Limb development
Encyclopedia
Tetrapod
Tetrapods are vertebrate animals having four limbs. Amphibians, reptiles, birds and mammals are all tetrapods; even snakes and other limbless reptiles and amphibians are tetrapods by descent. The earliest tetrapods evolved from the lobe-finned fishes in the Devonian...
s — animal
Animal
Animals are a major group of multicellular, eukaryotic organisms of the kingdom Animalia or Metazoa. Their body plan eventually becomes fixed as they develop, although some undergo a process of metamorphosis later on in their life. Most animals are motile, meaning they can move spontaneously and...
s with four limbs — is an area of active research in developmental biology
Developmental biology
Developmental biology is the study of the process by which organisms grow and develop. Modern developmental biology studies the genetic control of cell growth, differentiation and "morphogenesis", which is the process that gives rise to tissues, organs and anatomy.- Related fields of study...
. Limb formation begins in the limb field
Morphogenetic field
In developmental biology, a morphogenetic field is a group of cells able to respond to discrete, localized biochemical signals leading to the development of specific morphological structures or organs. The spatial and temporal extent of the embryonic fields are dynamic, and within the field is a...
, as a limb "bud." Fibroblast growth factor
Fibroblast growth factor
Fibroblast growth factors, or FGFs, are a family of growth factors involved in angiogenesis, wound healing, and embryonic development. The FGFs are heparin-binding proteins and interactions with cell-surface associated heparan sulfate proteoglycans have been shown to be essential for FGF signal...
(FGF) induces formation of an organizer, called the apical ectodermal ridge (AER), which guides further development and controls cell death. Apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
— programmed cell death — is necessary to eliminate webbing between digits.
The limb field is a region specified by expression of certain homeobox
Homeobox
A homeobox is a DNA sequence found within genes that are involved in the regulation of patterns of anatomical development in animals, fungi and plants.- Discovery :...
(Hox) genes and Tbx genes; Tbx5 for forelimb and Tbx4 for hindlimb. Establishment of the limb field requires retinoic acid
Retinoic acid
Retinoic acid is a metabolite of vitamin A that mediates the functions of vitamin A required for growth and development. Retinoic acid is required in chordate animals which includes all higher animals from fishes to humans...
signaling in the developing trunk of the embryo from which the limb emerges.
Limb formation results from series of epithelial-mesenchymal inductions between the limb bud mesenchymal cells and the overlying ectodermal cells. Cells from the lateral plate mesoderm
Lateral plate mesoderm
Lateral plate mesoderm is a type of mesoderm that is found at the periphery of the embryo.-Division into layers:It will split into two layers, the somatic layer/mesoderm and the splanchnic layer/mesoderm...
and the myotome
Myotome
In vertebrate embryonic development, a myotome is a group of tissues formed from somites that develop into the body wall muscle.Each myotome divides into a dorsal epaxial part and a ventral hypaxial part....
migrate to the limb field and proliferate to create the limb bud. The lateral plate cells produce the cartilaginous and skeletal portions of the limb while the myotome cells produce the muscle
Muscle
Muscle is a contractile tissue of animals and is derived from the mesodermal layer of embryonic germ cells. Muscle cells contain contractile filaments that move past each other and change the size of the cell. They are classified as skeletal, cardiac, or smooth muscles. Their function is to...
components. The lateral plate mesodermal cells secrete a fibroblast growth factor
Fibroblast growth factor
Fibroblast growth factors, or FGFs, are a family of growth factors involved in angiogenesis, wound healing, and embryonic development. The FGFs are heparin-binding proteins and interactions with cell-surface associated heparan sulfate proteoglycans have been shown to be essential for FGF signal...
(FGF7
FGF7
Keratinocyte growth factor is a protein that in humans is encoded by the FGF7 gene.-Further reading:...
and FGF10, presumably) to induce the overlying ectoderm
Ectoderm
The "ectoderm" is one of the three primary germ cell layers in the very early embryo. The other two layers are the mesoderm and endoderm , with the ectoderm as the most exterior layer...
to form an important organizing structure called the apical ectodermal ridge (AER).. The AER reciprocatively secretes FGF8 and FGF4 which maintains the FGF10 signal and induces proliferation in the mesoderm. The position of FGF10 expression is regulated by Wnt8c in the hindlimb and Wnt2b
WNT2B
Protein Wnt-2b is a protein that in humans is encoded by the WNT2B gene.-Further reading:...
in the forelimb. The forelimb and the hindlimb are specified by their position along the anterior/posterior axis
Anatomical terms of location
Standard anatomical terms of location are designations employed in science that deal with the anatomy of animals to avoid ambiguities that might otherwise arise. They are not language-specific, and thus require no translation...
and possibly by two T-box
T-box
T-box refers to a group of transcription factors involved in limb and heart development. In humans and some other animals, defects in the TBX5 gene expression can lead to finger-like thumbs and ventricular septal defects in which there is no separation between the left and right ventricle of the...
containing transcription factors: Tbx5 and Tbx4, respectively.
Programmed cell death removes the "webbing" between the digits and joints. BMP signaling induces cell death and Noggin blocks cell death in the digits.
Precartilage condensations
The limb's skeletal elements are prefigured by tight aggregates of mesenchymal cells called precartilage condensations. Mesenchymal condensation is mediated by extracellular matrixExtracellular matrix
In biology, the extracellular matrix is the extracellular part of animal tissue that usually provides structural support to the animal cells in addition to performing various other important functions. The extracellular matrix is the defining feature of connective tissue in animals.Extracellular...
and cell adhesion
Cell adhesion
Cellular adhesion is the binding of a cell to a surface, extracellular matrix or another cell using cell adhesion molecules such as selectins, integrins, and cadherins. Correct cellular adhesion is essential in maintaining multicellular structure...
molecules. Cartilage
Cartilage
Cartilage is a flexible connective tissue found in many areas in the bodies of humans and other animals, including the joints between bones, the rib cage, the ear, the nose, the elbow, the knee, the ankle, the bronchial tubes and the intervertebral discs...
differentiates from the condensations, giving rise to the skeletal primordia. In most tetrapod limb skeletons (though not in some amphibians), the cartilage skeleton is replaced by bone later in development.
Periodicities of the limb pattern
The limb is organized into three regions: stylopod, zeugopod, and autopod (in order from proximalAnatomical terms of location
Standard anatomical terms of location are designations employed in science that deal with the anatomy of animals to avoid ambiguities that might otherwise arise. They are not language-specific, and thus require no translation...
to distal
Anatomical terms of location
Standard anatomical terms of location are designations employed in science that deal with the anatomy of animals to avoid ambiguities that might otherwise arise. They are not language-specific, and thus require no translation...
). Within the distal two of these regions the skeleton contains a number of periodic and quasi-periodic pattern motifs. The zeugopod consists of two parallel elements along the anteroposterior (AP) axis and the autopod contains 3-5 (in most cases) elements along the same axis. The digits also have a quasi-periodic arrangement along the proximodistal (PD) axis, consisting of tandem chains of skeletal elements. The generation of the basic limb plan during development results from the patterning of the mesenchyme by an interplay of factors that promote precartilage condensation and factors that inhibit it.
The development of the basic limb plan is accompanied by generation of local differences between the elements. For example, the radius and ulna of the forelimb zeugopod are distinct from one another, as are the different fingers in the autopod. These differences can be treated schematically by considering how they are reflected in each of the limb's three main axes.
Axial patterning and related issues
Many investigations into the development of the limb skeletal pattern have been influenced by the "positional information" concept proposed by Lewis WolpertLewis Wolpert
Lewis Wolpert CBE FRS FRSL is a developmental biologist, author, and broadcaster.-Career:Wolpert was educated at the University of Witwatersrand , at Imperial College London, and at King's College London...
in 1971. In tune with this idea, efforts have been made to identify diffusive signaling molecules (morphogen
Morphogen
A morphogen is a substance governing the pattern of tissue development, and the positions of the various specialized cell types within a tissue...
s) that traverse orthogonal axes of developing limbs and determine locations and identities of skeletal elements in a concentration-dependent fashion.
Proximal/distal patterning
Hox genes contribute to the specification of the stylopod, zeugopod and autopod. Mutations in Hox genes lead to proximal/distalAnatomical terms of location
Standard anatomical terms of location are designations employed in science that deal with the anatomy of animals to avoid ambiguities that might otherwise arise. They are not language-specific, and thus require no translation...
losses or abnormalities. Three different models have been advanced for explaining the patterning of these regions.
Progress Zone model
The AER creates a zone of cell proliferation and lays down the limb from proximal to distal. The time cells leave the AER determines their positional value. Proximal structures are formed earlier than distal structures.The Progress Zone model was proposed 30 years ago but recent evidence has conflicted with this model.
Experimental evidence:
- Removing the AER at a later period of development results in less disruption of distal structures than if the AER was removed early in development.
- Grafting a new bud tip on top of an old bud tip results in a deletion and duplication of structures.
Early allocation and progenitor expansion model (or prespecification model)
Cells are specified for each segment in the early limb bud and this population of cells expand out as the limb bud grows. This model is consistent with the following observations. Cell division is seen throughout the limb bud. Cell death occurs within a 200 μm zone subjacent to the AER when it is removed; cell death forecloses some patterning. FGF-releasing beads are able to rescue limb development when the AER is removed by preventing this cell death.Experimental evidence:
- Labeled cells in different position of an early limb bud were restricted to single segments of the limb.
- Limbs lacking expression of required FGF4 & FGF8 showed all structures of the limb and not just the proximal parts.
More recently, however, the investigators primarily responsible for both the Progress Zone and Prespecification models have acknowledged that neither of these models accounts adequately for the available experimental data.
Activator-inhibitor (or reaction-diffusion) model
This model is based on the self-organizingSelf-organization
Self-organization is the process where a structure or pattern appears in a system without a central authority or external element imposing it through planning...
properties of excitable media
Excitable medium
An excitable medium is a nonlinear dynamical system which has the capacity to propagate a wave of some description, and which cannot support the passing of another wave until a certain amount of time has passed ....
described by Alan Turing
Alan Turing
Alan Mathison Turing, OBE, FRS , was an English mathematician, logician, cryptanalyst, and computer scientist. He was highly influential in the development of computer science, providing a formalisation of the concepts of "algorithm" and "computation" with the Turing machine, which played a...
in 1952. The excitable medium is the limb bud mesenchyme, in which cells interact by positively autoregulatory morphogens such as TGF-β and inhibitory siginaling pathways involving FGF
FGF
FGF may refer to:* Federación Gallega de Fútbol, the governing body of the sport of football in Galicia, Spain* Fédération Guinéenne de Football, the governing body of football in Guinea...
and Notch
Notch
Notch may refer to:* The nock of an arrow* Notch , a Hip hop, R&B, reggae, dancehall and reggaeton artist* Notch signaling pathway, a cell signaling system present in most multicellular organisms...
. Proximodistal and anteroposterior axes are not considered to be independently specified, but instead emerge by transitions in the number of parallel elements as the undifferentiated apical zone of the growing limb bud undergoes reshaping. This model only specifies a "bare bones" pattern. Other factors like Sonic hedgehog
Sonic hedgehog
Sonic hedgehog homolog is one of three proteins in the mammalian signaling pathway family called hedgehog, the others being desert hedgehog and Indian hedgehog . SHH is the best studied ligand of the hedgehog signaling pathway. It plays a key role in regulating vertebrate organogenesis, such as...
(Shh) and Hox proteins, primary informational molecules in the other models, are proposed instead to play a fine-tuning role.
Experimental evidence:
- Limb mesenchymal cells, when dissociated and grown in culture or reintroduced within ectodermal "hulls" can recapitulate essential aspects of pattern formationPattern formationThe science of pattern formation deals with the visible, orderly outcomes of self-organisation and the common principles behind similar patterns....
, morphogenesisMorphogenesisMorphogenesis , is the biological process that causes an organism to develop its shape...
and differentiation. - Peculiarities of the limb skeletal pattern in the mouse Doublefoot mutant are predicted outcomes of a Turing-type mechanism.
Anterior/posterior patterning
The Zone of Polarizing ActivityZone of polarizing activity
The Zone of Polarizing Activity is an area of mesenchyme that contains signals which instruct the developing limb bud to form along the anterior/posterior axis. Limb bud is undifferentiated mesenchyme enclosed by an ectoderm covering. Eventually, the limb bud develops into bones, tendons,...
(ZPA) in the limb bud has pattern-organizing activity by action of a morphogen gradient of Sonic hedgehog
Sonic hedgehog
Sonic hedgehog homolog is one of three proteins in the mammalian signaling pathway family called hedgehog, the others being desert hedgehog and Indian hedgehog . SHH is the best studied ligand of the hedgehog signaling pathway. It plays a key role in regulating vertebrate organogenesis, such as...
(Shh). Shh is both sufficient and necessary to create the ZPA and specify the anterior/posterior pattern in the distal limb (Shh is not necessary for the polarity of the stylopod). Shh is turned on in the posterior through the early expression of Hoxd genes, the expression of Hoxb8, and the expression dHAND. Shh is maintained in the posterior though a feedback loop between the ZPA and the AER. Shh induces the AER to produce FGF4
FGF4
Fibroblast growth factor 4 is a protein that in humans is encoded by the FGF4 gene.-Further reading:...
and FGF8
FGF8
Fibroblast growth factor 8 is a protein that in humans is encoded by the FGF8 gene.-External links:*...
which maintains the expression of Shh.
Digits 3,4 and 5 are specified by a temporal gradient of Shh. Digit 2 is specified by a long-range diffusible form of Shh and Digit 1 does not require Shh. Shh cleaves the Ci/Gli3 transcriptional repressor complex to convert the transcription factor Gli3 to an activator which activates the transcription of HoxD genes along the anterior/posterior axis
Anatomical terms of location
Standard anatomical terms of location are designations employed in science that deal with the anatomy of animals to avoid ambiguities that might otherwise arise. They are not language-specific, and thus require no translation...
. Loss of the Gli3 repressor leads to the formation of generic (non-individualized) digits in extra quantities.
Dorsal/ventral patterning
Dorsal/VentralAnatomical terms of location
Standard anatomical terms of location are designations employed in science that deal with the anatomy of animals to avoid ambiguities that might otherwise arise. They are not language-specific, and thus require no translation...
patterning arises from Wnt7a signals in the overlying ectoderm not the mesoderm. Wnt7a
WNT7A
Protein Wnt-7a is a protein that in humans is encoded by the WNT7A gene.-Further reading:...
is both necessary and sufficient to dorsalize the limb. Wnt7a also influences the anterior/posterior axis
Anatomical terms of location
Standard anatomical terms of location are designations employed in science that deal with the anatomy of animals to avoid ambiguities that might otherwise arise. They are not language-specific, and thus require no translation...
and loss of Wnt7a causes the dorsal side of limbs to become ventral sides and causes missing posterior digits. Replacing Wnt7a signals rescues this defect. Wnt7a is also required to maintain expression of Shh.
Wnt-7a also causes Lmx-1, a Lim Hox gene (and thus a transcription factor
Transcription factor
In molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
), to be expressed. Lmx-1 is involved in dorsalisation of the limb, which was shown by knocking out the Lmx-1 gene in mice . The mice lacking the Lmx-1 produced ventral skin on both sides of their paws. There are other factors thought to control the DV patterning; Engrailed-1 represses the dorsalizing effect of Wnt-7a on the ventral side of the limbs.