Sonic hedgehog (Shh) signaling is essential for the anterior-posterior (A-P) patterning of the embryonic limb bud. An additional role for Shh has now been identified in the mouse and chick, in which Shh promotes the proliferation of limb mesenchymal cells, which increases the number of digits that form.
What is the function of the SHH gene?
The SHH gene provides instructions for making a protein called Sonic Hedgehog. This protein functions as a chemical signal that is essential for embryonic development. Sonic Hedgehog plays a role in cell growth, cell specialization, and the normal shaping (patterning) of the body.
What is the role of AER in limb development?
The Apical Ectodermal Ridge (AER) is one of the main signaling centers during limb development. It controls outgrowth and patterning in the proximo-distal axis.
Which limb development signaling center expressed the Sonic Hedgehog?
The gene encoding the secreted protein Sonic hedgehog (Shh) is expressed in the polarizing region (or zone of polarizing activity), a small group of mesenchyme cells at the posterior margin of the vertebrate limb bud.
How does SHH act as Morphogen?
As a morphogen, Shh participates in the patterning of the developing spinal cord. Following closure of the neural tube, commissural (crossing; contralateral) and association (same side; ipsilateral) neurons develop in the dorsal half of the cord, while inter- and motor-neurons form in the ventral half of the cord.
What happens if AER is disrupted?
Organogenesis in Development When the AER is removed, distal limb structures fail to form, with the degree of severity in limb truncation correlating with the timing of AER removal (Saunders, 1948; Summerbell, 1974). These early studies established the AER as the major signaling center determining PD outgrowth.
Where does the AER form?
The apical ectodermal ridge (AER) is a structure that forms from the ectodermal cells at the distal end of each limb bud and acts as a major signaling center to ensure proper development of a limb.
What type of protein is SHH?
The SHH gene is a member of the hedgehog gene family with five variations of DNA sequence alterations or splice variants. SHH is located on chromosome seven and initiates the production of Sonic Hedgehog protein. This protein sends short- and long-range signals to embryonic tissues to regulate development.
Which signal initiates posterior digit development in the regenerating limb?
FGF signaling is initiated at the early stages of regeneration, which further activates HH signaling. Both FGF as well as HH signaling pathways directly and indirectly activate WNT signaling. These factors activate the genes required for cell cycle and growth during regeneration.
How do the AER and ZPA contribute to axis formation of the limb?
The ZPA (zone of polarizing activity) patterns the AP axis, and the AER (apical ectodermal ridge) maintains outgrowth of the limb bud, keeping underlying mesenchymal cells in the PZ (progress zone) in an undifferentiated state.
What protein does the SHH gene encode?
sonic hedgehogThe SHH gene encodes sonic hedgehog, a secreted protein that is involved in establishing cell fates at several points during development.
Why is it called sonic hedgehog pathway?
The mammalian hedgehog protein, sonic hedgehog (SHH) was named by a postdoc in Cliff Tabin's lab who took inspiration from a comic book his daughter was reading. SHH is involved in the hedgehog signalling pathway, which is a key regulator of all bilateral animal development.
What defines a morphogen?
A morphogen is defined as signaling molecules (proteins or otherwise) that act over long distances to induce responses in cells based on the concentration of morphogen that the cells interact with (Rogers and Schier, 2011). From: Cell Polarity in Development and Disease, 2018.
How do the AER and ZPA contribute to axis formation of the limb?
The ZPA (zone of polarizing activity) patterns the AP axis, and the AER (apical ectodermal ridge) maintains outgrowth of the limb bud, keeping underlying mesenchymal cells in the PZ (progress zone) in an undifferentiated state.
What plays an important role in development and allows for limbs digits to form?
The mesodermal cells induce the overlying ectoderm to form a structure called the apical ectodermal ridge (AER) that will be responsible for the sustained outgrowth and development of the limb.
What is the progress zone of the chick limb bud?
The progress zone is a layer of mesodermal cells immediately beneath the apical ectodermal ridge in the developing limb bud. The fate of the mesodermal cells is thought to be patterned by the length of time spent in the progress zone during limb outgrowth.
Why is the SHH signaling pathway important?
The SHH signaling pathway is essential for proper formation of the limb skeleton, as is required for the survival and expansion of distal chondrogenic progenitor cells. At the same time, SHH is important to specify digit identities along the anterior-posterior axis.
What happens when you lose activity in the SHH pathway?
Upon gain or loss of activity of the SHH pathway, bones are gained, lost or malformed, and such deregulation underlies the aetiology of various human congenital limb defects.
How does Shh affect the signaling pathway?
Another mechanism that influences the range of Shh signaling is the binding of Shh to cell surface and extracellular proteins. A generic response to Shh in all tissues is transcriptional up-regulation of genes encoding cell surface proteins such as Ptch1 and Hhip that bind Shh. The resultant increase in their expression in response to Shh creates negative feedback loops, that not only limit the spread of Shh by sequestering it at the cell surface, but also, in the case of Ptch1, because it inhibits Smoothened activity, dampens activation of the Shh pathway. In mice in which Ptch1 is conditionally inactivated in the limbs ( Butterfield et al., 2009 ), and therefore the signaling pathway is activated independently of Shh, the hind-limbs have extra digits, but the fore-limbs have fewer digits. This difference between hind-limbs and fore-limbs appears to be due to the timing of activation of the signaling pathway, which is earlier in the mutant fore- limbs ( Zhulyn et al., 2014 ).
Where is Shh expressed in chick wing buds?
Shh is expressed at the posterior margin of chick leg buds for a similar duration to its expression in chick wing buds. Furthermore, it has been demonstrated by treating leg buds with cyclopamine that the positional values that specify the three anterior digits of the chick leg are promoted in response to paracrine Shh signaling in an identical fashion to those that specify the three digits of the chick wing ( Towers et al., 2011 ). However, the positional value for the most posterior digit 4 is promoted in response to autocrine Shh signaling (Figure 2B ). Thus, when Shh signaling was attenuated in the chick leg bud by cyclopamine 4 h after onset of Shh expression, two toes with digit 1 identities arose—one from the polarizing region, the other from adjacent anterior tissue, while when Shh signaling was attenuated after 8 h, three digits develop, toes with digit 2 identities from the polarizing region and adjacent cells and a toe with a digit 1 identity from cells further away, and so on, until by 16 h, all the antero-posterior positional values in the leg bud have been specified ( Towers et al., 2011 ). These observations show that although it takes slightly longer to specify antero-posterior positional values in the leg compared to the wing, this process is nevertheless accomplished in the early leg bud, and, as in the wing bud, some considerable time elapses before these positional values are interpreted (Figure 2B ). It should be noted that, in the Ozd chicken mutant, a single digit 1 forms in the leg ( Ros et al., 2003 ).
What molecule mimics polarizing region grafts?
The first defined molecule found to mimic the duplicating activity of polarizing region grafts was the vitamin A derivative, retinoic acid ( Tickle et al., 1982, 1985) but it was subsequently shown that retinoic acid acts indirectly ( Noji et al., 1991; Wanek et al., 1991) by inducing Shh expression ( Riddle et al., 1993 ). There is now good evidence that Shh acts in a concentration-dependent fashion to induce digit duplications. When Shh –expressing cells, or beads soaked in bacterially produced ShhN protein (the active N-terminal fragment produced by autocatalytic cleavage of the large precursor Shh protein), are placed at the anterior margin of a chick wing bud, the extent of digit duplication depends on the number of Shh –expressing cells grafted or the concentration of ShhN protein in which the beads are soaked ( Yang et al., 1997 ). Fewer Shh -expressing cells or lower concentrations of Shh elicit duplication of only the anterior digit 1 ( Yang et al., 1997 ). Grafts of Shh -expressing cells that induce full digit duplications were also shown to result in two ulnae developing in the forearm together with a duplicated pattern of muscles ( Duprez et al., 1999 ).
How is positional value determined in developing limbs?
As indicated in the models outlined above, positional values in developing limbs are specified by paracrine Shh signaling, in which Shh acts as a long-range graded signal and in a concentration/time dependent fashion, or by the duration of autocrine Shh signaling. Therefore, the crucial questions are how a graded distribution of Shh arises, how the range of Shh signaling is controlled and how cells measure the concentration of Shh and the duration of Shh signaling.
What is the function of polarizing region in chick wing bud?
Experiments in which the polarizing region was grafted to the anterior margin of another chick wing bud showed that polarizing region signaling also plays a role in controlling the width of the limb bud and that widening of the bud is required to specify a complete set of new antero-posterior positional values ( Tickle et al., 1975; Smith and Wolpert, 1981 ). The earliest detected effect of a polarizing region graft was an increase in cell proliferation in adjacent mesenchyme in the host wing bud ( Cooke and Summerbell, 1980 ). In addition, it was proposed that the polarizing region controls the production of a factor by the mesenchyme that maintains the apical ectodermal ridge over the region of the wing bud that will give rise to distal structures including the digits ( Zwilling and Hansborough, 1956 ). The apical ectodermal ridge is a signaling region that rims the bud and is required for proximal-distal patterning and outgrowth and the laying down of structures along this axis; the extent of the apical ectodermal ridge across the antero-posterior axis controls the width of the wing bud and determines the number of digits that can form. The effects of the polarizing region on the apical ectodermal ridge also link antero-posterior and proximo-distal pattern formation. This explains the observation that polarizing region grafts made at later stages of development affect the antero-posterior pattern of more-distal structures ( Summerbell, 1974 ).
What is the Sonic hedgehog gene?
Over 20 years ago the first evidence was presented that Sonic hedgehog ( Shh ), an orthologue of the Drosophila Hedgehog ( Hh) gene, encodes the long sought after morphogen that specifies antero-posterior pattern in developing vertebrate limbs ( Riddle et al., 1993 ).
Does limb bud affect Shh?
Many studies have provided information about the expression of individual genes that are affected by Shh signaling in the limb. For example, changes in gene expression have been observed in chick limb buds treated with Shh or cyclopamine, and in mouse limb buds in which Shh or Gli3 is functionally inactivated, or in which Gli3 processing does not occur, e.g., mutants with defective cilia. Microarray analyses have been carried out in both chick and mouse limbs ( Vokes et al., 2008; Bangs et al., 2010 ). It has been estimated from one microarray study that 10% of the genes expressed in the early limb bud (about 1,000 genes) are downstream of Shh signaling ( Bangs et al., 2010 ). Putative direct targets of Gli3 repression have been identified by ChIP seq analysis of limb bud nuclear extracts using transgenic mice expressing a tagged form of the Gli3 protein ( Vokes et al., 2008 ). Further analysis has involved RNAseq ( Lewandowski et al., 2015 ).
Objectives
1. To learn about the early development of the limb bud and the regions that are most critical in the organization and outgrowth of the limbs, including the apical ectodermal ridge, the progress zone, and the zone of polarizing activity.
I. Introduction
Limb formation begins relatively late in gestation after the basic body plan has taken shape. The limb buds are first visible along the embryonic flanks in the middle (upper) and end (lower) of the fourth week ( Fig. 8.12/9.12 ). The activation of mesenchymal cells derived from lateral mesoderm (to form the limb field) appears to be a crucial step.
II. Initiation of Limb Bud Formation
It is not currently known what is the primary signal for initiation of limb buds, but based on model systems that can be manipulated experimentally FGF10 is a good candidate.
III. Apical Ectodermal Ridge and the Progress Zone Theory
The AER is required for limb outgrowth. Removal of the AER results in a stunted limb whose development is arrested shortly thereafter. However, segments that have begun to form continue to do so reasonably normally.
IV. The Anterior-Posterior Axis and the Zone of Polarizing Activity
As mentioned above, the ZPA, located in the posterior mesoderm, is responsible for establishing polarization along the anterior-posterior axis. Shh produced by the ZPA is necessary for its function. Shh production is sustained by FGF produced by the AER ( Fig. 8.16/9.17).
V. Dorsal-Ventral Polarity
Limbs are different on their dorsal and ventral surfaces. The skin on the palm of your hand, for example, is different from that on the back of your hand. This dorsal-ventral polarity of the limb is directed by the ectoderm. It is possible to detach the ectoderm from the mesoderm in a limb bud.
VI. Positional Information
The differentiation of the cells within the limb is dependent on their precise position and the growth factor signals they receive once their fate is restricted. The limb has served as a classical example of how gradients of signals from centers such as the ZPA and AER help establish positional information in the limb field.
Why is Shh only induced in the anterior region?
Shh is only induced in the anterior region because of signals from the AER. Experiments done by Heikinheimo et al. show that when the AER is removed, beads that express FGF are sufficient to induce Shh signaling in the ZPA. Thus, the likely signaling factor from the AER is FGF.
What is the role of Shh in ZPA?
Shh may be a critical signal regulating ZPA function, but the genes involved in Shh signaling are under the control of several other factors that are needed for ZPA maintenance and function including Hand2 and Hoxb-8. Retinoic acid, an important signaling molecule needed throughout embryogenesis, acts through the Hox genes. It was originally postulated that retinoic acid acts to induce the Hoxb-8 gene, but this hypothesis has not been supported by genetic studies in mouse embryos lacking retinoic acid synthesis that still express Hoxb-8 in the limb. Hoxb-8 signaling is active in the early embryo, beginning at the posterior end of the lateral plate mesoderm and extending to the anterior region. As Hoxb-8 spreads to more anterior regions, Shh is induced in the area that will become the ZPA. Shh is only induced in the anterior region because of signals from the AER. Experiments done by Heikinheimo et al. show that when the AER is removed, beads that express FGF are sufficient to induce Shh signaling in the ZPA. Thus, the likely signaling factor from the AER is FGF.
What is ZPA in a limb?
The zone of polarizing activity (ZPA) 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, muscles and joints. Limb bud development relies not only on the ZPA, but also many different genes, signals, and a unique region of ectoderm called the apical ectodermal ridge (AER). Research by Saunders and Gasseling in 1948 identified the AER and its subsequent involvement in proximal distal outgrowth. Twenty years later, the same group did transplantation studies in chick limb bud and identified the ZPA. It wasn't until 1993 that Todt and Fallon showed that the AER and ZPA are dependent on each other.
How do GLI transcription factors work?
Activation of Gli zinc-finger transcription factors occurs through the Hedgehog signaling pathway. There are three Gli factors that are essential for limb development: Gli1, Gli2 and Gli3. Without Shh, Gli2 and Gli3 are processed to a repressor form and travel to the nucleus to repress the Shh response. But when Shh is present, unprocessed Gli2 and Gli3 are able to pass into the nucleus and stimulate expression of Shh target genes, including Gli1. Studies in mice show that Gli3 knockouts have polydactyly digits. Fundamentally, Shh acts to remove repression of Gli3. When Shh diffuses from the ZPA, it predominates in the posterior region of the limb bud, activating Gli3 in the posterior region, while the repressor is still active in the anterior region. This leads to activation of other genes such as Hox genes, FGF genes and BMP genes in the posterior region, setting up digit patterning. BMP, plays a role in limb morphology, specifically, digit positioning, but the specific regulation of BMP is unclear.
What is the function of transcription factors in the limb bud?
Specifically, proteins called transcription factors (TF) help control the rate at which a gene is transcribed . The limb bud expresses a TF called ALX4 at the anterior part of the mesoderm, with the TF HOXB8 being expressed at the posterior portion. The Alx4 region, the medial region, and the Hox8 expressing area meet at a proximal area where the AER develops. The ZPA forms where the Hox8 region joins the AER.
Which genes are likely to be controlled by Shh within the ZPA?
In particular, the Hox genes A and D are likely to be controlled by Shh within the ZPA. Three phases of activation of the Hox genes results in patterning of the limb parallel to the expression of the Hox genes in a nested pattern.
Is Shh required for ZPA?
The idea that Shh is required for proper ZPA signaling and anterior/posterior limb formation needed to be tested. Riddle et al. took Saunders and Gasselings findings to the next step and proved that Shh is the morphogen within the ZPA that is required for anterior posterior patterning. By isolating the Shh gene and implanting it into the anterior limb bud, mirror image digit duplications formed.
Introduction
Specification of antero-posterior Pattern
Interaction Between Positional Information and A Turing-Type Mechanism
Mechanisms of Shh Signaling
Initiation of Shh Expression
Termination of Shh Expression
Response to Shh Signaling in The Limb
Clinical Aspects of Shh Signaling in The Limb
Shh Signaling and Limb Regeneration
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Evolutionary Aspects of Shh Signaling in The Limb