GLI3
and Shh interact to pattern the vertebrate limb
A Webpage Project for Developmental Biology
Taught
by Dr. Grant Mastick
A review of:
Progression of vertebrate limb development
through shh-mediated counteraction of gli3
Presented
by Gabrielle Bunker
Photo courtesy of Rachael
This
webpage was designed as part of the course requirement for Developmental
Biology. Papers were selected by each
student for review based on the paper's relevance to the study of developmental
biology. This webpage represents a
summation of the important information in the article Progression
of Vertebrate Limb Development through Shh-mediated counteraction of Gli3 by
Pascal te Weschler et al. The paper
provides an important link in vertebrate limb development that was previously
attributed to an erroneous mechanism
INTRODUCTION
·
Limb development consists of five sequential
stages, all of which take place in the lateral plate mesoderm at the flank of
the embryo.
o Limb
field allocation- Limb locations are set.
o Induction
of limb budding- Designated limb cells begin dividing more rapidly than
neighboring non-limb LPM.
o Limb
outgrowth and apical ectodermal ridge induction- The apical ectodermal ridge
forms. It runs along the
anterior-posterior axis of the limb bud and keeps limb cells dividing
(outgrowth) after they are initiated.
It is also responsible for dorso-ventral patterning.
o Patterning-
Anterior-posterior patterning and proximal-distal patterning, both of which are
controlled by signaling between the AER and the zone of polarizing
activity. The ZPA is located in the
posterior mesenchyme of the embryo and organizes the anterior-posterior axis.
o Cell
differentiation- Limb cells differentiate into bones, muscles, nerves, blood
vessels, hair, feathers, and scales.
·
In the patterning stage, the gene Shh is
absolutely required for the growth and patterning of distal and intermediate
limb structures; it can also mediate the ZPA, and is the only factor able to do
so (Capdevila and Belmonte 2001).
Figure
1- locations of AER and ZPA. Modified
from Capdevila and Belmonte 2001.
BACKGROUND
INFORMATION
·
In a wild-type embryo, Shh prevents processing of
Gli3 protein. When Gli3 is processed,
as happens in Shh-/- mouse embryos, it is broken down into small fragments that
have repressor activity. When it is not
processed (and thus left whole), as happens in wild-type Shh mouse embryos, it
acts as a transcriptional factor. In
wild type Shh mouse embryos, an anterior-posterior gradient of whole to
processed Gli3 results because Shh is produced in the anterior end of the
embryo. Normal limb development
proceeds.
·
Hox a11 and d11 are vital to pattern the
forearm(zeugopod) and Hox a13 and d13 are vital to pattern the digits
(autopod).
·
Prior to this experiment, it was believed that
anterior ectopic Shh signaling was directly responsible for digit duplications,
otherwise known as polydactyly (te Weschler et al 2002).
EXPERIMENTAL
SYSTEMS
1. The
researchers analyzed limb development of embryos that had genotypes* Shh+/+
Gli3 +/+, Shh -/- Gli3 +/+, Shh +/+ Gli3-/-, Shh -/- Gli3 +/-, and Shh-/-
Gli3-/-.
Figure
2- Gli3 and Shh. (a) represents a wild type embryo with an anterior-posterior
gradient of whole to processed Gli3.
(b) Represents a Shh-/- Gli3+/+ embryo where Gli3 is processed
everywhere.
2. The
researchers disrupted* one Gli3 allele in Shh-/- Gli3 +/+ embryos to produce Shh-/-
Gli3 +/-.
o They
then observed levels of genes Hox a11, d11, a13 and d13 before and after
disruption.
o They
also observed phenotype for developmental restoration.
ADVANTAGES
Mouse development is
representative of vertebrate development
The mutant phenotypes
were very easy to identify
There is no question
that these are developmental genes
DISADVANTAGES
The first of the two
experimental systems is purely correlational and the second is uses block-it
design.
RESULTS
1. Analysis
of limb development
·
Shh+/+, Gli3+/+
o Normal
phenotype
·
Shh-/-, Gli3+/+
o One
zeugopod (fused forearm) and no autopod (digit arch) formed
·
Shh+/+, Gli3-/-
o Polydactyl
limbs form
·
Shh-/-, Gli3+/-
o Two
zeugopods and undeveloped digits form
·
Shh-/-, Gli3-/-
o Polydactyl
and indistinguishable from Shh+/+ Gli3-/- embryos
Figure
3- Wild type mouse limb. Modified from
te Weschler et al, 2002.
Figure
4- Shh+/+ Gli3-/- and Shh-/- Gli3 -/-. Note
the phenotypic similarity between the two.
Modified from te Weschler et al, 2002
2. Disruption
of one Gli3 allele in Shh-/- Gli3 +/+ embryos and observation of the effects on
Hox genes (remember- we're talking about processed Gli3 protein with
repressor capabilities here because Shh is homozygous mutant)
·
Before
o Hox
a11- expressed
o Hox
d11- down-regulated
o Hox
a13- low expression
o Hox
d13- low expression
·
After Gli3 disruption
o Hox
a11- no effect- it functioned to begin with
o Hox
d11- expression is restored somewhat- its anterior boundary is near where it is
in wild-type embryos
o Hoxa13-
restored to intermediate levels
o Hoxd13-
remains low
·
Phenotypically speaking, disruption of one Gli3
allele restores distal limb development and especially improves zeugopodal development.
CONCLUSIONS
1. Analysis
of limb development
·
Polydactyly, which was previously attributed to
Shh signaling, is actually a result of Gli3 activity independent of Shh. Phenotypically indistinguishable polydactyly
occurs in Shh+/+Gli3-/- and Shh-/- Gli3-/- mice.
·
Shh is necessary to repress Gli3 protein
processing and Gli3 functions as either a necessary transcriptional factor when
whole or as a disrupting repressor when processed.
·
Shh mutations cause a different kind of limb
mutation seen in phenotypes of Shh-/- Gli3+/+ and Shh-/- Gli3+/- embryos.
Figure 5- Shh-/- Gli3+/+ and
Shh-/- Gli3 +/- genotypes. Shh causes a
phenotypic mutation much different from the Gli3 polydactyl mutant. Modified from te Weschler et al, 2002.
2. Disruption
of one Gli3 allele and observation of Hox genes
·
Two conclusions that are not mutually exclusive
of each other can be reached from this experiment:
o Processed
Gli3 protein interferes with Hox d11 and a13 because when one Gli3 allele is
interrupted expression of those two Hox genes increases.
o Whole
Gli3 protein is necessary for normal Hox a13 and d13 expression because d13
showed no improvement when Gli3 was interrupted and a13 showed intermediate
improvement.
SIGNIFICANCE
·
This experiment reforms an old idea about the
role of anterior ectopic Shh signaling in polydactyly in limb development.
·
It provides a new mechanism for polydactyly
involving Gli3 activity that is independent from Shh signaling.
·
It put together a pathway from Shh-/- to
processed Gli3 and identified the effect this path has on four Hox genes: a11,
d11, a13, and d13.
FUTURE
DIRECTIONS
·
Study Gli3's specific effect as a transcription
factor with Hox a13 and d13.
·
Study Gli3's specific effect as a repressor with
Hox d11 and a13.
·
Investigate the purpose of the anterior-posterior
gradient of whole to processed Gli3 protein.
REFERENCES
Review article
This review article detailed all the
known mechanisms of vertebrate limb development in terms of a discussion of the
genetics behind the five stages of developing limbs: limb field allocation,
induction of limb buds, limb outgrowth and apical ectodermal ridge induction,
anterior-posterior and proximal-distal patterning, and cell differentiation.
Primary research article
This research article delineated the
interactions between Shh and Gli3 in vertebrate limb patterning, specifically
concerning polydactyl limb formation.
Recently, it was believed that polydactylous limbs arose from ectodermic
anterior Shh signaling, but the authors find that Gli3 deficient mice have
polydactylous limbs independent of Shh signaling.
Related articles
This article states that Shh expression
is necessary to ensure normal limb development proceeds. The authors found that Shh mutants developed
limbs that had a posterior defect in the zeugopod and all of the digits except
one.
* Specific mechanisms (e.g. antibody
labeling) for the determination of genotype and disruption of Gli3 were not
discussed in the article.