In Drosophila, the gene bicoid functions as the anterior body pattern organizer. Embryos lacking maternally expressed bicoid fail to develop anterior segments, including the head and thorax. In wild-type eggs, Bicoid mRNA is localized in the anterior pole region and the Bicoid protein forms an anterior-to-posterior concentration gradient. bicoid activity is required for transcriptional activation of zygotic segmentation genes and the translational suppression of uniformly distributed maternal Caudal mRNA in the anterior region of the embryo. caudal as well as other homeobox genes and members of the Drosophila segmentation gene cascade have been found to be conserved in animal evolution. In contrast, bicoid homologs have been identified only in close relatives of the schizophoran fly Drosophila. This poses the question of how the bicoid gene evolved and adopted its unique function in organizing anterior-posterior polarity. bicoid has been cloned from a basal cyclorrhaphan fly, Megaselia abdita (Phoridae, Aschiza). The cyclorrhaphan flies are divided into two subordinate groups: the Aschiza and the Schizophora. The phorid Megaselia is an aschizan fly. Therefore, it is different from the monophyletic group of schizophoran flies that includes Drosophila and the few other species where, to date, bicoid has been identified. Megaselia-bicoid (Ma-bcd) transcripts accumulate first in the oocyte, where a transient ring-shaped pattern is observed. Later, during oogenesis, transcripts are expressed in the nurse cells; they accumulate in the anterior region of the oocyte and transiently at its posterior pole. In the early embryo, Ma-bcd transcripts spread from the anterior pole forming an enlarging anterior cap until the onset of cellularization. Subsequently, transcripts disappear rapidly. No zygotic expression is observed during embryogenesis. These findings establish identical expression patterns for bicoid and Ma-bcd in Drosophila and Megaselia (Stauber, 1999).

A comparison of the Ma-bcd homeodomain and homeodomain proteins of Drosophila indicates that aside from bicoid, Ma-bcd is most similar to zerknüllt (48.3%), whereas the similarity to homeodomains encoded by the other members of the Drosophila Hox-C is less pronounced (45.0%-36.7%). The homeodomain of Ma-bcd is related only distantly to the homeodomains encoded by orthodenticle and the Drosophila homologs of goosecoid and Ptx1 (38.3%-33.3%), which have been classified in the past as bicoid-related genes. These proteins share common DNA-binding properties that depend on the diagnostic lysine in position 50 of the homeodomain. Also notable is the fact that the zerknüllt homologs of other insects and their orthologs in various animal classes (the Hox3 genes of chordates, ribbonworm, and spider) show a higher degree of similarity to the Ma-bcd homeodomain than do the bicoid-related genes. These observations suggest that, in spite of the considerable sequence divergence exhibited by the Drosophila genes, bicoid and zerknüllt are closely related (Stauber, 1999).

To address the hypothesis that bicoid and zerknüllt are the closest relatives among Hox genes, the Megaselia zerknüllt gene (Ma-zen) was cloned to determine if Ma-zen provides a link between bicoid and the Hox3 genes of the vertebrate Hox clusters. Ma-zen was isolated by a PCR approach and the genomic DNA encompassing the transcription unit was isolated. Whole-mount in situ hybridization of Megaselia embryos reveals that Ma-zen transcripts are expressed only zygotically. They form a restricted pattern at the dorsal side of the blastoderm embryo, covering the area of the amnioserosa precursor cells, and disappear in the extended germ-band stage. Thus, Ma-zen is expressed like zerknüllt in Drosophila. This suggests that Ma-bcd and Ma-zen have separate functions in Megaselia, similar to the functions of their homologs in Drosophila (Stauber, 1999).

Sequence comparison of Ma-bcd and Ma-zen proteins clearly establishes a sister relationship between the two proteins. Evidence for this is based on the following findings. The homeodomain of Ma-bcd shows a higher sequence similarity to Ma-zen (50.0%) than to any other nonorthologous homeodomain. In addition, molecular phylogenetic trees involving the homeodomains of the Hox complex genes of Drospohila resolve with high confidence when the Ma-bcd and Ma-zen sequences, instead of the bicoid and zerknüllt sequences, are used for the analysis. It is important to note that the Drosophila homeodomains of the Hox complex evolve very slowly (except fushi tarazu) and can be assumed to be identical or almost identical in amino acid sequence in Megaselia and Drosophila. The alignment of the Ma-bcd and Ma-zen proteins shows conservation of sequences not only in the homeodomain but also N-terminal to it. The conserved sequences in front of the homeodomain are not evident from the comparison of Drosophila bicoid and zerknüllt. Thus, the sister-gene relationship of bicoid and zerknüllt revealed by the Megaselia genes remains hidden when the obviously more diverged sequences of the Drosophila genes are compared (Stauber, 1999).

The newly established sister-gene relationship implies that bicoid genes, like the zerknüllt genes, are direct homologs of the Hox3 genes in the Hox-C of noninsect animal classes. Thus, bicoid is a Hox gene in the phylogenetic sense, and the location of bicoid in the Hox-C of Drosophila is an ancestral trait. The consistent failure to isolate bicoid from insects other than flies, which has been attempted in various laboratories, suggests a recent origin for the bicoid gene. The fact that Ma-bcd is more similar to the zerknüllt genes of higher insects than to other Hox3 homologs is consistent with the assumption that bicoid originated recently during insect radiation (Stauber, 1999).

bicoid is expressed in the anterior egg region, where it exerts its role in patterning the anterior body of the larval fly. In contrast, zerknüllt and its orthologs function in extraembryonic anlagen. Although the extraembryonic anlage in flies, the amnioserosa, is located at the dorsal side of the blastoderm fate map, extraembryonic anlagen in other insects, such as the beetle Tribolium, are formed in an anterior egg position (see Tribolium early embryonic development). This suggests that initially the sister genes bicoid and zerknüllt may have been coexpressed in the anterior egg region. The subsequent recruitment of bicoid in patterning the embryo, instead of determining the dorsally shifted extraembryonic anlagen, changed the selection conditions for the gene. Ensuing adaptations must have resulted in a new set of target genes, as reflected by the characteristic lysine in Bicoid's homeodomain position 50 that specifies DNA recognition. The newly acquired functions of Bicoid entrained a significant change in the developmental mechanism of axis specification and now furnish an outstanding model of molecular evolution in a patterning process (Stauber, 1999).

The Drosophila gene bicoid functions at the beginning of a gene cascade that specifies anterior structures in the embryo. Its transcripts are localized at the anterior pole of the oocyte, giving rise to a Bicoid protein gradient, which regulates the spatially restricted expression of target genes along the anterior-posterior axis of the embryo in a concentration-dependent manner. The morphogen function of Bicoid requires the coactivity of the zinc finger transcription factor Hunchback, which is expressed in a Bicoid-dependent fashion in the anterior half of the embryo. Whereas hunchback is conserved throughout insects, bicoid homologs are known only from cyclorrhaphan flies. Thus far, identification of hunchback and bicoid homologs rests only on sequence comparison. In this study, double-stranded RNA interference (RNAi) was used to address the function of bicoid and hunchback homologs in embryos of the lower cyclorrhaphan fly Megaselia abdita (Phoridae). Megaselia-hunchback RNAi causes hunchback-like phenotypes as observed in Drosophila, but Megaselia-bicoid RNAi causes phenotypes different from corresponding RNAi experiments in Drosophila bicoid mutant embryos. Megaselia-bicoid is required not only for the head and thorax but also for the development of four abdominal segments. This difference between Megaselia and Drosophila suggests that the range of functional bicoid activity has been reduced in higher flies (Stauber, 2000).

Ma-bcd RNAi caused a reduction of anterior segments including the cephalopharyngeal head skeleton. The strongest phenotypes lack the head, thorax, and three to four abdominal segments, which are replaced by a mirror image duplication of the remaining abdomen. Ectopic gastrulation movements at the anterior pole can be delayed with respect to gastrulation movements at the posterior pole, resulting in asymmetric germ band extension. The relative positions of the remaining abdominal segments at different developmental stages and the unambiguous identification of the reduced abdominal segment 9 suggest that abdominal segments 1, 2, and 3 and the dorsal part of abdominal segment 4 are missing. Dorsally, the symmetry plane lies most likely in abdominal segment 5, whereas in the nerve cord, it lies in the fourth abdominal neuromere. Thus, such embryos resemble a bicaudal rather than the bicoid phenotype. Injection of bicoid dsRNA into Megaselia embryos does not produce specific defects. In less than 1% of injected embryos presumably unspecific head defects but no duplications were observed. These results indicate that Ma-bcd RNAi in Megaselia embryos causes the specific deletion of the anterior abdominal segments, which is not observed in the corresponding RNAi experiments with Drosophila or with bicoid- or hunchback-deficient Drosophila embryos. It is important to note, however, that in Drosophila, a symmetrical bicaudal-like phenotype had been observed when the combined activities of bicoid and hunchback are repressed in the anterior half of the embryos, indicating synergistic effects of both genes. It was therefore asked whether coinjection of Ma-bcd and Ma-hb dsRNAs into Megaselia embryos results in a more than additive extension of anterior deletions as compared with single dsRNA injections. The phenotypes obtained after combined Ma-bcd and Ma-hb dsRNA injections are similar to the sum of effects observed in independent Ma-bcd dsRNA and Ma-hb dsRNA injections. In addition, the ventral nerve cord is more disorganized and interrupted between the symmetrical abdominal halves, and the gut is reduced in size. These results suggest only a weak synergistic effect of Ma-bcd and Ma-hb in the abdomen (Stauber, 2000).

The members of the evolutionarily conserved Hox-gene complex are required for specifying segmental identity during embryogenesis in various animal phyla. The Hox3 genes of winged insects have lost this ancestral function and are required for the development of extraembryonic epithelia, which do not contribute to any larval structure. Higher flies (Cyclorrhapha) such as Drosophila melanogaster contain Hox3 genes of two types, the zerknüllt type and the bicoid type. The zerknüllt gene is expressed zygotically on the dorsal side of the embryo and is required for establishing extraembryonic tissue. Its sister gene bicoid is expressed maternally and the transcripts are localized at the anterior pole of the mature egg. Bicoid protein, which emerges from this localized source during early development, is required for embryonic patterning. All known direct bicoid homologs are confined to Cyclorrhaphan flies. This study describes Hox3 genes of the non-Cyclorrhaphan flies Empis livida (Empididae), Haematopota pluvialis (Tabanidae), and Clogmia albipunctata (Psychodidae). The gene sequences are more similar to zerknüllt homologs than to bicoid homologs, but they share expression characteristics of both genes. It is proposed that an ancestral Hox3 gene had been duplicated in the stem lineage of Cyclorrhaphan flies. During evolution, one of the gene copies lost maternal expression and evolved as zerknüllt, whereas the second copy lost zygotic expression and evolved as bicoid. These findings correlate well with a partial reduction of zerknüllt-dependent extraembryonic tissue during Dipteran evolution (Stauber, 2002).

Clogmia zerknüllt (Ca-zen) is strongly expressed in the germ-line cells of ovarian egg chambers. The transcripts are detected in the nurse cells and the oocyte, and seem to be evenly distributed in the early embryo. To corroborate maternal expression of Ca-zen, a Northern blot analysis was performed with mRNA prepared from ovaries. A single band of expected size is obtained after hybridization with a Ca-zen probe. Zygotic expression of Ca-zen starts during cellularization of the blastoderm in an anterior and dorsal domain, which corresponds to the anlage of extraembryonic tissue. Extraembryonic Ca-zen expression is maintained during gastrulation, but no zygotic Ca-zen expression outside the extraembryonic anlage/tissue is observed. The expression pattern of Ca-zen differs in two important ways from conserved zerknüllt expression in Cyclorrhaphan flies: (1) Ca-zen is expressed maternally, whereas in Cyclorrhapha zerknüllt genes are expressed strictly zygotically; (2) zygotic Ca-zen expression extends to the anterior tip of the cellular blastoderm, whereas zerknüllt expression in Cyclorrhapha is restricted to a narrow dorsal strip at the same developmental stage. Thus, Ca-zen in Clogmia combines expression characteristics of bicoid and zerknüllt in Cyclorrhapha (Stauber, 2002).

The maternal expression of all three newly identified Hox3/zerknüllt homologs implies selection for this trait in lower Diptera and release from a corresponding specific constraint in Cyclorrhapha. To understand better the changing developmental constraints during the evolution of Diptera, embryonic development throughout this taxon was compared. Embryonic development in Diptera seems rather uniform and resembles that of Drosophila. However, an important difference within Diptera occurs with respect to extraembryonic tissue organization. The establishment of extraembryonic tissue requires the activity of a Hox3/zerknüllt gene not only in Drosophila, but most likely in all winged insects. In species of several insect orders and, in particular, in all non-Cyclorrhaphan flies analyzed so far, including the three species of this study, extraembryonic tissue consists of an amnion and a serosa. These two epithelia do not contribute to the embryo proper but transiently wrap the embryo. In contrast, Cyclorrhapha including Megaselia and Drosophila develop without such wrapping, and the extraembryonic tissue is reduced to a transient dorsal epithelium, termed amnioserosa and, as recently discovered in Drosophila, some additional cells surrounding the yolk. Thus, the transition in extraembryonic tissue organization in the stem lineage of Cyclorrhaphan flies occurs in a period when maternal expression is lost in the zerknüllt-type Hox3 genes (Stauber, 2002).

On the basis of these findings, it is proposed that bicoid and zerknüllt evolved in the stem lineage of Cyclorrhaphan flies from a Hox3 gene with maternal and zygotic expression, which is still found in non-Cyclorrhaphan Diptera. In the common progenitor, zygotic activity was required for extraembryonic development, a feature conserved by the Cyclorrhaphan zerknüllt genes. Maternal activity of Hox3/zerknüllt homologs is not understood currently; some better understanding will require adopting methods for gene inactivation in non-Cyclorrhaphan Diptera. However, because maternal expression of Hox3/zerknüllt homologs is conserved in all non-Cyclorrhaphan Diptera analyzed so far, maternal activities of these genes are important for development. An understanding is lacking of how maternal Hox3/zerknüllt activity turned into maternal bicoid activity, one of the problems being the different DNA- and RNA-binding properties of Bicoid compared with all other Hox genes. In Drosophila, ectopic expression of zerknüllt induces extraembryonic tissue and ectopic expression of bicoid induces anterior embryonic structures. Thus, both genes have counteracting effects and cannot convert their respective activities in the same spatial domain of the embryo. Therefore, a separation of the functional expression domains of bicoid and zerknüllt in time and space, as well as selective loss of maternal versus zygotic enhancer elements, seems to be an important prerequisite for subsequent divergent evolution of both genes in Cyclorrhapha. It is suggested that anterior localization of Bicoid, which is based on specific sequence elements in the 3' untranslated region of the gene, and respecification of anterior blastoderm toward an embryonic fate were important steps toward this goal. In summary, the key features of this model are as follows: a single Hox3 gene with maternal and zygotic activity is present in the stem lineage of Diptera; it was duplicated in the stem lineage of Cyclorrhapha, giving birth to maternal bicoid and zygotic zerknüllt. The functional evolution of Bicoid-specific DNA- and RNA-binding properties became possible after the reduction of the extraembryonic anlage/tissue. It will be challenging to test this model at the levels of genomics, developmental genetics, and morphology (Stauber, 2002).

In Drosophila, a gradient of Bicoid protein (Bcd) originates from prelocalized mRNA at the anterior pole of the egg and establishes developmental programs including those for the larval head and thorax in a concentration-dependent manner. Orthologous proteins have been reported only from cyclorrhaphan flies. However, a Bcd-like determinant has been postulated for a large variety of insects including leafhoppers, beetles and midges. It therefore remains to be determined whether diverged Bicoid orthologs exist in other insect orders. bcd encodes a homeodomain-containing transcription factor, and is located immediately upstream of zerknüllt (zen) in the Hox gene complex (Hox-C) of various drosophilids. Based on these findings it has been suggested that bcd originated as a result of a gene duplication involving zen. However, it has been difficult to test this hypothesis due to rapid sequence evolution of bcd and zen. Only recently has sequence analysis of a bcd homolog from the basal cyclorrhaphan fly Megaselia (Phoridae) provided direct support for a sister-gene relationship of bcd and zen, and implicated the position of bcd upstream of zen in the Hox-C as ancestral (Brown, 2001 and references therein).

The linkage of bcd and zen was tested in the blowflies Calliphora erythrocephalaand Lucilia sericata (Calliphoridae, Diptera) where bcd homologs had been identified. Using degenerate PCR primers zen homeoboxes were isolated from both species. Specific nested primer pairs, when used in long range PCR, amplify single genomic DNA fragments of 12kb (Calliphora) and 16kb (Lucilia), linking the homeoboxes of the bcd and zen homologs. The identity of each fragment has been verified by Southern-blot hybridization and terminal sequencing. Thus, bcd is linked to a Hox class 3 gene in blowflies (Calyptratae) as it is in drosophilids (Acalyptratae), and the 5' to 3' orientation of bcd and zen with respect to one another is conserved. This linkage was probably inherited from the common ancestor of the monophyletic Schizophora (Acalyptratae and Calyptratae), which comprise the majority of family-level diversity of Diptera. These observations strongly support the hypothesis that bcd arose as a tandem duplication of zen within the Hox-C. It is concluded that analysis of the relevant Hox-C portion of selected species provides a means to map the origin of Bcd on the phylogenetic tree (Brown, 2001).

In the red flour beetle Tribolium castaneum, a holometabolous insect distantly related to flies, orthologs of the eight arthropod homeotic genes, as well as ftz and zen are arrayed in the same order as their Drosophila counterparts, an order that has been maintained for over 300 million years. To determine whether a highly diverged Bcd ortholog is located in its predicted position in the Tribolium Hox-C, a BAC clone spanning the region from the 5' exon of mxp/Hox2 to Tcftz was screened. This sequence was analyzed using BLAST, and the Baylor College of Medicine genefinder program to predict open reading frames and putative transcription units. A second zen gene was found immediately downstream of the one previously identified. These genes are most likely the result of an independent duplication in the lineage leading to Tribolium. Although known transcription units were faithfully predicted, no other homeodomain-encoding sequences were found. The Tribolium Hox-C does not, therefore, contain a Bcd ortholog in the interval between mxp/Hox2 and TcDfd/Hox4 (Brown, 2001).

Conservation of the relative positions of zen and bcd in blowflies and drosophilid fruitflies, combined with the absence of a bcd gene in the corresponding position in the Tribolium Hox-C, provide direct support for the hypothesis that bcd originated recently, presumably after the basal radiation of holometabolous insects. As the homeotic complexes of additional holometabolous insects are analyzed for bcd, an understanding of the origin of bcd will be refined (Brown, 2001).

The conclusion that bcd emerged after the basal radiation of holometabolous insects does not necessarily imply that beetles and more primitive insects develop without an anterior determinant. In fact, several observations have been taken as evidence for such a factor in beetle and leafhopper development. If these inferences are correct the results presented here suggest that bcd functionally replaces an ancestral anterior determinant (Brown, 2001).

A nine-nucleotide motif, YUGUUYCUG, (BLE1) is common to the 3' untranslated regions of four sequenced messages of Drosophila Bicoid and Nanos mRNAs and Xenopus An2 and Vg1 mRNAs. Bicoid mRNA specifically lacking this nonamer is partially mislocalized. In contrast, nonamer deletion is inconsequential to message stability (Gottlieb, 1992).

Maternal activation of gap genes in the hover fly Episyrphus

The metameric organization of the insect body plan is initiated with the activation of gap genes, a set of transcription-factor-encoding genes that are zygotically expressed in broad and partially overlapping domains along the anteroposterior (AP) axis of the early embryo. The spatial pattern of gap gene expression domains along the AP axis is generally conserved, but the maternal genes that regulate their expression are not. Building on the comprehensive knowledge of maternal gap gene activation in Drosophila, loss- and gain-of-function experiments were used in the hover fly Episyrphus balteatus (Syrphidae) to address the question of how the maternal regulation of gap genes evolved. It was found that, in Episyrphus, a highly diverged bicoid ortholog is solely responsible for the AP polarity of the embryo. Episyrphus bicoid represses anterior zygotic expression of caudal and activates the anterior and central gap genes orthodenticle, hunchback and Krüppel. In bicoid-deficient Episyrphus embryos, nanos is insufficient to generate morphological asymmetry along the AP axis. Furthermore, torso transiently regulates anterior repression of caudal and is required for the activation of orthodenticle, whereas all posterior gap gene domains of knirps, giant, hunchback, tailless and huckebein depend on caudal. It is conclude that all maternal coordinate genes have altered their specific functions during the radiation of higher flies (Cyclorrhapha) (Lemke, 2010).

Therefore, Episyrphus and other lower cyclorrhaphan flies establish global AP polarity only through bicoid and lack sizable input of nanos, although endogenous nanos activity in these species might stabilize the AP axis by repressing anterior development. Despite the absence of a redundant maternal system to generate global AP polarity, Eba-bcd appears to be a less potent transcriptional activator than Bicoid. In contrast to Drosophila, gap gene activation at the anterior pole of the Episyrphus embryo requires a strong contribution of the terminal system, whereas the posterior domains of knirps and giant are strictly dependent on caudal and do not appear to receive a significant activating input by Eba-bcd. Thus, rather than a strong activation potential, the exclusive control of the central Eba-Kr domain by Eba-bcd appears to be the crucial difference to Drosophila, which renders AP polarity in the Episyrphus embryo entirely dependent on bicoid (Lemke, 2010).

The molecular characterization is described of the paired-type homeobox gene D-Ptx1 of Drosophila, a close homolog of the mouse pituitary homeobox gene Ptx1 and the unc-30 gene of C. elegans, characterized by a lysine residue at position 9 of the third alpha-helix of the homeodomain. D-Ptx1 is expressed at various restricted locations throughout embryogenesis. Initial expression of D-Ptx1 in the posterior-most region of the blastoderm embryo is controlled by fork head activity in response to the activated Ras/Raf signaling pathway. During later stages of embryonic development. D-Ptx1 transcripts and protein accumulate in the posterior portion of the midgut, in the developing Malpighian tubules, in a subset of ventral somatic muscles, and in neural cells. Phenotypic analysis of gain-of-function and lack-of-function mutant embryos show that the D-Ptx1 gene is not involved in morphologically apparent differentiation processes. It is concluded that D-Ptx1 is more likely to control physiological cell functions than pattern formation during Drosophila embryogenesis (Vorbruggen, 1997).

Ptx1, a bicoid-related homeo box transcription factor involved in transcription of the pro-opiomelanocortin gene

The pituitary gland contains six distinct hormone-producing cell types that arise sequentially during organogenesis. The first cells to differentiate are those that express the pro-opiomelanocortin (POMC) gene in the anterior pituitary lobe. The other lineages, which appear later, include cells that are dependent on the POU factor Pit-1 and another POMC-expressing lineage in the intermediate pituitary lobe. Using AtT-20 cells as a model for early expression of POMC in the anterior pituitary, a regulatory element has been defined that confers cell specificity of transcription and a cognate transcription factor has been cloned. This factor, Ptx1 (pituitary homeo box 1), contains a homeo box related to those of the anterior-specific genes bicoid and orthodenticle in Drosophila, and Otx-1 and Otx-2 in mammals. Ptx1 activates transcription upon binding a sequence related to the Drosophila Bicoid target sites. Ptx1 is the only nuclear factor of this DNA-binding specificity that is detected in AtT-20 cells, and it is expressed at high levels in a subset of adult anterior pituitary cells that express POMC. However, Ptx1 is expressed in most cells of Rathke's pouch at an early time during pituitary development and before final differentiation of hormone-producing cells. Thus, Ptx1 may have a role in differentiation of pituitary cells; its early expression pattern suggests that it may have a role in pituitary formation. In the adult pituitary gland, Ptx1 appears to be recruited for cell-specific transcription of the POMC gene (Lamonerie, 1996).

Ptx1, a homeo box gene related to bicoid and orthodenticle and Otx-1 and Otx-2 in mammals, activates transcription upon binding a sequence related to the target sites bicoid and orthodenticle in Drosophila

Ptx1 is a member of the small bicoid family of homeobox-containing genes; it was isolated as a tissue-restricted transcription factor of the pro-opiomelanocortin gene. The homeodomain of Ptx1 contains a lysine at position 9 of the recognition helix (position 60 of the homeodomain). This residue is strategically placed in the major groove of DNA and it is a major determinant of DNA-binding specificity recognizing the CC doublet of the target site. This lysine residue defines the bicoid subfamily of homeoboxes, including Otx1 and 2 and Goosecoid. Ptx1 expression during mouse and chick embryogenesis was determined by in situ hybridization in order to delineate its putative role in development. In the head, Ptx1 expression is first detected in the ectoderm-derived stomodeal epithelium at E8.0. Initially, expression is only present in the stomodeum and in a few cells of the rostroventral foregut endoderm. A day later, Ptx1 mRNA is detected in the epithelium and in a streak of mesenchyme of the first branchial arch, but not in other arches. Ptx1 expression is maintained in all derivatives of these structures, including the epithelia of the tongue, palate, teeth and olfactory system, and in Rathke's pouch. Expression of Ptx1 in craniofacial structures is strikingly complementary to the pattern of goosecoid (See Drosophila Goosecoid) expression. Gsc labelling in the mandibular component is confined to a central stripe of mesenchyme whereas Ptx1 labelling is observed more laterally.

Similarly, the epithelium of the first arch, a site of strong Ptx1 expression, is not labelled by the Gsc probe. Ptx1 is expressed early (E6.8) in posterior and extraembryonic mesoderm, and in structures that derive from these. The restriction of expression to the posterior lateral plate is later evidenced by exclusive labelling of the hindlimb but not forelimb mesenchyme. In the anterior domain of expression, the stomodeum is shown by fate mapping to derive from the anterior neural ridge (ANR) which represents the most anterior domain of the embryo. The concordance between these fate maps and the stomodeal pattern of Ptx1 expression supports the hypothesis that Ptx1 defines a stomodeal ectomere that lies anterior to the neuromeres that have been suggested to constitute units of a segmented plan directing head formation. Drosophila Gsc is expressed in the stomodeal invagination, while vertebrate Gsc is not. Based on these gene expression patterns, it is thought that the vertebrate stomodeum is an evolutionary innovation, assuring the ventral placement of the mouth (Lanctot, 1997).

Pituitary homeobox 1 (Ptx1) is a homeodomain-containing transcription factor acting on transcription of all pituitary hormone genes. Its expression is first detected in the stomodeal ectoderm and is maintained in all derivatives of this structure, including Rathke's pouch. Ptx1 is expressed in all pituitary cells but it is differentially expressed in different lineages at both the messenger RNA and protein levels. On day 12.5 of mouse embryonic development, cells expressing the highest levels of Ptx1 are restricted to the forming pars tuberalis, also called the rostral tip, a region where the first alpha-glycoprotein subunit-expressing cells appear. Coimmunolocalization studies reveal that alpha-glycoprotein subunit-positive cells express the highest levels of Ptx1 throughout development and in the adult gland. The quantitative differences in Ptx1 expression in pituitary cell lineages may relate to a role in cell proliferation, lineage commitment, and/or the control of organ development (Lanctot, 1999a).

Ptx1 belongs to an expanding family of bicoid-related vertebrate homeobox genes. These genes, like their Drosophila homolog, seem to play a role in the development of anterior structures and, in particular, the brain and facies. The chromosomal localization of mouse Ptx1 is reported, and the cloning, sequencing, and chromosomal localization of the human homolog PTX1. The putative encoded proteins share 100% homology in the homeodomain and are 88% and 97% conserved in the N- and C-termini, respectively. Intron/exon boundaries are also conserved. Murine Ptx1 was localized, by interspecific backcrossing, to Chromosome (Chr) 13 within 2.6 cM of Caml. The gene resides centrally on Chr 13 in a region syntenic with human Chr 5q. Subsequent analysis by fluorescent in situ hybridization places the human gene, PTX1, on 5q31, a region associated with Treacher Collins Franceschetti Syndrome. Taken together with the craniofacial expression pattern of Ptx1 during early development, the localization of the gene in this chromosomal area is consistent with an involvement in Treacher Collins Franceschetti Syndrome (Crawford, 1997).

The Ptx1 (pituitary homeobox 1) homeobox transcription factor was isolated as a transcription factor of the pituitary POMC gene. In corticotrope cells that express POMC, cell-specific transcription is conferred in part by the synergistic action of Ptx1 with the basic helix-loop-helix factor NeuroD1. Since Ptx1 expression precedes pituitary development and differentiation, its expression and function in other pituitary lineages was investigated. Ptx1 is expressed in most pituitary-derived cell lines, as is the related Ptx2 gene. However, Ptx1 appears to be the only Ptx protein in corticotropes and the predominant one in gonadotrope cells. Most pituitary hormone-coding gene promoters are activated by Ptx1. Thus, Ptx1 appears to be a general regulator of pituitary-specific transcription. In addition, Ptx1 action is synergized by cell-restricted transcription factors to confer promoter-specific expression. Indeed, in the somatolactotrope lineage, synergism between Ptx1 and Pit1 is observed on the PRL promoter, and strong synergism between Ptx1 and SF-1 is observed in gonadotrope cells on the betaLH promoter but not on the alphaGSU (glycoprotein hormone alpha-subunit gene) and betaFSH promoters. Synergism between these two classes of factors is reminiscent of the interaction between Drosophila Fushi tarazu and Ftz-F1. Antisense RNA experiments performed in alphaT3-1 cells that express the alphaGSU gene show that expression of endogenous alphaGSU is highly dependent on Ptx1, whereas many other genes are not affected. Interestingly, the only other gene found to be highly dependent on Ptx1 for expression was the gene for the Lim3/Lhx3 transcription factor. Thus, these experiments place Ptx1 upstream of Lim3/Lhx3 in a cascade of regulators that appear to work in a combinatorial code to direct pituitary-, lineage-, and promoter-specific transcription (Tremblay, 1998).

Human Ptx3 is a member of the Bicoid-related subgroup of transcription factors that includes Drosophila Bicoid, Orthodenticle and Goosecoid. The mesencephalic dopaminergic (mesDA) system regulates behavior and movement control and has been implicated in psychiatric and affective disorders. Ptx3 is uniquely expressed in the neurons of this system. Its expression starting at E11.5 in the developing mouse midbrain correlates with the appearance of mesDA neurons. The number of Ptx3-expressing neurons is reduced in Parkinson patients, and these neurons are absent from 6-hydroxydopamine-lesioned rats, an animal model for this disease. Thus, Ptx3 is a unique transcription factor marking the mesDA neurons at the exclusion of other dopaminergic neurons, and it may be involved in developmental determination of this neuronal lineage (Smidt, 1997).

Genetic analysis of mouse mutants has demonstrated the importance of the homeobox genes Rpx, Lhx3 and Pit1 for anterior pituitary gland development. Pit1 mutations have also been identified in several human families with multiple pituitary hormone deficiencies. To identify additional homeobox regulators of pituitary development, an adult pituitary gland cDNA library was screened for homeobox sequences. The identification of a novel bicoid-related homeodomain gene is reported, expressing two alternatively spliced mRNA products that encode proteins of 271 and 317 amino acids, respectively. The proteins have been named Ptx2a and Ptx2b since they are highly related to Ptx1/P-OTX. Ptx2 is expressed in both developing and adult pituitary gland, eye and brain tissues, suggesting an important role in development and maintenance of anterior structures. Ptx2 was mapped close to Egf on mouse chromosome 3, in a region having extensive synteny homology with HSA 4q. These data make the human Ptx2 homolog a candidate gene for Rieger syndrome, an autosomal-dominant disorder with variable craniofacial, dental, eye and pituitary anomalies (Gage, 1997).

Signaling molecules such as Activin, Sonic hedgehog, Nodal, Lefty, and Vg1 have been found to be involved in determination of left-right (L-R) asymmetry in the chick, mouse, or frog. However, a common signaling pathway has not yet been identified in vertebrates. Pitx2, a bicoid-type homeobox gene expressed asymmetrically in the left lateral plate mesoderm, may be involved in determination of L-R asymmetry in both mouse and chick. Since Pitx2 appears to be downstream of lefty-1 in the mouse pathway, whether mouse Lefty proteins could affect the expression of Pitx2 in the chick was examined. The results indicate that a common pathway from lefty-1 to Pitx2 likely exists for determination of L-R asymmetry in vertebrates (Yoshioka, 1998).

Left-right asymmetry in vertebrates is controlled by activities emanating from the left lateral plate. How these signals get transmitted to the forming organs is not known. A candidate mediator in mouse, frog and zebrafish embryos is the homeobox gene Pitx2. It is asymmetrically expressed in the left lateral plate mesoderm, tubular heart and early gut tube. Localized Pitx2 expression continues when these organs undergo asymmetric looping morphogenesis. Ectopic expression of Xnr1 in the right lateral plate induces Pitx2 transcription in Xenopus. Misexpression of Pitx2 affects situs and the morphology of organs. These experiments suggest a role for Pitx2 in promoting looping of the linear heart and gut (Campione, 1999).

The cloning and temporal and spatial expression patterns are reported for a novel homeobox gene Backfoot (BFT for the human gene; Bft for the mouse gene) whose expression reveals an early molecular distinction between forelimb and hind limb. BFT was identified as a sequence-specific DNA-binding protein. In addition to the homeodomain, it shares a carboxyl-terminal peptide motif with other paired-like homeodomain proteins. Northern hybridization analysis of RNAs from human tissues reveals that human BFT is highly expressed in adult skeletal muscle and bladder. During midgestation embryogenesis, mouse Bft is expressed in the developing hind limb buds, mandibular arches, and Rathke's pouch. The expression of Bft begins prior to the appearance of hind limb buds in mesenchyme but is never observed in forelimbs. At later stages of limb development, the expression is progressively restricted to perichondrial regions, most likely in tendons and ligaments. The timing and pattern of expression suggest that Bft plays multiple roles in hind limb patterning, branchial arch development, and pituitary development. Bft is likely identical to a mouse gene, Ptx1, that was recently isolated by Lamonerie (1996) and that has been suggested to play a role in pituitary development (Shang, 1997).

Pitx1 is a Bicoid-related homeodomain factor that exhibits preferential expression in the hindlimb, as well as expression in the developing anterior pituitary gland and first branchial arch. Pitx1 gene-deleted mice exhibit striking abnormalities in morphogenesis and growth of the hindlimb, resulting in a limb that exhibits structural changes in tibia and fibula as well as patterning alterations in patella and proximal tarsus to more closely resemble the corresponding forelimb structures. Deletion of the Pitx1 locus results in decreased distal expression of the hindlimb-specific marker, the T-box factor Tbx4. On the basis of similar expression patterns in chick, targeted misexpression of chick Pitx1 in the developing wing bud causes the resulting limb to assume altered digit number and morphogenesis, with Tbx4 induction. It is hypothesized that Pitx1 serves to critically modulate morphogenesis, growth, and potential patterning of a specific hindlimb region, serving as a component of the morphological and growth distinctions in forelimb and hindlimb identity. Pitx1 gene-deleted mice also exhibit reciprocal abnormalities of two ventral and one dorsal anterior pituitary cell types, presumably on the basis of its synergistic functions with other transcription factors, and show defects in the derivatives of the first branchial arch, including cleft palate, suggesting a proliferative defect in these organs analogous to that observed in the hindlimb (Szeto, 1999).

In spite of recent breakthroughs in understanding limb patterning, the genetic factors determining the differences between the forelimb and the hindlimb have not been understood. The genes Pitx1 and Tbx4 encode transcription factors that are expressed throughout the developing hindlimb but not forelimb buds. Misexpression of Pitx1 in the chick wing bud induces distal expression of Tbx4, as well as HoxC10 and HoxC11, which are normally restricted to hindlimb expression domains. Wing buds in which Pitx1 is misexpressed develop into limbs with some morphological characteristics of hindlimbs: the flexure is altered to that normally observed in legs; the digits are more toe-like in their relative size and shape, and the muscle pattern is transformed to that of a leg (Logan, 1999).

The restricted expression of the Ptx1 (Pitx1) gene in the posterior half of the lateral plate mesoderm has suggested that it may play a role in specification of posterior structures, in particular, specification of hindlimb identity. Ptx1 is also expressed in the most anterior ectoderm, the stomodeum, and in the first branchial arch. Ptx1 expression overlaps with that of Ptx2 in stomodeum and in posterior left lateral plate mesoderm. Targeted inactivation of the mouse Ptx1 gene severely impairs hindlimb development: the ilium and knee cartilage are absent and the long bones are underdeveloped. Greater reduction of the right femur size in Ptx1 null mice suggests partial compensation by Ptx2 on the left side. The similarly sized tibia and fibula of mutant hindlimbs may be taken to resemble forelimb bones: however, the mutant limb buds appear to have retained their molecular identity as assessed by forelimb expression of Tbx5 and by hindlimb expression of Tbx4, even though Tbx4 expression is decreased in Ptx1 null mice. The hindlimb defects appear to be, at least partly, due to abnormal chondrogenesis. Since the most affected structures derive from the dorsal side of hindlimb buds, the data suggest that Ptx1 is responsible for patterning of these dorsal structures and that as such it may control development of hindlimb-specific features. Ptx1 inactivation also leads to loss of bones derived from the proximal part of the mandibular mesenchyme. The dual role of Ptx1 revealed by the gene knockout may reflect features of the mammalian jaw and hindlimbs that were acquired at a similar time during tetrapod evolution (Lanctôt, 1999b).

Ptx1 mutation affects mandibular structures whose function has changed during transition from a reptilian to a mammalian jaw joint. These correspond to the tympanic and gonial bones that derive from the angular bone of primitive tetrapods, and to the proximal part of the mandibular bone, which may be considered an extension of the ancestral dentary bone. The hindlimb defects may also represent the loss of a function acquired during limb evolution. Some fossil snakes as well as some pythons have vestigial hindlimbs that resemble those of the Ptx1 -/- mice in that they have tibia and fibula of similar size, simple joint structure, and a small ilium. The association of hindlimb and mandibular deficiencies in Ptx1 null mice may reflect the parallel evolution of these structures (Lanctôt, 1999).

The Bicoid-related transcription factor Pitx2 is rapidly induced by the Wnt/Dvl/ß-catenin pathway and is required for effective cell-type-specific proliferation by directly activating specific growth-regulating genes. Wnt signaling, in acting upstream of Pitx2, directly induces Pitx2 gene expression, based on the recruitment of LEF1 to evolutionary-conserved sites in the Pitx2 gene 5'-regulatory regions, with a regulated exchange of HDAC1 for ß-catenin occurring on these Pitx2 sites. Regulated exchange of HDAC1/ß-catenin converts Pitx2 from repressor to activator, analogous to control of TCF/LEF1. Pitx2 then serves as a competence factor required for the temporally ordered and growth factor-dependent recruitment of a series of specific coactivator complexes that prove necessary for Cyclin D2 gene induction (Kioussi, 2002).

Pituitary gland development serves as an excellent model system in which to study the emergence of distinct cell types from a common primordium in mammalian organogenesis. The role of the morphogen Sonic hedgehog (SHH) in outgrowth and differentiation of the pituitary gland has been investigated using loss- and gain-of-function studies in transgenic mice. Shh is expressed throughout the ventral diencephalon and the oral ectoderm, but its expression is subsequently absent from the nascent Rathke's pouch as soon as it becomes morphologically visible, creating a Shh boundary within the oral epithelium. Oral ectoderm/Rathke's pouch-specific 5' regulatory sequences (Pitx1HS) from the bicoid related pituitary homeobox gene (Pitx1) were used to target overexpression of the Hedgehog inhibitor Hip (Huntingtin interacting protein) to block Hedgehog signaling. It was found that SHH is required for proliferation of the pituitary gland. In addition, evidence is provided that Hedgehog signaling, acting at the Shh boundary within the oral ectoderm, may exert a role in differentiation of ventral cell types (gonadotropes and thyrotropes) by inducing Bmp2 expression in Rathke's pouch, which subsequently regulates expression of ventral transcription factors, particularly Gata2. Furthermore, the data suggest that Hedgehog signaling, together with FGF8/10 signaling that arises from the dorsally located infundibulum, synergizes to regulate expression of the LIM homeobox gene Lhx3, which has been proved to be essential for initial pituitary gland formation. Thus, SHH appears to exert effects on both proliferation and cell-type determination in pituitary gland development (Treier, 2001).

Activation of the Wnt pathway results in rapid recruitment of the Pitx2 gene and binding of Pitx2 to promoters of specific growth control genes. This linkage between the Wnt pathway and Pitx2 gene expression provides an insight into the molecular mechanisms of cell type-specific proliferation, based on the required actions of Pitx2 to activate specific, critical growth-control gene targets acting in G1. Based on in vivo studies, as well as the actions in pituitary and muscle cell models, Pitx2 is required for normal proliferation when expressed in heterologous cells; Pitx2 can actually inhibit proliferation. It is speculated that this may occur by squelching coregulatory factors required by DNA binding transcription factors that exert analogous functions to Pitx2. To subserve its proliferative effects, Pitx2 must bind to its cognate DNA sites and requires an N-terminal activation domain, but not the C terminus. Together, these data suggests that three independent events underlie Pitx2-dependent activation of cell type-specific proliferation: Wnt-dependent activation of Pitx2; Wnt and growth factor-dependent relief of Pitx2 repression function; and serial recruitment of a series of specific coactivator complexes that act in a promoter-specific manner, analogous to effects of β-catenin on LEF1 (Kioussi, 2002).

bicoid: Biological Overview | Regulation | Targets of Activity | Protein Interactions | Miscellaneous Interactions | Developmental Biology | Effects of Mutation | References

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