A Xenopus gene, Xbap, is closely related to Drosophila bagpipe. Xbap, like bagpipe is expressed in the developing musculature of the midgut, suggesting that this developmental role of bagpipe is evolutionarily conserved. However, a second, novel role in development is suggested by the observation that Xbap is expressed in the region of the developing facial cartilage, a neural crest derivative. Xbap expression marks the precursors to the basihyobranchial, palatoquadrate, and possible Meckel's cartilages. In adults, Xbap is expressed in kidney, pancreas, spleen and stomach, with slightly lower levels in the intestine, skeletal muscle and tongue. Heart, liver, and lung contain little or no Xbap mRNA. This vertebrate bagpipe sequence is expressed in both mesodermally and neural crest-derived tissues (Newman, 1997).

In Drosophila, dorsal mesodermal specification is regulated by the homeobox genes tinman and bagpipe. Vertebrate homologs of tinman and bagpipe have been isolated in various species. Moreover, in vertebrates, there are at least four different genes related to tinman, which indicates that this gene has been duplicated during evolution. One of the murine homologs of tinman is the cardiac homeobox gene Csx or Nkx2.5. Gene targeting of Csx/Nkx2.5 shows that this gene is required for completion of the looping morphogenesis of the heart. However, it is not essential for the specification of the heart cell lineage. Early cardiac development might therefore be regulated by other genes, which may act either independently or in concert with Csx/Nkx2.5. Possible candidates might be other members of the NK2 class of homeobox proteins, like Tix/Nkx2.6, Nkx2.3, nkx2.7, or cNkx2.8. This study reports that murine Tix/Nkx2.6 mRNA has been detected in the heart and pharyngeal endoderm. Xenopus XNkx2.3 and chicken cNkx2.3 are expressed in the heart as well as in pharyngeal and gut endoderm. In contrast, murine Nkx2.3 is expressed in the gut and pharyngeal arches but not the heart. In zebrafish and chicken, two new NK-2 class homeoproteins, nkx2.7 and cNkx2.8, have been identified. Zebrafish nkx2.7 is expressed in both the heart and pharyngeal endoderm. In chicken, cNkx2.8 is expressed in the heart primordia and the primitive heart tube and becomes undetectable after looping. No murine homologs of nkx2.7 or cNkx2.8 have been found so far. The overlapping expression pattern of NK2 class homeobox genes in the heart and the pharynx may suggest a common origin for these two organs. In the Drosophila genome, the tinman gene is linked to bagpipe, another NK family gene. A murine homolog of bagpipe, Bax/Nkx3.1, is expressed in somites, blood vessels, and the male reproductive system during embryogenesis (this study), suggesting that this gene's function may be relevant for the development of these organs. A bagpipe homolog in Xenopus, Xbap, is expressed in the gut musculature and a region of the facial cartilage during development (Tanaka, 1998).

Mouse and human homologs of the bagpipe gene have been isolated and designated as Bapx1 and BAPX1, respectively. Bapx1 encodes a predicted protein of 333 amino acids, and has significant regions of homology outside the homeodomain with members of the NK homeobox gene superfamily. Protein sequence alignment identifies a conserved region (LTPFSIQAILN) near the N-terminal region of the predicted protein sequence, which has been referred to as the TN-domain. There is also another conserved domain (NK2-SD) located C-terminal to the homeobox. Bapx1 maps to the proximal end of chromosome 5 in mouse, near the Msx1 gene. The syntenic region in human corresponds to a chromosomal region containing loci for several skeletal disorders. Bapx1 is first detectable in embryos just prior to axis rotation in lateral plate mesoderm (splanchnic mesoderm) adjacent to the endodermal lining of the prospective gut, and in the most newly formed somites in the region corresponding to the presclerotome, the precursor of the vertebrae. Thus, Bapx1 is one of the earliest developmental markers for the sclerotome portion of the somite and the gut mesentery. Bapx1 continues to be expressed well into organogenesis in lateral plate mesoderm surrounding the midgut and hindgut, and in essentially all cartilaginous condensations that will subsequently undergo endochondral bone formation. The expression pattern of Bapx1 in murine embryos suggests that there are evolutionary conserved mechanisms of visceral mesoderm development across the animal kingdom, and that the mammalian Bapx1 gene may have recently acquired an additional developmental role in skeletal patterning (Tribioli, 1997).

The axial structures, consisting of the notochord and the neural tube, play an essential role in the dorsoventral patterning of somites and in the differentiation of their many cell lineages. The role of the axial structures in the mediolateral patterning of the somite has been investigated by using a newly identified murine homeobox gene, Nkx-3.1 (homologous to Drosophila bagpipe), as a medial somitic marker in explant in vitro assays. Nkx-3.1 is dynamically expressed during somitogenesis only in the youngest, most newly-formed somites at the caudal end of the embryo. The expression of Nkx-3.1 in pre-somitic tissue explants is induced by the notochord and maintained in newly-differentiated somites by the notochord and both ventral and dorsal parts of the neural tube. By exposing explants to either COS cells transfected with a Shh expression construct or to recombinant Shh, it is shown that Sonic hedgehog (Shh) is one of the signaling molecules that can reproduce the effect of the axial structures. Shh can induce and maintain Nkx-3.1 expression in pre-somitic mesoderm and young somites, but not in more mature, differentiated ones. The effects of Shh on Nkx-3.1 expression are antagonized by a forskolin-induced increase in the activity of cyclic AMP-dependent protein kinase A. The expression of the earliest expressed murine myogenic marker, myf 5, is also regulated by the axial structures, but Shh, by itself, is not capable of inducing or maintaining it. It is suggested that the establishment of somitic medial and lateral compartments and the early events in myogenesis are governed by a combination of positive and inhibitory signals derived from the neighboring structures, as has previously been proposed for the dorsoventral patterning of somites (Kos, 1998).

The mouse Bapx1 gene is homologous to the Drosophila homeobox containing bagpipe (bap) gene. A shared characteristic of the genes in these two organisms is expression in gut mesoderm. In Drosophila, bap functions to specify the formation of the musculature of the midgut. To determine the function of the mammalian cognate, a mutation was targeted into the Bapx1 locus. Bapx1, similar to Drosophila, does have a conspicuous role in gut mesoderm; however, this appears to be restricted to development of the spleen. In addition, Bapx1 has a major role in the development of the axial skeleton. Loss of Bapx1 affects the distribution of sclerotomal cells, markedly reducing the number that appear ventromedially around the notochord. Subsequently, the structures in the midaxial region, the intervertebral discs, and centra of the vertebral bodies, fail to form. Abnormalities are also found in those bones of the basal skull (basioccipital and basisphenoid bones) associated with the notochord. It is clear that the structure of the notochord is also abnormal in Bapx-/- animals. At E14.5, the periodic notochordal pattern is disrupted, and by E18.5 the enlarged nucleus pulposus of each intervertebral disc is missing. Shh expression was examined in the notochord at E11.5 (a stage relevant to Shh signaling) and no differences in expression were observed. Because Bapx1 is not detectably expressed in the notochord, it is suggest that the disruption of the notochord is caused by lack of surrounding Bapx1 expressing sclerotomal cells. It has been suggested that these sclerotomal-derived cells are necessary for the modeling of the notochordal pattern, perhaps by signaling back to the notochord. It is postulated that Bapx1 confers the capacity of cells to interact with the notochord, effecting inductive interactions essential for development of the vertebral column and chondrocranium (Lettice, 1999).

The Drosophila bap gene affects specification of the visceral mesoderm resulting in reduction or loss of visceral musculature. Bapx1 is expressed broadly in the splanchnic mesoderm, which in addition to spleen development contributes to the gut musculature. However, Bapx1 does not have an overt effect on formation of gut musculature, suggesting there is no equivalent function for the mouse and Drosophila genes. Bapx1 and bap, therefore, affect cognate tissue types, i.e., gut mesoderm, but with differing consequences. In addition to a restricted role in splanchnic mesoderm, Bapx1 is also expressed in the limb and Meckel's cartilage, where there is no apparent phenotype. Thus the possibility exists that other bagpipe-related genes are present that compensate for the loss of Bapx1 (Lettice, 1999).

In aging men, the prostate gland becomes hyperproliferative and displays a propensity toward carcinoma. Although this hyperproliferative process has been proposed to represent an inappropriate reactivation of an embryonic differentiation program, the regulatory genes responsible for normal prostate development and function are largely undefined. The murine Nkx3.1 homeobox gene is the earliest known marker of prostate epithelium during embryogenesis and is subsequently expressed at all stages of prostate differentiation in vivo as well as in tissue recombinants. A null mutation for Nkx3.1 obtained by targeted gene disruption results in defects in prostate ductal morphogenesis and secretory protein production. Notably, Nkx3.1 mutant mice display prostatic epithelial hyperplasia and dysplasia that increases in severity with age. This epithelial hyperplasia and dysplasia also occurs in heterozygous mice, indicating haploinsufficiency for this phenotype. Because human NKX3.1 is known to map to a prostate cancer hot spot, it is proposed that NKX3.1 is a prostate-specific tumor suppressor gene and that loss of a single allele may predispose to prostate carcinogenesis. The Nkx3.1 mutant mice provide a unique animal model for examining the relationship between normal prostate differentiation and early stages of prostate carcinogenesis (Bhatia-Gaur, 1999).

The Bapx1 homeobox gene, a member of the NK gene family, is one of the earliest markers for prechondrogenic cells that will subsequently undergo mesenchymal condensation, cartilage production and, finally, endochondral bone formation. In addition, Bapx1 is an early developmental marker for splanchnic mesoderm, consistent with a role in visceral mesoderm specification, a function performed by its Drosophila homolog bagpipe. The human homolog of Bapx1 has been identified and mapped to 4p16.1, a region containing loci for several skeletal diseases. Bapx1 null mice are affected by a perinatal lethal skeletal dysplasia and asplenia, with severe malformation or absence of specific bones of the vertebral column and cranial bones of mesodermal origin, with the most severely affected skeletal elements corresponding to ventral structures associated with the notochord. Evidence is provided that the failure of the formation of skeletal elements in Bapx1 null embryos is a consequence of a failure of cartilage development, as demonstrated by downregulation of several molecular markers required for normal chondroblast differentiation [alpha 1(II) collagen, Fgfr3, Osf2, Indian hedgehog, Sox9], as well as a chondrocyte-specific alpha 1 (II) collagen-lacZ transgene. The cartilage defects are correlated with failed differentiation of the sclerotome at the time when these cells are normally initiating chondrogenesis. Loss of Bapx1 is accompanied by an increase in apoptotic cell death in affected tissues, although cell cycling rates are unaltered (Tribioli, 1999).

Shh acts early in the development of the axial skeleton, to induce a prochondrogenic response to later BMP signaling. Somitic expression of the transcription factor Nkx3.2, homolog of Drosophila bagpipe, is initiated by Shh and sustained by BMP signals. Misexpression of Nkx3.2 in somitic tissue confers a prochondrogenic response to BMP signals. The transcriptional repressor activity of Nkx3.2 is essential for this factor to promote chondrogenesis. Conversely, a 'reverse function' mutant of Nkx3.2 that has been converted into a transcriptional activator inhibits axial chondrogenesis in vivo. It is concluded that Nkx3.2 is a critical mediator of the actions of Shh during axial cartilage formation, acting to inhibit expression of factors that interfere with the prochondrogenic effects of BMPs (Murtaugh, 2001).

Transient Shh signals from the notochord and floor plate confer a competence in somitic tissue for subsequent BMP signals to induce chondrogenesis. It has therefore been proposed that Shh induces a factor(s) that renders somitic cells competent to chondrify in response to subsequent BMP signals. Forced expression of Nkx3.2 (Drosophila homolog: Bagpipe), a transcriptional repressor induced by Shh, is able to confer chondrogenic competence in somites. Administration of Shh or forced Nkx3.2 expression induces the expression of the transcription factor Sox9 in the somitic tissue. Forced expression of Sox9 can, in turn, induce robust chondrogenesis in somitic mesoderm, provided that BMP signals are present. In the presence of BMP signals, Sox9 and Nkx3.2 induce each other's expression. Thus, Nkx3.2 may promote axial chondrogenesis by derepressing the expression of Sox9 in somitic mesoderm. Furthermore, forced expression of either Sox9 or Nkx3.2 not only activates expression of cartilage-specific genes in somitic mesoderm, but also promotes the proliferation and survival of the induced chondrocytes in the presence of BMP signals. However, unlike Nkx3.2, Sox9 is able to induce de novo cartilage formation in non-cartilage-forming tissues. These findings suggest that Shh and BMP signals work in sequence to establish a positive regulatory loop between Sox9 and Nkx3.2, and that Sox9 can subsequently initiate the chondrocyte differentiation program in a variety of cellular environments (Zeng, 2002).

The homeobox containing transcription factors Nkx3.1 and Nkx3.2 (Bapx1) are transiently coexpressed in somites during early embryonic mouse development. Targeted disruption of the Nkx3.2 (Bapx1) gene in mice results in limited defects of chondrocranial bones and the axial skeleton, particularly pronounced in cervical vertebrae. In contrast, inactivation of the Nkx3.1 gene causes no apparent skeletal phenotype despite its early expression in sclerotomal cells. These observations suggest that both genes might fulfill partially overlapping functions during development of the vertebral column. To test this hypothesis Nkx3.1/Nkx3.2 double homozygous mutants were generated. The simultaneous loss of both genes causes embryonic lethality between E12.5 and E17.5. Double mutants exhibit enhanced defects of vertebrae compared with Bapx1-deficient animals. In vertebral anlagen, sclerotomal cells condensing around the notochord are almost completely lost during early embryogenesis of double null mutants. Defects appeared most severe in the cranial region and less prominent in thoracic and lumbar regions. The reduction of chondrogenic cells results in the incomplete formation of vertebral bodies, missing major parts of their ventro-medial aspects. The enhanced skeletal phenotype of double null mutants compared to the single Bapx1 mutation demonstrates that Nkx3.1 contributes to the formation of the axial skeleton in addition to the Bapx1 gene. Moreover, both genes seem to collaborate in a yet unknown vital function in the mouse embryo (Herbrand, 2002).

The paired-box transcription factors Pax1 and Pax9 synergistically act in the proper formation of the vertebral column. Nevertheless, downstream events of the Pax1/Pax9 action and their target genes remain to be elucidated. By analyzing Pax1;Pax9 double mutant mice, it has been found that expression of Bapx1 in the sclerotome requires the presence of Pax1 and Pax9, in a gene dose-dependent manner. By using a retroviral system to overexpress Pax1 in chick presomitic mesoderm explants, it has been shown that Pax1 can substitute for Shh in inducing Bapx1 expression and in initiating chondrogenic differentiation. Furthermore, Pax1 and Pax9 can transactivate regulatory sequences in the Bapx1 promoter and they physically interact with the Bapx1 promoter region. These results strongly suggest that Bapx1 is a direct target of Pax1 and Pax9. Together, it is concluded that Pax1 and Pax9 are required and sufficient for the chondrogenic differentiation of sclerotomal cells (Rodrigo, 2003).

The middle ear apparatus is composed of three endochondrial ossicles (the stapes, incus and malleus) and two membranous bones, the tympanic ring and the gonium, all of which which act as structural components to anchor the ossicles to the skull. Except for the stapes, these skeletal elements are unique to mammals and are derived from the first and second branchial arches. In combination with goosecoid (Gsc), the Bapx1 gene defines the structural components of the murine middle ear. During embryogenesis, Bapx1 is expressed in a discrete domain within the mandibular component of the first branchial arch and later in the primordia of middle ear-associated bones, the gonium and tympanic ring. Consistent with the expression pattern of Bapx1, mouse embryos deficient for Bapx1 lack a gonium and display hypoplasia of the anterior end of the tympanic ring. At E10.5, expression of Bapx1 partially overlaps that of Gsc and although Gsc is required for development of the entire tympanic ring, the role of Bapx1 is restricted to the specification of the gonium and the anterior tympanic ring. Thus, simple overlapping expression of these two genes appears to account for the patterning of the elements that compose the structural components of the middle ear and suggests that they act in concert. In addition, Bapx1 is expressed both within and surrounding the incus and the malleus. Examination of the malleus shows that the width, but not the length, of this ossicle is decreased in the mutant mice. In non-mammalian jawed vertebrates, the bones homologous to the mammalian middle ear ossicles compose the proximal jaw bones that form the jaw articulation (primary jaw joint). In fish, Bapx1 is responsible for the formation of the joint between the quadrate and articular (homologs of the malleus and incus, respectively) enabling an evolutionary comparison of the role of a regulatory gene in the transition of the proximal jawbones to middle ear ossicles. Contrary to expectations, murine Bapx1 does not affect the articulation of the malleus and incus. This change in role of Bapx1 following the transition to the mammalian ossicle configuration is not due to a change in expression pattern but results from an inability to regulate Gdf5 and Gdf6, two genes predicted to be essential in joint formation (Tucker, 2004).

The mechanism by which left-right (LR) information is interpreted by organ primordia during asymmetric morphogenesis is largely unknown. Spleen and pancreatic laterality has been shown to be dependent on a specialised, columnar mesodermal-derived cell layer referred to as the splanchnic mesodermal plate (SMP). At early embryonic stages, the SMP is bilateral, surrounding the midline-located stomach and dorsal pancreatic bud. Under control of the LR asymmetry pathway, the left SMP is maintained and grows laterally. Mice carrying the dominant hemimelia (Dh) mutation lack the SMP. Significantly, the mice are asplenic and the pancreas remains positioned along the embryonic midline. In the absence of Fgf10 expression, the spleno-pancreatic mesenchyme and surrounding SMP grow laterally but contain no endodermal component, showing that leftward growth is autonomous and independent of endoderm. In the Bapx1-/- mutants, the SMP is defective. Normally, the SMP is a source for both Fgf9 and Fgf10; however, in the Bapx1 mutant, Fgf10 expression is downregulated and the dorsal pancreas remains at the midline. It is concluded that the SMP is an organiser responsible for the leftward growth of the spleno-pancreatic region and that Bapx1 regulates SMP functions required for pancreatic laterality. It is suggested that Bapx1 and Dh mutants are good models for heterotaxy syndromes that include asplenia (double right isomerism). Neither mouse mutant shows cardiac, lung or liver malformations and, therefore, they provide insights into laterality disorders in which only a restricted number of organ systems are affected. It is proposed that the developmental mechanism that drives asymmetric organ morphogenesis in spleen and pancreas differs from that responsible for lobation of the lung and morphogenesis of the heart tube and that it is dependent on a mesodermal-derived structure, the SMP (Hecksher-Sørensen, 2994).

Bagpipe homologs and pharyngeal development

A new Caenorhabditis elegans NK-2 class homeobox gene (Drosophila homologs tinman and bagpipe) has been identified and designated ceh-24. CEH-24 is one of four NK-2 class homeodomain proteins in C. elegans. Distinct cis-acting elements generate a complex neuronal and mesodermal expression pattern. A promoter-proximal enhancer mediates expression in a single pharyngeal muscle, the donut-shaped m8 cell located at the posterior end of the pharynx. A second mesodermal enhancer is active in a set of eight nonstriated vulval muscles used in egg laying. Activation in the egg laying muscles requires an 'NdE-box' consensus motif (CATATG) that is related to, but distinct from, the standard E-box motif bound by the MyoD family of transcriptional activators. Ectodermal expression of ceh-24 is limited to a subset of sublateral motor neurons in the head of the animal; this activity requires a cis-acting activator element that is distinct from the control elements for pharyngeal and vulval muscle expression. Activation of ceh-24 in each of the three cell types coincides with the onset of differentiation. Using a set of transposon-induced null mutations, it has been shown that ceh-24 is not essential for the formation of any of these cells. Although ceh-24 mutants have no evident defects under laboratory conditions, the pattern of ceh-24 activity is apparently important for Rhabditid nematodes: the related species C. briggsae contains a close homolog of C. elegans ceh-24, including a highly conserved and functionally equivalent set of cis-acting control signals (Harfe, 1998).

In Xenopus, one bagpipe-related gene, Xbagpipe (Xbap) has previously been described. The isolation of a second Xenopus homologue named zampogna (zax) is described. zax is transcribed within the muscular layer of the forming midgut as well as in the embryonic head, where zax transcripts mark both the pharyngeal endoderm and the future infrarostral cartilage (Newman, 1999). P>A conserved endothelin 1 signaling pathway patterns the jaw and other pharyngeal skeletal elements in mice, chicks and zebrafish. In zebrafish, endothelin 1 (edn1 or sucker) is required for formation of ventral cartilages and joints in the anterior pharyngeal arches of young larvae. Genetic analyses in the zebrafish is presented of two edn1 downstream targets -- the bHLH transcription factor Hand2 and the homeobox transcription factor Bapx1 -- that mediate dorsoventral (DV) patterning in the anterior pharyngeal arches. edn1-expressing cells in the first (mandibular) and second (hyoid) pharyngeal arch primordia are located most ventrally and surrounded by hand2-expressing cells. Along the DV axis of the early first arch primordia, bapx1 is expressed in an intermediate domain, which later marks the jaw joint; this expression requires edn1 function. bapx1 function is required for formation of the jaw joint, the joint-associated retroarticular process of Meckel's cartilage, and the retroarticular bone. Jaw joint expression of chd and gdf5 also requires bapx1 function. Similar to edn1, hand2 is required for ventral pharyngeal cartilage formation. However, the early ventral arch edn1-dependent expression of five genes (dlx3, EphA3, gsc, msxe and msxb) are all present in hand2 mutants. Further, msxe and msxb are upregulated in hand2 mutant ventral arches. Slightly later, an edn1-dependent ventral first arch expression domain of gsc is absent in hand2 mutants, providing a common downstream target of edn1 and hand2. In hand2 mutants, bapx1 expression is present at the joint region, and expanded ventrally. In addition, expression of eng2, normally restricted to first arch dorsal mesoderm, expands ventrally in hand2 and edn1 mutants. Thus, ventral pharyngeal specification involves repression of dorsal and intermediate (joint region) fates. Together these results reveal two critical edn1 effectors that pattern the vertebrate jaw: hand2 specifies ventral pharyngeal cartilage of the lower jaw and bapx1 specifies the jaw joint (Miller, 2003).

In vertebrate embryos, streams of cranial neural crest (CNC) cells migrate to form segmental pharyngeal arches and differentiate into segment-specific parts of the facial skeleton. To identify genes involved in specifying segmental identity in the vertebrate head, a screen was performed for mutations affecting cartilage patterning in the zebrafish larval pharynx. The positional cloning and initial phenotypic characterization of a homeotic locus discovered in this screen is presented. A zebrafish ortholog of the human oncogenic histone acetyltransferase MOZ (monocytic leukemia zinc finger) is required for specifying segmental identity in the second through fourth pharyngeal arches. In moz mutant zebrafish, the second pharyngeal arch is dramatically transformed into a mirror-image duplicated jaw. This phenotype resembles a similar but stronger transformation than that seen in hox2 morpholino oligo (hox2-MO) injected animals. In addition, mild anterior homeotic transformations are seen in the third and fourth pharyngeal arches of moz mutants. moz is required for maintenance of most hox1-4 expression domains and this requirement probably at least partially accounts for the moz mutant homeotic phenotypes. Homeosis and defective Hox gene expression in moz mutants is rescued by inhibiting histone deacetylase activity with Trichostatin A. Although early patterning of the moz mutant hindbrain is found to be normal, a late defect is found in facial motoneuron migration in moz mutants. Pharyngeal musculature is transformed late, but not early, in moz mutants. Relatively minor defects are detected in arch epithelia of moz mutants. Vital labeling of arch development reveals no detectable changes in CNC generation in moz mutants, but later prechondrogenic condensations are mispositioned and misshapen. Mirror-image hox2-dependent gene expression changes in postmigratory CNC prefigure the homeotic phenotype in moz mutants. Early second arch ventral expression of goosecoid (gsc) in moz mutants and in animals injected with hox2-MOs shifts from lateral to medial, mirroring the first arch pattern. bapx1, which is normally expressed in first arch postmigratory CNC prefiguring the jaw joint, is ectopically expressed in second arch CNC of moz mutants and hox2-MO injected animals. Reduction of bapx1 function in wild types causes loss of the jaw joint. Reduction of bapx1 function in moz mutants causes loss of both first and second arch joints, providing functional genetic evidence that bapx1 contributes to the moz-deficient homeotic pattern. Together, these results reveal an essential embryonic role and a crucial histone acetyltransferase activity for Moz in regulating Hox expression and segmental identity, and provide two early targets, bapx1 and gsc, of moz and hox2 signaling in the second pharyngeal arch (Miller, 2004).

Serotonin 2B receptor signaling is required for craniofacial morphogenesis and jaw joint formation in Xenopus; 5-HT2B signaling is required to define and sustain the Xbap expression necessary for jaw joint formation

Serotonin (5-HT) is a neuromodulator that plays many different roles in adult and embryonic life. Among the 5-HT receptors, 5-HT2B is one of the key mediators of 5-HT functions during development. Xenopus laevis was used as a model system to further investigate the role of 5-HT2B in embryogenesis, focusing on craniofacial development. By means of gene gain- and loss-of-function approaches and tissue transplantation assays, it was demonstrated that 5-HT2B modulates, in a cell-autonomous manner, postmigratory skeletogenic cranial neural crest cell (NCC) behavior without altering early steps of cranial NCC development and migration. 5-HT2B overexpression induced the formation of an ectopic visceral skeletal element and altered the dorsoventral patterning of the branchial arches. Loss-of-function experiments revealed that 5-HT2B signaling is necessary for jaw joint formation and for shaping the mandibular arch skeletal elements. In particular, 5-HT2B signaling is required to define and sustain the Xbap expression necessary for jaw joint formation. To shed light on the molecular identity of the transduction pathway acting downstream of 5-HT2B, the function was analyzed of phospholipase C beta 3 (PLC) in Xenopus development, and it was shown that PLC is the effector of 5-HT2B during craniofacial development. These results unveiled an unsuspected role of 5-HT2B in craniofacial development and contribute to understanding of the interactive network of patterning signals that is involved in the development and evolution of the vertebrate mandibular arch (Reisoli, 2010).

It is tempting to speculate that endothelin receptor and serotonergic receptor 2B signaling cooperate in reinforcing the PLC pathway and in modulating Xbap expression during first arch NCC patterning. However, although endothelin signaling is able to modulate both Xbap and XHand2 gene expression, 5-HT2B signaling seems to be required to maintain Xbap expression in the domain of the mandibular arch that gives rise to the jaw joint. In 5-HT2B morphants, in fact, XHand2 mRNA expression is unaltered and, consequently, the distal part of Meckel's cartilage develops normally. As the regulatory network involved in the specific expression of Bapx1 in vertebrates is conserved, perhaps 5-HT2B signaling controls Bapx1 expression in mammalian embryos as well. This aspect, to be verified and further explored, could be of interest particularly in the light of the possible involvement of BAPX1 in human birth defects, such as those of the oculo-articular-vertebral spectrum, which involve alterations in the first and second branchial arch derivatives, and considering the wide spectrum of serotonergic drugs available and commonly used in therapy including during pregnancy (Reisoli, 2010).

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