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BLOCKS OF ANALYSIS
Signal Transduction Pathways and Sonic Hedgehog
Signal transduction pathways are essential for cell to cell communication. They play a critical role during the
development of most organisms. The major components of a signal transduction pathway are the signaling
molecule, the receptor, and usually several second messenger molecules that transduce the extracellular
signal into an intracellular response. This case study focuses on one particular signal transduction pathway
that includes the secreted signaling protein Sonic hedgehog, and how signaling abnormalities can have a
significant adverse impact on the development of the organism.
Sonic hedgehog was discovered to be a homologue of the Drosophila segment polarity hedgehog (hh) gene.
This gene was already well characterized and had been shown to encode a secreted signaling protein
involved in Drosophila development. There are three different mammalian homologues: Sonic hedgehog,
desert hedgehog, and Indian hedgehog (Echelard et al., 1993). Sonic hedgehog is expressed in the neural
tube floorplate, the Hensen node, the early gut endoderm, the posterior of the limb buds, and the
notochord.
The Sonic hedgehog protein is a secreted intercellular signaling molecule. Its precursor form is autocatalytically
cleaved into an N-terminal (Shh-N) and a C-terminal (Shh-C) domain. During catalysis, a molecule of
cholesterol is covalently attached to Shh-N which performs the signaling function. Shh-C carries out the
cleavage reaction and the cholesterol attachment.
Shh-N has also been shown to be palmitoylated.
Shh-N binds to the transmembrane protein Patched
(Ptc). This then derepresses a Ptc-interacting
membrane protein called Smoothened (Smo),
resulting in the activation of GLI, a transcription
factor. GLI, in turn, is thought to control the
ultimate expression of the nuclear targets of Sonic
hedgehog (Walterhouse et al., 1999; Muenke and
Beachy, 2000; Figure 1).
The Development of the Brain and
Holoprosencephaly Holoprosencephaly (HPE)
is a congenital malformation of the head that results from the failure of the forebrain to divide properly
into the mature regions of the brain. The frequency of this disorder is 1 in 250 embryos and 1 in 16,000
newborns (Roessler et al., 1996). The severity of this condition has a dramatic range, from very mild forms
that are difficult to notice to extremely severe forms that are lethal. The different forms of HPE can be
categorized into 3 major groups: alobar, semilobar, and lobar. In alobar HPE, there is no subdivision of the
prosencephalon. In semilobar HPE, there are rudimentary cerebral lobes, but often no olfactory tracts or
bulbs. Finally, in lobar HPE, the brain has distinct cerebral lobes, although they may be reduced in size. In
addition, there may be other abnormalities of the olfactory tracts, olfactory bulbs, and the corpus callosum.
In the most severe form of alobar HPE, the brain is small with a single cerebral ventricle. A nose-like
proboscis is present over a single, medial eye, giving rise to the description cyclopia. The birth of children and
animals with a single eye throughout history has spawned many myths, including the Greek cyclops of
Homer’s Odyssey (Cohen and Sulik, 1992). Another severe form of holoprosencephaly is called ethmocephaly.
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Here, the two eyes are separated, but only by the proboscis. Another variation is cebocephaly, in which the
eyes are still spaced closely together, but are located above a nose with a single nostril.
The moderate forms of holoprosencephaly are characterized by a prosencephalon that has split somewhat
but still not to a normal degree. In the most severe form within this category, the eyes are still close-set, the
nose
is flat and there is a medial cleft lip. At the next most severe level, there are still close-set eyes, with a bilateral
cleft lip and a flat process representing the philtrum.
The mildest forms of HPE include phenotypes such as closely or
widely spaced eyes, dental abnormalities, or an impaired sense of
smell (Cohen and Sulik, 1992; Muenke, 1994). The range of HPE
phenotypes is continuous over the entire spectrum. Examples of
some of the cases described above are shown in Figure 2. The
degree of craniofacial abnormality usually, but not always, reflects
the degree of brain abnormality. Almost all patients display some
form of developmental delay. Those children born with severe
HPE usually die before their first birthday (Muenke and Beachy,
2000).
To understand how holoprosencephaly could develop, one must
understand the fundamentals of normal brain development. The
early brain starts as a simple tube. It initially subdivides into three
brain vesicles: the forebrain (prosencephalon), the midbrain
(mesencephalon), and the hindbrain (rhombencephalon). Later in
development, the prosencephalon is subdivided into the
telencephalon and the diencephalon, and the rhombencephalon
gives rise to the metencephalon and myelencephalon. A critical step is the cleavage of the tissue along several
different axes Fig. 2. Holoprosencephalic Phenotypes (from Muenke, into distinct functional regions. Along the sagittal axis, the 1994)of
holoprosencephaly phenotypes, from the most Shown here are examples of the wide spectrum prosencephalon cleaves into cerebral
hemispheres. Transversely, it severe forms on the top to more mild forms towards is divided into the telencephalon and
the diencephalon. Finally, the bottom. along the horizontal axis it is cleaved into olfactory and optic bulbs (Cohen
and Sulik, 1992). The optic vesicles that form are normally well separated from each other by the developing
structures of the ventral forebrain.
In the severe forms of HPE, the ventral forebrain structures do not form and the optic vesicle arises as a single,
central evagination. The nasal structures are abnormally positioned due to the subsequent growth of the single
eye in the face. The prechordal plate mesoderm appears to be especially critical for the induction of ventral
forebrain structures. Sonic hedgehog is expressed in the prechordal plate mesoderm. The prosencephalic neural
plate is also important for proper development of these regions (Muenke and Beachy, 2000). Th e abnormal
developmental events leading to human holoprosencephaly occur within the first 4 weeks of gestation (Golden,
1999).
Holoprosencephaly can be either sporadic or inherited. In families with this condition, the entire range of
phenotypes may be observed. The inheritance patterns may be autosomal dominant, autosomal recessive, and
possibly X-linked as well. Teratogens have also been shown to cause holoprosencephaly, including alcohol
and the alkaloids of the plant Veratum califonicum. Certain compounds that affect cholesterol biosynthesis can
also lead to holoprosencephaly, possibly reflecting the attachment of cholesterol to Shh during the signaling
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process. Maternal diabetes is also a risk factor for holoprosencephaly (Muenke, 1994; Golden, 1999; Muenke
and Beachy, 2000).
In 1996, mutations in the Sonic hedgehog gene were linked to human holoprosencephaly (Roessler et al.,
1996; Belloni et al., 1996). In a follow-up study, a mutational analysis of the Sonic hedgehog gene in 344
unrelated patients with HPE was performed (Nanni et al., 1999). Of these 344 patients, 23 (6.7%) had
mutations in the Shh gene, with 14 representing patients with a family history of HPE and the other 9
representing cases of sporadic HPE. At the molecular level, the mutations occurred throughout the gene and
no specific genotype-phenotype correlation was apparent in this study. Complicating the issue is the fact that
approximately 30% of HPE carriers are clinically normal. This makes the provision of effective genetic
counseling for these families a critical issue.
Holoprosencephaly is known to occur in many different species of animals, suggesting that animal model
research will provide a great deal of insight into the mechanisms by which Shh regulates early brain
development (Muenke, 1994). “Knock-out” mice that lack Sonic hedgehog gene function exhibit cyclopia
and ventral cell types within the neural tube are absent (Chiang et al., 1996). Zebrafi sh also require the
activity of Sonic hedgehog for normal forebrain development (Golden, 1999). Surgical and molecular
experiments performed on chick embryos also showed similar results (Hu and Helms, 1999).
In addition to Sonic hedgehog, it is likely that there are at least 11 other genes involved in the development
of HPE, including ZIC2, SIX3, and TGIF (Brown et al., 1998; Wallis et al., 1999; Nanni et al., 1999; Muenke
and Beachy, 2000). ZIC2 is a member of the Drosophila odd-paired family of zinc-finger transcription factors.
ZIC2 is expressed in the dorsal neural tube, the eye and the distal limb (Brown et al.,
1998). SIX3, a homolog of the Drosophila optix gene, functions in midline forebrain and eye formation. It is
expressed in the rostral, anterior region of the neural plate, the midline ventral forebrain, the optic recess,
and the developing retina (Wallis et al., 1999). TG-Interacting Factor (TGIF) is a homeodomain protein that
may repress retinoic acid regulated gene transcription. Retinoic acid is a teratogen known to cause HPElike
abnormalities (Muenke and Beachy, 2000). Scientists are still looking for the many additional genes believed
to be linked to holoprosencephaly.
Ethical Precedents for Organ/Body Donation
Our current state of progress in science and medicine would never have been achievable without the
donation of cadavers and body tissues. Nevertheless, approaching a grieving family about the possible
donation of a loved one’s body or organs is always a delicate proposition.
Complicating the matter even further is the wide diversity of cultural and religious attitudes towards the postmortem use of the human body for medical or research purposes. For many people, the body is not just
utilitarian in purpose, but is considered sacred. Many religious groups, both historical and current, believe
that the integrity of the body is essential for salvation after death. This was a prevalent viewpoint during the
Middle Ages. Attitudes only began to change during the Renaissance, when both artists and scientists began
to study both the external and internal anatomy of the human body. Eventually, by the early nineteenth
century, the cadaver was a standard tool in medical training and practice. However, due to continued public
sentiment about the sanctity of the human body, the supply of cadavers was generally drawn from “social
outcasts” during this period of history (Nelkin and Andrews, 1998a).
Many modern day cultures or religious groups have certain beliefs about the treatment of the body. Often
there is a belief in the requirement for an “intact body” to be preserved for a later resurrection. Among
Orthodox Jews, the body must be buried whole, even to the extent that a body part removed during life
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(such as an amputated leg) must be stored until that person’s death to be buried with the remainder of the
body. Many Native Americans also have strong beliefs about maintaining the integrity of the body, as do
people of Islamic faith. Nevertheless, many faiths also place high value on the saving of human life and love
of one’s neighbor, which, for many theologians, justifies the use of human tissues for research and
educational purposes (Nelkin and Andrews, 1998a; Campbell, 1998).
The current feverish pace of scientific and medical advancement is only increasing the commercial value of
human bodies as research material. Physicians and scientists involved in a commercially linked collaboration
using these donated tissues may have certain conflict-of-interest issues to deal with. Problem areas include
the purchase of patients’ tissues by scientists, the profit that scientists may someday earn off of research
done on these tissues, and outright theft involving such tissues. The acquisition of royalty rights for the
patient or patient’s family from commercial products made from donated tissues is a growing issue. Two
key pieces of legislation that address the use of cadavers and organs are the Uniform Anatomical Gift Act
(1968) and the National Organ Transplantation Act (1984). Legislation concerning the use of tissues, cells,
and DNA, however, is still in its infancy (Nelkin and Andrews, 1998a; Nelkin and Andrews, 1998b). The
need for a more comprehensive ethical and legal framework is essential as these issues continue to arise and
be disputed in the courts, the medical community, and the media.
Consent from the family or the deceased by prior decree is a fundamental principle in research ethical
practice and law. Occasionally, the wishes of the family and the deceased may contradict each other. If the
wishes of the deceased are clearly known, such as in the case of a valid written testament, these wishes
should not be superseded by the family’s desires. The only instances where the state may override the
family or the deceased person’s wishes to autopsy or otherwise study the body is when the cause of death
must be known (as in a possible criminal action) or when the well-being of the community is at stake.
Whether or not consent is given may depend on the nature of the research being done. For instance,
members of various ethnic groups or genders may be concerned that the study of IQ or crime in relation to
their “group” may result in a stigmatization of that group (Nelkin and Andrews, 1998a).
Perhaps the gravest danger in not respecting the concerns of the family and the community is that the
integrity of the research institutions will be damaged. Continued advancement of biological research is
dependent upon the support of the community. A lack of respect for the concerns of the community by
scientists will eventually translate into an erosion of support for scientifi c and medical advancement, in
spirit,
in legislation, and in taxpayer-supported funding.
REFERENCES
The journal articles marked with an asterisk are comprehensive reviews on the genetic causes of
holoprosencephaly and are recommended as a good starting point for further research. I would recommend
placing them on reserve in the library, as the journals they are published in are not necessarily found in many
smaller college libraries.
Books
Gilbert, S.F. 1997. Developmental Biology, 5th ed. Sinauer Associates, Inc.: Sunderland, Mass.
Purves, D., G.J. Augustine, D. Fitzpatrick, L.C. Katz, A.-S. LaMantia, and J.O. McNamara. 1997. Neuroscience.
Sinauer Associates, Inc.: Sunderland, Mass.
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Wolpert, L., R. Beddington, J. Brockes, T. Jessell, P. Lawrence, and E. Meyerowitz. 1998. Principles of
Development. Oxford University Press: Oxford.
Journal Articles
Belloni, E., M. Muenke, E. Roessler, G. Traverso, J. Siegel-Bartelt,A. Frumkin, H.F. Mitchell, H.
DonisKeller, C. Helms, A.V. Hing, H.H.Q. Heng, B. Koop, D. Martindale, J.M. Rommens, L.-C.
Tsui, and S.W. Scherer. 1996. Identifi cation of Sonic hedgehog as a candidate gene responsible for
holoprosencephaly. Nature Genet. 14:353-356.
Brown, S.A., D. Warburton, L.Y. Brown, C.-Y. Yu, E.R. Roeder, S. Stengel-Rutkowski, R.C.M. Hennekam,
and M. Muenke. 1998. Holoprosencephaly due to mutations in ZIC2, a homologue of Drosophila
oddpaired. Nature Genet. 20:180-183.
Campbell, C.S. 1998. Religion and the body in medical research. Kennedy Inst. Ethics J. 8:275-305.
Chiang, C., Y. Litingtung, E. Lee, K.E. Young, J.L. Corden, H. Westphal, and P.A. Beachy. 1996. Cyclopia
and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383:407-413. Cohen Jr.,
M.M., and K.K. Sulik. 1992. Perspectives on holoprosencephaly: Part II. Central nervous system,
craniofacial anatomy, syndrome commentary, diagnostic approach, and experimental studies. J Craniofac.
Genet. Dev. Biol. 12: 196-244.
Echelard, Y., D.J. Epstein, B. St. Jacques, L. Shen, J. Mohler, J.A. McMahon, and A.P. McMahon. 1993.
Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of
CNS polarity. Cell 75:1417-1430.
* Golden, J. A. 1999. Towards a greater understanding of the pathogenesis of holoprosencephaly. Brain and
Development 21: 513-521.
Hu, D. and J.A. Helms. 1999. Th e role of Sonic hedgehog in normal and abnormal craniofacial
morphogenesis. Development 126: 4873-4884.
Muenke, M. 1994. Holoprosencephaly as a genetic model for normal craniofacial development. Seminars in
Developmental Biology 5: 293-301.
* Muenke, M., and P.A. Beachy. 2000. Genetics of ventral forebrain development and holoprosencephaly.
Current Opinion in Genetics and Development 10: 262-269.
Muenke, M. and P.A. Beachy. 2001. Holoprosencephaly. In Th e Metabolic and Molecular Bases of Inherited
Disease. 8th Ed. Scriver, C.R., A.L. Beaudet, W.S. Sly, D. Valle, B. Childs, and B. Vogelstein, eds. New
York: McGraw-Hill.
Nanni, L., J.E. Ming, M. Bocian, K. Steinhaus, D.W. Bianchi, C. de Die-Smulders, A. Giannotti, K.
Imaizumi, K.L. Jones, M. Del Campo, R.A. Martin, P. Meinecke, M.E.M. Pierpont, N.H. Robin, I.D.
Young, E. Roessler, and M. Muenke. 1999. Th e mutational spectrum of the Sonic Hedgehog gene in
holoprosencephaly: SHH mutations cause a signifi cant proportion of autosomal dominant
holoprosencephaly. Human Molecular Genetics 8: 2479-2488.
Nelkin, D., and L. Andrews. 1998a. Do the dead have interests? Policy issues for research after life. American
Journal of Law and Medicine 24: 261-291.
Nelkin, D. and L. Andrews. 1998b. Homo Economicus: Commercialization of Body Tissue in the Age of Biotechnology.
Hastings Center Report. 28:30-39.
O’Neill, O. 1996. Medical and scientifi c uses of human tissue. Journal of Medical Ethics 22: 5-7.
Roessler, E., E. Belloni, K. Gaudenz, P. Jay, P. Berta, S.W. Scherer, L.-C. Tsui, and M. Muenke. 1996.
Case Teaching Notes for “Sonic Hedgehog” by Lauren E. Yaich
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Mutations in the human Sonic Hedgehog gene cause holoprosencephaly. Nature Genet. 14: 357-360.
Wallis, D.E., E. Roessler, U. Hehr, L. Nanni, T. Wiltshire, A. Richieri-Costa, G. Gillessen-Kaesbach, E.H.
Zackai, J. Rommens, and M. Muenke. 1999. Mutations in the homeodomain of the human SIX3 gene
cause holoprosencephaly. Nature Genet. 22: 196-198.
Walterhouse, D.O., J. Yoon, and P.M. Iannaccone. 1999. Developmental pathways: Sonic
hedgehogPatched-GLI. Environmental Health Perspectives 107: 167-171. Web Sites
Th e Carter Center for Brain Research in Holoprosencephaly and Related Malformations
http://www.stanford.edu/group/hpe
National Institute of Neurological Disorders and Stroke
http://www.ninds.nih.gov/health_and_medical/disorders/holoprosencephaly.htm
Online Mendelian Inheritance in Man http://www.ncbi.nlm.nih.gov/omim Zygote
http://zygote.swarthmore.edu
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Acknowledgements: Th is case study was developed with support from Th e Pew Charitable Trusts and the National Science
Foundation as part of the Case Studies in Science Workshop held at the State University of New York at Buff alo on May 22-26,
2000. Image credit: Fig. 2 from Muenke, M. 1994. Holoprosencephaly M. 1994. Holoprosencephaly as a genetic model for normal
craniofacial development. Seminars in Developmental Biology 5: 293-301. Used with permission from Academic Press Ltd.
Copyright held by the National Center for Case Study Teaching in Science, University at Buff alo, State University of New
York. Originally published April 19, 2001. Please see our usage guidelines, which outline our policy concerning permissible
reproduction of this work.
Case Teaching Notes for “Sonic Hedgehog” by Lauren E. Yaich
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