Drosophila Homeobox Gene Research Articles

Expression of the Drosophila homeobox gene, Distal-less, supports an ancestral role in neural development.

Distal-less (Dll) encodes a homeodomain transcription factor expressed in developing appendages of organisms throughout metazoan phylogeny. Based on earlier observations in the limbless nematode Caenorhabditis elegans and the primitive chordate amphioxus, it was proposed that Dll had an ancestral function in nervous system development. Consistent with this hypothesis, Dll is necessary for the development of both peripheral and central components of the Drosophila olfactory system. Furthermore, vertebrate homologs of Dll, the Dlx genes, play critical roles in mammalian brain development. Using fluorescent immunohistochemistry of fixed samples and multiphoton microscopy of living Drosophila embryos, we show that Dll is expressed in the embryonic, larval and adult central nervous system and peripheral nervous system (PNS) in embryonic and larval neurons, brain and ventral nerve cord glia, as well as in PNS structures associated with chemosensation. In adult flies, Dll expression is expressed in the optic lobes, central brain regions and the antennal lobes. Characterization of Dll expression in the developing nervous system supports a role of Dll in neural development and function and establishes an important basis for determining the specific functional roles of Dll in Drosophila development and for comparative studies of Drosophila Dll functions with those of its vertebrate counterparts.

Homeodomain proteins: an update.

Here, we provide an update of our review on homeobox genes that we wrote together with Walter Gehring in 1994. Since then, comprehensive surveys of homeobox genes have become possible due to genome sequencing projects. Using the 103 Drosophila homeobox genes as example, we present an updated classification. In animals, there are 16 major classes, ANTP, PRD, PRD-LIKE, POU, HNF, CUT (with four subclasses: ONECUT, CUX, SATB, and CMP), LIM, ZF, CERS, PROS, SIX/SO, plus the TALE superclass with the classes IRO, MKX, TGIF, PBC, and MEIS. In plants, there are 11 major classes, i.e., HD-ZIP (with four subclasses: I to IV), WOX, NDX, PHD, PLINC, LD, DDT, SAWADEE, PINTOX, and the two TALE classes KNOX and BEL. Most of these classes encode additional domains apart from the homeodomain. Numerous insights have been obtained in the last two decades into how homeodomain proteins bind to DNA and increase their specificity by interacting with other proteins to regulate cell- and tissue-specific gene expression. Not only protein-DNA base pair contacts are important for proper target selection; recent experiments also reveal that the shape of the DNA plays a role in specificity. Using selected examples, we highlight different mechanisms of homeodomain protein-DNA interaction. The PRD class of homeobox genes was of special interest to Walter Gehring in the last two decades. The PRD class comprises six families in Bilateria, and tinkers with four different motifs, i.e., the PAIRED domain, the Groucho-interacting motif EH1 (aka Octapeptide or TN), the homeodomain, and the OAR motif. Homologs of the co-repressor protein Groucho are also present in plants (TOPLESS), where they have been shown to interact with small amphipathic motives (EAR), and in yeast (TUP1), where we find an EH1-like motif in MATα2.Electronic supplementary materialThe online version of this article (doi:10.1007/s00412-015-0543-8) contains supplementary material, which is available to authorized users.

Regulation and Functions of the lms Homeobox Gene during Development of Embryonic Lateral Transverse Muscles and Direct Flight Muscles in Drosophila

BackgroundPatterning and differentiation of developing musculatures require elaborate networks of transcriptional regulation. In Drosophila, significant progress has been made into identifying the regulators of muscle development and defining their interactive networks. One major family of transcription factors involved in these processes consists of homeodomain proteins. In flies, several members of this family serve as muscle identity genes to specify the fates of individual muscles, or groups thereof, during embryonic and/or adult muscle development. Herein, we report on the expression and function of a new Drosophila homeobox gene during both embryonic and adult muscle development.Methodology/Principal FindingsThe newly described homeobox gene, termed lateral muscles scarcer (lms), which has yet uncharacterized orthologs in other invertebrates and primitive chordates but not in vertebrates, is expressed exclusively in subsets of developing muscle tissues. In embryos, lms is expressed specifically in the four lateral transverse (LT) muscles and their founder cells in each hemisegment, whereas in larval wing imaginal discs, it is expressed in myoblasts that develop into direct flight muscles (DFMs), which are important for proper wing positioning. We have analyzed the regulatory inputs of various other muscle identity genes with overlapping or complementary expression patterns towards the cell type specific regulation of lms expression. Further we demonstrate that lms null mutants exhibit reduced numbers of embryonic LT muscles, and null mutant adults feature held-out-wing phenotypes. We provide a detailed description of the pattern and morphology of the direct flight muscles in the wild type and lms mutant flies by using the recently-developed ultramicroscopy and show that, in the mutants, all DFMs are present and present normal morphologies.Conclusions/SignificanceWe have identified the homeobox gene lms as a new muscle identity gene and show that it interacts with various previously-characterized muscle identity genes to regulate normal formation of embryonic lateral transverse muscles. In addition, the direct flight muscles in the adults require lms for reliably exerting their functions in controlling wing postures.

The cap 'n' collar family member NF-E2-related factor 3 (Nrf3) is expressed in mesodermal derivatives of the avian embryo

NF-E2-related factor 3 (Nrf3) is a recently identified member of a family of transcription factors homologous to the Drosophila "cap 'n' collar" or CNC protein. The cnc gene is located immediately 3' to the Drosophila homeobox gene cluster and has been shown to regulate at least one of those genes, deformed. Likewise, human and mouse CNC homologues are located immediately 3' to each of the four Hox complexes, although no genetic interactions have yet been demonstrated in vertebrates. Work presented here demonstrates that Nrf3, adjacent to the Hox A cluster, is expressed during early development of the chicken embryo. Expression begins in the presumptive heart myocardium from the time of cardiac tube fusion through the looping process. Nrf3 transcripts then disappear from the heart and are next observed in the myotomal compartment of maturing somites, restricted to the medial portion along the rostrocaudal axis and fading after muscle precursors migrate. Central nervous system expression appears gradually and persists at low levels in ventricular neuroepithelial cells until at least embryonic day 6. Strong expression is observed in the early epiphysis, in the collecting ducts of the developing kidney and in individual cells of the yolk sac, underlying blood islands. This is the first description using in situ hybridization of the expression of a CNC family member and its dynamics through the course of early development.

Nk6, a novel Drosophila homeobox gene regulated by vnd

Nk( x)-type homeobox genes are an evolutionarily conserved family that regulate diverse developmental processes. Here we describe a novel Drosophila gene, Nk6, which encodes an Nk-type transcription factor most homologous to vertebrate Nkx6.1 and Nkx6.2. The homeodomains and NK decapeptide domains of all three proteins are highly conserved. Nk6 is expressed in the embryonic brain, ventral nerve cord, hindgut, and internal head structures. Nerve cord expression is in midline precursors, several ventral and intermediate column neuroblasts, and later in neurons but not glia, similar to the known expression of Nkx6 genes in the neural tube. We show genetically that Nk6 is positively regulated, directly or indirectly, by vnd in brain precursors. In vnd mutants, head neuroectoderm Nk6 expression is abolished where it is normally co-expressed with vnd. Conversely, vnd-overexpression leads to ectopic Nk6 expression in the brain. These findings further highlight the importance of interactions between Nk( x)-type genes in regulating their expression.

CDX-1 and CDX-2 are expressed in human colonic mucosa and are down-regulated in patients with Hirschsprung’s disease associated enterocolitis

Caudal type homeobox gene-1 and -2 (CDX-1 and CDX-2), homologues of the Drosophila homeobox gene caudal, encode transcription factors in endoderm derived tissues of the intestine. CDX genes control proliferation and differentiation of intestinal mucosal cells and colon cancer cells. Hirschsprung’s Disease (HD) or congenital intestinal aganglionosis, a major developmental anomaly of intestine, which causes functional intestinal obstruction, is frequently associated with enterocolitis. Aetiology of HD-associated enterocolitis (HDEC) remains obscure. Reduction of gut mucosal enteroendocrine cells, and inefficient transfer of the secretory immunoglobulin A across the gut mucosal cell were shown to be associated with enterocolitis in HD patients suggesting that mucosa may directly involve in the pathophysiology of HDEC. This study aims to ascertain whether the CDX-1 and CDX-2 genes, that control the proliferation and differentiation of mucosal cells, play a role in HDEC. Using semi-quantitative reverse transcription–polymerase chain reaction (RT–PCR) and in situ hybridisation, we analysed the expression of CDX-1 and CDX-2 genes in colon specimens of normal controls, necrotising enterocolitis (NEC) infants, and HD patients with and without enterocolitis. We showed for the first time that CDX-1 and CDX-2 genes were expressed in the colonic mucosal epithelium in normal, NEC and in HD infants. However, the expressions of both genes were reduced in patients with HDEC. Our findings suggest that reduced expression of CDX-1 and CDX-2 genes in mucosa may be associated with the development of HDEC.

A cluster of Drosophila homeobox genes involved in mesoderm differentiation programs

Although genes involved in common developmental programs are usually scattered throughout the metazoan genome, there are some important examples of functionally interconnected regulatory genes that display close physical linkage. In particular the homeotic genes, which determine the identities of body parts, are clustered in the Hox complexes and clustering is thought to be crucial for the proper execution of their developmental programs. Here we describe the organization and functional properties of a more recently identified cluster of six homeobox genes at 93DE on the third chromosome of Drosophila. These genes, which include tinman, bagpipe, ladybird early, ladybird late, C15, and slouch, all participate in mesodermal patterning and differentiation programs and show multiple regulatory interactions among each other. We propose that their clustering, through unknown mechanisms, is functionally significant and discuss the similarities and differences between the 93DE homeobox gene cluster and the Hox complexes. BioEssays 23:125–133, 2001. © 2001 John Wiley & Sons, Inc.

A cluster of Drosophila homeobox genes involved in mesoderm differentiation programs.

Although genes involved in common developmental programs are usually scattered throughout the metazoan genome, there are some important examples of functionally interconnected regulatory genes that display close physical linkage. In particular the homeotic genes, which determine the identities of body parts, are clustered in the Hox complexes and clustering is thought to be crucial for the proper execution of their developmental programs. Here we describe the organization and functional properties of a more recently identified cluster of six homeobox genes at 93DE on the third chromosome of Drosophila. These genes, which include tinman, bagpipe, ladybird early, ladybird late, C15, and slouch, all participate in mesodermal patterning and differentiation programs and show multiple regulatory interactions among each other. We propose that their clustering, through unknown mechanisms, is functionally significant and discuss the similarities and differences between the 93DE homeobox gene cluster and the Hox complexes.

Specificity and Efficiency of Cre-Mediated Recombination in Emx1–cre Knock-in Mice

Emx1 is a mouse homologue of the Drosophila homeobox gene empty spiracles and its expression is restricted to the neurons in the developing and adult cerebral cortex and hippocampus. We reported previously the creation of a line of transgenic mice in which the cre gene was placed directly downstream of the putative Emx1 promoter using ES cell technology. We showed that Cre protein was present in the cerebral cortex of the transgenic mice and was able to mediate loxP-specific recombination in vitro. In the present study, the specificity and efficiency of the cre-mediated recombination were determined using three independent lines of reporter mice and a combination of histochemical staining, neuronal culture, and Southern detection of the genomic DNA. Our results showed that the recombination was highly efficient in all three lines of reporter mice tested and confirmed that the deletion was restricted to the neurons in the cerebral cortex and hippocampus. Furthermore, we have determined that the recombination efficiency in the cerebral cortex was 91%. Our results suggest that Emx1 is not expressed in every neuron in the developing and adult cerebral cortex. This line of cre mice should contribute to the studies of cortical development and plasticity.

The Drosophila homeobox gene optix is capable of inducing ectopic eyes by an eyeless-independent mechanism.

optix is a new member of the Six/so gene family from Drosophila that contains both a six domain and a homeodomain. Because of its high amino acid sequence similarity with the mouse Six3 gene, optix is considered to be the orthologous gene from Drosophila rather than sine oculis, as previously believed. optix expression was detected in the eye, wing and haltere imaginal discs. Ectopic expression of optix leads to the formation of ectopic eyes suggesting that optix has important functions in eye development. Although optix and sine oculis belong to the same gene family (Six/so) and share a high degree of amino acid sequence identity, there are a number of factors which suggest that their developmental roles are different: (1) the expression patterns of optix and sine oculis are clearly distinct; (2) sine oculis acts downstream of eyeless, whereas optix is expressed independently of eyeless; (3) sine oculis functions synergistically with eyes absent in eye development whereas optix does not; (4) ectopic expression of optix alone, but not of sine oculis can induce ectopic eyes in the antennal disc. These results suggest that optix is involved in eye morphogenesis by an eyeless-independent mechanism.

Structure, mapping and expression of the human gene encoding the homeodomain protein, SIX2.

Vertebrate genes with sequence similarity to the Drosophila homeobox gene, sine oculis (so), constitute the SIX family. There is notable expression of members of this family in anterior neural structures, and several SIX genes have been shown to play roles in vertebrate and insect development, or have been implicated in maintenance of the differentiated state of tissues. Mutations in three of these genes in man (SIX5, SIX6 and SIX3) are associated with severe phenotypes, and therefore, the cloning of other human genes from this family is of interest. We have cloned and characterised the gene that encodes human SIX2, elucidated its gene structure and conducted expression studies in a range of tissues. SIX2 is widely expressed in the late first-trimester fetus, but has a limited range of expression sites in the adult. The expression pattern of SIX2 and its localisation to chromosome 2p15-p16 will be of use in assessing its candidacy in human developmental disorders.

Characterisation of the promoter region of the zebrafish six7 gene

The Drosophila homeobox gene sine oculis and its murine homologue Six3 have both been shown to have regulatory functions in eye and brain development. In zebrafish, three Six3-related genes with conserved expression during early eye and head formation have been identified. One of these, six7, is first expressed at the gastrula stage in the involuting axial mesoderm, and later in the overlying neuroectoderm from which the forebrain and optic primordium develop. To elucidate the mechanisms regulating six7 expression, we isolated a 2.7-kb fragment of the 5′-flanking region. Three sequentially deleted fragments of this upstream region were used to produce GFP reporter constructs for analysis of tissue-specific expression in zebrafish embryos. The results show that a 625-bp upstream fragment is sufficient to direct strong expression of the reporter during gastrulation and early neurulation. The proximal part of the promoter contains binding sites for various constitutive transcription factors and an additional upstream element that was shown to be critical in directing expression to the anterior region of the zebrafish brain.

Emx1-Specific Expression of Foreign Genes Using “Knock-in” Approach

Emx1 is a mouse homologue of the Drosophila homeobox gene empty spiracles. Its expression is limited to the neurons in developing and adult cerebral cortex and hippocampus. Because of the highly restricted expression pattern of the Emx1 gene, it would be quite desirable to characterize the promoter of the Emx1 for directing foreign gene expression in the transgenic mouse. We report here that we have achieved the Emx1-specific expression in transgenic mice by inserting the lacZ reporter and cre genes directly into the exon 1 of the Emx1 gene using embryonic stem (ES) cell technology. The distribution of the β-galactosidase activity in the transgenic mice was consistent with the published results obtained using in situ hybridization and immunohistochemistry. Furthermore, we have demonstrated that Cre protein was present in the cerebral cortex of the transgenic mice and was able to mediate loxP-specific recombination in vitro. The creation of this line of cre transgenic mice, and the demonstration that the insertion site located in the exon 1 of the Emx1 gene could render foreign genes a specific expression pattern restricted to the developing and adult cerebral cortex and hippocampus, should be conducive to further studies of the effect of a gene mutation or overexpression upon the development and plasticity of cerebral cortex and hippocampus.

Cardiac enhancer activity of the homeobox genetinman depends on CREB consensus binding sites inDrosophila

The Drosophila homeobox gene tinman plays a critical role in subdividing the early mesoderm. In particular, tinman is absolutely required for formation of the heart and visceral mesoderm. tinman expression is initiated throughout the mesoderm of the trunk region under the control of the bHLH transcription factor encoded by the twist gene, a determinant of all mesoderm. Later, tinman expression is restricted to the dorsal portion of the mesoderm, a process that is directed by decapentaplegic (dpp) whose product (a TGF-beta-related protein) is secreted by the overlaying ectoderm. Further restriction of tinman expression to the cardiac progenitors, in which it will persist throughout development, involves the secreted segmentation gene product encoded by wingless (wg, a Drosophila Wnt gene). Here, we show that strong early expression depends on the synergistic action of an enhancer element at the 5' end of the gene in conjunction with an element in the first intron. Moreover, two distinct enhancer regions are responsible for tinman expression in the heart: one region confers expression in the heart-tube-associated pericardial cells, the other element drives expression in the contractile, myocardial cells. The latter element contains two CREB consensus binding sites that are essential for cardiac-specific expression. genesis 26:55-66, 2000.

Cardiac enhancer activity of the homeobox gene tinman depends on CREB consensus binding sites in Drosophila

Summary: The Drosophila homeobox gene tinman plays a critical role in subdividing the early mesoderm. In particular, tinman is absolutely required for formation of the heart and visceral mesoderm. tinman expression is initiated throughout the mesoderm of the trunk region under the control of the bHLH transcription factor encoded by the twist gene, a determinant of all mesoderm. Later, tinman expression is restricted to the dorsal portion of the mesoderm, a process that is directed by decapentaplegic (dpp) whose product (a TGF-β-related protein) is secreted by the overlaying ectoderm. Further restriction of tinman expression to the cardiac progenitors, in which it will persist throughout development, involves the secreted segmentation gene product encoded by wingless (wg, a Drosophila Wnt gene). Here, we show that strong early expression depends on the synergistic action of an enhancer element at the 5′ end of the gene in conjunction with an element in the first intron. Moreover, two distinct enhancer regions are responsible for tinman expression in the heart: one region confers expression in the heat tube associated pericardial cells, the other element drives expression in the contractile, myocardial cells. The latter element contains two CREB consensus binding sites that are essential for cardiac-specific expression. genesis 26:55–66, 2000. © 2000 Wiley-Liss, Inc.

Delineation of multiple deleted regions in 7q in myeloid disorders.

Loss of chromosome material due to deletions of the long arm of chromosome 7, del(7q), is a consistent finding in all types of myeloid disorders, invariably associated with a poor prognosis. Two different segments, 7q22 and 7q32-q33, have been implicated as critical regions of gene loss associated with these disorders. In the present study, we used fluorescence in situ hybridization (FISH) to characterize the 7q22 breakpoint of an apparently balanced t(7;7)(p13;q22) in an acute myeloid leukemia patient. FISH analysis on bone marrow metaphases from this patient revealed that the sequence corresponding to a series of three ordered cosmids from 7q22 was deleted from one of the der(7) chromosomes. These cosmids contain the human homologue of the Drosophila homeobox gene cut (CUTL1) and span a region of approximately 150 kb. Although the proximal boundary of the deleted segment could not be exactly defined, we estimate the size of this deletion to be approximately 500 kb. Subsequently, we carried out FISH studies using the CUTL1 cosmids on a further 16 patients with deletions of 7q and myeloid disorders. The sequence corresponding to at least two of the cosmids was deleted from the del(7q) in 11 out of 14 cases with a proximal breakpoint within 7q22. Further detailed FISH mapping in this series of 17 patients has identified two other nonoverlapping commonly deleted segments at 7q31-q32 and 7q33, respectively. These data confirm and refine other studies, implying that several different genes on 7q may be involved in the pathogenesis of myeloid diseases. Genes Chromosomes Cancer 25:384-392, 1999.

Six class homeobox genes in Drosophila belong to three distinct families and are involved in head development

The vertebrate Six genes are homologues of the Drosophila homeobox gene sine oculis ( so), which is essential for development of the entire visual system. Here we describe two new Six genes in Drosophila, D-Six3 and D-Six4, which encode proteins with strongest similarity to vertebrate Six3 and Six4, respectively. In addition, we report the partial sequences of 12 Six gene homologues from several lower vertebrates and show that the class of Six proteins can be subdivided into three major families, each including one Drosophila member. Similar to so, both D-Six3 and D-Six4 are initially expressed at the blastoderm stage in narrow regions of the prospective head and during later stages in specific groups of head midline neurectodermal cells. D-Six3 may also be essential for development of the clypeolabrum and several head sensory organs. Thus, the major function of the ancestral Six gene probably involved specification of neural structures in the cephalic region.

A Double Interaction Screen identifies positive and negative ftz gene regulators and Ftz-interacting proteins

Regulatory genes directing embryonic development are expressed in complex patterns. The Drosophila homeobox gene fushi tarazu ( ftz) is expressed in a striped pattern that is controlled by several discrete and large cis- regulatory elements. One key cis-element is the ftz proximal enhancer which is required for stripe establishment and which mediates autoregulation by direct binding of Ftz protein. To identify the trans-acting factors that regulate ftz expression and autoregulation, we developed a modified yeast two hybrid screen, the Double Interaction Screen (DIS). The DIS was designed to isolate both DNA binding transcriptional regulators that interact with the proximal enhancer and proteins that interact with Ftz itself when it is bound to the enhancer. The screen identified two candidate Ftz protein cofactors as well as activators and repressors of ftz transcription that bind directly to the enhancer. One of these (Tramtrack Ttk) was previously shown to bind to at least five sites in the proximal enhancer; genetic studies suggested that Ttk acts as a repressor of ftz in the embryo. Here we show that, in yeast cells, Ttk protein strongly activates transcription, suggesting that yeast may be missing a necessary co-repressor which is present in Drosophila embryos. Further, we have characterized the activity of a second candidate ftz repressor isolated in the screen – the product of the pair-rule gene sloppy paired – a member of the forkhead family. We show that Slp1 is a DNA binding protein. We have identified a high affinity binding site for Slp1 in the ftz proximal enhancer. Slp1 represses transcription via this binding site in yeast cells, consistent with its role as a direct repressor of ftz stripes in interstripe regions during late stages of embryogenesis. The DIS should be a generally useful method to identify DNA binding transcriptional regulators and protein partners of previously characterized DNA binding proteins.

Dorsoventral patterning in the Drosophila central nervous system: the intermediate neuroblasts defective homeobox gene specifies intermediate column identity.

One of the first steps in neurogenesis is the diversification of cells along the dorsoventral axis. In Drosophila the central nervous system develops from three longitudinal columns of cells: ventral cells that express the vnd/nk2 homeobox gene, intermediate cells, and dorsal cells that express the msh homeobox gene. Here we describe a new Drosophila homeobox gene, intermediate neuroblasts defective (ind), which is expressed specifically in the intermediate column cells. ind is essential for intermediate column development: Null mutants have a transformation of intermediate to dorsal column neuroectoderm fate, and only 10% of the intermediate column neuroblasts develop. The establishment of dorsoventral column identity involves negative regulation: Vnd represses ind in the ventral column, whereas ind represses msh in the intermediate column. Vertebrate genes closely related to vnd (Nkx2.1 and Nkx2.2), ind (Gsh1 and Gsh2), and msh (Msx1 and Msx3) are expressed in corresponding ventral, intermediate, and dorsal domains during vertebrate neurogenesis, raising the possibility that dorsoventral patterning within the central nervous system is evolutionarily conserved.

Drosophila engrailed can substitute for mouse Engrailed1 function in mid-hindbrain, but not limb development.

The Engrailed-1 gene, En1, a murine homologue of the Drosophila homeobox gene engrailed (en), is required for midbrain and cerebellum development and dorsal/ventral patterning of the limbs. In Drosophila, en is involved in regulating a number of key patterning processes including segmentation of the epidermis. An important question is whether, during evolution, the biochemical properties of En proteins have been conserved, revealing a common underlying molecular mechanism to their diverse developmental activities. To address this question, we have replaced the coding sequences of En1 with Drosophila en. Mice expressing Drosophila en in place of En1 have a near complete rescue of the lethal En1 mutant brain defect and most skeletal abnormalities. In contrast, expression of Drosophila en in the embryonic limbs of En1 mutants does not lead to repression of Wnt7a in the embryonic ventral ectoderm or full rescue of the embryonic dorsal/ventral patterning defects. Furthermore, neither En2 nor en rescue the postnatal limb abnormalities that develop in rare En1 null mutants that survive. These studies demonstrate that the biochemical activity utilized in mouse to mediate brain development has been retained by Engrailed proteins across the phyla, and indicate that during evolution vertebrate En proteins have acquired two unique functions during embryonic and postnatal limb development and that only En1 can function postnatally.