Interactive Fly, Drosophila



Aristaless-related proteins and development patterning

cDNA clones have been isolated encoding a novel protein, Alx-4, that contains a paired-type homeodomain. Analysis of the homeodomain sequence shows that Alx-4 belongs to a family of genes that are related to the Drosophila gene aristaless, and includes the mammalian genes Alx3, Cart-1, MHox, and S8. The expression of Alx-4 during development was examined by Northern blot and whole mount in situ hybridization. Antibodies were generated to recombinant Alx-4 protein and Alx-4 protein has been identified in nuclear extracts prepared from mouse embryos. The expression pattern of Alx-4 suggests that it may play a role in the patterning of structures derived from craniofacial mesenchyme, the first branchial arch, and the limb bud. The results provide a starting point for the analysis of a new member of the family of paired type homeodomain proteins (Qu, 1997a).

Correct development of the limb is dependent on coordination between three distinct signaling centers. Recently, fibroblast growth factor-4 has been identified as a crucial determinant of AER function, which directs limb bud outgrowth, and Sonic hedgehog has been identified as a signaling molecule that mediates ZPA function, which specifies anterior-posterior patterning in the developing limb bud. In addition, Shh and FGF-4 reciprocally reinforce each other's expression via a positive feedback loop, providing a molecular basis for the coordination of limb bud outgrowth and anterior-posterior patterning. The mechanisms by which these signaling centers come to occupy their normal positions in the posterior limb bud during development are not understood. Alx-4 encodes a paired-type homeodomain protein related to Drosophila aristaless. Axl-4, like Aristaless, lacks a paired domain. Alx-4 is expressed in several populations of mesenchymal cells, including mesenchymal cells in the anterior limb bud and the craniofacial region. Mice homozygous for targeted disruption of the Alx-4 gene have multiple abnormalities, including preaxial polydactyly. The polydactyly is associated with the formation of an ectopic anterior ZPA, as indicated by anterior expression of Sonic hedgehog, HoxD13 and fibroblast growth factor-4. The expression of other candidate regulators of anterior-posterior positional information in the limb bud, including HoxB8 and Gli3, is not altered in Alx-4 mutant embryos. Chromosomal mapping experiments have shown Alx-4 is tightly linked to Strong's luxoid, a polydactylous mouse mutant. The results identify Alx-4 as a determinant of anterior-posterior positional identity in the limb and a component of a regulatory program that restricts ZPA formation to the posterior limb bud mesenchyme (Qu, 1997b).

Mutations that affect vertebrate limb development provide insight into pattern formation, evolutionary biology and human birth defects. Patterning of the limb axes depends on several interacting signaling centers; one of these, the zone of polarizing activity (ZPA), comprises a group of mesenchymal cells along the posterior aspect of the limb bud that expressrd sonic hedgehog (Shh) and plays a key role in patterning the anterior-posterior (AP) axis. The mechanisms by which the ZPA and Shh expression are confined to the posterior aspect of the limb bud mesenchyme are not well understood. The polydactylous mouse mutant Strong's luxoid (lst) exhibits an ectopic anterior ZPA and expression of Shh that results in the formation of extra anterior digits. The Strong's luxoid mutant, lst, was first noted in 1946 as a polydactylous mouse (originally called the Springville mouse) that arose in the course of 3-methylcholanthrene mutagenesis experiments performed by Leonnel Strong in his investigations of chemical induction of mammary tumors. A second allele, designated lstJ, arose spontaneously at The Jackson Laboratory. Both lst alleles exhibit semidominant inheritance, such that heterozygotes have preaxial polydactyly that is most often manifest as a broadened hallux or a single additional anterior triphalangeal digit on the hindlimb. A new chlorambucil-induced deletion allele, lstAlb, is described that uncovers the lst locus (reveals a 1st phenotype in double heterozygotes). Integration of the lst genetic and physical maps suggests the mouse Aristaless-like4 (Alx4) gene, which encodes a paired-type homeodomain protein that plays a role in limb patterning, as a strong molecular candidate for the Strong's luxoid gene. In genetic crosses, the three lst mutant alleles fail to complement an Alx4 gene-targeted allele. Molecular and biochemical characterization of the three lst alleles reveal mutations of the Alx4 gene that result in loss of function. Alx4 haploinsufficiency and the importance of strain-specific modifiers leading to polydactyly are indicative of a critical threshold requirement for Alx4 in a genetic program operating to restrict polarizing activity and Shh expression in the anterior mesenchyme of the limb bud, and suggest that mutations in Alx4 may also underlie human polydactyly (Qu, 1998).

Proper functioning of the ZPA demands that it be precisely localized in time and space. The identification of loss-of-function mutations in Alx4 mice, together with the expression of Alx4 in the anterior limb bud, demonstrate that Alx4 function is not required for the formation of the normal posterior ZPA; in contrast, Alx4 is required to repress both ZPA formation and Shh expression in the anterior aspect of the limb bud. This requirement for Alx4 function is similar to the requirement for Gli3 gene activity to repress ectopic Shh expression and ZPA formation. Based on molecular marker and genetic analysis, Alx4 currently cannot be positioned in a genetic pathway restricting anterior Shh expression. Three different lines of evidence provide insight into the refinement of ZPA activity and subsequent Shh expression and serve to provide a framework for viewing Alx4 function:

    (1) Signaling by retinoids is implicated, as the engraftment of beads soaked in RA antagonists to the posterior aspect of the limb bud results in decreased Shh expression, and engraftment of RA-soaked beads to the anterior limb bud results in an ectopic anterior domain of Shh expression in the limb bud and the formation of an ectopic anterior ZPA. Although the mechanism by which retinoids exert this effect is unclear, it may relate to the known ability of RA to regulate AP positional specification by controlling axial Hox gene expression. Hoxb-8 expression in chick limb buds is altered in response to RA bead grafts, and misexpression of Hoxb-8 in the anterior mouse forelimb bud results in anterior Shh expression and polydactyly. Ectopic forelimb expression of Hoxb-8 is not detected in either Alx4 lst or Alx4 tm1qw mutant limb buds, suggesting that Alx4 is not required for restriction of Hoxb-8 expression. Whether Alx4 is a target of patterning by retinoids or Hox genes remains to be determined.

    (2) The continued expression of Shh in the posterior mesoderm is contingent on the expression of Wnt-7a in the dorsal ectoderm and FGFs in the AER; this regulation presumably serves to coordinate patterning along the three axes. Prior genetic studies of the interaction of lst with recessive formin limb deformity mutant alleles have indirectly suggested a dependence of the ectopic ZPA on a preaxial extension of the AER and its reciprocal signaling for Shh maintenance. Formin mutations cause an inability to form or maintain a proper apical ectodermal ridge that subsequently leads to a failure to maintain Shh expression, resulting in oligosyndactyly. When Alx4 lst is placed in trans to mutant formin alleles, the semidominant polydactyly of Alx4 lst heterozygotes is suppressed and a wild-type limb results. The genetic evidence that the Alx4 alleles are likely to represent severe hypomorphic or null alleles suggests that lst and formin are operating on distinct developmental pathways. Presumably, Alx4 loss leads to ectopic Shh expression in the anterior limb bud mesenchyme, which is dependent on formin function for the anterior expansion of the AER, which may be required to support ectopic Shh expression.

    (3) The large number of mouse and human polydactylous conditions suggests that the genetic mechanisms that restrict Shh from the anterior mesoderm of the limb bud are complex. Prior studies examining genetic interactions between lst and the polydactylous mutants luxate and luxoid led to a view that the phenotypic consequences were additive.

With respect to Gli3 Xt and Alx4, it is not clear whether they participate in hierarchical or parallel pathways. At the level of RNA in situ expression analysis, neither Alx4 nor Gli3 expression are significantly altered in the respective mutant backgrounds, suggesting they function in separate biochemical pathways to repress Shh expression in the anterior limb bud mesenchyme. Whether Alx4 expression is altered in the limb buds of other preaxial polydactylous mutants remains to be examined (Qu, 1998 and references).

During vertebrate evolution the transition of fins into limbs has been proposed to have involved a developmental elaboration of digits as novel distal autopod elements. The molecular changes underlying this evolutionary process are not clear, but may reflect distal cell growth in response to distal Hox gene expression. Regardless of mechanism, the fossil record suggests that the ancestral Devonian tetrapods had limbs with up to eight digits. The digits of polydactylous limbs of Acanthostega and Ichthyostega appear at some level to resemble the mirror-image polydactyly seen in Alx4 alleles. This resemblance suggests that during the fin-to-limb transition, a branch of ancestral tetrapod limbs were in fact polydactylous due to the presence of symmetric anterior and posterior ZPAs. Subsequently, during evolution of the pentadactyl limb, the presence of an anterior ZPA would have become actively repressed. Based on this notion, the Alx4 mutant alleles would represent atavistic mutations, suggesting that during tetrapod evolution Alx4 may have been recruited or co-opted from a role in axial patterning as part of a genetic program to repress anterior ZPA formation. Alternatively, as may be argued from the restriction of Shh to the proximal posterior region of the fin bud in the teleost zebrafish, the polydactylous specimens in the fossil record may represent the 'capture' of an evolutionary offshoot and Alx4 expression in the anterior limb bud may instead be ancestral. An ancestral role for Alx4 is also suggested by the expression of Drosophila relatives of Alx4 and Gli3 (aristaless and cubitus interruptus, respectively) in the anterior compartment of the wing imaginal disc. Whereas in AP patterning of the fly wing ci appears to act downstream of hedgehog, the function of aristaless and its relationship to hedgehog and AP patterning is less well defined. Analysis of molecular markers such as Shh, Gli3 and Alx4 expression in zebrafish and in more primitive species such as lungfish and coelacanths should provide information by which to evaluate these speculations (Qu, 1998 and references).

Strong's Luxoid (lstD) mice have a 16 bp deletion in the homeobox region of the Alx-4 gene. This deletion, which leads to a frame shift and a truncation of the Alx-4 protein, could cause the polydactyly phenotype observed in lstD mice. The chick homolog of Alx-4 was cloned and its expression was investigated during limb outgrowth. Chick Alx-4 displays an expression pattern complementary to that of Shh, a mediator of polarizing activity in the limb bud. Local application of Sonic hedgehog and fibroblast growth factor, in addition to ectodermal apical ridge removal experiments suggest the existence of a negative feedback loop between Alx-4 and Shh during limb outgrowth. Analysis of polydactylous mutants indicate that the interaction between Alx-4 and Shh is independent of Gli3, a negative regulator of Shh in the limb. These data suggest the existence of a negative feedback loop between Alx-4 and Shh during vertebrate limb outgrowth (Takahashi, 1998).

A subset of the vertebrate family of paired (prd)-class homeobox genes is characterised by the presence of a sequence encoding a conserved protein domain of unknown function near the carboxy terminus. Since this sequence is also found in the Drosophila transcription factor Aristaless, which also contains a prd-class homeodomain, the C-terminal domain has been termed the aristaless domain. At least fourteen different vertebrate genes have been described that encode an aristaless domain, all of them also encoding a prd-class homeodomain. The family of aristaless-domain-containing transcription factors differs also from paired and its vertebrate homologs (the Pax proteins) in that at position 50 of the homeodomain, which is important for its DNA-binding specificity, a lysine (Ptx) or glutamine (others) is present, instead of a serine. The aristaless-related genes thus form a group with distinct molecular characteristics. A subset of the aristaless-related genes is expressed mainly in mesoderm and neural crest-derived mesenchyme; some of them have been shown to be involved in aspects of skeletogenesis. This subset includes the related genes Prx1 and Prx2 (formerly known as MHox and S8, respectively), which share extensive additional sequence similarities, and the likewise related genes Alx3, Alx4 and Cart1 (ten Berge, 1998 and references).

Prx1 and Prx2 are expressed in very similar patterns predominantly in mesenchyme. Prx1 loss-of-function mutants show skeletal defects in skull, limbs and vertebral column. Mice in which Prx2 is inactivated by a lacZ insertion have no skeletal defects, whereas Prx1/Prx2 double mutants showed many novel abnormalities in addition to an aggravation of the Prx1 single mutant phenotype. Defects are found in the external, middle and inner ear; reduction or loss of skull bones; a reduced and sometimes cleft mandible, and limb abnormalities, including postaxial polydactyly and bent zeugopods. Although mutations in 5' Hoxd or Hoxa genes can result in limb abnormalities like those found in the Prx1/Prx2 mutants, no abnormal Hoxd11 or Hoxd13 expression is found in Prx1/Prx2 double mutant- limb buds. It is possible that the Prx genes are downstream of the Hoxd genes, or that they regulate similar target genes involved in limb morphogenesis. Either a single incisor, or a complete incisor loss is seen in the lower jaw, and ectopic expression of Fgf8 and Pax9 is found medially in the mandibular arch of double mutants. A novel method to detect beta-galactosidase activity in hydroxyethylmethacrylate sections allows detailed analysis of Prx2 expression in affected structures. The results suggest a role for Prx genes in mediating epitheliomesenchymal interactions in inner ear and lower jaw. In addition, Prx1 and Prx2 are involved in interactions between perichondrium and chondrocytes that regulate their proliferation or differentiation in the bones of the zeugopods Other members of the aristaless-related gene family Alx3, Alx4 and Cart1 are expressed in the distal part of the mandibular arch much like Prx1 and Prx2Alx4 and Cart1, but neither show strong mandibular abnormalities. As in the case of Prx1 and Prx2, redundancy of these genes may prevent a stronger phenotype of the mandible and combined mutants may prove insightful with respect to pharyngeal arch development (ten Berge, 1998).

The aristaless-related homeobox genes Prx1 and Prx2 are required for correct skeletogenesis in many structures. Mice that lack both Prx1 and Prx2 functions display reduction or absence of skeletal elements in the skull, face, limbs and vertebral column. A striking phenotype is found in the lower jaw, which shows loss of midline structures, and the presence of a single, medially located incisor. Development of the mandibular arch of Prx1-/-Prx2-/- mutants was investigated to obtain insight into the molecular basis of the lower jaw abnormalities. In mutant embryos a local decrease in proliferation of mandibular arch mesenchyme is observed in a medial area. Interestingly, in the oral epithelium adjacent to this mesenchyme, sonic hedgehog (Shh) expression is strongly reduced, indicative of a function for Prx genes in indirect regulation of Shh. Wild-type embryos that were exposed to the hedgehog-pathway inhibitor, jervine, partially phenocopy the lower jaw defects of Prx1-/-Prx2-/- mutants. In addition, this treatment leads to loss of the mandibular incisors. A model is presented that describes how loss of Shh expression in Prx1-/-Prx2-/- mutants leads to abnormal morphogenesis of the mandibular arch. According to this model, Prx1 and Prx2 expression in mandibular mesenchyme is required to stimulate the expression of Shh in the medial domain of the oral epithelium via an as yet unknown intermediate. The Shh protein then promotes cell proliferation in part of the underlying mesenchyme, which is required for correct morphogenesis. In the Prx1-/-Prx2-/- mutant, reduction of Shh expression leads to reduction of mesenchymal proliferation in the medial-oral region. This lack of proliferation causes a malformation of the mandibular processes such that the oral region develops in a more medial position, and the aboral region in a more lateral position. Consequently, the oral expression domains of Dlx2, Alx3 and Fgf8 shift medially, while the aboral domains of Dlx2 and Alx3 shift laterally. Fgf8 expression in the medial region subsequently induces the formation of the medial incisor (ten Berge, 2001).

A group of mouse aristaless-related genes has been implicated in functions in the development of the craniofacial skeleton. An Alx3 mutant allele has been generated in which the lacZ coding sequence is inserted in-frame in the Alx3 gene and the sequences encoding the conserved protein domains are deleted. Mice homozygous for this null allele are indistinguishable from wild-type mice. Compound mutants of Alx3 and Alx4, however, show severe craniofacial abnormalities which are absent in Alx4 single mutants. Alx3/Alx4 double mutant newborn mice have cleft nasal regions. Most facial bones and many other neural crest derived skull elements are malformed, truncated or even absent. The craniofacial defects in Alx3/Alx4 double mutant embryos become anatomically manifest around embryonic day 10.5, when the nasal processes appear to be abnormally positioned. This most probably leads to a failure of the medial nasal processes to fuse in the facial midline and subsequently to the split face phenotype. A significant increase in apoptosis localised in the outgrowing frontonasal process was detected in embryonic day 10.0 double mutant embryos; this apoptosis is proposed to be the underlying cause of the subsequent malformations (Beverdam, 2001).

Calvarial bones (skull vault) form by direct ossification of mesenchyme. This requires condensation of mesenchymal cells which then proliferate and differentiate into osteoblasts. Congenital hydrocephalus (ch) mutant mice lack the forkhead/winged helix transcription factor Foxc1. In ch mutant mice, calvarial bones remain rudimentary at the sites of initial osteogenic condensations. In this study, the ossification defect in ch mutants has been localized to the calvarial mesenchyme, which lacks the expression of transcription factors Msx2 and Alx4. This lack of expression is associated with a reduction in the proliferation of osteoprogenitor cells. BMP induces Msx2 in calvarial mesenchyme. BMP also induces Alx4 in this tissue. BMP-induced expression of Msx2 and Alx4 requires Foxc1. It is therefore suggested that Foxc1 regulates BMP-mediated osteoprogenitor proliferation and that this regulation is required for the progression of osteogenesis beyond the initial condensations in calvarial bone development (Rice, 2003).

Dynamic control of head mesoderm patterning

The embryonic head mesoderm gives rise to cranial muscle and contributes to the skull and heart. Prior to differentiation, the tissue is regionalised by the means of molecular markers. This pattern is shown to be established in three discrete phases, all depending on extrinsic cues. Assaying for direct and first-wave indirect responses, it was found that the process, analyzed in the chicken, is controlled by dynamic combinatorial as well as antagonistic action of retinoic acid (RA), Bmp and Fgf signalling. In phase 1, the initial anteroposterior (a-p) subdivision of the head mesoderm is laid down in response to falling RA levels and activation of Fgf signalling. In phase 2, Bmp and Fgf signalling reinforce the a-p boundary and refine anterior marker gene expression. In phase 3, spreading Fgf signalling drives the a-p expansion of bHLH transcription factor MyoR (musculin) and Tbx1 expression along the pharynx, with RA limiting the expansion of MyoR. This establishes the mature head mesoderm pattern with markers distinguishing between the prospective extra-ocular and jaw skeletal muscles, the branchiomeric muscles and the cells for the outflow tract of the heart (Bothe, 2011).

Expression of Fgf and Bmp responsive molecules indicated that the anterior head mesoderm receives Fgf and Bmp signals for the first time during phase 2 when Alx4 and MyoR are upregulated. Suppression of Bmp signalling prevented, and elevated Bmp signalling advanced, Alx4 activation. Thus, Bmp is necessary and sufficient to control Alx4. MyoR, however, was repressed by suppression of either Bmp or Fgf signalling. Elevation of Bmp or Fgf signalling promoted MyoR, albeit only at the stage at which the gene is normally expressed; premature MyoR expression could only be achieved by combinatorial application of Bmp and Fgf. Thus, combined Fgf and Bmp activity is required to activate MyoR (Bothe, 2011).

Expression analysis showed that the onset of MyoR is rather sudden. The bead implantation experiments indicated that in the anterior head mesoderm, Fgf enhanced the expression of Bmp responsive genes and Bmp upregulated genes indicative of active Fgf signalling. This suggests that Bmp and Fgf reinforce each other, possibly creating the appropriate setting to activate MyoR. Studies on mouse mutants placed Pitx2 upstream of MyoR. Thus, it is conceivable that, in addition to Bmp and Fgf, the earlier activation of Pitx2 is a further prerequisite for the activation of MyoR (Bothe, 2011).

In the posterior head mesoderm, Bmp strongly suppressed Tbx1. Fgf signalling, however, was unaffected, suggesting that Bmp controls the anterior border of Tbx1 expression, possibly directly targeting Tbx1. Tbx1, by contrast, has recently been suggested to suppress Bmp signalling by preventing Smad1-Smad4 interaction. This suggests that Tbx1 indirectly controls the extension of Bmp dependent markers (Bothe, 2011).

When Bmp and Fgf signalling commences in the anterior head mesoderm, Fgf signalling levels increase significantly in the posterior domain, owing to the positive Fgf-Tbx1 feedback loop. After applying Fgf to the anterior head mesoderm, i.e. elevating the Fgf level beyond that which is normally found there, it was noticed that Pitx2 and Alx4 expression declined. Thus, although Fgf is necessary for the activation of MyoR, high Fgf levels prevent the molecular set-up of the anterior head mesoderm. This infers that, whereas Bmp controls the anterior border of the posterior head mesoderm marker, Fgf controls the posterior border of the two anterior markers Pitx2 and Alx4 (Bothe, 2011).

In phase 3, extension of MyoR and Tbx1 expression is concomitant with the spread of high-level Fgf signalling along the floor of the pharynx. Fgf application was found to accelerate the MyoR-Tbx1 spread, and suppression of Fgf signalling prevented it. This suggests that Fgf signalling is key to establishing the final head mesoderm pattern. Notably, MyoR remained sensitive to RA. In the embryo, however, the site of RA production continuously recedes posteriorly during phases 2 and 3, suggesting that the posterior extension of MyoR expression occurs at a rate set by RA (Bothe, 2011).

The anteriorly spreading Fgf signals will eventually reach the Pitx2-Alx4 domain. Both genes were negatively regulated by high Fgf levels in phases 1 and 2; yet, in phase 3 the genes remain expressed. Likewise, Tbx1 spreads anteriorly although this territory is controlled by Bmp. Notably, Fgf levels vary along the anteroposterior extent of the pharynx; at HH13, for example, Fgf signalling appears lower in the anterior compared with the posterior pharyngeal arches. Thus, it is possible that in the anterior head mesoderm, Fgf levels might remain low enough to allow Pitx2 and Alx4 expression, but rise sufficiently to override the Bmp effect on Tbx1. Conversely, the Fgf levels in the posterior head mesoderm might by so high that MyoR expression can spread, whereas Pitx2 and Alx4 remain repressed. It cannot be excluded that additional signals restrict Pitx2 and Alx4 expression. Yet, the spread of MyoR outside of the Pitx2 territory indicates that in phase 3 MyoR expression has become independent from its former upstream regulator (Bothe, 2011).

RA, Bmp and Fgf signalling play multiple roles during development. RA, in many settings, promotes cell differentiation; in the head, RA first suppresses cardiac markers to set the posterior limit of the heart field, but then specifies the sinoatrial region of the heart. Moreover, RA has the capacity to provide cells with a more posterior positional identity. Bmp is a crucial regulator of cardiac development and has been suggested to recruit head mesodermal cells into the cardiac lineage. Fgf promotes the secondary heart field and keeps cells proliferative and undifferentiated. Therefore whether the observed changes in head mesodermal marker expression occurred because of cell recruitment into cardiac lineage, premature differentiation or posteriorisation was tested. RA or Fgf treatment was found not to change cell fate or differentiation status. Bmp induced cardiac marker gene expression only when applied during phase 0. When applied in phase 1, i.e. just before Bmp signalling is normally activated in the head mesoderm, Bmp did not induce cardiac markers unless the dosage was increased. This suggests that, possibly, cardiac induction can occur from exposure to higher Bmp levels and/or longer exposure times. Taken together, this study suggests that RA, Bmp and Fgf specifically control head mesoderm patterning with the cells remaining undifferentiated and competent to enter any of the possible mesodermal lineages (Bothe, 2011).

Aristaless homologs and neural patterning

Cutaneous sensory neurons that detect noxious stimuli project to the dorsal horn of the spinal cord, while those innervating muscle stretch receptors project to the ventral horn. DRG11, a paired homeodomain transcription factor related to Drosophila Aristaless, is expressed in both the developing dorsal horn and in sensory neurons, but not in the ventral spinal cord. Mouse embryos deficient in DRG11 display abnormalities in the spatio-temporal patterning of cutaneous sensory afferent fiber projections to the dorsal, but not the ventral spinal cord, as well as defects in dorsal horn morphogenesis. These early developmental abnormalities lead, in adults, to significantly attenuated sensitivity to noxious stimuli. In contrast, locomotion and sensori-motor functions appear normal. Drg11 is thus required for the formation of spatio-temporally appropriate projections from nociceptive sensory neurons to their central targets in the dorsal horn of the spinal cord (Chen, 2001).

The formation of the neural circuits that mediate pain sensation is an important subject in neural development, yet remarkably little is known about the molecular mechanisms that control this process in vivo. The only published mutations that affect the development of nociceptors are those in the genes encoding NGF, its receptor trkA, the bHLH transcription factor NGN1, and the POU-domain transcription factor Brn3.0. None of these mutations, however, affects the initial establishment of connections between the DRG and the dorsal horn. NGF and trkA are required for the survival of sensory neurons long after they have differentiated and extended axons to their targets, while Brn3.0 appears to control the expression of neurotrophin receptors. NGN1, by contrast, controls the initial determination of trkA+ sensory neuron precursors. Thus, the early projection defect seen in Drg11-/- embryos is distinct from other mutations affecting the development of nociceptive circuits, and may provide a useful point of entry for studies of the cell-intrinsic control of this process (Chen, 2001).

The earliest detectable cellular defect in Drg11-/- mice is an abnormal projection of primary sensory afferent fibers to the dorsal horn, at E13.5. Because Drg11 is expressed in the sensory ganglia and spinal cord at this stage, it is not clear whether this initial projection defect reflects an intrinsic function for the gene in the DRG, the dorsal horn, or both. However, the severity of the projection defect, as detected by calbindin-28K staining, appears similar at cervical and thoraco-lumbar levels, while the subsequent defects in dorsal horn development are more prominent caudally. These observations suggest that the morphological abnormalities in the dorsal horn may develop independently of the projection defect. Consistent with this, similar morphological abnormalities, including a reduction in small, darkly staining neurons and a shortening of the dorsal funiculus, are seen in mutants lacking Lmx1b, a LIM homeodomain transcription factor expressed in the dorsal horn but not in sensory neurons. In Lmx1b-/- embryos, expression of Drg11 is lost in the spinal cord but not in the DRG. This observation supports the idea that the dorsal horn defects in Drg11-/- mice may reflect an intrinsic function for the gene in the spinal cord. Whether the projection defect reflects, conversely, an intrinsic role for DRG11 in sensory neurons or, rather, a requirement in the dorsal horn that is independent of axial position will require site-specific knockouts of Drg11 (Chen, 2001).

The absence of afferent fiber ingrowth to the dorsal horn in E13.5 Drg11-/- embryos reflects a delay and not a total arrest: by E16.5 calbindin+ and trkA+ fibers have penetrated the spinal gray matter. However the increased density of these fibers medially, and increased frequency of midline crossing, suggests that the abnormal trajectory reflects more than a simple deletion of afferent projections to the lateral-most dorsal horn. Rather, both the timing and the spatial distribution of cutaneous afferent projections into the spinal gray matter are abnormal in Drg11-/- embryos (Chen, 2001).

The apparent lateral-to-medial shift in the distribution of cutaneous afferents in Drg11-/- mice may reflect alterations in the somatotopic organization of these projections. Cutaneous afferents with distal (or ventral) peripheral targets project to more medial regions of the dorsal horn, while those with more proximal (or dorsal) peripheral targets project laterally. This medio-lateral somatotopy is already evident from the earliest stages of afferent fiber penetration to the dorsal horn. The fact that Drg11 is expressed more abundantly in the lateral than in the medial dorsal horn, taken together with the apparent medial bias of afferent fibers in the mutant, suggests that the gene may be involved in some aspect of medio-lateral patterning that underlies such somatotopy. However, it is important to note that the loss of PKCgamma+ neurons in Drg11-/- mice is observed throughout the medio-lateral extent of lamina IIi. This may explain why deficiencies in pain sensitivity are detected distally as well as proximally in adult Drg11-/- mice (Chen, 2001).

Behavioral tests in adult Drg11-/- animals reveal a significantly reduced sensitivity to noxious stimuli across a broad range of modalities, including mechano-, thermo-, and chemo-sensitivities. By contrast, locomotion and sensorimotor function appeared normal. Consistent with this behavioral data, a dramatic cell loss is observed in the lateral regions of the dorsal horn, as well as a reduction in afferent innervation in laminae I and II. This innervation primarily represents C- and Adelta fibers. Given this neuronal and afferent fiber loss, it is somewhat surprising that the reduction of pain sensitivity in adult Drg11-/- mice is not more complete. However, because the remaining afferent fibers mediate apparently normal synaptic transmission with their surviving second-order targets, the incomplete loss of sensitivity to noxious stimuli may simply reflect a reduced volume of synaptic information transmitted in the dorsal horn of Drg11-/- mice (Chen, 2001).

What is the connection between the early developmental defects observed in Drg11-/- embryos and the anatomical deficiencies seen in adults? The cell and afferent fiber loss in the lateral dorsal horn of adult Drg11-/- mice is consistent with the pattern of defects seen in embryos. The more general loss of PKCgamma+ neurons may, however, reflect an independent, later action of DRG11 to control the differentiation or survival of these cells. In contrast to these embryonic and perinatal defects, the loss of sensory neurons is only observed in adult DRG. This suggests either that this sensory neuron deficit is secondary to the earlier defects or that Drg11 has a later, independent function in these peripheral neurons (Chen, 2001).

Vertebrate epibranchial placodes give rise to visceral sensory neurons that transmit vital information such as heart rate, blood pressure and visceral distension. Despite the pivotal roles they play, the molecular program underlying their development is not well understood. The zebrafish mutation no soul, in which epibranchial placodes are defective, disrupts the forkhead-related, winged helix domain-containing protein Foxi1. Foxi1 is expressed in lateral placodal progenitor cells. In the absence of foxi1 activity, progenitor cells fail to express the basic helix-loop-helix gene neurogenin that is essential for the formation of neuronal precursors, and the paired homeodomain containing gene phox2a that is essential for neuronal differentiation and maintenance. Consequently, increased cell death is detected, indicating that the placodal progenitor cells take on an apoptotic pathway. Furthermore, ectopic expression of foxi1 is sufficient to induce phox2a-positive and neurogenin-positive cells. Taken together, these findings suggest that Foxi1 is an important determination factor for epibranchial placodal progenitor cells to acquire both neuronal fate and subtype visceral sensory identity (Lee, 2003).

Visceral sensory neurons include the geniculate, petrosal and nodose ganglia, which have distinct but also overlapping connectivity patterns. Fate mapping experiments show that they all derive from the epibranchial placodes. However, in the no soul mutant, a differential effect on these neurons is seen: whereas the geniculate and petrosal neurons fail to develop, the nodose ganglia are partially spared. Interestingly, nodose ganglia are also less affected in mice with targeted disruption of ngn 2 and phox2a. These analyses suggest that although the three distal ganglia share a developmental origin, different mechanisms may operate in their determination. Interestingly, it was observed that unlike geniculate and petrosal ganglia, which express phox2a prior to phox2b, nodose ganglia initiate phox2b expression prior to that of phox2a. Therefore, it is possible that the commitment and differentiation of at least subsets of nodose ganglion are under the control of yet unidentified regulatory hierarchies. Alternatively, other neural progenitor populations are able to compensate for the loss of epibranchial placode-derived nodose progenitor cells (Lee, 2003 and references therein).

Phox2b regulates neuronal cell cycle exit and identitiy

In the vertebrate neural tube, cell cycle exit of neuronal progenitors is accompanied by the expression of transcription factors that define their generic and subtype specific properties, but how the regulation of cell cycle withdrawal intersects with that of cell fate determination is poorly understood. Here it is shown by both loss- and gain-of-function experiments that the neuronal-subtype-specific homeodomain transcription factor Phox2b drives progenitor cells to become post-mitotic. In the absence of Phox2b, post-mitotic neuronal precursors are not generated in proper numbers. Conversely, forced expression of Phox2b in the embryonic chick spinal cord drives ventricular zone progenitors to become post-mitotic neurons and to relocate to the mantle layer. In the neurons thus generated, ectopic expression of Phox2b is sufficient to initiate a program of motor neuronal differentiation characterized by expression of Islet1 and of the cholinergic transmitter phenotype, in line with previous results showing that Phox2b is an essential determinant of cranial motor neurons. These results suggest that Phox2b coordinates quantitative and qualitative aspects of neurogenesis, thus ensuring that neurons of the correct phenotype are generated in proper numbers at the appropriate times and locations (Dubreuil, 2000).

The period of acquisition of neuronal identity in the neural precursors starts before and extends beyond completion of the final mitosis. This has been studied most intensively during motor neuron development. Three general classes of motor neurons can be distinguished: the somatic motor (sm) neurons that innervate most skeletal muscles in the body, the branchiomotor (bm) neurons that innervate the branchial arch-derived muscles of the face and jaw, and visceral motor (vm) neurons that innervate sympathetic and parasympathetic ganglia. The dividing progenitors of most sm and of the spinal vm neurons, i.e. of the motor neurons that send axons ventrally from the neural tube, express the LIM homeobox genes Lhx3 (Lim3) and Lhx4. In embryos lacking both genes, post-mitotic spinal motor neurons are still generated in proper numbers, but acquire identities reminiscent of cranial bm or vm neurons. Conversely, ectopic expression of Lhx3 in the hindbrain leads to the generation of ventrally projecting neurons at the expense of a cranial vm phenotype, but does not seem to influence the number of neurons generated. Likewise, the homeobox gene MNR2 (potential Drosophila homolog: CG8254) is switched on by chick motor neuron progenitors just before their final division. When misexpressed in the spinal cord region of the chick neural tube, ectopic MNR2 is able to induce an sm differentiation program, characterized by expression of the Islet1 and HB9 transcriptional regulators, which are required for post-mitotic sm differentiation. However, timing and extent of neurogenesis appear to be controlled by an MNR2-independent process. In the mouse, an MNR2 homolog has not been found, and the closely related HB9 gene may have subsumed MNR2 function in this species. In mice lacking HB9, motor neurons transiently express interneuron markers and acquire inappropriate subtype identities, but they are still generated in correct numbers. Upstream of these transcription factor genes, another set of homeodomain proteins subdivides the VZ into different progenitor domains and is responsible for conferring distinct progenitor identities. Nkx6.1 in particular is expressed in the motor neuron progenitors and induces a sm phenotype when misexpressed in the dorsal spinal cord. However, no effects of Nkx6.1 on neurogenesis per se have been reported. Thus, although exit from the cell cycle and acquisition of subtype-specific (as well as generic) neuronal identity appear tightly coordinated in time, how this is achieved has not been elucidated (Dubreuil, 2000 and references therein).

In the embryonic mouse and chick hindbrain, the closely related paired-type homeobox genes Phox2a and Phox2b are expressed by all bm and vm, but not by sm neurons. Phox2b, but not Phox2a, is already expressed in the dividing progenitors and expression of both genes persists in the differentiating mantle layer neurons. The analysis of the Phox2b knockout phenotype has uncovered two successive functions of this transcription factor in hindbrain motor neurons. In the progenitors, Phox2b activity is required for high level expression of the bHLH family member Mash1. In the post-mitotic precursors, it is necessary for all further differentiation, both generic and type-specific. In the absence of Phox2b, post-mitotic motor neurons are not generated in proper numbers in the VZ. Conversely, forced expression of Phox2b in the presumptive spinal cord of chick embryos, where it is normally not expressed, promotes expression of early post-mitotic markers and migration into the mantle layer. In addition, Phox2b induces ectopic expression of motor neuron markers in the dorsal spinal cord. Together, these results suggest that Phox2b coordinately regulates the decisions to exit the cell cycle and to become a particular type of neuron (Dubreuil, 2000).

Neurogenesis in the vertebrate neural tube is marked by a rapid succession of events in precursor cells. Before the first post-mitotic neurons are generated, different homeodomain transcription factors already subdivide the VZ into distinct progenitor domains. Once neurogenesis is under way, the progenitors committed to become a neuron complete their last mitosis close to the lumen in most regions of the neural tube. They then migrate away from the VZ while transiently activating genes such as Math3/NeuroM or the Ebf family members (homologs of Drosophila Knot/Collier), which mark the earliest steps of generic neuronal differentiation. This is accompanied or soon followed by expression of downstream transcription factor genes that specify neuronal sub-type identity (Dubreuil, 2000).

In mice lacking Phox2b, there is a massive reduction in post-mitotic mantle layer cells in the region where the bm neurons of the facial nerve normally develop. Depletion of the mantle layer is not due to an early death of mantle layer cells, as observed for example in Islet1 knockout embryos. Expression of Delta1/Dll1, the earliest known marker expressed by VZ cells after completion of their last S phase, is defective in the VZ of mutant embryos. Since this defect is not due to a Phox2b requirement for the survival of Dll1 + cells, this suggests that Phox2b positively regulates cell cycle exit of the progenitors in which it is expressed. Gain-of-function experiments in the chick neural tube fully confirm the role of Phox2b as a positive regulator of cell cycle exit. Forced expression of Phox2b reduces the numbers of BrdU-incorporating and PCNA-expressing cycling cells and upregulates Delta1/Dll1 and NeuroM/Math3, which are transiently expressed by early post-mitotic precursors on their way out of the VZ. Hence Phox2b clearly acts on progenitor cells in the VZ. Lack of Phox2b has the opposite effect: the number of Delta1/Dll1 + cells in the VZ is reduced by half and NeuroM/Math3 expression is extinguished (Dubreuil, 2000).

Two days after transfection, most cells ectopically expressing Phox2b have relocated to the mantle layer and express the generic neuronal marker class III beta-tubulin, an effect not seen in control transfections. Hence, Phox2b expression is sufficient to drive cells to become post-mitotic, to start a neuronal differentiation program and to migrate to the mantle layer. In the absence of Phox2b, neuroepithelial cells are still able to become post-mitotic, albeit at reduced rates. This argues that rather than being absolutely required for cell cycle withdrawal, Phox2b positively regulates the rate at which it occurs. At what step in the pathway does Phox2b act? The bHLH transcription factors with proneural activity are another class of transcriptional regulators that appear to control timing and extent of neuronal differentiation. In Phox2b knockout embryos, Mash1, a homolog of Drosophila proneural genes, is expressed at strikingly reduced levels in the bm progenitors and is downregulated prematurely in the sympathetic ganglion primordia. Thus, one possibility is that Phox2b acts by maintaining or boosting expression of proneural genes, a possibility corroborated by the finding that Phox2b upregulates expression of Delta1/Dll1, which is thought to be a target of transcription factors with proneural activity. Alternatively, Phox2b may function downstream of, or in parallel with, proneural gene products, by promoting expression of bHLH transcription factors that lie further downstream in the regulatory cascade or by cooperating with bHLH factors in the control of cell cycle regulators. Finally, Phox2b acts in the context of the Delta-Notch lateral inhibition system. As judged by the reduction in Hes5 expression, Notch signaling is activated to a lesser degree in the Phox2b mutant territory, probably as a consequence of reduced expression of the Notch ligand Dll1. This should initiate a feedback loop, counteracting the negative effect of Phox2b deficiency on Dll1 expression. The gain-of-function experiments should initiate a feedback loop that opposes the effect of Phox2b misexpression. It is thus easy to understand that in Phox2b mutants not all cells remain cycling progenitors, and that not all cells misexpressing Phox2b can be driven to express Delta1 or NeuroM at any one time (Dubreuil, 2000).

Analysis of the loss-of-function phenotype has shown that Phox2b is absolutely required for the generation of the bm and vm neurons in which it is expressed. Phox2b is also sufficient to induce characteristics of a motor neuronal phenotype in the lateral spinal cord, where motor neurons normally never develop. The ectopic Phox2b-expressing cells switch on the motor neuronal markers Islet1/2 and ChAT, but not Islet2, which is specific for sm neurons. Forced expression of Phox2b also transiently activates Phox2a (whose expression in motor neurons is confined to bm and progenitor cells). Phox2b appears sufficient to activate a differentiation program resembling that of bm or hindbrain vm neurons, but in the absence of specific markers these two fates cannot be distinguished (Dubreuil, 2000).

Motor neurons are normally generated only in the ventral neural tube in response to Sonic hedgehog signaling from the notochord and floor plate. Generation of alar plate neurons depends on BMPs and related molecules that are expressed in the dorsalmost regions of the neural tube, and exposure of progenitors to BMPs inhibits motor neuron generation. Like MNR2 and Hb9, Phox2b is able to override such negative control of motor neuronal differentiation in the dorsal spinal cord. In the hindbrain, however, the dorsal Phox2b-expressing precursors do not give rise to motor neurons, but to sensory relay neurons, to neurons of the reticular formation and to noradrenergic neurons. Other studies have shown that forced expression of Phox2b or Phox2a in the chick neural crest pathway or of Phox2a in the zebrafish embryo generates ectopic noradrenergic neurons. One possibility thus is that Phox2b must cooperate with other factors to specify dorsal sub-type identities in the hindbrain, and that these factors are lacking in the spinal cord (Dubreuil, 2000).

Taken together, these studies show that a single transcription factor is able both to increase cell cycle exit of neuronal progenitors and to initiate at least some aspects of sub-type- specific differentiation of the neurons thus generated. Transcriptional regulators, which assume this role in neuronal types other than bm and vm neurons, remain to be identified (Dubreuil, 2000).

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