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Differentiation of smooth muscle cells and
enteric neurons in zebra fish.
Sarah Beckman
Advisor: Ken Wallace
Goal:
In our research, we will focus on development of the smooth muscle and enteric neurons
of the zebra fish intestine, specifically FKBP and BMP pathways. Our aim is to show
that FKBP is involved in smooth muscle development in the intestines of zebra fish.
Also, to identify the role of BMP signaling in formation of the enteric system and
intestinal smooth muscle development.
Introduction:
Zebra fish, Danio rerio, are freshwater fish that originated in East India. They are useful
to study developmental biology because they develop outside of the mother’s body and
they are clear, allowing the researcher to watch them develop under a microscope. In
addition, zebra fish are vertebrates, which make them more similar to humans than
invertebrates such as Drosphilla. Because of their size, they are easy to maintain in large
numbers and readily reproduce under laboratory conditions. The zebra fish model has
relatively few cells compared to other vertebrates, thus making it a comparatively simple
model for study. Also, it is easy to introduce foreign genetic material and perform other
experimental manipulations because there is direct access to the developing embryo.
Figure 1: Danio rerio
source: www.thetropicaltank.co.uk/.../ zebdano1.jpg
We will specifically be studying the digestive tract of zebra fish. Digestive tract
development is similar among many species. The digestive system plays an essential role
in vertebrate physiology. In addition to being the site of nutrient digestion and adsorption,
the digestive organs provide a barrier to environmental toxins, provide essential immune
function, and have important roles in metabolism and salt-water absorption. (Wallace
2002).
The gastro intestinal (GI) tract is innervated by the autonomic nervous system, which can
be divided into the extrinsic and intrinsic, or enteric, nervous system. The extrinsic
nervous system can be further divided into parasympathetic and sympathetic branches.
The parasympathetic innervation is supplied by the vagus and pelvic nerves.
Sympathetic innervation is supplied by nerves which run between the spinal cord and
prevertebral ganglia and between these ganglia and organs of the gut. The enteric neural
system is defined as the system of neurons and their supporting cells that is present
within the wall of the gastrointestinal tract (Newgreen, 2001). The enteric nervous system
is grouped into several distinct networks, two of the most important being the myenteric
and submucosal plexuses. Processes from the neurons of the plexuses do not just
innervate target cells such as smooth muscle, secretory cells, and adsorptive cells. They
also connect to sensory receptors and are interwoven with processes from other neurons
located both inside and outside the plexuses. Thus, integration of activities can take place
entirely within the enteric nervous system (Johnson, 2001). Another important aspect of
the GI tract is smooth muscles.
Smooth muscle is responsible for the contractibility of organs such as the gastrointestinal
tract. The contractions of smooth muscles are far slower and sustained longer than in
skeletal muscles. Smooth muscle cells are generally smaller than skeletal muscles,
between 4 – 10 um wide and 50-100 um long. The cells of smooth muscles are grouped
into branching bodies that are surrounded by connective tissue sheets. Individual cells
are generally coupled to one another so that contractions of a bundle of muscle are
synchronous. Not every smooth muscle cell is innervated, thus neurotransmitters act on
only a few of the cells and the message must be communicated form one cell to the next.
(Johnson, 2001)
Figure 2: Smooth muscle tissue
Source: www.mhhe.com/biosci/ ap/histology_mh/smmusc.jpg
FKBP is a protein which has been shown to be responsible for smooth muscle
differentiation in the gut of amniotes such as chickens (Bitar K N, 2003). The general
class of FKBPs are immunophilins. Immunophilins were initially identified by their high
binding affinity for the immunosuppressant drugs cyclosprina A and FK506, the
macrolide antibiotic produced by Streptomyces tskubaenisis. Most immunophilins
however, do not appear to be involved in immunosuppressive activities. They appear to
function in a wide range of cellular activities including folding, assembly and trafficking
of proteins. (Patterson, et all. 2000)
BMPs were originally discovered due to their role in bone formation, thus they are
known as bone morphogenic proteins. However, bone formation is only one of their
many functions. They have been found to regulate cell division, apoptosis, cell migration
and differentiation (Gilbert, 20003). BMP may play a role in migration as well as
proliferation of the enteric precursors.
They act as extracellular ligands which bind to receptors in the cell membrane. A ligand
is the general term for any molecule that binds specifically to a receptor sit of another
molecule. Ligand binding generally causes a receptor protein to undergo a change in
conformation, which causes it to react with another cellular molecule. (Campbell et all.,
2003). Binding of BMP causing phosphorylation of Smad proteins (see figure 3) which
act as transcription factors in the nucleus of the cell (Gilbert, 2003).
Figure 3: The Smad pathway
source: info.med.yale.edu/.../ courses/class4/img020.gif
One of the reasons that we study the smooth muscle of the intestine and enteric neurons is
because of maladies such as Hirschprung’s disease (HSCR). This disease is a birth defect
affecting the bowel, and is due to an absence, or near absence, or enteric neuron system
ganglia in the contracted terminal region. HSCR occurs in around 1/5000 live births, and
mutations in a number of genes are involved in HSCR, but these genes account for < 50%
of the cases. This suggests there are still unknown genes that, when mutated, predispose
to HSCR (Newgreen, 2001).
Methods:
I have done some preliminary research using the procedures outlined below.
Probe for hybridization
To make a probe plasmid DNA is combined with appropriate enzymes and buffer to lyse
the section of DNA that codes for the protein of interest. This mixture incubates for one
hour at 37 C. Next an extraction is performed using phenol and chloroform to separate
out then DNA from the rest of the material in the mixture. Next a transcription reaction
is carried out to transform DNA to RNA. In this reaction is DNA, buffer, neucleotide
mix, DTT, polymerase, and water. This mixture is incubated for 2 hours ate 37 C. Next
the RNA mixture is run through a centri-step spin column to purify the RNA and the
RNA solution is diluted with 100 ul hybe solution.
RNA in situ hybridization
The embryos are rehydrated with PBST and then incubated for 20 minutes with PBST.
Next, they are washed and set in pre-hybe solution for 30 minutes at 55 C. Finally the
probe is added and they embryos are incubated overnight at 55 C. The next day the
embryos are washed for 30 minutes with .2X SSC and then washed 3 times with varying
concentrations of SSC and PBST. Next, the embryos are incubated with PBST and 5%
goat serum, and a 1:2000 dilution of Dig AP Antibody for 2 hours and then washed and
incubated overnight at 4 C. On the third day, the embryos are washed with AP buffer and
then incubated in the buffer for 1 hour. Finally, NBT and BCIP are added to the embryos
and buffers and rocked covered in foil until a pattern develops. Pattern formation takes
approximately 45 minutes.
Sectioning
Hybridized embryos are placed in plastic blocks. Sections of these blocks can be cut off
to view horizontal sections of the embryos to better view staining.
Timeline:
Spring Semester 2006
– Preliminary research
Summer 2006
– REU off campus
Fall 2006
-- BMP
-- heat shock inhibition
-- section Smad
-- FKBP
-- morpholina inhibition
Spring 2006
-- further research and writing of thesis
References:
Bitar, K. N. (2003). Function of gastrointestinal smooth muscle: from signaling to
contractile proteins. Am J Med 115 Suppl 3A, 15S-23S.
Burns, A.J., (2005) Migration of neural crest-derived enteric nervous system precursor
cells to and within the gastrointestinal tract. Int J Dev Biol 49, 143-150.
Campbell, N. A., Reece, J. B. (2002). “Biology” Pearson Education Inc : San Francisco.
Chalazonitis, A., D'Autreaux, F., Guha, U., Pham, T. D., Faure, C., Chen, J. J., Roman,
D., Kan, L., Rothman, T. P., Kessler, J. A., and Gershon, M. D. (2004). Bone
morphogenetic protein-2 and -4 limit the number of enteric neurons but promote
development of a TrkC-expressing neurotrophin-3-dependent subset. J Neurosci
24, 4266-82.
Fukuda, K., Tanigawa, Y., Fujii, G., Yasugi, S., and Hirohashi, S. (1998). cFKBP/SMAP;
a novel molecule involved in the regulation of smooth muscle differentiation.
Development 125, 3535-42.
Gilbert, Scott F. (2003). “Developmental Biology” Sinaur Associates Inc : Sunderland,
Massachusetts.
Chalazonitis, A., D'Autreaux, F., Guha, U., Pham, T. D., Faure, C., Chen, J. J., Roman,
D., Kan, L., Rothman, T. P., Kessler, J. A., and Gershon, M. D. (2004). Bone
morphogenetic protein-2 and -4 limit the number of enteric neurons but promote
development of a TrkC-expressing neurotrophin-3-dependent subset. J Neurosci
24, 4266-82.
Johnson, L. R. (2001). “Gastrointestinal Physiology” Mosby, Inc.: St. Louis, Missouri.
Newgreen, D., and Young, H. M. (2002). Enteric nervous system: development and
developmental disturbances--part 1. Pediatr Dev Pathol 5, 224-47.
Patterson, C.E., Schaub, T., Coleman, E. J., Davis, E.C. (2000). Developmental
Regulation of FKB65 An ER-localized Extracellular Matrix Binding- Protein.
Mol Biol of the Cell 11, 3925-3935.
Stewart, D. R., and von Allmen, D. (2003). The genetics of Hirschsprung disease.
Gastroenterol Clin North Am 32, 819-37
Sukegawa, A., Narita, T., Kameda, T., Saitoh, K., Nohno, T., Iba, H., Yasugi, S., and
Fukuda, K. (2000). The concentric structure of the developing gut is regulated by
Sonic hedgehog derived from endodermal epithelium. Development 127, 1971-80.
Taraviras, S., and Pachnis, V. (1999). Development of the mammalian enteric nervous
system. Curr Opin Genet Dev 9, 321-7.
Wallace, K. N., and Pack, M. (2003). Unique and conserved aspects of gut development
in zebrafish. Dev Biol 255, 12-29