blocked with 5% milk in 1X PBS for one hour at room temperature with shaking. Primary Ptc

antibody (1:1000 in 5% milk) and Smo antibody (1:500 in 5% milk) were added to the blots and

then incubated for one hour at room temperature with shaking. Then, the blots were rinsed three

times for 20 minutes each in 0.1% Tween20 in 1X PBS and anti rabbit HRP-linked antibody was

added (1:2000 in 1X PBS, 5% milk, and 0.1% Tween20). The blots were then rinsed and

visualized with chemilluminescence. The chemilluminescent procedure was carried out

according to the Thermo Scientific Pierce ECL Western Blotting Substrate protocol. The gel

was imaged using the BioRad Molecular Imager® ChemiDoc™ XRS+ Imaging System.

2.5 MTT Cell Viability Assay

Following the elimination of glial cells, neurons extracted from the SCG of E21 rat pups

were treated under conditions for five days. Then, MTT (500 µg/mL) was added to the cells and

they were incubated at 37°C for 2 hours. Then cells were lysed with DMSO and loaded into a

96-well plate. The plate was loaded into the BioRad Benchmark Plus Microplate Reader and

Microplate Manager software measured absorbance at 562 nm.

2.6 Statistical Analysis

Data were statistically analyzed using a one-way ANOVA followed by Tukey’s Test on

Sigma Plot. Data was also quantified by Image J and reported using mean ± SEM. Experiments

were a replicate of three experiments counting 100 cells.

3. Results

3.1 Shh induces dendritic growth in sympathetic neurons

Previous studies have shown that sympathetic neurons are a good model system to study

dendritic growth because they are multipolar in vivo, however they do not extend dendrites in

vitro without the presence of specific growth factors (14). This observation was confirmed in

our study where neurons from the superior cervical ganglia (SCG) of E21 rats grown in control

medium, following the elimination of glial cells, showed an average of (0.14 ± 0.04) dendrites

(Fig. 1A, D). Since the EC50 of recombinant Shh-N was shown to be 0.1-0.4 µg/mL, we used a

10-fold excess, which was similar to what was used in previous studies (15). When cells were

treated with Shh at 0.5 µg/mL, there was an induction of dendritic growth and cells showed an

average of 1.27 ± 0.07 dendrites per cell (Fig. 2A). In addition to affecting the number of

dendrites, Shh also increases the dendritic arbor of these neurons and gives a bigger dendritic

tree (Fig. 2B). The dendrites are much longer in sympathetic neurons when treated with Shh at

0.5 µg/mL (131 ± 10.6 µm) than in control cells (0.00 ± 0.00 µm). Since Shh shows an increase

in the number of dendrites per cell and an increase in dendritic arbor compared to control

conditions, Shh works as an inducer of dendritic growth in sympathetic neurons.

However, Shh is not the most powerful inducer of dendritic growth. Previous studies

have shown that one of the most powerful regulators of dendritic growth in sympathetic neurons

belong to a family called the Bone Morphogenetic Proteins (BMPs), which belong to the

transformation growth factor family (TGF-β) (16). In this study, sympathetic neurons treated

with submaximal concentrations of BMP-7 (5 ng/mL) showed 2.21 ± 0.10 dendrites per cell,

which was slightly more than Shh (0.5 µg/mL) treated cells, which showed 1.27 ± 0.07 dendrites

per cell (Fig. 2A). Cells treated with a higher concentration of Shh at 1 µg/mL appeared similar

to cells treated with BMP-7 at 5 ng/mL (Fig. 1B, E, C, F). However, cells treated with maximum

concentrations of BMP-7 (50 ng/mL), showed 3.80 ± 0.14 dendrites per cell. Therefore, Shh is

not a strong inducer of dendritic growth, especially at maximum concentrations of BMP-7, but it

does indeed potentiate growth.

Previous studies have shown that BMPs and Shh work with each other in other systems

such as setting up the identity of spinal neurons in embryonic development (15). Therefore,

neurons were treated with these molecules together at low concentrations to determine if there

was an interaction between BMPs and Shh to induce dendritic growth. At submaximal

concentrations of BMP-7 (5 ng/mL), the number of dendrites per cell with BMP-7 and Shh

together (2.47 ± 0.01 dendrites per cell) compared to BMP-7 alone (2.21 ± 0.10 dendrites per

cell) was not statistically significant (Fig. 2A). At maximal concentrations of BMP-7 (50

ng/mL) with Shh, the number of dendrites were similar to that were observed with BMP-7 alone.

Thus, although Shh induces dendritic growth on its own, it did not significantly enhance BMP-7

induced dendritic growth. The lack of an additive effect suggests that both pathways are not

independent but that both molecules seem to be funneling into the same pathway.

The treatment of sympathetic neurons with increasing concentrations of Shh at 0.03

µg/mL, 0.1 µg/mL, 0.3 µg/mL, and 1 µg/mL show a dose dependent increase in the number of

dendrites extended by the neurons from 1.60 ±0.12 dendrites per cell at 0.03 µg/mL to 2.07 ±

0.10 dendrites per cell at 1 µg/mL (Fig. 3).

3.2 The effect of Shh is specific to dendrites

To determine if the effects of Shh were specific to dendrites and did not affect cell

survival, the MTT viability assay was done on cells treated with different conditions. Cells

treated with control media showed absorbance values of 0.89 ± 0.11 (Fig. 4). Cells treated with

Shh (1µg/mL) alone did not show a significant decline from control media and were around

(0.71 ± 0.003). BMP-7 (1 ng/mL) and Shh together showed a higher absorbance (1.80 ± 0.58)

compared to BMP-7 (1.05 ± 0.12) or Shh alone. However, the variation in the data was not

statistically significant. This indicates that Shh’s effect on dendritic growth did not affect cell

health suggesting that its effects are specific to dendrites and dendritic growth.

3.3 Ptc and Smo are present in sympathetic neurons

To determine if Shh was working through its canonical pathway to induce dendritic growth

in sympathetic neurons, we first looked for the presence of the Ptc and Smo receptors in

sympathetic neurons. During Shh signaling, the secreted Shh protein leaves the cell and binds to

the Patched (Ptc) receptor of a neighboring cell. This activates the Smoothened (Smo) G-protein

coupled receptor, which in turn activates a family of Gli transcriptional activators that enter the

nucleus and turn on the transcription of genes (15). Therefore, the presence of Ptc and Smo in

sympathetic neurons would be indicative of the presence of Shh signaling at a later stage of

embryonic development.

To identify the presence of Ptc and Smo, the neurons were treated with either control

medium or with BMP-7 and then immunostained using antibody against Ptc or Smo. Our results

show that Ptc and Smo were present in both control cells and BMP-7 treated cells. Ptc staining

appeared in the cell body (Fig. 5A, B), and Smo staining was present in the cell body and

dendrites (Fig. 5C, D).

Since the Shh signaling components were present in sympathetic neurons, the next step

was to determine if sympathetic neurons expressed Shh protein. Upon treating cells with either

control medium or BMP-7 (50 ng/mL) and immunostaining with mouse antibody to Shh, we

found Shh staining in the cytoplasm of the cell body under control conditions and with BMP-7

(Fig. 6A, B). The presence of Shh in sympathetic neurons in vitro suggest that these neurons had

the ability to make Shh and secrete it to neighboring cells where Shh binds to its Ptc and Smo

receptors, thus initiating its signaling pathway.

3.4 Inhibiting Smo alters BMP induced dendritic growth

To further confirm if Shh works through its canonical pathway, we used cyclopamine, an

inhibitor of Smo, and therefore, Shh signaling. Cyclopamine is a teratogen that inhibits the Shh

pathway by binding to Smo, thus causing cyclopia and other birth defects (17). Humans or mice

lacking Shh develop cyclopia due to a failure of separation of the lobes of the forebrain (15). To

determine if Shh is working through its normal signaling pathway, cells were treated with Shh (1

µg/mL) and cyclopamine (100 nM). Cells treated with Shh showed an average of 1.69 ± 0.09

dendrites per cell whereas cells treated with Shh and cyclopmaine together showed an average of

1.25 ± 0.10 dendrites per cell (Fig. 7). Therefore, cyclopamine inhibits dendritic growth,

showing nearly as many dendrites as the control (1.10 ± 0.12). This suggests that the induction

of dendritic growth by Shh was mediated through a pathway involving Smo receptor.

3.5 Shh does not affect the nuclear translocation of Smad-1 proteins

Since Shh is working through its normal signaling pathway, and the effect of BMP-7 and

Shh together does not seem to be additive, we looked at how the BMP and Shh pathways interact

with each other during dendritic growth. In the BMP signaling pathway, BMP family members

bind to BMP receptors (BMPRI and BMPRII). When BMP binds, the receptors phosphorylate

the Smad-1 or Smad-5 proteins. The phosphorylated Smads leave the receptor and combine with

Smad-4 downstream. These Smad-1/Smad-4 and Smad-5/Smad-4 complexes enter the nucleus

and turn transcription on or off at specific genes (18). Therefore, the nuclear translocation of

phosphorylated Smad-1 protein was used to determine if Shh induces the BMP-7 signaling

pathway. Neurons exposed to control media showed Smad-1 staining in the cytoplasm, with no

staining in the nucleus (Fig. 8A). Neurons treated with BMP-7 showed nuclear staining (Fig.

8B). However, the neurons treated with Shh at 1 µg/mL did not show nuclear staining for

phosphorylated Smad-1, as evidenced by the staining in the cytoplasm and the dark, unstained

nucleus of the cell (Fig. 8C), indicating that Smad-1 translocation was unaffected by the presence

of Shh.

3.5 Shh pathway is downstream from the BMP-7 signaling pathway in sympathetic

neurons

Since Shh does not seem to be influencing the BMP pathway, we looked at whether the

BMP-7 pathway was inducing the Shh pathway by treating neurons with both cyclopamine (100

nM) in conjunction with BMP-7 (5 ng/mL). Compared to BMP-7 alone, which showed 2.12 ±

0.10 dendrites per cell (Fig. 10), cells with both cyclopamine and BMP-7 showed a decrease in

dendritic growth with 1.24 ± 0.01 dendrites per cell (Fig. 9D, 10). These results show that

blocking Shh signaling resulted in an inhibition of BMP-7 induced dendritic growth.

Furthermore, there was a higher amount of Smo protein (86 kDa) in control lysates

compared to lysates from BMP-7 treated cells, suggesting that Smo receptor many be a potential

point of interaction between the two pathways.

4. Discussion

Our results show that Sonic Hedgehog induces dendritic growth in sympathetic neurons.

This study is the first study showing an effect of this family on dendritic growth. Shh, however,

is a mild inducer of dendritic growth in these neurons. While the average number of dendrites in

Shh treated and control neurons varied between different experiments (Fig. 2B and 7), the same

general trends were seen. The average number of dendrites was always higher in neurons that

had been treated with Shh compared to neurons under control conditions. Variability in the

number of dendrites can be due to factors such as cell plate density, maturity of rat pups upon

dissection, and inherent differences between superior cervical ganglia in individual rat pups.

One possible reason for the mild induction of dendritic growth by Shh was the

concentration of Shh used in this study. In this study, the dendritic growth observed with the

Shh dose curve was still increasing at 1 µg/mL and has not yet leveled off at its maximum

concentration (Fig. 3). It would therefore be interesting to look at the effects of higher

concentrations of Shh to see if we can induce more dendrites and potentiate dendritic growth in

the presence of submaximal concentrations of BMP-7. Furthermore, our data indicates that there

is an endogenous concentration of Shh in sympathetic neurons in vitro with the absence of glial

cells. Therefore, it would be interesting to look at whether glial cells also produce Shh to see if

the endogenous concentration of Shh is actually greater in vivo thus having a greater effect on

dendritic growth in the animal.

In addition, this study shows that Ptc and Smo receptors are present in sympathetic

neurons, and the inhibition of Smo reduces Shh’s effects on dendritic growth. This indicates that

Shh is working through its normal signaling pathway to induce dendritic growth in sympathetic

neurons. Our data also shows that BMP is inducing the Smo receptor levels, however, this may

be due to the fact that Smo is also located in the dendrites, which are extended in the presence of

BMP. Therefore, to further confirm if BMP is inducing the Shh pathway, it would valuable to

look at the location of Gli, the downstream signaling component in the Shh pathway, to see if

BMP is inducing the Shh pathway downstream in the neuron.

Previous studies have shown that Shh and BMP interact in other neuronal systems. Shh

and BMP work against each other in the patterning of the frontonasal process during

development (19). On the other hand, both earlier BMP signaling and later Hh signaling are

needed to induce satb2 expression in the palate and jaw (20). In our study, BMP induces the Shh

signaling pathway but Shh does not affect the BMP signaling pathway because the translocation

of Smad-1, the downstream signaling component in the BMP pathway, was unaffected by the

presence of Shh. Therefore, it would be interesting to look at the interaction between Gli and

Smad-1 to help understand how these two pathways specifically interact at the downstream level

to control the growth of dendrites. Furthermore, studies have identified specific genes that are

regulated by BMP-7 in sympathetic neurons during primary dendritic growth (16). Therefore, it

would be helpful to determine if these BMP-7 regulated genes are also targets of the Shh

pathway, which would show if these two molecules are interacting at a transcriptional level.

In summation, this study shows that Shh induces dendritic growth, Shh and its signaling

components are present in sympathetic neurons, and the Shh signaling pathway is induced by

BMP-7. These discoveries provide a more complete understanding of how the dendritic arbor of

a sympathetic neuron is generated. Such an understanding can shed light on developing

treatments to recreate the dendritic arbor of damaged neurons that have lost their dendrites and,

therefore, their function. Such damaged neurons are seen in patients with neurodegenerative

diseases and neuronal injuries. Therefore, this study provides relevant information to help

develop treatments for neurodegenerative disease and neuronal injury.

Following the elimination of glial cells, cultures of sympathetic neurons from E21 rat

pups were treated for 5 days with either control medium (A, D), BMP-7 at 5 ng/mL (B, E), or

with Shh at 1 µg/mL (C, D). Panels A-C are the phase contrast images under 20X magnification

of the cells D-F that are the fluorescent images immunostained with phosphorylated

neurofilament antibody (SMI-32) against dendritic proteins. A and D are control, B and E are

BMP-7 (5 ng/mL), and C and F are Shh (1 µg/mL) treated cells.

(B) (A)          (C)  

(D)    

(E)   (F)  

 

Figure  1:    The  effect  of  Shh  on  dendritic  growth  

0  

50  

100  

150  

200  

250  

Control   Shh  0.5  μg/mL   BMP-­7  5ng/mL   Shh  0.5  μg/mL+BMP-­7  5ng/mL  

Total  Dendritic  Arbor  (μm)  

Treatment  

The  Effect  of  Shh  on  Dendritic  Arbor  

Dendritic  Arbor  (μm)  

                                                                                             Figure  2:    The  effect  of  Shh  on  dendritic  growth  

0  

0.5  

1  

1.5  

2  

2.5  

3  

3.5  

4  

4.5  

Control   Shh  -­  N  0.5  μg/mL  

BMP-­7  5  ng/mL  

Shh  -­N  0.5  μg/mL  +  BMP-­7  5  

ng/mL  

BMP-­7  50  ng/mL  

Shh  -­N  0.5  μg/mL  +  BMP-­7  50  

ng/mL  

Number  of  dendrites/cell  

Treatment  

The  Effect  of  Shh  on  Dendritic  Growth  

(A)  

(B)  

                           *  

                   *  

Following the elimination of glial cells, cultures of sympathetic neurons from E21 rat pups were treated under conditions for 5 days. The neurons were immunostained with phosphorylated neurofilament antibody (SMI-32) against dendritic proteins. Cells were counted using microscopy under UV light with a secondary fluorescent antibody. The dendritic arbor of the cells was quantified using Image J. The changes in the number of dendrites per cell are shown in (A) and the changes to the dendritic arbor are shown in (B). The data are expressed as mean ± SEM (N ≈ 100) for panel A and dendritic arbor size per cell (N ≈ 50) for panel B. * Denotes treatments that are statistically significant to control as deduced by ANOVA followed by Tukey’s Test (p < 0.05).  

Following the elimination of glial cells, the sympathetic neurons from E21 SCG treated

with Shh at increasing concentrations ranging from 0.03 µg/mL to 1 µg/mL for 5 days. The

number of dendrites per cells was measured at different doses after 5 days of treatment. The

graph shows the number of dendrites per cell versus the concentration of Shh. The data is

represented as mean ± SEM.

Shh  Dose  Curve  

Figure  3:    The  effect  of  Shh  on  dendritic  growth  is  dose  dependent  

Following the elimination of glial cells, sympathetic neurons were treated with control

medium, BMP-7 (1 ng/mL), Shh (1 µg/mL), and BMP-7 and Shh together for five days. Then,

cells were treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for

two hours and then lysed with DMSO. The absorbance of the lysed cells was measured at 562

nm using Microplate Manager software and Benchmark Reader. The data is represented as mean

± SEM (n = 2). Data was analyzed with a one-way ANOVA followed by Tukey’s Test showing

no statistical significance.

0  

0.5  

1  

1.5  

2  

2.5  

Control   BMP-­7  1  ng/mL   Shh  1  μg/mL   BMP-­7  1  ng/mL+Shh  1μg/mL  

Absorbance  at  562  nm  (arbitrary  units)  

Treatment  

MTT  Cell  Viability  Assay  

Absorbance  at  562  nm  

Figure  4:    Measuring  cell  viability  by  the  absorbance  of  MTT  product  in  cells  at  562  nm  

Following the elimination of glial cells, cultures of sympathetic neurons from E21 rat

pups were treated with control medium in (A) and (C), and with BMP-7 at 50 ng/mL in (B) and

(D). The cultures were then immunostained with either Smo antibody (1:250 dilution in 5%

BSA) shown in panels C and D or Ptc antibody (1:500 dilution in 5% BSA) shown in panels A

and D. Neurons were visualized by microscopy under UV light with a secondary fluorescent

antibody. All photographs were taken at 20 X magnification. Ptc and Smo were present in

control medium and in the presence of BMP-7. Ptc staining appears in the cell body, and Smo

staining is present in the cell body and dendrites.

(B)                  (A)    

           (C)    

Figure  5:    The  Ptc  and  Smo  receptors  of  the  Shh  signaling  pathway  are  present  in  sympathetic  neurons    

(D)  

 

Following the elimination of glial cells, sympathetic neurons obtained from E21 rat pups

were treated under control conditions (A) and with BMP-7 at 50 ng/mL (B) for five days. The

cells were then immunostained with mouse antibody to Shh (1:250 dilution in 5% BSA). When

seen at 20 X magnification, cells treated with control media and cells treated with BMP-7 at 50

ng/mL show the appearance of Shh in the cytoplasm of the cell body where the staining is

present.

(A)     (B)    

Figure  6:  Shh  is  present  in  sympathetic  neurons  

Following the elimination of glial cells, sympathetic neurons from E21 rat pups were

treated with either control medium, Shh at 1µg/mL, cyclopamine at 100 nM, and cyclopamine at

100 nM with Shh at 1 µg/mL for five days. The neurons were then immunostained with the

SMI-32 antibody against dendritic proteins. The cells were counted using microscopy under UV

light with a secondary fluorescent antibody. The changes in the number of dendrites per cell (N

≈ 100) are shown in Figure 6. * Denotes treatments that are statistically significant to each other

as deduced by ANOVA followed by Tukey’s Test (p < 0.05).

0  

0.2  

0.4  

0.6  

0.8  

1  

1.2  

1.4  

1.6  

1.8  

2  

Control Shh-N 1µg/mL Cyc 100 nM Cyc 100 nm + Shh-N 1µg/mL

Number  0f  dendrites/cell  

Treatments  

The  Effect  of  Shh  and  Cyclopamine  on  Dendritic  Growth  

Number  of  dendrites/cell  

                               *    

*

Figure  7:  Cyclopamine  inhibits  Shh    

Cultures of sympathetic neurons from E21 rat pups were treated with control medium

(A), BMP-7 at 50 ng/mL (B), and Shh at 1 µg/mL (C) after the elimination of glial cells. Cells

were then immunostained with rabbit antibody to Smad-1 and viewed under 20 X magnification.

The control and Shh treated cells have staining in the cytoplasm while the nucleus remains dark

and unstained. The cells treated with BMP-7 have staining in the nucleus and cytoplasm.

(A)     (B)      

(C)      

Figure  8:    Smad-­‐1  translocation  in  sympathetic  neurons  treated  with  BMP-­‐7  and  Shh  

Indicates  the  nucleus.  

Following the elimination of glial cells, cultures of sympathetic neurons from E21 rat

pups were treated for 5 days with either control medium (A), BMP-7 at 50 ng/mL (B),

cyclopamine at 100 nM (C), or with cyclopamine and BMP-7 together (D). Panels A-D are

fluorescent images immunostained with phosphorylated neurofilament antibody (SMI-32)

against dendritic proteins at 20X magnification.

(A)     (B)      

(C)      

(D)      

Figure  9:    Cyclopamine  decreases  dendritic  growth  at  maximal  concentrations  of  BMP-­‐7  

 

0  

0.5  

1  

1.5  

2  

2.5  

Control   Cyclopamine  100  nM  

BMP-­7  5  ng/mL   BMP-­7  5  ng/mL  +  Cyc  100  nM  

Number  of  dendrites/cell  

Treatment  

The  Effect  of  Cyclopamine  on  Dendritic  Growth  

Following the elimination of glial cells, sympathetic neurons from E21 rat pups were

treated with control medium (A), BMP-7 at 5 ng/mL (B), Cyclopamine at 100 nM (C), and

Cyclopamine at 100 nM with BMP-7 at 5 ng/mL (D) for five days. The neurons were then

immunostained with the SMI-32 antibody against dendritic proteins. The cells were counted

using microscopy with under UV light with a secondary fluorescent antibody. All photographs

were taken under 20 X magnification. The changes in the number of dendrites per cell (N ≈ 100)

are shown. * Denotes treatments that are statistically significant as deduced by ANOVA

followed by Tukey’s Test (p < 0.05).

(A)  

Figure  10:    Cyclopamine causes a decrease in dendritic growth at submaximal concentrations of BMP-7

 

                                               *  

3.51 µg of control and BMP-7 protein was loaded onto a 4-12% Bis-Tris gel and

immunoblotted onto nitrocellulose membranes. The blot was treated with rabbit antibody

against Smo and visualized by ECL using the BioRad Molecular Imager® ChemiDoc™ XRS+

Imaging System. The band for Smo appears at 86 kDa as expected.

Figure  11:    Western  Blot  Gel  shows  protein  levels  of  Smo  at  86  kDa  

110  kDa  

               80  kDa  86  kDa  

Control   BMP-­‐7  

60 kDa

 

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