Analysis of the effects of Streptomyces hyaluronidase on

formation of the neural tubeGary C. Schoenwolf and Marilyn Fisher

From the Department of Anatomy, University of UtahJournal of Embryology and Experimental Morphology

Bethany RovnackBethany RovnackDecember 4, 2003December 4, 2003

       Neurolation is the complete development of the neural tube

The process that initiates the formation

of the nervous system

Neurolation occurs in 3 steps:

1. Formation of the Neural Plate

2. Bending of the Neural FoldsWith Formation of the Neural Groove and Neural Folds

Elevation of the Neural Folds

3. Convergence of the Neural Folds

Fusion of the Neural Folds

 

Background:

•Experimentation on the development of the neural tube began about

50 years ago•Different hypotheses have been

presented and debated•Intrinsic: concentrated to neural plate

•Extrinsic: forces outside the neural plate

• Studied the role of Hyaluronic Acid is contained within the extracellular matrix

• It is found under neural folds of both avian and mammal embryos

• Hyaluronic acid makes up about 84% of the glycosominoglycans (GAGs) made by the tissues when the neural tube is first starting to develop

Chick embryo ofthirty-three

hours’ incubation

Background cont…

•Hyaluronic acid exists naturally in all living organisms and is a universal

component in the extracellular matrix.

• It provides a cushion effect

•Provides protection.

• Same chemical structure bacteria as well as in human beings.

• It is produced by tissues in early embryos, such as the neural tube.

•Hyaluronic Acid plays an important role in tissue hydration, lubrication and cellular function.

•It is able to hold more water than any other natural substance.

•Due to its hydrating properties, it increases smoothness, and softening and decreased wrinkles where it is found.

Reasonable to Conclude that…Because Hyaluronic Acid is involved in hydration, with increased

volume in the neural folds within the extracellular matrix, the force produced will would aid in the production of the neural tube

(neurolation).

Background Continued…

Would Neurolation occur normally in embryos that lacked Hyaluronic acid?

Hypothesis:If there was a decrease in the amount

of Hyaluronic acid in the embryo, then neurolation would not occur normally, leading to an increase in the number of

neural tube defects (NTD).

      Chick Embryos in ovo treated with Streptomyces hyaluronidase

To Test the Hypothesis:

Materials & Methods: Embryos (stages 8-9) studied for formation of spinal cord• Special precautions were taken to study effects only caused by Streptomyces hyaluronidase only• Other side effects could hinder the study - they were taken into account

FYI: Hyaluronic Acid is part of the family of enzymes, which inhibits the growth of the glycosaminoglycan chain(GAGs) through the plasma membrane into the extracellular matrix.

•All neural tube defects found in the embryo’s developing spinal cords were those treated with Streptomyces hyaluronidase due to the failure of increase in neural folds

1. White Leghorn Chicken eggs were incubated @ 38˚C2. A small window was cut through the part of the shell that

covered the embryo using standard techniques3. The following was injected into the sub-blastodermic space of the 4

different test embryos:(a) 20ul SH (200 TRU/ml in 0.9% saline) – Batch 1 & 2(b) 20ul SH in a solution of 3 parts: 0.9% saline to 1 part albumen, trypsin(c) 20ul Hyaluronic Acid digested with

40 TRU, SH & boiled 5 min to destroy the enzyme, 0.9% saline(d) 3 parts 0.9% saline to 1 part albumen

 

4. All embryos were illuminated with fiber optics 5. The “windows” were sealed with scotch tape6. Returned to incubator for an additional 24h.

Assay for Protease Activity

Reason: An assay was done to determine the level of protease contamination contained within the batches of Streptomyces hyaluronidase used in the treatment of the embryos.

Fibrinogen/agar-coated Plate I was prepared using the following procedure:1.) 1g agar in 75 ml – 0.05m Tris-Buffer 2.) 0.014m Calcium Lactate was boiled for 15 min, or until agar

dissolved3.) Cooled to 60ºC

Fibrinogen/agar-coated Plate II was prepared using the following procedure:1.) 80mg Fibrinogen in 25ml of 0.025m Tris-Buffer2.) Centrifuged3.) Slowly added to the agar

Both mixtures were incubated for 1h in 80 ºC

Poured into a 5ml/dish

- cooled to room temperature

studied right away storied in the refrigerator

- Small wells were made in the newly formed agar/fibrinogen gels using a glass using a glass pipette.

-Filled with 10ul of enzyme solution- Incubated @ 37ºC for 24-36h

-Prepared for Processing of Microscopy

Agents Tested Concentrations Detectable LysisStreptomyces Hyaluronidase 200 TRU/ml No

Pronase or Trypsin 100ug/ml Yes10ug/ml Yes1ug/ml Yes

0.1ug/ml Barely (@36h)0.01ug/ml No0.001ug/ml No

Pronase or Trypsin + 100ug/ml YesAlbumen 10ug/ml No

1ug/ml No0.1ug/ml No

Summary of the various concentrations of enzymes tested Summary of the various concentrations of enzymes tested for proteolytic Activityfor proteolytic Activity

Discussing the Effects:

• Most embryos treated with Streptomyces hyaluronidase experienced NTD

•The frequency found in this particular study was related to the batch of enzymes used

•NTD formed in 60% of the embryos treated with E1• 94% of the embryos treated with E2

Experienced neural tube defects •However, no major difference could be detected in

the two batches of enzymes (E)•They both appeared to be equally effective in their

activity within the matrix

Histology  Gross Features of NTD:

Treatment of embryos with Streptomyces hylaronidase resulted in embryos experiencing “non-closure” type NTDs:

(1) Failure of convergence    

(2) Unsuccessful fusion along the spinal cord

(3) The amount of enzymatic activity

Distinguishing Characteristics

• Reduction in the size of the extracellular space

• Marked dilation of the embryonic blood vessels

• Presence of intracellular inclusions

Solutions injected sub-

blastodermically

# if embryos with closed neural

tubes

# of embryos with neural tube

defects

% of embryos with neural tube

defects

Hyaluronidase

(1)24 36 60

Hyaluronidase

(2)3 51 94

Hyaluronidase

(2) + saline0 16 100

Trypsin

(0.1ug/ml)14 2 13

Hyaluronic Acid digested with hyaluronidase (2) and boiled for 5m

24 3 11

Saline/Albumen Mixture

64 10 14

Fig. 30: EMF-induced abnormalities in chicken embryos. (Farrell et al, 1997)

Pictures (a)-(d) show normal, unexposed embryos following a 48 hour incubation. The spinal cord is the "tail" of the embryo, and the neurall tube inside this appears completely normal; so does the developing brain. Picture (h) is a transverse section at the level of the hindbrain for embryo (a) as indicated by line (h) in picture (a). This brain is normal.

Pictures (e)-(g) and (i) show neural tube and brain defects induced by electromagnetic fields. In (e)-(g) arrows indicate neural folds of open neural tubes in the spinal cord. This is an abnormality known in humans as spina bifida. The hindbrain shown in (i) of embryo (g) is abnormal - totally flattened - compared to the normal brain shown in (h).

1. Elevation of the Neural Folds2. Convergence of the Neural Folds3. Fusion of the Neural Folds across the

Dorsal midline~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

CONSEQUENCES:•The depletion of SH has no effect on elevation

•However, convergence is blocked completely when Streptomyces hylaronidase is absence

•As well as the inhibition of fusion of the neural folds

Conclusion:

The results presented supportthe idea that neurolation is a complex process involving several different components. The important role of the extracellular

matrix is key in the data shown above. The experiments displayed here show that the effects of neurolation due to the

injections were not solely due to the protease contaminate nor the Hyaluronic Acid. This is supported by the data of those embryos

that were treated with the Hyaluronic Acid and did not experience any deficiencies. Only 14% of the control group experienced NTD. The hypothesis stated in the literature

supports the theory that multiple factors are most likely the main factor of neurolation.

Analysis of the effects of Streptomyces hyaluronidase on the formation of the neural tubeBy Gary C. Shoenwolf and Marilyn FisherFrom the Department of Anatomy, University of UtahJournal of Embryology and Experimental Morphology73, 1-15. 1983