Limb development

The limb development is an area of developmental biology and forms the basis for insight into the molecular and cellular mechanisms for form-finding in the organogenesis of vertebrates. The limb development seeks to explain the progressive shaping of the skeletal pattern in the limbs of the tetrapods ( land vertebrates with four limbs).

6.1 Self-organizing properties of the mesenchymen Präknorpelgewebes - model of Newman and Müller 2005
6.2 The model of Chaturvedi, Hentschel, Alber, Newman 2005
6.3 The model of Zhu, Zhang, Alber, Newman 2010
6.4 BMP receptor - interaction model of Badugu, Iber et al. 2012
6.5 Hox genes regulate finger spacing and number in a Turing model 2012

Overview of the overall process

The incipient development of the extremity occurs in the extremities field. The extremities field is a region in the right and left flank of the embryo. The limb development begins in that grows not yet differentiated, uniform mesenchymes cellular tissue of the lateral mesoderm of the somites and the ectoderm lying above everts to a paddle-shaped limb bud. It leads to the formation of an organizer, the apical ectodermal rim ( AER), which is a long, bead-like structure on the flattened tip of the bud, which controls the further growth of the bud in the direction away from the body (Fig. 4, 8b). Cells in the bud condense first, then differentiate into many steps in cartilage, chondrogenesis and form in their early phase genetic and epigenetic patterns, which later form the bone of the upper arm ( Stylopod ), forearm ( Zeugopod ) and hand ( autopod ). The exact localization and form finding of these discrete, skeletal elements within the growing bud are the real object of research for limb development. In Towers and Tickle puts it in 2009: " Our knowledge of this process is still rather fragmented and many of the proposed models remain controversial ."

The following models are outlined which have arisen since the end of the 1960s and molecular insights into the development of the first chicken wing ( Fig. 2) and later in the anterior limb of the mouse ( Fig. 1a, 1b, 3 ) provided. There are also models listed (including the morphogen gradient model), which can be regarded as outdated, but still appear in newer textbooks. After more than 50 years of research it is not clear in what form cells in the bud position information can be obtained that allow them to differentiate at the exact locations to be determined in cartilage tissue and thus to enable the patterning of the Wirbeltierextremität. It can be assumed that there are no such position information. Recent computer simulation models that operate at the cellular level and not at the genetic level, work without location information and instead focus on Turing and Gierer - Meinhardt -based pattern formation processes. The challenges are therefore now in the integration of molecular models with epigenetic ( cellular ) pattern formation by means of computer simulations.

Figure 3 components and axes in the limb of the mouse

Figure 4 AER - Apical ectodermal sidebar. Organizer region (signal center ) for the control of the proximo - distal and antero -posterior Auswuches the bud

Figure 5 ZPA. Zone of polarizing activity. Organizer region decisive for the pattern formation of the fingers or toes

Terms (phenotype )

Important phenotypic terms to the understanding of the limb and its development on the example of the front limbs of man are:

Upper arm ( Stylopod ) (Fig. 3 )
Elle ( ulna ) and radius (radius ) = ( Zeugopod ) (Fig. 3 )
Hand ( carpals and metacarpal ) with fingers (digits ) = ( autopod ) (Fig. 3). The autopod is the real innovation in the vertebrate limb.
AER apical ectoderm rim ( Fig. 4, 8b): organizer region (signal center ) for proximo - distal limb bud formation
ZPA zone of polarizing activity (Fig. 5): organizer region at the posterior end of the limb; responsible for the antero -posterior axis formation. Influenced the number and identity of fingers.
Morphogen: diffusibles molecule that determines an organizer region. It forms a concentration gradient and obtained directly or indirectly unique cell responses at different concentrations. Examples include retinoic acid ( RA) or Sonic hedgehog ( SHH).

Timing and duration of the limb skeleton formation

The development of the extremities ( wings and legs ) in the chick takes approximately from the third to the sixth day, with a total development time of 21 days. In the mouse, the protuberance of the bud begins after about 9.5 days; bone formation of the extremities is complete after four days around the middle of the 13th day of development (Fig. 1b) with a total development time of also about 21 days. In humans (Fig. 6), the limb development ( 6-8 mm embryo) starts from the fifth week of pregnancy. The manual development of foot development goes ahead about two days. In the eighth week, arms, hands, legs and feet are formed largely (embryo: about 3 cm), the apoptosis of separating the fingers and toes is then carried out.

Axial pattern and periodicity

The following main axes are distinguished in the extremity on the example of the human hand:

Proximo - distal axis: from shoulder to fingertip
Anterior- posterior axis: from the fifth finger to the thumb
Dorso - ventral axis: by hand inside to the outside

The development of the limb occurs primarily along the proximo - distal, antero -posterior and dorso- ventral axis. The processes along these three axes are partially, but not independently. The formation of the axes is achieved by coordinated interactions between signaling centers. These are the AER ( apical ectodermal ridge ), which controls the proximo - distal growth, the ZPA (zone of polarizing activity ) for the antero- posterior axis and the ectoderm of the bud, which controls the dorsal- ventral axis. The growth of the bud in proximo -distal direction is much more pronounced than in the anteroposterior direction. Models treat the proximo - distal either primary development process, such as the progression zone model or the antero- posterior process as the morphogen gradient model. The integration of the two axes in models is not yet well advanced.

The proximal extremity is divided distally into three regions: upper arm (English Stylopod, Latin humerus ), forearm (English Zeugopod, lat foreleg ) and hand / foot, autopod (Latin podium car ). In the distal direction to double and then a multiplication of the bone takes place. The Stylopod consists of an Zeugopod the two bones ( ulnar and radius). The autopod has three ( chicken wings ) to five elements ( mouse ) along the antero -posterior axis. Fingers and toes also show quasi-periodic pattern in the form of tandem -scale limbs along the proximo - distal axis. The elements at the same level of the proximo - distal axis have different identities, so the ulna and radius, but also the individual fingers or toes.

Deterministic models

The classical models of limb development and their successors systems are based on the solution of the problem of pattern formation of genes and gene regulation and thus of deterministic processes. The shape formation is to be explained from the lowest, the molecular biological level of organization as a hierarchical process chain. You want to analyze the interactions reinforced the different signaling pathways, as well as understand their downstream effects today. An overview of the current state of research on the integration of transcriptional networks in the Wirbeltierextremität give Rabinowitz and Vokes (2012 ). The main genregulatorisch based models provide Towers and Tickle (2009) together.

The two necessary properties, which have the models in this group are the presence of a gradient and cell information. The cell can interpret the different strength and duration- different chemical signals of the gradient and specifically react to it. She receives a specific identifiable cell information by the gradient. On the basis of this ability, they can differentiate into different tissues, such as bone, cartilage, muscle or connective tissue.

Morphogen gradient model (French flag model) by Wolpert 1969

The Mophogen gradient model proposed by Lewis Wolpert in 1969 as a model for limb development in the chick. Wolpert postulated that the segregated from the ZPA of origin, in a progressive direction of expression along the AP axis spatially decreasing concentration of a time not yet known morphogen for the fate of the cells in range of this morphogen ( cellfate ) and thus is responsible for the finger construction education.

The morphogen forms a Verlaufsgradienten and supplies in this way the coordinates and location information for the cells so that they can differentiate at precisely determined locations in cartilage and thus occurs the pattern formation of the fingers or toes. Afterwards, the diffusing protein Sonic hedgehog ( Shh ) was identified as the likely morphogen. Wolpert assumed that the decreasing morphogen concentration with threshold effects, is thus interpreted as a discrete increments. He has marked blue-white- red Such threshold levels, which the model also the name French flag model earned (Fig. 7). The explanation of the action of the morphogen was carried out by Wolpert first on the spatial concentration, but was later extended to include on the temporal concentration of SHH in the direction of the antero -posterior axis, whereby, however, the original model was modified by Wolpert. Verlaufsgradient of acting in a manner that is still unknown, one of the cells. You can, according to the theory, interpret its concentration and duration as position values , encode molecular and ultimately translate into the required finger anatomy.

The basic problem in Wolpert's model can be seen that a problem ( position information ) with another problem ( morphogen ) is explained, for which no explanation is provided. The model does not explain, secondly, as a local, self-reinforcing source can be generated for the morphogen. This statement will be delivered only by Gierer - Meinhardt 1972. The fingers should thirdly in this morphogen gradient model actually be arranged in a circle around the ZPA, which is itself a three-dimensional, oval structure. However, the fingers are aligned in a plane. This may not be the model. Fourth, the morphogen gradient model originally advocated direct action of a morphogen to generate precise position information has been provided as the sole explanation recently questioned, not only by Wolpert itself: A morphogen can not sufficiently reliable in its newer perspective, the precision and robustness produce, both are required for pattern formation. The coordination processes have been found not to be today .. Fifth, are signals for position information, as Wolpert introduced himself in his model much finer. This model can be regarded as obsolete for the reasons mentioned from today's perspective.

Progression zone model by Summerbell and Wolpert 1976

The classic progression zone model was introduced in 1976 as an extension to the morphogen gradient model (Fig. 8). Put simply arise after this model successively the structures of Stylopeds, then the Zeugopods and finally the fingers or toes. The specific cell and tissue formation should be explained that initially held with progressive proliferation of the bud along the proximo - distal axis by the AER cells in the adjacent growth zone "neutral ", that is not or not yet differentiate. The later cells leaving the growth phase, the more distal structures arise from them, depending on whether shorter or longer cells remain in the growth zone, until they leave, it is decided differently about their fate. The cells are therefore a subject to " internal clock " with which they can measure their whereabouts in the progression zone. The progression zone model is thus the first model, which brings the time factor combined with Morphogensignalen. This model is confirmed by removal of the AER at an early stage, which further leads to staying away parts of the Stylopods and Zeugopods, so only a stump of the arm formed, while its removal at a late stage of development, the parts mentioned are trained, but not the autopod and thus not the fingers. The mechanism for generating the proximo - distal structures to explain the autonomy of undifferentiated cells in the Progessionszone the Progessionszonen model.

The progression zone model is not able, for example, to explain the regeneration of a salamander leg. There would have to be set again after cutting the leg the clock again. The model was also recently found by two studies in question, who deny the cells of said autonomy, at least the autonomy to generate the proximo - distal structure based on the assumed "internal clock ". The authors, who came independently to similar empirical results, explain the progression of the proximo - distal structure not with cell autonomy, but with opposite cell signals, the proximal retinoic acid ( RA) who sends in the bud distally, and the distal FGF activity, which, starting from the AER acts in the opposite direction. The result according to the authors a dynamic balance between these signals. This controls the participation of appropriate Hoxgene the proximo - distal development of the Zeugopods and Stylopods. In this case, the researchers were able, for example, to produce desired distal structures in vivo by recombinant transplanted tissue proximal to places that were not exposed to RA expression. Conversely, could a full proximo - distal axis with distal tissue generated in vitro by the cultured cells were exposed to exogenous RA influence the overdriven the distal signals.

Multistage gene activation by Meinhardt 1983/2009 and proximo - distal differentiation front model by Tabin and Wolpert 2007

Already 1982/83 drew Hans Meinhardt that cells can not be promoted at once, but gradually, irreversibly more proximal or more distal specification. This gene activation was already seen as a space- dependent process. Then there is a feedback from the proximo - distal reached state of mesenchymal cells on the strength of a posteriorisierenden signal which is formed in the ectodermal AER. It was assumed that initially a rudimentary pattern is formed (for example, 1112 of the full sequence if the proximo - distal structures is referred to as 1-6). If enough cells of this type are formed by proliferation of type 2 cells, then the produced in the AER signal increases to the extent that cells are made of Type 3. Only if enough type -3 cells are then formed, type -4 cells ( Fig. 10) are formed.

This model is compatible with the progression zone model. It is a feature of the model that after amputation at any point the missing structure may be re- formed, hence the name model Bootstrap. The model also allows that distal structures can be formed too early, for example, if the spread of the molecules is disturbed by dead cells. Such a pattern corresponds to the thalidomide deformity. Meinhardt has his theory refined 2009: ". A higher organism is much too complex that it could be generated by a single morphogen gradient " Only gene activations, which run sequentially switch -like, with the participation of a gradient form progressively getting sharper boundaries and positions for skelettäre form: the consequence of sequential gene expression sets are always finer pattern and position limits. The boundaries between regions where different genes are active, may be new organizer regions to form the pattern of limbs, wings, etc. ".

Simulation: Stepped gene activation under the influence morphogen signal ( and Fig.10 )

Such limits could, for example, the different reactions to be SHH (concentration, duration, or both together), which develop between the toes of the mouse, as they Harp et al. Have described 2004. Position information are transmitted by the concentration and / or temporal effect of a single morphogen as unique coordinates to the cells in the classical sense Wolpert, there is no longer here. Rather, the morphogen sequentially activates several stepped expression processes in the course of which it only comes in clear positions and phänotpyischen gradients.

For Tabin and Wolpert, the progression zone model is not the high amount of molecular gene expression data meet that have been generated in the past few years. An example of this is the influence of Hoxgenen who finds no access to the previous models. Therefore, the authors presented taking into account the existing data before an alternative framework for the proximo - distal patterning. After that there are several different gene expression domains, each with specific groups of interacting genes in successive stages of bud growth. At the proximal border, can not be received from the AER- FGF signaling, a Differentiationsfront is formed. This limit prefigures the proximo - distal sequence that appear in the individual elements of chondrogenesis by little. So the result is a formation of several progenitor pools for each segment ( Zeugopod, Stylopod and autopod ). The Differenziationsfront model describes thus very similar to what Meinhardt earlier showed.

Self-regulated system of interdependent cell signaling feedback loops by Bénazet, Zeller et al. 2009

Bénazet, Zeller et al., University of Basel, claiming to present the first integrated model, the well-known and new pathways in positive and negative epithelial-mesenchymal feedback loops linked and thus has an integration of existing models. However, this applies to earlier models, such as by Tabin and Wolpert 2007 already. The team led by Zeller shows a threefold system consisting of an initiation phase, a propagation phase and a Terminationsphase. Gremlin1 whose antagonist BMP, FGF, and SHH are the pillars of the three subsystems. The model is in the initiation phase before the earlier models and explains first the conditions to ensure that AER and ZPA can establish and maintain their function. The authors speak of a Prepatterning at this stage, wherein to the early time position information " checked " and updated continuously in the course of growth. Your exact fixation occurs only in later stages of development, but prior to the differentiation of the cells. Thresholds as in the morphogen gradient model are not required for this.

The model is understood as a systemic approach. The spatial aspect is but little addressed. A general problem for understanding how structures are formed leg, is that there is little site-specifically activated genes. From the model it is not clear from this, such as the fingers are formed in space and time. A simulation has not yet been carried out, and also not possible. Here the resultant at the ETH Zurich in 2012 BMP receptor model goes much further. Is noteworthy, however, that both models that are even developed in cooperation between the two teams, and both the early phase to explain the structure formation of the limb, cite different control loops for the same facts. The Shh signaling pathway about which team Zeller has a high value in more than one phase, is seen from the other team in respect Litingtung et al 2002 as expendable.

Car Regulatory Models

The evolutionary developmental biology provides patterning processes not primarily at the genetic or Genregulationsebene but in interactive interplay of genetic with higher organizational level ( cells, tissues ). Basically, for all the models listed below are the work of Alan Turing ( 1952). and Gierer and Meinhardt (1972). Turing mechanisms may produce irregular patterns; but only the dynamic processes with self-organization and scaling described by Gierer and Meinhardt are the epigenetic mechanism for rule -like morphological form-finding, including the limb. Was pointed parallel to the gene- centered approaches to epigenetic possibilities of limb development early on. Younger models go from here no longer of chemical diffusion processes, but by cell -cell responses and can depict realistic based on this biological activator inhibitor - processes. Despite this, today there are still considerable difficulties to reproduce the activator -inhibitor molecular processes empirically adequate. In the following, a set of such models is presented. The ersen three are further developments of the mathematical model of comprehensive LALI Hentschel et al., 2005. LALI stands for the extended Turing model type Local auto -activation, lateral inhibition within the meaning of Gierer Meinhardt.

Self-organizing properties of the mesenchymen Präknorpelgewebes - model of Newman and Müller 2005

Newman and Müller have the classic models a new view of the emergence of the hand ( Origination and innovation) compared with that of both moves only the genetic, epigenetic processes and the interaction into view ( Fig. 11). A handful of cellular and molecular core processes of mesenchymen tissue forms the basis for the operations. Existence and transmission of position information in the individual cells are not required. Thus, this approach provides a first symstemische approach by the interdependent interaction of the system components, genes, gene products, cells and tissue is treated. Epigenetic processes are according to this view, not only for the explanation of the evolutionary emergence of the hand ( Origination ) but also for the explanation of their development in recent creatures fundamental and indispensable.

Three main properties of the epigenesis of the system development are formulated:

The autonomy properties of its components ( cell behavior, tissue geometry, etc. ),
The ability of the mesenchymen tissue in the condensation to self-organization and the formation of spatio- temporal patterns,
The non- linear response ability of cells and tissue to minor changes, such as genetic mutation, alteration epigetischer parameters or environmental influences. Exceeding ( thresholds ) may be responsible for discrete phenotypic variation or innovation.

The model of Chaturvedi, Hentschel, Alber, Newman 2005

This model uses computer simulations that can reproduce by cell and cell communication processes with reaction-diffusion equations organogenesis hand starting and for the first time in three dimensions the formation of fingers grasp ( Fig. 12). The model is based on the original, but modified reaction-diffusion equation set of Hentschel et al.

The following model variables and methods are used for the spatio-temporal regulated condensation in the model of Hentschel include:

Molecular level ( nucleus ),
Subzellularebene ( mitochondria),
Cell level,
Cellular and tissue level,
Tissue level,
Organ level

Chaturvedi the model is based on a relatively small molecular processes, but uses a plurality of cellular and supra - cellular mechanisms. It can represent with this approach skelettäre coarse spatial structures. On the integration of the mode of action of Gli3, Shh and Wnt -7 that control the limb geometry and zones are working, which will lead to further realism of the models. Organizer regions in the bud, such as AER and ZPA, but also the growth zone are highlighted priori. Your creation is therefore not reproduced. Your specific location in the bud plays no role in the model. This model consists of computing capacity reasons, not primarily on molecular processes. Its inner core is the cell with the CPM software application.

The model of Zhu, Zhang, Alber, Newman 2010

2010, a simulation model has been published which provides advanced insights into limb development and also can reveal evolutionary differences.

The model is as the previous one on the model of Hentschel et al ( 2005), also based in order to activator inhibitor equations and thus to self- organization capability which is established outside of genetic information in the mesenchyme. It requires no location information a priori. Activators and inhibitors are similar to the model of Newman and Mueller ( 2005). On the problem that to date, no single morphogen can be empirically identified as an inhibitor and that little earlier, the molecular identity of a long - long-range inhibitor is called elusive, Zhu Amongst others an unspecified. An inhibitor must be provided that the model can not be represented differently. The center of this model, a Fibroblast Growth Factor (FGF ), a proximo -distal gradient as an important player is based on the AER. Further, BMP activity of underlying molecular " Prepattern " dictates is configured by only the cellular pattern. Only through such a multi-stage, at the molecular level is not yet transparent process now of basically laying diffusion curve for the patterning is determined.

In the simulation model (see Video figure 13) growth and condensation follow the progression zone model. Clearly the AER ( right), a narrow progression zone (center) and the growth zone can be identified.

The model generates a two-dimensional pattern, and is further simplified in comparison with the models of Hentschel, Newman Müller and Chaturvedi the number of equations is reduced to two reaction-diffusion equations. Progress can be achieved in the sense that the different zones of the bud as AER, frozen zone, growth zone can be distinguished. Different cellular processes that occur empirically, are incorporated or are held constant in the model. An extension of the AER during the growth of the bud is included. One starts with a curved apical contour. Generally speaking, the model takes into account the change in shape ( reshaping ) of bud tissue.

Compared to the previous models, asymmetries can be represented in the autopod, ie differences in the skeletal elements in the number of toes or their length, as empirically for different species actually occur. Shh and Hoxaktivitäten are taken into account. Evolutionary transitions can be produced. The pattern of results from this model do not arise on the lower, genetic organization level. Rather, the Turing equations reflect the self-organization of the cellular tissue of the bud, ie autonomous features that only come into existence at this level. Hoxgene, Genregulierungen, especially transcription factors contribute towards the parameters of the equations. They distract and thus refine the spatial orientation of the bone, but are not actually shaping factors. These are epigenetically. The evolutionary species-specific or different length, thickness, and orientation of the bone is achieved by means of so-called change kinetic reaction parameters that represent the rates of production of the morphogen or the activator are on the association rate constant of activator and inhibitor.

Zhu, among others simulate both the proximo - distal outgrowth of the bud and the antero -posterior patterning of the Autopods, ie the number of toes. These are positively correlated with increasing width of the Autopods. For widening the Autopods a parameter is used, which can be seen as a simulation of the Shh expression of ZPA. The simulation based on the continuous changes in gene and cell level, ie forms of discrete elements.

Critical to the model one can evaluate the severity of bone formation, which ( Fig. 13 ) is directly seen in the simulation of the progression zone, not very realistic in comparison to the earlier Figure 2, which also comes from Newman. The specification process of the bones ( cartilage formation ) thus runs more gradual. The toes are not formed simultaneously ( Fig. 2). It is positive that the Zhu model contains both a proximo - distal and antero- posterior growth and a latter also allows Polydaktylieformen as part of the widening of the bud.

BMP receptor - interaction model of Badugu, Iber et al. 2012

The latest model comes from the Department of Biosystems Science and Engineering at ETH Zurich. The first time, the following approaches are integrated: (A) A Turing model ( rather LALI model) with molecular, BMP receptor - based interactions as well as FGFs, (B) a growing domain, (C) a realistic 2D buds geometry of the mouse. The model is based on the BMP signaling pathway at the earliest recognizable interface for the onset of condensation. Shh, Gli and Gremlin are not needed in the model. The simulation is compatible with a large number of empirical experiments at the bud as well as several pre-and postaxial Polydaktyliemutanten. It is noteworthy that the statements made in this model are not identifiable comparable to the model of Bénazet, Zeller et al., Which was created at the University of Basel in 2009. The problem with this model is that the BMP receptor is not a diffuser. But it builds the model to centrally. Furthermore, the pattern image generated in the model does not resemble the empirical picture of Prächondrogeniesierung the vertebrate hand as shown in Figure 2

Hox genes regulate finger spacing and number in a Turing model 2012

A new model is Turing - processes to the previously used basis of the Hox genes. Previously only gradients in Turing models seen, so diffusing substances that act as an activator or inhibitor. It is, therefore, a new view that non-diffusible transcription factors such as the Hoxa and Hoxd - group play a significant role in pattern formation. This is exactly what has Sheth et al. 2012 empirically demonstrated. Thereafter, the expression of these in the development of late- hand distal expressed genes is inversely proportional to the number of fingers: its reduction increases the number of fingers. Serves the reduction in parallel with the reduction of Gli3R, polydactyly is even more pronounced. Hox genes are said saw responsible for the wavelength of the finger pitches. Under the stated conditions, up to 14 fingers can be generated on one hand in the mouse and in the empirical model.

Critical appraisal of the models from the present perspective and future research agenda

For a long time with the two classical models, the morphogen gradient model and the progression zone model, separate considerations on the structure of the palms exist, once the declaration by the processes along the proximo - distal axis, and by the which first independent statement the ZPA processes along the antero -posterior axis. However, none of the models can adequately explain by itself the spatial formation of identities in the bud, and especially the Autopods. Each of the models is indeed confirmed by empirical cut-and- pace trials. Final analysis, however - one follows the logic of these models - the coordinates only be determined exactly when the cells in question both her place on the proximo - distal axis and the on the anteroposterior axis, and strictly speaking the on the dorso- ventral axis may be communicated. There must be in embryogenesis hand processes that make it possible for cells to interpret position points on all three axes, therefore from this point of view.

The ongoing difficulties uncovering position signals empirically led other scientists to more holistic, systemic explanation of structure formation, at least in the direction of the two principal axes. Further developments in this area are characterized by the focus on cross-disciplinary, computer-assisted ( in silico ) research. The challenges lie in the following areas:

1 Improved synthesis of molecular to systemic models.

Second simulation of the behavior of the bud in the dynamic growth process. Both morphogens and reaction-diffusion systems behave in growing domains unlike in static.

3 transition to stochastic compared to the previously often deterministic models regarding gene expression, cell processes, etc. ..

4 Proof of the robustness of the cell processes used, so their immunity to stochastic noise ( noise).

5 Analysis of the scaling problem, that is, that due to different sizes of buds (domains) produce different patterns. Neither scale the strip in the French flag model uniformly nor results in the same number of strips in the activator -inhibitor systems with larger domain.

6 Empirical determination of the activator and in particular a long broad inhibitor for skelettäre form finding. Both quantities are essential in activator inhibitor systems.

7 Empirical determination of the molecular basis for possible position information, if actually present ( receptors).

8 Determination of suitable methods to make the distribution of SHH and other morphogens in the developing bud real-time quantitative imaging.

9 Integration of the dorso- ventral axis, and their molecular - cellular base as a prerequisite for empirically -based three-dimensional models in which all hand limbs lie in a plane.

10 When formed from joints ( joints ), if that is not alone in the places the case in which it comes to periodicity ( elbow, knee, wrist and )? Fingers and phalanges of the toes are also connected by joints.

Methods of empirical research

The external location of the extremities allows a variety of manipulation of the embryo, the results are easily visible. Genetic changes do not lead to death of the experimental animal in most cases. For these reasons, the extremity of the vertebrate stands out as an excellent model system. The first decades were primarily in experimental transplantation experiments on embryos. First grafts were removed from the resulting bud and planted in other places again. When the ZPA and AER and their positions were known, it has taken this organizer regions of other animals and eg the anterior ZPA addition to the implanted posterior again. In addition, these components have been removed to former / the later time points or in larger / smaller doses and / or implanted. Thus it was hoped that findings about changes in skeletal formation. In recent times, by molecular biology operates with in-situ hybridization and in particular to gene knockout. By switching off of genes can be involved in the development of the function of the limb. This is also called gain of function or loss of function experiments. Thus, the experiment of Litingtung et al. Out in 2002 to the surprising finding that a double knock-out of Sonic hedgehog and Gli3 (Shh - / -, Gli3 - / -) a polydactyl hand generated, so you could not expect. Both genes are, therefore, unnecessary for the finger formation, when they are turned off together. The above experiments yield no or only limited to information about how to interact with important signaling pathways and downstream effects which they produce. It is controversial whether the factors in the phenotypic pattern formation (eg finger) can be recognized in this way at all.

Sonic hedgehog - key gene for limb development?

There was no other gene or protein more scientific papers have been written in connection with the development of Wirbeltierextremität as a hedgehog on Sonic. Hedgehog signaling pathway is a highly conserved in evolution and plays a central role in a number of pattern formation, both in vertebrates and insects. Numerous experiments have been made with the CPA to determine its mode of operation before it was known that the morphogen in the ZPA. As Tabin 1993 he discovered that Sonic hedgehog is the morphogen, which is expressed in the cells of the ZPA and diffuses in the extracellular matrix of the autopod from posterior to anterior, concentrating on the study of the mechanism of action of Sonic hedgehog was even higher. The proliferation of cells in the course of Shh expression, and therefore the anterior -posterior growth of the bud, as a consequence of the ZPA has been significantly ( Fig. 14). After Tickle SHH is now confirmed as an important morphogen for pattern formation in the autopod. His far-reaching effect is dependent on both the dose and the duration of the diffusion. The ZPA will then check both the number and identity of the fingers. Most thumb- sided malformations of the hands ( preaxial polydactyly ) are associated with changes in expression of Shh.

Today, it is no longer assumed that the diffusing action of SHH may be sufficiently accurate to be able to afford the positioning of the cells ( cartilage and adjacent tissue or skeletal exact form ) alone can. It is possible that SHH itself does not act in a graded effect, but that of his production of another substance is induced, which has the corresponding gradient effect and passes. Or there may be several consecutive Patterningsprozessen, refine the structures and positions. The model of Badugu et al. (2012 ) to explain the digits Patternings works without Shh.

Sonic hedgehog was also one of the first genes for which an in mammals and fish alike highly conserved non- coding cis- regulatory element ( ZRS ) was found, which controls the Shh expression in detail. Mutations in the ZRS lead to präaxialer polydactyly. Finally, the sonic hedgehog signaling system was among the first of which has been described, that it produces a stochastic, and thus genetic switch threshold effects in the extremities in the regulation of development of Gli ..

In addition to its antero- posterior effect in the autopod Shh interacts via the ZPA with the AER and maintains its function during the growth of the bud permanently maintained. Conversely, Shh is not expressed when the AER is eliminated. FGF4 acts on the maintenance of Shh and Shh to maintain FGF4. Without this interaction fingers are reduced to the first finger either or missing entirely. However, this relationship does not mean that Shh is directly responsible for the number and identities of the distal elements, as will be seen by Tabin and McMahon, 2008. It is not even clear message whether a correlation exists between the duration of the SHH-signaling activity, which is required to specify a finger, and the order in which the condensation takes place cartilage. SHH has from today's perspective, primarily two Hauptfunktionben: It controls the antero -posterior identity of the fingers and the number of cells and thus the growth of the bud. Only in an indirect way it comes about SHH to threshold effects of finger formation. On the mode of action and role of SHH for the digit patterning is still subject of intensive research.

Robustness versus plasticity of the number of toes

The Wirbeltierextremität is a very robust anatomical shape. At the same time it is characterized by an extreme evolutionary adaptability, as can be seen in its various forms in vertebrate species ( Fig. 14). Both properties appear intuitively contradictory. However, Andreas Wagner was able to show that robust forms with distinct gene networks better conditions for variation and innovation have to be less robust forms.

The pentadaktyle upper limit of the hand ( five fingers ) is still not fully understood. Early types of dinosaurs (eg Acanthostega ) are interpreted today because of the similarity of the toes rather than polydactyl, because as seven or achtzehig. There are no vertebrate species, the standard has more than five fingers or toes on a limb. Panda and Mole have deformed metacarpal bone with finger character. Exceptions with selective influence of man are certain populations of the cat (Maine Coon ) and the Norwegian Lundehund ( polydactyly ). Galis leads Entwicklungsconstraints as a reason that a larger number of fingers is inhibited