Hox genes and pattern development of vertebrates
Pattern formation: harmonious arrays of different elements, such as the array
of fingers on the hand, body pattern (head, trunk, and tail), or limb patterns.
Pattern formation is best understood in Drosophila, where most genes that
contribute to the body plan are described.
Anteroposterior axis in vertebrates is
specified by a group of genes called
homeobox genes. There are many
similarities to Drosophila.
The dorsoventral axis in vertebrates is
also specified by genes that have
counterparts in Drosophila.
Interestingly, although vertebrates and
invertebrates share a similar body
plan, it is inverted.
Vertebrate limb development is
controlled by multiple genes,
including homeobox genes.
Development depends on the process
of reciprocal interaction between
ectoderm and mesenchyme.
Homeobox genes
The homeobox: many genes that control pattern formation share a consensus
sequence of 180 nucleotides. Genes with a homeobox are highly conserved in
plants and animals and are called homeobox genes.
Homeodomain: the protein encoded by a homeobox gene contains a 60 amino
acid domain encoded by the homeobox. All homeodomain containing proteins
are transcription factors that bind to gene specific promoters or enhancers.
The homeodomain has three alpha
helices. The recognition helix (a3) aligns
in the major groove of DNA
TALE proteins: (Exd) an atypical family
of homeodomain proteins that help to
target specific homeodomain
transcription factors to their correct cis
elements.
They bind to DNA first, they alter the
DNA conformation slightly, and this
assists in binding of other
homeodomain transcription factors.
TALE proteins also stabilize bound
homeodomain proteins.
Homeobox genes are often clustered on one chromosome
Homeobox genes exhibit two patterns of localization:
1. Some are scattered throughout the genome.
2. Hox genes: other homeobox genes are clustered within a small region and in
a very specific sequence (Hox genes form a Hox complex). These Hox genes
are highly conserved in organisms ranging from flies to humans.
How did Hox genes
evolve?
Hox complexes: arose by
repeated duplication and
mutation of an ancestral
homeobox gene. This
formed an ancestral HOX
complex.
In some organisms,
including most
vertebrates, the HOX
complex has been
duplicated four times.
Hox genes are organized into paralogy and orthology groups
Paralogy group: are simply the natural clusters of Hox genes on the
chromosome. Vertebrates have 4, flies have 1. They are named a, b, c, and d.
Orthology group: sequence comparisons of each gene shows that certain
genes are closely related. These are the genes in most vertical columns (for
example, Hox a9, b9, c9 and d9). Orthologous Hox genes from different species
can replace one another in function (Hox d4 substitutes for Dfd in Drosophila).
Genes of any
orthology group have
corresponding
positions within each
complex (if clusters
are aligned in rows,
the orthology groups
form columns. Genes
are numbered
consecutively from 113.
Hox genes specify anteroposterior body pattern
The physical order of Hox genes within the complex is related to their order of
expression along the anteroposterior axis of the embryo!!
Genes at the 3’ end are expressed in the anterior and genes at the 5’ end are
expressed progressively further posteriorly.
Hoxb cluster is expressed in the central nervous system. Each gene is first
expressed at a sharply defined point and expression continues posteriorly and
gradually tapers off.
Rhombomeres: the pattern of Hox gene expression often coincide with
repetitive bulges in the sides of the rhomencephalon.
mouse
What accounts for the pattern of Hox gene expression?
mouse
Enhancer sharing: the sequential order of Hox genes within the complex may
result from the fact that all genes in the complex share a common enhancer
element. If any gene is removed, or if the complex is broken apart, the removed
genes may not be expressed properly.
Thus, the sequential order has been preserved during evolution of different
organisms.
The dorsoventral body plan:
are frogs just upside down flies?
The dorsoventral body pattern of vertebrates and invertebrates are similar but
inverted. Invertebrates, such as lobsters and flies, have the central nervous
system on the ventral side and the heart is positioned dorsally.
How is the dorsoventral axis
established?
Vertebrates: goosecoid stimulates
noggin and chordin, which help
induce the dorsal pattern. BMP-4 and
xolloid induce the ventral pattern.
Invertebrates: decapentaplegic (dpp)
and tolloid induce dorsal
development such as heart. Short
gastrulation protein (sog) induces
ventral development of CNS.
Dpp and BMP-4 are similar and
belong to the TGF-b family. Sog and
chordin also share sequence
similarity.
Are sog and chordin interchangable in flies and frogs?
If the body pattern of flies and frogs is established by similar signals, one
might expect ectopic expression of sog from flies to act as Spemann’s
organizer and to be able to organize neural tube in a frog embryo. Injection of
sog RNA into the early frog gastrula causes formation of a second blastopore.
Frog embryos can be completely ventralized by exposure to UV radiation (no
CNS develops). However, the development of UV-treated embryos can be
completely rescued by injection of sog RNA.
Injection of noggin RNA from frogs into Drosophila embryos prevented the
development of normal dorsal structures such as amnioserosa and dorsal
ectoderm, but induced a second set of neurons.
Thus, the signals for dorsoventral pattern formation are similar in flies and
frogs. However, it is a mystery why they become inverted.
Hox gene expression contributes to limb development
Limb buds: the first stage of limb development occurs at 5 weeks in humans
when small paddle shaped limb buds form.
Apical ectodermal ridge (AER): a specialized structure formed by the
ectodermal covering of the limb bud. It is a ridge that runs anterior to posterior.
Progress zone: underneath the AER lies a zone of mesenchyme that actively
proliferates to form the limb. Somites contribute mesenchyme to form muscles
and lateral plate mesoderm forms cartilage and connective tissue.
The limb is formed by
differential growth of
mesenchyme cells, by
programmed cell death
(between digits), and
specific patterns of
differentiation induced by
Hox genes and other factors.
How does the limb know
where to form? How do arms
become different than legs?
How does the limb develop
its three axis?
Limb position is determined by FGF and Hox genes
The position of limb development depends on signals from other tissues.
Fibroblast growth factor (FGF): these growth factors are produced by
mesenchyme (FGF-10) and epidermis (FGF-8) to induce limb formation. If a
small bead containing FGF is implanted under the skin, an extra limb develops.
If the bead is implanted in the flank near the anterior, it forms a wing. If the
bead is implanted posteriorly, it forms a leg. Knock out mice lacking FGF-10 fail
to develop limbs and have no apical ectodermal ridge or zone of polarizing
activity.
Hox gene expression is directly changed by FGF beads.
Under normal conditions, wing expresses Hox d9
Flank expresses Hox b9 and c9.
Leg expresses Hox b9, c9, and d9.
Ectopic development of wing on the flank results in loss of Hox b9 and
Hox c9, whereas, ectopic development of leg is induced by induction of
all three Hox genes.
Signals from somites and lateral plate mesoderm
specify the dorsoventral limb axis
Limb bud mesoderm is first induced by signals from the adjacent somites. This
mesoderm then induces ectoderm to form the AER.
The somite continues to induce ectoderm to form the dorsal surface of the
limb. The lateral plate mesoderm induces the ventral portion of limb ectoderm.
Radical fringe: a gene
expressed in the dorsal
ectoderm that provides a
dorsalizing function and
helps to induce the AER.
Wnt-7a: another gene
expressed in the dorsal
ectoderm. It stimulates the
underlying mesenchyme to
produce Lmx-1, an
important signal for dorsal
differentiation.
Engrailed: a gene
expressed in the ventral
ectoderm that provides a
ventralizing function. It also
inhibits expression of
radical fringe and Lmx-1.
The AER develops at the
junction between areas that
express radical fringe and
engrailed.
Sonic hedgehog induces the anteroposterior pattern
of fingers and toes
If a small piece of the chicken posterior wing bud is grafted to the anterior
portion of a second embryo, it causes extra digits in a mirror image pattern.
Normally, II, III, and IV develop. Now IV, III, II, III, IV form.
Zone of polarizing activity: the ability of a tissue to establish the posterior
position of the anteroposterior pattern (rear of limb bud).
Sonic hedgehog: shh is
expressed at high level in
the zone of polarizing
activity, suggesting it’s
importance.
To test this, purified shh
DNA was transfected into
cells and the cells were
transplanted into the wing
bud.
The exact same result
occurred, thus, shh is
important for generating
wing pattern.
Reciprocal interaction is critical for wing development
Interactions occur between the AER on the surface and limb bud mesenchyme
that lies underneath in the progress zone.
Limb bud mesenchyme induces formation of the AER from limb bud ectoderm.
If the ectoderm of a limb bud is removed at an early stage of development, the
mesoderm induces a new AER. If the AER is removed during a later stage, the
limb mesenchyme stops growing and the limb is truncated.
If the limb bud mesenchyme is removed or replaced with other tissue, the AER
quickly degenerates.
The AER serves a permissive rather than an instructive role. If you reverse its
orientation, digits will develop normally. If you replace wing AER with leg AER,
the wing develops normally.
The mesenchyme is the instructive influence for limb development.