"Read More About It" Pages - Biology 376 - Fall 2007

Dec. 12 presentations

The Zinc finger domain of Wilms' tumor 1 suppressor gene (WT1) behaves as a dominant negative, leading to abrogation of WT1 oncogenic potential in breast cancer cells

Presented by Alaina Vrontikis

Wilms' tumor is a type of cancer in the kidney which affects pluripotent embryonic precursor cells. The Wilms' tumor 1 suppressor gene (WT1) was first identified as a mutated gene in Wilms' tumor that had lost its ability to suppress the tumor. However, recent data has shown that the wild type WTI is expressed in most of Wilms' tumors and many other cancers, including breast cancer. This alluded to WT1 behaving as an oncogene. The purpose of this study was to further understand the WT1 protein and its role in cancer.

The WT1 protein has an N-terminal domain which is involved in the repression and activation of transcription and a C-terminal that has four C2H2 zinc finger domains involved in DNA/RNA binding and protein protein interactions. There are four main types of WT1 due to alternate splicing. Experiments using zinc finger only forms of WT1 and full length forms were used test the effects on the WT1 promoter and breast cancer cell growth because it has been seen that WT1 can behave as an activater or repressor. This was done using luciferase assays, chromatin immunoprecipitation reactions, electrophoresis, northern and western blotting, and colony, cell division, and apoptotic assays. The results demonstrated that WT1 is regulated by the zinc finger domain of WT1 and the and the expression of WT1 can be inhibited by WT1-ZF. Also, that in breast cancer cells that express WT1, WT1-ZF behaves as a dominant negative and inhibits cell growth and survival. It was also found that different forms of WT1 can be more or less repressive than others. The ability of WT1 to be a repressor or activator depends upon the context of the cell.

The study concludes that WT1-ZF behave as a dominant negative of WT1 in WT1-expressing breast cancer cells and that WT1 is important in the growth of some forms of breast cancer and in regulating apoptosis, the cell cycle, and self-renewal capacity.

Links related to the paper:

The Drosophila melanogaster gene brain tumor negatively regulates cell growth and ribosomal RNA synthesis

Presented by Tyonna Respicio

Cell growth depends on protein synthesis, regulation of translation, and regulation of growth. There can also be a regulation of cell growth through control of ribosome synthesis. Ncl-1 is s gene found in C. Elegans that is known to negatively regulate ribosome synthesis. It can regulate RNA synthesis and inhibit cell grown. Ncl-1 mutant worms are larger than wild type worms in that they have larger cells with enlarged nucleoli due to the fact that nothing is regulating the synthesis of ribosomal RNA (rRNA). This leads to high numbers of ribosomes. Drosophila melanogaster tumor suppressor brain tumor (brat) is a gene in Drosophila that is most similar to ncl-1. Brat and ncl-1 are similar in structure as well as function. Brat also is known to regulate cell growth and rRNA synthesis. It is a tumor suppressor gene as is ncl-1. Tumor suppressor genes function to inhibit cell proliferation or promote apoptosis. Apoptosis is programmed cell death. If a tumor suppressor gene loses its function it promotes cancer and cells will begin to grow uncontrollably. The mutants of these tumor suppressor genes are recessive. There must be a complete knock out of both good copies of the gene in order to see a mutant.

In this experiment the researchers wanted to prove that the function of the brat gene in Drosophila is to negatively regulate cell growth and rRNA. They showed that brat and ncl-1 are functionally homologous by showing that brat can functionally replace the ncl-1 gene in C. Elegans. They also compared the cells sizes in brat mutant and brat wild type. The brat mutant cells were larger than the wild type cells because of the excess cell growth. It was shown that brat mutant cells have enlarged nucleoli and excess rRNA. On the other end instead of looking at the loss of the function of brat, they also looked at overexpression of brat. They found that brat overexpression inhibits organ growth. Overexpression of brat also inhibits cell growth and results in enlarged cells. Lastly the researchers found that brat inhibits rRNA accumulation and suggest that brat negatively regulates the level of cellular rRNA. All of the experiments the researchers did supports the fact that brat represses cell growth and ribosome synthesis. It is a tumor suppressor gene and without its proper function cells will grow and become cancerous.

Links related to the paper:

Protein expression and intracellular localization of prostate apoptosis response-4 (Par-4) are associated with apoptosis induction in nasopharyngeal carcinoma cell lines

Presented by Colin Williams

The focus of this article was the role of the proapoptotic gene “Prostate apoptosis response-4 (Par-4)” in the selective induction of cell death in most cancer cells. Because this research was done in Taiwan, they focused on a cancer prevalent in South China and Southeast Asia called Nasopharyngeal carcinoma (NPC). The cause of this cancer has been previously linked to the Epstein-Barr virus (EBV) as well as genetic and other environmental factors such as diet and exposure to carcinogens, but the molecular mechanisms remain unknown. Previous genetic research has linked the loss of chromosome 3p and the amplification of chromosome 12 to the development of NPC. This research prompted Jeng-Woei Lee and associates to look at the gene Par-4 because it is found on chromosome 12 and is a known factor in cell apoptosis.

Their research showed that Par-4 interacts with many different proapoptotic proteins, many of which we have previously discussed; such as, WT1, PKC, p62, DLK/ZIP, F-actin and the Raf-MAPK-ERK pathway. They found that the overexpression of Par-4 is sufficient for induction of apoptosis in most cancer cells, but not all of them. In some cases, Par-4 must be accompanied by a secondary apoptotic signal. It was also shown that localizaton of Par-4 within the cell was a factor in regulating apoptosis.

Links related to the paper:

Common Activation of Canonical Wnt Signaling in Pancreatic Adenocarcinoma

Presented by Jessica McEwen

Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer in western societies. It is known that formation of PDA begins with mutations that cause deregulation of Notch and Sonic Hedgehog pathways. Since Sonic Hedgehog (Shh) regulates Wnt signaling in other organs this study investigated if Wnt signaling is active in PDA cells and how this affects tumor growth.

In canonical Wnt signaling soluble Wnt ligand binds the frizzled receptor. This causes a signaling cascade that inhibits B-catenin phosphorylation. B-catenin (which is normally present in the cell membrane) that is non-phosphorylated moves through the cytoplasm to the nucleus. In the nucleus B-catenin binds TCF-LEF transcription factors which activate transcription of Wnt target genes. Thus the presence of B-catenin in the cytoplasm or nucleus of a cell is indicative of Wnt signaling.

The researchers began by examining 136 human PDA samples for Wnt signaling. They used B-catenin as a marker for pathway activation. They found a significant number of tumors with B-catenin present in the cell nucleus (13%) or cytoplasm (65%) indicating active Wnt signaling. The researchers then used genetically modified mice that were susceptible to forming tumors similar to PDA to further investigate Wnt pathway activity. They found increased Wnt signaling in the mouse model tumors. In an additional mouse model, forced expression of Shh in pancreatic cells caused Wnt signaling. Thus they concluded that increased Shh signaling can induce increased Wnt signaling.

Finally the researchers investigated how inhibition of Wnt signaling affected tumor growth and survival. Four different PDA cell lines were grown in culture. Then three inhibitors of Wnt signaling (Icat, dn-Lef-1 and anti-B-catenin siRNA) were added to the cell cultures. All three inhibitors caused a significant decrease in cell proliferation as well as an increase in apoptosis. This proves that Wnt signaling is important for tumor cell growth and survival.

Links related to the paper:

Interplay between the tumor suppressor p53 and TGF beta signaling shapes embryonic body axes in Xenopus

Presented by Joey Schneider

The Transforming Growth Factor-b superfamily (TGF-b) signaling pathway begins with the activation of the Smad family of transcription factors. The TGF-b ligand binds to the type II TGF-b receptor which allows for the receptor to bind type I TGF-b receptor. Type I receptor is then phosphorylated by the type II receptor. This then activates a group of proteins called Smads. The activated Smads act as transcription factors in the nucleus. Most TGF-b isoforms prevent cell proliferation.

In this study, the researchers examined the function of p53 in xenopus as a transcription factor that could possible contribute to the formation of the anterior-posterior axis, instead of as a tumor suppressor. When p53 was coexpressed with noggin into the animal cap of a developing xenopus embryo, target genes indicated that posterior neural structures had developed. In the absence of p53, the only gene induced was Otx2, a forebrain marker. However, since p53 also induces the formation of mesoderm, it was uncertain whether the posteriorizing characteristics of p53 were due to primary protein interaction or by secondary interactions of the mesoderm.

The researchers then focused on the function of p53 in the mesoderm. They found that p53 interacts with the TGF-b family to regulate the expression of homeobox genes Mix 1, Mix 2, and Xhox3. In humans, the majority of TGF-b1 induced genes had a p53 binding site. This is significant because TGF-b1 was identified on its ability to induce malignancy in certain mammalian cells. However, at least in xenopus, p53 does not interact with all genes stimulated by Smads from the TGF-b signaling pathway. Goosecoid, an organizer specific homeobox gene, is not induced by p53 and shows that there is some specificity in this pathway. What is interesting about this research is that p53 acts is downstream of TGFb ligand-induced receptor activation. The functions of other downstream genes may be important for the other functions of p53 such as cell cycle arrest and apoptosis.

Links related to the paper:

Treatment of Terminal Peritoneal Carcinomatosis by a Transducible p53-Activating Peptide

Presented by Charlene Trinidad

Because late-stage cancers have shown an increased resistance to current chemotherapy and treatment, there has been an increased need to develop a therapeutic strategy to induce apoptosis (programmed cell death) of such cancer cells. The p53 tumor suppressor protein plays a fundamental role in inducing such apoptosis and growth arrest in response to cellular stress. However, some cells have mutations in the p53 pathway, making them resistant to the inhibition of tumors cells and instead leading to the unrestrained development of "immortal" cancer cells. It is practically a universal characteristic of cancer cells to have mutations in the p53 pathway. Therefore, an effort to restore p53 function should hold promising results in inducing apoptosis of cancerous cells.

This paper focuses on the efforts of Steven Dowdy and his colleagues to restore p53 protein function in tumor cells. Because the cell membrane only allows passage of small molecules, a technique was developed by Dowdy which allowed the large p53C' (synthesized peptides) to enter cancer cells. Such peptides which are capable of traversing the plasma membrane are referred to as protein transduction domains (PTDs). Though such PTDs successfully solve the problem of macromolecule delivery into the plasma membrane, they show increased susceptibility to degradation. Therefore, a transducible D-isomer RI-TATp53C' peptide was created, which selectively activates the p53 protein in cancer cells. Experiments were done which proved that RI-TATp53C’ peptide treatment directly inhibited overall tumor proliferation.

These studies led to the hypothesis that reactivation of endogenous p53 protein will be an effective means of treating cancer. Interestingly enough, when studies were done on mouse strains that modeled human metastatic disease, they found that restoring p53 protein function in tumor cells both dramatically increases lifespan in mice and also eliminates disease. They were then able to conclude that linking PTD’s to p53C’ and to other p53-activating peptides may be an effective therapeutic strategy in curing a significant amount of human cancers.

Links related to the paper:

Abrogation of TGF-beta signaling induces apoptosis through the modulation of MAP kinase pathways in breast cancer cells

Presented by Lisa Tsoi-A-Sue

Transforming Growth Factor beta has the ability to alter the activity of MAP kinases. The purpose of the experiment was to determine the role of autocrine TGF-beta signaling through MAP kinases in controlling cell survival of human breast cancer MCF-7 cells. TGF-beta is able to activate three major MAPK family members:- Erks, p38 and c-Jun N-terminal Kinases (JNK). Activation of MAPKs can have either anti-apoptotic or pro-apoptotic effects which is important in tumor progression. TGF-beta plays a role in carcinogenesis by suppressing tumors in early epithelial carcinogenesis but has the ability to promote cancer in the later stages of tumor progression.

Telomerase-immortalized human mammary epithelial cells (HMECs)and human breast cancer MCF-7 cells were used to determine the effects of altered MAP kinases by TGF-beta in promoting cell survival and determining whether it is naturally promoted in mammary epithelial cells. Blocking autocrine TGF-beta signaling was shown to increase the apoptosis in MCF-7 cells but not in HMECs. This is observed as that Erk is down-regulated while p38 in MCF-7 cells is up-regulated. In HMECs, the operational autocrine TGF-beta pathway is blocked but there is little effect on cell apoptosis. This means that in HMECs, blockage of the TGF-beta signaling was not able to alter MAPK activation and thus shows that this survival promoting activity of autocrine TGF-beta is an acquired function that occurs during carcinogenesis. The MAP Kinase Pathway is altered by the expression of DNRII cells in MCF-7 cells. MCF-7 cells are promoted in the presence of autocrine TGF-beta signal through alteration of the MAPK pathway (via expression of DNRII) by keeping active Erk high and active p38 low, which would prevent apoptosis of the MCF-7 cells. This presents the possibility of inducing apoptosis in breast cancer cells by targeting the TGF-beta pathway.

Links related to the paper:

Gain of Function of a p53 Hot Spot Mutation in a Mouse Model of Li-Fraumeni Syndrome

Presented by Ashley Pisani

We all have many cancerous cells living in our body at every moment of the day, but what separates one with cancer from one without is correct function of cell cycles and programmed cell death. Li-Fraumeni Syndrome is the mutation seen in many humans tumors and occurs because of a mutation in the p53 tumor suppressor gene. This mutation is inherited, and depending on how many copies of the mutation you are born with the more susceptible one becomes. A mouse model was used to further study the consequences of mutations in the p53 gene. This was done by making mice with a nucleotide 515 substitution in the p53 gene, which has been seen most in human cancers.

Through studying mice this paper showed that mutations in the p53 gene are actually two types of mutations; Gain-of-function or Dominant negative mutations. It was seen that there was a greater than normal wildtype function ( gain-of-function phenotype) in the p53 gene with a single nucleotide 515 switch. They contained a higher transforming potential then compared to mice with knock out for gene p53. They also studied the p63 and p73 which may have been inactivated by mutant p53 which binds and inhibits there function. This is a Dominant negative mutation, when p53 proteins that come from mutant alleles block the function of the normal p53, p63, and p73 proteins. The overall conclusion of the paper is that the p53 mutant with the single nucleotide substitution becomes stable in tumors and no longer activates programmed cell death.

This paper overall supports other studies that show mutations of a single substitution of allele from G-to-A base cause a gain-of-function mutation that is stabilized and may lead to metastasis. By studying this mutation in mice it is hoped that an antibody will be able to be made against mutations in the p53 gene, or at least some sort of therapy to lower tumor formation and induce cell death.

Links related to the paper:

Key apoptosis regulating proteins are down-regulated during postnatal tissue development

Presented by Gaby Zapata

Apoptosis is the regulated cell death that occurs in cells as a way to create a balance of the turnover of cells. When cells do not die at the rate that they are supposed to, cancers develop, and if cells die faster than they are supposed to, they cause neurodegenerative disorders. The process of apoptosis occurs by a cascade of cystein proteases called caspases. These caspases are enzymes that are originally expressed as inactive and once an extrinsic or intrinsic event occurs, they are then activated. The activation of the caspases then sets off a series of events that ultimately lead to cell death in a very organized fashion. In the extrinsic pathway, and external ligand binds to the cell surface receptor and recruitment and activation of caspses-8 occurs which then activates caspase-3 which is the executioner responsible for cell death. The intrinsic pathways are initiated by the cytochrome c release from the mitochondria that causes the Apoptotic Activating Factor 1 (Apaf-1) to be activated. The Apaf-1 activation then recruits and activates caspase-9 which then activates the executioner caspase-3 and cell death occurs. The release of cytochrome c is controlled by a family of proteins known as BcI-2. Through knockout models, this intrinsic pathway has been shown to be essential for the correct development in the mouse.

A group at the laboratory of Dr Cotter at the University of Cork Ireland had previously showed that postnatally the eye continues to develop and in order to do so it down regulates the key proteins of the intrinsic apoptotic pathway. The researchers postulate that this might be a defense mechanism to protect the eye from loosing irreplaceable cells. In this paper, they further investigate if the down-regulation of the intrinsic apoptotic pathway will also occur in distinct tissues like the brain, skeletal muscle, and the heart. What they found was that the Apaf-1, caspase-3 and Bin are all down-regulated in the neocortex of the adult brain, adult skeletal muscle and the heart. They also found that all three of these proteins are expressed in the IGL of the adult cerebellum and the adult thymus, which means that the apoptotic pathway is not turned down.

The investigators were able to demonstrate this by looking at the expression patterns of these apoptotic factors (Bin, Apaf-1, caspase-3 and caspase-9) in the tissue of the brain, muscle, and heart. This process was done by Immunoblots which allows the investigators to separate proteins by size on a gel and then detect specific proteins using specific antibodies against each of the proteins observed. Different stages of prenatal development were compared to the adult mouse to determine when the given proteins were down-regulated or not. The results were also confirmed through Immunohistochemical which takes thin slices of the tissue stained with specific antibodies to determine where in the tissues the proteins are located. The study concluded that all three of the proteins (Bin, Apaf-1, caspase-3 and caspase-9 are down-regulated in the adult brain, heat, and muscle but not in the IGL cerebellum or in the thymus.

Links related to the paper:

Epistatic and independent functions of Caspase-3 and Bcl-X in developmental programmed cell death.

Presented by Brian Beckord

Apoptosis, programmed cell death, is a critical factor in the developing mammalian nervous system. Apoptsois performs a variety of tasks in the developing CNS including; controlling neuronal population size to match their corresponding peripheral targets, facilitating region specific apoptosis which leads to proper development of nervous system structure, and regulating the size of the progenitor pool. The mechanisms involved in apoptosis are regulated by various apototic factors including proapoptotic factors; Bax, caspase-9 and caspase-3 and a critical antiapoptotic factor Bcl-X. In studies performed, Bcl-x deficient mice die as embryos and illicit gross neuronal cellular death. Caspase-3 and caspase-9 deficient mice depict decreased neuronal apoptosis during nervous system development, resulting in morphologic abnormalities. Because csapase-3 is located epistatically downstream from Bcl-x, the research aimed to determine whether Bcl-acts to directly block the apoptotic functions of caspase-3 or if these factors act independently. This study examines the relationship and specific function of the apoptotic factors Bcl-x and caspase-3

In order to determine the relationship and role of Bcl-x and caspase-3, the researchers generated double Bcl-x/caspase-3 mutants by inserting a caspase-3 mutation into Bcl-x deficient mice, thus allowing one to observe whether caspase-3 deficiency would decrease the phenotypic outcome of the, upstream, Bcl-x deficiency. The researchers found that the caspase-3 deficiency led to the prevention of a Bcl-x deficient increase in apoptosis of neuronal cells, but did not change an increased hematopoietic stem cell death or embryo lethality. The caspase-3 deficiency was found to independently control the level of neuronal progenitor cell apoptosis. Thus, the observed effects of Bcl-x and caspase-3 depict a mechanism in the central nervous system in which apotosis serves to regulate the progenitor cell population.

Links related to the paper:

Bcl-2 constituvtively suppresses p53-dependent apoptosis in coloredctal cancer cells

Presented by Nick Chalmers

The fundamental problem with cancer and cancer cells is unregulated cell division and apoptosis, programmed cell death. Many mutations in specific regions of DNA, especially DNA mismatch repair regions, can cause inappropriate expression levels of certain key genes that regulate cell death and apoptosis. By silencing certain anti-apoptotic genes in colorectal cancer cells, apoptosis can be induced. By studying and searching for genes that control the cell death process scientists can identify target genes to fight cancer. This paper focuses on the Bcl-2 gene control over p53 and its effects on cell death regulation.

By using colorectal cancer cells this paper studied the relationship between the Bcl-2 and p53 genes. It was determined that by silencing the Bcl-2 gene it caused massive p53 dependent apoptosis. Using RNA interference the Bcl-2 and Bcl-xl were silenced in both p53+/+ and p53-/- cells and showed that p53 was necessary to induce cell death. Other apoptotic pathways were also investigated as well as other necessary factors such as the Bax and Caspase 2 genes. This paper showed that both Bax and Caspase-2 are necessary for apoptosis regardless of Bcl expression. Based on these results there are new possible anti-cancer therapeutic applications. Possibly by targeting and silencing Bcl-2 in colorectal cancer cells, or possibly by silencing genes that effect Bcl-2 and p53, such as Bax and Caspase-2, cancerous cells could be induced to undergo apoptosis.

Links related to the paper:

Nov. 19, 2007 Presentations

BNDF gene replacement reveals multiple mechanisms for establishing neurotrophin specificity during sensory nervous system development

Presented by Robyn Forsman

Neurotrophins are required for the development of the peripheral nervous system. The neurotrophic family is made up of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4). These neurotrophins control neuron survival, target innervation and synaptogenesis. Neurotrophic factors are secreted by target cells and prevent the neurons from initiating programmed-cell death. The functions of these neurotrophins are mediated by their interactions with tyrosine kinase (Trk) receptors. The two neurotrophins that were observed in this study were brain derived neurotrophic factor and neurotrophin 3. Brain derived neurotrophic factor binds to the TrkB receptor and neurotrophin 3 binds the TrkC receptor but can also interact with TrkA receptor and the same receptor used by BDNF, the TrkB receptor.

In this study they replaced the BDNF gene with that of NT3 (BDNF^NT3/NT3) to observe the specificity and selective roles of BDNF and NT3 during development. In different sensory systems the BDNF^NT3/NT3 mice expressed differences in the ability of NT3 in promoting survival, short-range innervation and synaptogenesis. They ran this experiment in the vestibular system and in the gustatory system. In the vestibular system they found that TrkB or TrkC receptors are sufficient in promoting neuron survival but that TrkB is required for proper innervation and synaptogenesis. In the gustatory system the actions of BDNF are unable to be replaced with NT3. They think this is because taste neurons have a temporary selective expression of TrkB. Therefore NT3 can be replaced for BDNF but only in certain parts of the peripheral nervous system and there is no general mechanism for neurotrophin specificity.

Links related to the paper:

Ethanol exposure affects gene expression in the embryonic organizer and reduces retinoic acid levels.

Presented by Victoria Nguyen

Fetal Alcohol Syndrome (FAS) is a severe form of Fetal Alcohol Spectrum Disorder (FASD), which causes abnormalities to the fetus due to alcohol (ethanol) exposure. Alcohol consumption during pregnancy can cause severe neurological and growth defects in developing embryos, such as mental and growth retardation, microcephaly, skeletal defects and dysmorphia. Different animal models (mouse, frog, chick, etc...) have been used to study the effects of ethanol and the mechanism pathways of the malformations.

In this experiment, Xenopus was used to study the effects of alcohol exposure during organogenesis, a crucial period of embryonic development. During this study, researchers exposed Xenopus at different embryonic stages to different levels of ethanol exposure. A closer examination of the embryonic stage will reveal the correlation between retinoic acid (RA) levels, ethanol, and organizer specific genes in Spemann's organizer. They found that ethanol exposed embryos were most sensitive between late blastula and early/mid gastrula stages. During this time, the formation and development of the Spemann's organizer was also occurring and overlapped with the embryo's sensitivity. Researchers noted that ethanol inhibited RA signaling pathways that were crucial in the function of Spemann's organizer. Ethanol reduces the level of RA in the embryo, causing it to be severely deformed.

Links related to the paper:

Hoxd and Gli3 Interactions Modulate Digit Number in the Amniote Limb

Presented by April Krogstad

Pentadactyly is the ancestral amniote digit formula despite the multiple variations in many tetrapods. In humans, the most common alteration of the limbs is changes in the number of digits, most commonly polydactyly. It is known that the ZPA through production of Shh controls limb patterning including alterations in the number and identity of the digits. Shh prevents the processing of Gli3, causing it to act as a transcriptional repressor. It has been suggested that the polydactyly characteristics of Gli3 mutants depends on the anterior up-regulation of 5'Hoxd genes. To test this hypothesis they generated a mutant for Gli3 and the three most 5'-located Hoxd genes.

Results of their experiment showed that the development of mutant limbs indicated a genetic interaction between Gli3 and the posterior Hoxd genes resulting in an increase in the number of digits. The phenotype of the combined mutation also showed that the polydactyly of the Gli3 mutant limbs did not require the three most 5' Hoxd genes and suggested that posterior Hoxd genes may actually have some negative effect in determining digit number in the absence of Gli3. The polydactyly of the mutants was shown to be a type two polydactyly because of symmetric unidentifiable digits and disruption of Gli3 function. Results also showed Shh signaling to be irrelevant for the mutant phenotype.

The results of a gene expression analysis showed that the mutant limb developed with a uniform expression of Hoxd9, Hoxd10, and Hoxa13 across the distal limb mesoderm in a pattern similar to Gli3 in the mutant limb. Their data suggests, like the currently accepted idea, that the recruitment of the HoxA and Hox D clusters in developing appendages may have mediated the evolutionary transition from polydactyly to pentadactyly in ancestral tetrapods. They believe that this recruitment may have carried the implementation of Shh activation and overall that the recruitment of the HoxD cluster had to include a correct balance between anterior and posterior products as well as with Gli3.

Links related to the paper: