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Keywords:

  • Pax genes;
  • differentiation;
  • brain development;
  • progenitor cell;
  • lineage specification;
  • neurogenesis;
  • myogenesis;
  • transcription factors

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. Pax EXPRESSION DURING DEVELOPMENT
  5. Pax EXPRESSION IN PROGENITOR CELLS OF ADULT TISSUE
  6. COMMON ANCESTRAL Pax GENE: ONE GENE, ONE FUNCTION?
  7. Pax DOWNSTREAM TARGETS
  8. SUMMARY
  9. FUTURE DIRECTIONS
  10. REFERENCES

Pax transcription factors are critical for the development of the central nervous system (CNS) where they have a biphasic role, initially dictating CNS regionalization, while later orchestrating differentiation of specific cell subtypes. While a plethora of expression, misexpression, and mutation studies lend support for this argument and clarify the importance of Pax genes in CNS development, less well understood, and more perplexing, is the continued Pax expression in the adult CNS. In this article we explore the mechanism of action of Pax genes in general, and while being cognizant of existing developmental data, we also draw evidence from (1) adult progenitor cells involved in regeneration and tissue maintenance, (2) specific expression patterns in fully differentiated adult cells, and (3) analysis of direct target genes functioning downstream of Pax proteins. From this, we present a more encompassing theory that Pax genes are key regulators of a cell's measured response to a dynamic environment. Developmental Dynamics 237:2791–2803, 2008. © 2008 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. Pax EXPRESSION DURING DEVELOPMENT
  5. Pax EXPRESSION IN PROGENITOR CELLS OF ADULT TISSUE
  6. COMMON ANCESTRAL Pax GENE: ONE GENE, ONE FUNCTION?
  7. Pax DOWNSTREAM TARGETS
  8. SUMMARY
  9. FUTURE DIRECTIONS
  10. REFERENCES

Within the developing embryo, members of the highly conserved Pax gene family are initially considered to play an intrinsic role in regionalization and at later stages in the specification of specific subtypes of cells (Stoykova and Gruss,1994; Kawakami et al.,1997). Pax expression continues in the adult, in both stem/progenitor cell populations and, intriguingly, in mature cells (Seale et al.,2000; Shin et al.,2003; Thompson et al.,2004,2007,2008; Maekawa et al.,2005; Thomas et al.,2007; Fedtsova et al.,2008). The reason for persistent expression in mature cells, particularly in the central nervous system (CNS), has perplexed us and instigated our quest to better define the role of Pax genes. Based on its locality, a cell will have a particular combinatorial gene expression signature which dictates its unique identity and therefore its function. Here we hypothesize that Pax genes are one of the many key regulators of a cell's measured response, or lack thereof, to a dynamic environment. Supporting evidence for our proposal comes from myriad observations indicating that; (1) during early development, Pax expression is concentrated regionally in response to heightened environmental signals, and later in specific subpopulations of differentiating cells in response to local signals (Monsoro-Burq et al.,1996; Tanabe and Jessell,1996; Ericson et al.,1997; Marcelle et al.,1999; Timmer et al.,2002; Taneyhill and Bronner-Fraser,2005), (2) Pax expression typically continues in adult tissue progenitor cells characterized by their ability to respond to environmental signals (Seale et al.,2000,2003; Kohwi et al.,2005; Lang et al.,2005; Maekawa et al.,2005), (3) adult differentiated cells re-express Pax genes when they are required to respond to environmental cues (Kioussi et al.,1995; Imgrund et al.,1999; Thomas et al.,2007), (4) Pax genes originate from a common ancestral gene and thus have a residual common function (Balczarek et al.,1997), and (5) many identified target genes of Pax transcription factors function in modulating cell responsiveness to stimuli (Bernier et al.,2001; Bouchard et al.,2005; McCann et al.,2007; Wang et al.,2007; White and Ziman,2008).

Pax EXPRESSION DURING DEVELOPMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. Pax EXPRESSION DURING DEVELOPMENT
  5. Pax EXPRESSION IN PROGENITOR CELLS OF ADULT TISSUE
  6. COMMON ANCESTRAL Pax GENE: ONE GENE, ONE FUNCTION?
  7. Pax DOWNSTREAM TARGETS
  8. SUMMARY
  9. FUTURE DIRECTIONS
  10. REFERENCES

While Pax genes are expressed throughout the embryo, most are involved in patterning of the CNS and it is here, in development of the neural tube/CNS, that Pax genes provide an exquisite example of their function—the coordination of cellular responses to regionally secreted factors leading to the production of appropriately positioned and differentiated cell types (Chalepakis et al.,1993; Stoykova and Gruss,1994; Tanabe and Jessell,1996). Traditionally the restricted dorsoventral expression patterns of Pax genes, from the very earliest stages of neural tube formation, determine neural tube progenitor cell fate. Specifically, dorsal cell types are thought determined by Pax3 and Pax7 in response to secretion of bone morphogenetic protein (BMP) from within the overlying ectoderm and roof plate, and more ventral cell types by Pax6 in response to Sonic Hedgehog (Shh) secretion from the notochord and floor plate (Jostes et al.,1990; Stoykova and Gruss,1994; Mansouri et al.,1996a; Monsoro-Burq et al.,1996; Tanabe and Jessell,1996; Ericson et al.,1997; Kawakami et al.,1997; Mansouri and Gruss,1998; Lee et al.,2000; Timmer et al.,2002; Bel-Vialar et al.,2007).

The segmented expression of Pax genes in response to secreted local cues (BMPs dorsally and Shh ventrally) continues as the brain differentiates from the neural tube. Pax3 and 7 define the dorsal mesencephalic (midbrain) region (Nomura et al.,1998; Matsunaga et al.,2001) and Pax6 the ventral mesencephalon and forebrain (Stoykova and Gruss,1994). Pax2 and 5 specify the midbrain primordium (Schwarz et al.,1999) and later Pax2, 5, and 8 coordinate the formation of the midbrain–hindbrain boundary together with secretory factor, FGF8 (Lun and Brand,1998; Schwarz et al.,1999; Picker et al.,2002).

As local inductive cues become more restricted during later stages of CNS development, Pax expression patterns change from region-specific to cell-specific. For example, Pax6 is expressed in the differentiating neural cells of the optic vesicles (Stoykova and Gruss,1994) in response to local signals from the overlying ectoderm (Bailey et al.,2004), while Pax7 expression continues in dorsal tectal/collicular neurons (Jostes et al.,1990; Kawakami et al.,1997; Thompson et al.,2004,2007,2008; Thomas et al.,2007) and Pax3 is found in Bergmann glia and cells surrounding the Purkinje cells (Stoykova and Gruss,1994).

Pax mutants also highlight the developmental significance of Pax genes in regulating cellular responsiveness and in the presence of unaltered regional cues, Pax-expressing cells which lack a functional Pax gene fail to differentiate into specific subtypes. For example, Pax2 null Zebrafish noi mutants fail to develop an isthmus (Brand et al.,1996), while Pax3 mutants have significant neural crest and neural tube/CNS defects (Auerbach,1954; Wildhardt et al.,1996) and Pax5 knockout mice exhibit defects in the midbrain–hindbrain region (Urbanek et al.,1994,1997). Similarly, Pax6 mutations result in severe defects in forebrain development and eye formation (Hill et al.,1991; Jordan et al.,1992; Callaerts et al.,1997; Mastick et al.,1997; Niimi et al.,1999,2002) while Pax7 knockout mice exhibit craniofacial defects (Hill et al.,1996; Mansouri et al.,1996b). Although hypomorphic mutant studies are beginning to elucidate a mode of action for Pax genes, earlier studies of Pax mutant animals were to some extent hindered by compensation through gene expression of other Pax family members, and, did not, we believe, fully characterize the role of individual Pax genes (Relaix et al.,2004; Zhou et al.,2008).

The dynamic spatiotemporal expression patterns led to the long standing dogma that Pax genes play a biphasic role during CNS development, namely regionalization followed by differentiation (Stoykova and Gruss,1994; Kawakami et al.,1997). However, a mechanism that supports this hypothesis has not been forthcoming, nor does it address the persistent expression of Pax genes in adult tissues, both within and outside the CNS. Here, after re-evaluation of expression patterns, particularly in adult tissues where Pax genes are expressed in both progenitor and differentiated cells, and analysis of downstream target genes, we put forward a more encompassing paradigm that Pax genes play a key role in the ability of the expressant cell to respond to local region-specific cues. If the theorized mechanism of action of Pax genes is correct, and Pax expression renders the expressant cells responsive to their environment, then Pax function could chaperone cells through a variety of functional outcomes which are determined by differing spatial and temporal regional cues.

Pax EXPRESSION IN PROGENITOR CELLS OF ADULT TISSUE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. Pax EXPRESSION DURING DEVELOPMENT
  5. Pax EXPRESSION IN PROGENITOR CELLS OF ADULT TISSUE
  6. COMMON ANCESTRAL Pax GENE: ONE GENE, ONE FUNCTION?
  7. Pax DOWNSTREAM TARGETS
  8. SUMMARY
  9. FUTURE DIRECTIONS
  10. REFERENCES

Like their developmental counterparts, progenitor cells that remain within adult tissues are programmed to respond appropriately to altered environmental signals that occur in response to tissue trauma or disease. Progenitor cell populations spend the vast majority of their lifespan in a quiescent limbo-state awaiting activation signals which lead to asymmetric proliferation and production of progeny able to either differentiate or regain quiescence. Compellingly, the progenitor cells within mature tissues which respond to local environmental cues and exhibit proliferative, anti-apoptotic, and often multipotential differentiation capacities commonly express Pax genes (Seale et al.,2000; Asakura et al.,2001; Collins et al.,2005; Hack et al.,2005; Lang et al.,2005; Bel-Vialar et al.,2007). We now suggest that these traits, necessary for any cell to maintain a responsive state, may be preferentially conferred to the adult tissue progenitor cells through Pax function.

Within the adult CNS for example, Pax6 expression continues in progenitor cells of the subventricular (SVZ) and subgranular germinal centers. New neurons are produced from these progenitors in response to the mitogenic signals of EGF, TGF, VEGF and NPY (Craig et al.,1996; Kuhn et al.,1997; Tropepe et al.,1997; Schanzer et al.,2004; Howell et al.,2005; Chiba et al.,2007). The majority of these progenitor cells typically express Pax6 during proliferation with expression down-regulated upon exit from the cell cycle and entry into a migratory phase (Kohwi et al.,2005; Maekawa et al.,2005). A subpopulation of these cells, however, retain Pax6 expression, migrate from the SVZ and subsequently populate the olfactory bulb. Interestingly, when Pax6-deficient neuroblasts are transplanted into the SVZ of adult wild-type mice, they display similar migratory and neuronal differentiation capabilities but undergo precocious differentiation resulting in cell fate switching and failure to generate specific dopaminergic subclasses (Kohwi et al.,2005). Thus, Pax6 expression appears crucial for progenitor-state retention and response to a distinct environment for determination of the appropriate subclass of neurons (Fig. 1).

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Figure 1. A: Schematic representation of adult mouse neuronogenesis, depicting proliferative Pax6-expressing cells within the subventricular zone (SVZ) of wild-type mice. B–D:Pax6 expression is down-regulated in most progenitors upon exit from the cell cycle, culminating in neuron formation (b); however, a subpopulation of neuroblasts retain Pax6 expression and migrate by means of the rostral migratory stream (c) to the olfactory bulb (OB), whereupon Pax6 expression is down-regulated resulting in formation of dopaminergic periglomerular cell neurons (d). E: By contrast, Pax6-deficient cells grafted into the wild-type environment produce progeny which are capable of migration to the OB and of neuronal differentiation, albeit precociously, but fail to produce neurons of the dopaminergic class (Kohwi et al.,2005). Thus, Pax6 expression maintains the cells in an undifferentiated phenotype until they reach the appropriate destination where they are involved in production of the appropriate subclass of neurons.

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To corroborate our theory further we also looked outside the CNS. A well-studied example, the skeletal muscle satellite cell, is considered an archetypal response cell that remains quiescent until environmental cues signal for their activation, proliferation and differentiation into new myoblasts (Fig. 2). Skeletal muscle satellite cells are defined by Pax7 expression from their initial emergence from the dermomyotome until the onset of their differentiation (Seale et al.,2000,2004; Zammit et al.,2002,2004,2006; Oustanina et al.,2004;). In Pax7−/− mice, the number of satellite cells is dramatically reduced (Seale et al.,2000) and those remaining do not respond appropriately to environmental cues, as evidenced by a 25–30% reduction in proliferation and a marked increase in apoptosis and cell cycle defects (Relaix et al.,2006). Fated Pax7 expressing satellite cells can be induced, in vivo and in vitro, to differentiate along alternate lineages, given appropriate environmental cues (Asakura et al.,2001; Wada et al.,2002; Shefer et al.,2004; Morrison et al.,2006), whereas forced Pax7 expression in satellite cell-derived myoblasts has been shown to delay their differentiation (Zammit et al.,2006). Thus, in the case of adult skeletal muscle satellite cells, Pax7 is a specific and critical regulator of survival, self renewal and plasticity in response to local environmental cues.

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Figure 2. The skeletal muscle satellite cell, defined by Pax7 expression, is considered an archetypal response cell that remains quiescent until environmental cues signal for their activation, proliferation, and differentiation into new myoblasts. This process is ablated in Pax7 knockout mice.

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Melanocytic stem cells of neural crest origin are a further example of Pax-expressing progenitor cells programmed to respond to changes in the adult tissue environment. Situated within a niche in the hair follicle, melanocytic progenitor cells function in a manner similar to skeletal muscle satellite cells. Normally quiescent, these Pax3 expressing stem cells receive signals for re-entry into the cell cycle after activation associated with injury or normal hair cycling (Nishimura et al.,2002). After cytokinesis, one daughter cell exits the cell cycle to remain in the niche, while the other migrates to the epithelium before differentiation (Nishimura et al.,2002; Blanpain et al.,2004). In fact, Pax3 defines a nodal point in melanocyte stem cell differentiation; while Pax3 activates the melanogenic cascade through activation of Microphthalmia-associated transcription factor (Mitf), it simultaneously acts downstream to compete with the Mitf protein for occupancy of Dopachrome tautomerase (Dct) enhancer (Lang et al.,2005). Differentiation occurs in response to Wnt- and β-catenin signaling, which displaces Pax3, allowing Dct transcription. Thus, it is clear that a tightly controlled pathway specifies melanocyte development and that the maintenance of melanoblasts in a committed but undifferentiated permissive state is determined by Pax3 and regulated by Wnt signaling-activated β-catenin (Fig. 3; Lang et al.,2005).

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Figure 3. Pax3 “Pan gene” functions: (Robson et al.,2006) Pax3 acts at a nodal point between the specification and maintenance of melanoblasts and their differentiation. A: Pax3 specifies melanoblasts from neural crest cells by physically interacting with the transcription factor Sox10 (Smit et al.,2000) and both collaborate in the activation of the melanocyte-specific transcription factor gene Mitf by binding to adjacent sites in the Mitf promoter (Bondurand et al.,2000; Lang et al.,2000). Together, Pax3, Sox10, and Mitf are responsible for the regulation of the melanocyte differentiation gene Dct. B: Pax3 forms a complex with groucho protein Grg4 on the Dct promoter, repressing Dct, thus maintaining cells in an undifferentiated state. C: Wnt signaling through frizzled (Fzd) receptors activates β-catenin, which displaces Pax3 and groucho, allowing Mitf and Sox10 to bind and activate Dct expression, leading, irrevocably, to melanocyte differentiation (Galibert et al.,1999; Lang et al.,2005).

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Pax Expression and Re-expression in Adult Differentiated Cells

While Pax genes play a fundamental role in regulating the responsiveness of progenitor cells in developing and adult tissues, the most valuable insight to the role of Pax genes has come from expression and re-expression in terminally differentiated cells after injury or disease (Table 1). Notably, this response is observed specifically in cells derived from those that expressed Pax genes during development.

Table 1. Role of Pax Genes
GeneSpeciesOrgan/tissueCellsProposed functionReference
  • aUnless indicated analysis indicates in vivo expression in adult differentiated cells and not cell lines or progenitor cells of adult tissue. GWGP, German Waltzing guinea pig.

  • *

    Examples of Pax expressant cells that respond to altered local environmental cues.

  • **

    Where indicated, the expression of Pax genes was identified by detection of RNA by RT-PCR or Northern blotting and not IHC and therefore cell classes were not determined and therefore may include expression in progenitor cells of adult tissues.

Pax2HumanEyeONH astrocytesNot specified(Yang and Hernandez,2003)
  Pancreas*Islet cellsRegulation of glucagon gene expression(Ritz-Laser et al.,2000)
  Kidney*Cells of the collecting duct & descending loop of HenleAnti-apoptotic/protection against osmotic stress in response to high NaCl concentration(Cai et al.,2005)
  Genital tractEpithelium of Fallopian and glandular tissueNot specified(Tong et al.,2007)
 MouseGenitourinary Tract*Epithelia of ductus deferens & epididymisRegulation of epithelial genes involved in sperm maturation and support(Oefelein et al.,1996)
  Genital tractsMale & femaleSequence specific DNA binding & subsequent modulation of promoter activity(Fickensher et al.,1993)
  BrainNuclei of midbrain, pons/medulla, cerebellumDifferentiation, maintenance and functional assembly of specific subsets of cells(Stoykova and Gruss,1994)
  Spine*Spinal interneurons (Bechade et al.,2002)
Pax3HumanCerebellumNot specified (RNA)**Speculated as a tumor suppressor gene(Tsukamoto et al.,1994)
  Esophagus   
  Stomach   
  Liver   
  Pancreas   
  SkinMelanocytesNot specified(Gershon et al.,2005)
 MouseBrainNuclei of pons/medulla, cerebellumDifferentiation, maintenance and functional assembly of specific subsets of cells(Stoykova and Gruss,1994)
 GWGPCochlearMelanocytesFormation of melanocytes to support development of marginal and basal cells for correct formation of stria vascularis of the cochlear, function in adult not known(Jin et al.,2007)
Pax4HumanPancreasβ-cells (islet)Not specified(Heremans et al.,2002)
   Islet CellsNot specified(Zalzman et al.,2003)
 RatPancreas*β-cells (islet)β-cell plasticity/anti-apoptotic/regulation of total β-cell population and thus islet mass (transactivates Bcl-xl and C-myc promoters)(Brun et al.,2004)
 MousePancreasNot specified (RNA)**Not specified(Kojima et al.,2003)
Pax5HumanBrainPAG of midbrainNot specified(Torlakovic et al.,2006)
   Medulla oblongataNot specified 
   Caudal nucleusNot specified 
  UterusSmooth MuscleNot specified 
  Genital tractsEpididymisMultiple steps of genital tract development 
   Endocervical glandsNot specified 
  Hematopoietic SystemSpleen, bone marrowSpeculate tumor suppressor activity(Kaneko et al.,1998)
 MouseBrainNuclei of midbrain, pons/medullaDifferentiation, maintenance and functional assembly of specific subsets of cells(Stoykova and Gruss,1994)
  Genital tract*TestisSpeculate spermatogenesis(Adams et al.,1992)
  Hematopoietic System*B-cell follicles of lymph node & spleenB-cell differentiation 
   *Mature B cellsMaintenance of specific differentiation status/tumor suppressor gene of the B lymphoid lineage(Cobaleda et al.,2007)
   *B cellsPax5-mediated gene repression is essential for normal homeostasis of hematopoietic development/down-regulation is required for plasma cell (terminal) differentiation(Delogu et al.,2006)
Pax6HumanPancreasβ-cells (islet)Not specified(Heremans et al.,2002)
   *Islet cellsNot specified(Zalzman et al.,2003)
  EyeRetinal ganglion cells Inner part of INLNot specified, suggested maintenance of eye tissue. Their recent experiments also demonstrate persistent expression in the mouse inner retina throughout life (Iseli et al, submitted)(Stanescu et al.,2007)
 RatPancreasIslet cellsFindings indicate differential requirements for Pax6 gene dosage in displaying function and maintaining architecture of adult pancreatic islets(Hamasaki et al.,2007)
  HippocampusHilar mature neuronsAstrocytesSpeculated that Pax6 may play a role in the maintenance of certain neuronal populations in the adult CNS(Nacher et al.,2005)
 MousePancreasNot specified (RNA)**Not Specified(Kojima et al.,2003)
   *Islet cells  
  CerebellumNot specified (RNA)**Results support a role in differentiation and maturation of mature cell subtypes(Beimesche et al.,1999)
  EyeSmooth muscle cells of iris sphincter and iris pigmented epitheliumRequired for the normal length of the adult iris(Davis-Silberman et al.,2005)
  BrainNuclei of diencephalon, telencephalon, mesencephalon, pons/medulla and cerebellumMaintenance of specific subsets of cells(Stoykova and Gruss,1994)
Pax7RatMidbrainSuperior colliculusNot specified(Thomas et al.,2007)
 MouseBrainDorsal midbrainNuclei of ventral midbrain, pons/medullaDifferentiation, maintenance and functional assembly of specific subsets of cells(Stoykova and Gruss,1994)
  MidbrainSuperior collicular neuronsNot specified(Thompson et al.,2004,2007,2008)
 ChickenBrain*Tectal neurons*Nuclei of Thalamus/Pons/MidbrainSpeculate maintenance of normal physiology(Shin et al.,2003)
   Cerebellar Bergmann glia  
Pax8HumanKidneyNot specified (RNA)**Not specified(Poleev et al.,1992)
 RatThyroid Not specified RNA)**Modulation of thyroid-specific gene expression in adulthood(Zannini et al.,1992)
  KidneyNot specified (RNA)**Not specified
  Ant. pituitaryNot specified (RNA)**Not specified 
 MouseKidneyNot specified (RNA)**Not specified(Plachov et al.,1990)
 ReviewThyroid*Follicular cellsWide temporal expression suggests recycling of the thyroid-specific transcription factors and thus control of different sets of target genes at diverse developmental stages(Damante et al.,2001)
Pax9HumanEsophagus*KeratinocytesNormal differentiation process of internal stratified squamous epithelia (esophageal keratinocytes)– increasing malignancy of dysplasias is associated with a progressive loss of PAX9 expression(Gerber et al.,2002)
 Human MouseEsophagusNot specified (RNA)**Formation and maintenance of internal stratified squamous epithelium in the anterior digestive tract, which is a different epithelial type to the caudal digestive tract (simple monolayered epithelium)(Peters et al.,1997)
  Anterior digestive tract*Tongue, oral epithelium, salivary gland, pharynx, esophagus            
  ThymusNot specified (RNA)**Not specified(Neubuser et al.,1995)

In the CNS for example, Pax7 expression persists in subtypes of differentiated tectal/superior collicular neurons which remain responsive to their environment, and show increased Pax7 expression during optic nerve innervation (Thomas et al.,2006,2007; Thompson et al.,2007). Even more perplexing is the observed re-expression of Pax7 in a large number of superior collicular neurons, leading to re-activation of developmental guidance cues such as ephrin-A2 after optic nerve deafferentation (Rodger et al.,2001; Thomas et al.,2004,2007).

Similarly, Pax6 gene expression remains (or becomes re-expressed) in differentiated retinal ganglion cells of lower vertebrates, conferring these animals with the plasticity to continuously grow and/or regenerate their optic nerve. This is in striking contrast to the adult mammalian retina, which does not possess the ability to undergo spontaneous optic nerve regeneration and in which the majority of retinal ganglion cells do not retain Pax6 expression (Ziman et al.,2001; Rodger et al.,2006).

Upon scrutiny, it is strikingly apparent that Pax expression persists within adult differentiated cells of most tissues where it is required to maintain correct tissue identity/performance (see Table 1). In general, cells that once expressed or currently express Pax genes may remain responsive to selective environmental cues and react in a manner in keeping with the proposed Pax function.

COMMON ANCESTRAL Pax GENE: ONE GENE, ONE FUNCTION?

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. Pax EXPRESSION DURING DEVELOPMENT
  5. Pax EXPRESSION IN PROGENITOR CELLS OF ADULT TISSUE
  6. COMMON ANCESTRAL Pax GENE: ONE GENE, ONE FUNCTION?
  7. Pax DOWNSTREAM TARGETS
  8. SUMMARY
  9. FUTURE DIRECTIONS
  10. REFERENCES

The observed capacity of numerous Pax genes to direct the response to an altered local environment demonstrates a conserved apparatus in which Pax genes provide (or are at least strongly linked to) a mechanism through which cells evoke a response to signaling, irrespective of cell type or state of maturation (Tomarev et al.,1996; Callaerts et al.,1999). Therefore, an intriguing concept of one overarching function for multiple Pax genes emerges. Pax genes have arisen from a single ancestral gene by gene and/or chromosome duplication during early metazoan history. Members of the Pax family are defined by the presence of a highly conserved 128 amino acid DNA binding domain with six alpha-helices (paired domain), and, in many cases, a complete or residual homeodomain (Balczarek et al.,1997). The paired domain is encoded for by the paired-box, from which the family name is derived (Balczarek et al.,1997). Indeed, the DNA binding paired domain that Pax proteins utilize for target gene selection remains strikingly highly conserved, and with conservation of this domain comes conservation of function. While higher vertebrates have nine Pax genes and Drosophila have ten (Bopp et al.,1989; Walther et al.,1991; Balczarek et al.,1997; Sun et al.,1997; Jun et al.,1998; Miller et al.,2000; Dominguez et al.,2004), the simplest of multicellular animals, the placazoans (specifically, Trichoplax adhaerens) who lack nerve or muscle cells and any kind of body symmetry, have a single Pax gene (Hadrys et al.,2005). Given that conserved functional domains of Pax genes predate the origin of nervous and muscular systems, Pax proteins must play a functional role more fundamental than the specification of heterogeneous cell types.

There is also substantive evidence of functional equivalence of protein products within paralogous Pax groups. For example, mutations in the Pax7 gene result in a less severe phenotype, thought due to overlapping spatiotemporal expression patterns of the paralogous Pax3 gene. To test this possibility, Relaix and colleagues (2004) used targeted deletion to insert the Pax7 coding region into the Pax3 locus, thus deleting Pax3 and replacing it with Pax7. The substituted Pax7 allele rescued Pax3 function in somite segmentation, epaxial and hypaxial dermomyotome development, neural tube closure, and neural crest development (Relaix et al.,2004), leading the authors to conclude that most of the functions of Pax3 and Pax7 were prefigured in the single ancestral Pax3/7 gene. This theory is further supported by up-regulation of Pax7 expression noted in Pax3 hypomorphic mice (Zhou et al.,2008).

Similarly, a Pax5 minigene knocked in to the Pax2 locus is sufficient to rescue the Pax2−/− mouse midbrain-hindbrain phenotype, which otherwise presents with an unformed posterior midbrain and cerebellum (Bouchard et al.,2000). These studies emphasize that a common conserved function has been maintained between paralogous Pax genes, undoubtedly predating gene duplication. Thus the primitive role played by Pax genes in coordinating signals for morphogenesis overarches the ability to direct the process itself and provides an explanation for the ability of any single Pax gene, within diverse cell types, to coordinate the cell's response to endogenous cues.

Pax DOWNSTREAM TARGETS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. Pax EXPRESSION DURING DEVELOPMENT
  5. Pax EXPRESSION IN PROGENITOR CELLS OF ADULT TISSUE
  6. COMMON ANCESTRAL Pax GENE: ONE GENE, ONE FUNCTION?
  7. Pax DOWNSTREAM TARGETS
  8. SUMMARY
  9. FUTURE DIRECTIONS
  10. REFERENCES

Any theory of Pax function would not be complete without an investigation of their direct downstream target genes. Although much is still unknown about downstream genes targeted by Pax transcription factors, what is known adds credence to a direct role in regulating the plasticity (responsiveness) of Pax-expressing cells. The recent identification of Pax7 targets in particular provides convincing rationale for our hypothesis, because many genes targeted by Pax7 are involved in regulating cellular responsiveness to environmental signals (Fig. 4; White and Ziman,2006,2008). In vivo assays performed during mouse embryonic development show that Pax7 targets a complex array of receptors and intracellular signaling pathways, including integral components of Ras-MAPK, JAK-STAT, and calmodulin-dependent signaling pathways (White and Ziman,2008). Importantly, this echoes multiple studies implicating Pax3 in the regulation of receptors and components of signaling pathways such as the secretory glycoprotein Wnt1 (Fenby et al.,2008), HGF / c-Met (Epstein et al.,1996; Mayanil et al.,2001; Relaix et al.,2004; Tomescu et al.,2004), c-RET (Lang et al.,2000; Lang and Epstein,2003), as well as both TGF-α (Barber et al.,2002) and TGF-β2 (Mayanil et al.,2006). Multiple isoforms of Pax3 are also known to independently regulate signaling and morphogen response pathways by means of the Shh functional homologue Dhh, Fgf17, Kitl, and Rac1 to control cell differentiation and proliferation (Wang et al.,2007).

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Figure 4. Biological processes and Molecular functions of Pax7 downstream target genes. Pax7 target genes identified by direct binding in vivo (White and Ziman,2008) or regulation in C2C12 myoblasts (McKinnell et al.,2008) were catalogued for GO Biological Process using the Panther database (Mi et al.,2007).

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The ability to regulate cell signaling in response to environmental signals is a property of many Pax proteins. For example, Necab is downstream of Pax6 and is part of a signal transduction pathway involved in retinal development (Bernier et al.,2001). Pax2 initiates expression of Fgf8, the isthmic organizer signal at the midbrain–hindbrain boundary, as well as key midbrain transcription factors En2 and Brn1 (Pou3f3); intracellular signaling modifiers Sef and Tapp1 (Bouchard et al.,2005) and GDNF (Brophy et al.,2001) are also regulated by Pax2 as is the negative regulator of Wnt signaling, SFRP2 (Brophy et al.,2003).

PAX5 controls cell adhesion, migration, and differentiation of pro-B cells by regulating signaling pathways at multiple levels. Downstream target genes encode cell surface proteins Cd19, Cr2 (CD21), Cd72, Cd791 (Igα), and Blnk, as well as nuclear proteins Vpreb3, Slamf6, Siglecg, Lcp2, and Prkd2, among others (Nutt et al.,1998; Horcher et al.,2001; Schebesta et al.,2002). In fact, continuous PAX5 activity is required not only for maintenance of appropriate B-cell signaling pathways, but also for repression of inappropriate signaling pathways at all stages of B-cell maturation (Schebesta et al.,2002). Finally, in the thyroid, PAX8 mediates TSH-induced regulation of thyroid cell differentiation and function by modulation of the expression of key genes such as those encoding thyroglobulin, thyroperoxidase, and the sodium/iodide symporter NIS (Zannini et al.,1992; Fabbro et al.,1998; Ferretti et al.,2005). Thus, it becomes clear that the panoply of demonstrated downstream targets include many signal transducing agents which provide insight into the observed responsiveness principally conferred to cells by Pax expression.

SUMMARY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. Pax EXPRESSION DURING DEVELOPMENT
  5. Pax EXPRESSION IN PROGENITOR CELLS OF ADULT TISSUE
  6. COMMON ANCESTRAL Pax GENE: ONE GENE, ONE FUNCTION?
  7. Pax DOWNSTREAM TARGETS
  8. SUMMARY
  9. FUTURE DIRECTIONS
  10. REFERENCES

The exploitation of technical advances has allowed a more sophisticated analysis of biological function within complex systems, imparting a clearer understanding of the governance of biological complexity. This new, more detailed knowledge of the function of multiple Pax genes within different cell types points quite clearly to a fundamental property of Pax genes in the regulation of a discerning responsiveness of Pax-expressing cells to their local environment. The observation of altered Pax expression within adult differentiated cells in response to injury and that Pax-expressing progenitor cells are associated with regenerative adult tissue types upholds the concept that Pax genes are key regulators of cellular responsiveness to local cues for healing. The demonstration that Pax proteins of various species target genes with a functional repertoire as diverse as lineage determination, anti-apoptosis, morphogenesis, maintenance of the state of differentiation, and cell signaling indicates that the role played by Pax genes is not merely linked to cellular ontogeny or age, but rather to a capability to respond to, and therefore survive, a changing tissue environment. As these properties are shared by multiple Pax genes across phylogeny, it would tend to indicate that their function has an analogous feature similar to that of the ancestral gene—conference to the expressant cell of a highly regulated transcriptional response to its altered regional environment.

FUTURE DIRECTIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. Pax EXPRESSION DURING DEVELOPMENT
  5. Pax EXPRESSION IN PROGENITOR CELLS OF ADULT TISSUE
  6. COMMON ANCESTRAL Pax GENE: ONE GENE, ONE FUNCTION?
  7. Pax DOWNSTREAM TARGETS
  8. SUMMARY
  9. FUTURE DIRECTIONS
  10. REFERENCES

Here, we have attempted to provide a comprehensive overview of Pax gene function, and from this we synthesize the proposal that members of the Pax gene family in general are key regulators of a cell's response to a dynamic environment. As with all new concepts, several questions remain unanswered. First and foremost, how do we dissect the dynamic overarching function of a complex multigene family? In answering this question, the revolution in accessibility to genomic analysis of transcription factor function (both ChIP-chip and expression microarray datasets for different tissues at different stages) will certainly prove invaluable. Testing the data generated will certainly require the skilful use of conditional knockouts (even of multiple Pax genes) to dissect the physiological relevance of Pax gene function.

An important focus for future investigations will be to identify upstream regulators of Pax genes; Pax expression by means of these upstream regulators in specific tissue types and at specific developmental stages will allow the assessment of changes in morphology in response to expression patterns of Pax. Equally important, is continued identification of the downstream targets of Pax genes as dysregulation of downstream Pax targets, in turn, can be directly analyzed in Pax mutant animal models. These investigations, however, need to be carefully designed with paralogous Pax cross-regulation in mind and be interpreted with care, as Pax function is often dosage dependent (Zhou et al,2008).

Furthermore, investigations of Pax mode of function would not be complete without characterization of cofactors which participate with Pax proteins during target gene regulation. It is well known that differential regulation of specific target genes appropriate to the given tissue type / developmental stage context is often dependent on concise availability of binding cofactors. Analysis and manipulation of Pax binding cofactors may shed light on the mechanisms by which Pax7, for example, directs a cell along a neurogenic rather than a myogenic lineage in the midbrain.

Finally, it will be necessary to examine the extent to which Pax gene expression in adult tissue confers responsiveness to environmental cues. Future investigations along these lines could benefit from the use of conditional and hypomorphic animal models for comparison with healthy adult Pax expressant tissue, where differences in regeneration can be analyzed across a spectrum of Pax perturbation.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. Pax EXPRESSION DURING DEVELOPMENT
  5. Pax EXPRESSION IN PROGENITOR CELLS OF ADULT TISSUE
  6. COMMON ANCESTRAL Pax GENE: ONE GENE, ONE FUNCTION?
  7. Pax DOWNSTREAM TARGETS
  8. SUMMARY
  9. FUTURE DIRECTIONS
  10. REFERENCES