ladybird early and ladybird late: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene names - ladybird early and ladybird late

Synonyms -

Cytological map position - 93D9--93E2

Function - transcription factor

Keyword(s) - segment polarity, CNS, PNS, brain, antenna

Symbols - lbe and lbl

FlyBase ID:FBgn0011278 and FBgn0008651

Genetic map position - 3-[72]

Classification - homeobox

Cellular location - nuclear



NCBI links: Ladybird early Precomputed BLAST | Entrez Gene


Ladybird late Precomputed BLAST | Entrez Gene


BIOLOGICAL OVERVIEW

The ladybird early and ladybird late genes are homeobox genes arranged in tandem, located in the Drosophila 93E homeobox gene cluster. The pattern of lbe and lbl transcription is complex; in a critical expression domain it overlaps that of wingless in the epidermis. This overlap suggests that lbe and lbl contribute to the segmentation process and that they may more properly belong to the family of segment polarity genes (Jagla, 1994).

While early wingless transcription is dependent on Hedgehog, Hedgehog receptors Patched and Smoothened, and a signal transduction apparatus including Fused and Cubitus interruptus, subsequent expression is maintained by a wingless autoregulation pathway involving Armadillo, Shaggy/Zeste white and cAMP-dependent Protein kinase A. Regulation of wingless expression is even more complex, since wingless expression in the ventral epidermis is maintained by Gooseberry, and in the dorsal epidermis by Lbe and Lbl. winglessseems to have become the prototype for genes subject to complex multistage temporal regulation.

The belief that lbe and lbl are targets of wingless signaling and that they also regulate wingless transcription is based on genetic and transcriptional evidence. lbe and lbl are transcribed in a pattern that matches the expression of wingless. lbe and lbl transcription requires wingless as well as Armadillo, a component of the wingless pathway.

In order to test the influence of lbe and lbl on wingless expression, embryos lacking lbe and lbl have been tested for wingless expression. These embryos lack Wg protein in the labrum and anal plate and have reduced levels of Wg in the dorsal epidermis. Their cuticular denticles are reduced number and have abnormal pigmentation. The abnormal denticles are restricted to those regions posterior to the segmental border row of cells (the primary row) and include only the more posterior tertiary and quaternary cell types (Bokor, 1996 and Jagla, 1997a). It therefore appears that lack of lbe and lbl produces a partial segment polarity phenotype, in which some elements of segment polarity are normal and others are not (Jagla, 1997a).

What is the relationship between lbe and lbl defects and those caused by gooseberry mutation? gooseberry is required for the maintenance of wingless transcription after this transcription is initially established. Jagla (1997a) argue that gsb is required for the maintenance of wg only in the ventral epidermis, and that lbe and lbl are required for late wg expression in the dorsal and in the terminal epidermis. Embryos carrying a null mutation in lbe and lbl do not show a wg-like cuticle phenotype in the ventral epidermis, probably because wg expression in this region is maintained by gsb (Li, 1993).

The embryonic heart precursors of Drosophila are arranged in a repeated pattern of segmental units. There is growing evidence that the development of individual elements of this pattern depends on both mesoderm intrinsic patterning information and inductive signals from the ectoderm. ladybird early and ladybird late, are involved in the cardiogenic pathway in Drosophila. At early stage 12, lb genes are expressed in clusters of about four cells per hemisegment in the developing heart region. These cells represent a segmental subset of tinman-expressing heart progenitors, which form a continuous row at the dorsal crest of the mesoderm at this stage. even-skipped expression begins at a slightly earlier time than lb in similar clusters of cells. It appears that two cells from each segmental eve cluster develop into a particular type of pericardial cells, termed e-PCs. Double stainings for lb and eve expression demonstrate that the e-PC progenitors are distinct from the lb-expressing heart progenitors and located posteriorly adjacent to them in each segment. Similar stainings of embryos at later stages show that the lb-expressing cells give rise to a subpopulation of cardioblasts (CBs) and a second type of pericardial cells, termed l-PCs. Cell rearrangements during stage 12, which involve a 90° clockwise rotation of the heart progenitor clusters within each segment, place the lb-expressing cells at the dorsal side and move the eve-expressing cells ventrally to them. This morphogenetic process results in a dorsal row of cardioblasts and ventrolaterally adjacent rows of pericardial cells on either side of the embryo. At stage 14, generally four out of six cardioblasts per hemisegment express both tin and mef-2. Double stainings with Lb antibodies show that the two anterior tin- and mef-2-expressing cardioblasts in each hemisegment co-express lb. In addition, tin and lb are co-expressed in the l-PCs, which are located ventrally below the cardioblasts. However, lb is not expressed in the e-PCs, which are found in more lateral positions at this stage. These results indicate a diversification among cardioblasts of each segment, as well as among the pericardial cells, that is already apparent during stage 11 (Jagla, 1997b).

LBE and LBL are required for anal plate formation. The anal plate does not develop in lbe and lbl mutants. The activator of lbe and lbl gene activity in the anal plate is most likely the homeotic gene forkhead, which governs terminal development. Alternatively, the homeobox gene caudal, required for the anal pads, tuft and anal sense organ formation, could be part of the genetic circuitry that switches on the wg-ladybird autoregulatory loop in the terminal region (Jagla, 1997a).

The analysis of ladybird regulation of epidermal development suggests that a temporal hierarchy of cuticle and denticle development exists in the epidermis and that lbe and lbl are involved in regulating the later stages of this process.

Genome-wide view of cell fate specification: ladybird acts at multiple levels during diversification of muscle and heart precursors

Correct diversification of cell types during development ensures the formation of functional organs. The evolutionarily conserved homeobox genes from ladybird/Lbx family were found to act as cell identity genes in a number of embryonic tissues. A prior genetic analysis showed that during Drosophila muscle and heart development ladybird is required for the specification of a subset of muscular and cardiac precursors. To learn how ladybird genes exert their cell identity functions, muscle and heart-targeted genome-wide transcriptional profiling and a chromatin immunoprecipitation (ChIP)-on-chip search were performed for direct Ladybird targets. The data reveal that ladybird not only contributes to the combinatorial code of transcription factors specifying the identity of muscle and cardiac precursors, but also regulates a large number of genes involved in setting cell shape, adhesion, and motility. Among direct ladybird targets, bric-a-brac 2 gene was identified as a new component of identity code and inflated encoding αPS2-integrin playing a pivotal role in cell-cell interactions. Unexpectedly, ladybird also contributes to the regulation of terminal differentiation genes encoding structural muscle proteins or contributing to muscle contractility. Thus, the identity gene-governed diversification of cell types is a multistep process involving the transcriptional control of genes determining both morphological and functional properties of cells (Junion, 2007).

Uncovering how the cell fate-specifying genes exert their functions and determine unique properties of cells in a tissue is central to understanding the basic rules governing normal and pathological development. To approach the cell fate determination process at a whole genome level a search was performed for transcriptional targets of the homeobox transcription factor Lb known to be evolutionarily conserved and required for specification of a subset of cardiac and muscular precursors. To this end the targeted expression profiling and the novel ChIP-on-chip method ChEST were combined. The data revealed an unexpectedly complex gene network operating downstream from lb, which appears to act not only by regulating components of the cell identity code but also as a modulator of pan-muscular gene expression at fiber-type level. Of note, the role of Drosophila lb in regulating segment border muscle (SBM) founder motility appears reminiscent of the role of its vertebrate ortholog Lbx1, known to control the migration of leg myoblasts (Vasyutina, 2005) in mouse embryos (Junion, 2007).

Earlier genetic studies revealed that within the same competence domain the cell fate specifying factors acted as repressors to down-regulate genes determining the identity of neighboring cells. Consistent with this finding, lb was found to repress msh and kruppel (kr) during diversification of lateral muscle precursors and even skipped (eve) within the heart primordium. This study found that additional identity code components are regulated negatively by lb. In the lateral muscle domain lb acts as a repressor of the MyoD ortholog nau and the NK homeobox gene slou, both known to be required for the specification of a subset of somatic muscles. This suggests that a particularly complex network of transcription factors (Ap, Msh, Kr, Nau, Slou) controls the specification of individual muscle fates in the lateral domain. Interestingly, none of these factors is coexpressed with lb in the SBM, which appears to be a functionally distinct muscle requiring a specific developmental program. Besides factors with well-documented roles in diversification of muscle fibers, the global approach identified a few novel potential players in the muscle identity network. Among them are expressed in somatic muscle precursors the Pdp1 gene encoding Par domain factor and the CG32611 gene containing a zinc finger motif (Junion, 2007).

Interestingly, in the cardiac domain the data demonstrate that lb is able to positively regulate the expression of tin and the effector of RTK pathway pointed (pnt), both involved in cardiac cell fate specification. These findings are consistent with earlier observations that the forced lb expression leads to the ectopic tin-positive cells within the dorsal vessel. Also, during early cardiogenesis lb directly represses bric a brac 2 (bab2), which emerges as a novel component of the genetic cascade controlling the diversification of cardiac cells. Thus, the ability of Lb to act either as repressor or as activator suggests a context-dependent interaction with cofactors. Of note, several miroarray identified Lb targets have also been found in the RNAi-based screen for genes involved in heart morphogenesis (Junion, 2007).

The data indicate that lb exerts its muscle identity functions via regulation of pan-muscular genes that control cell movements, cell shapes and cell-cell interactions including myoblast fusion, myotube growth, and attachment events (Junion, 2007).

Regulation of if provides an example of how the cell-type-specific fine-tuning of expression can be achieved for genes expressed in all muscle precursors. It was reported previously (Junion, 2005) that if is directly regulated by dMef2 via two intronic CRMs, which most probably ensure a generic muscle-specific if expression. Here, a distinct, Lb-dependent if CRM was identified, able to drive expression in a restricted subset of muscles including the SBM, thus indicating that the if transcription is regulated by a coordinated action of generic and muscle-type-specific CRMs. Identification of Lb-dependent CRM close to if also indicates that lb may contribute to the dMef2-regulated feed-forward loops (Sandmann, 2006). Such a modular regulation of transcription levels would provide an efficient way for precise, fiber-type-specific setting of muscle genes activity. As shown for if in the SBM context, this regulation is expected to be direct via the identified here Lb-binding module. The data also suggest that a similar muscle-type-specific transcriptional regulation can be achieved in an indirect mode. For example, the pan-muscular kettin/sls gene encoding a giant protein required for the formation and maintenance of normal sarcomere structure is regulated by three distinct dMef2-binding CRMs able to drive expression in a muscle-type-specific manner (Junion, 2005). Among them, CRM 1 located upstream the gene was found to drive expression in a subset of ventral and lateral muscles including SBM (Junion, 2005). This study identifies sls as an Lb target in the microarray screen, and demonstrates that in embryos overexpressing Lbe the Sls expression is up-regulated. These findings suggest that Lb can act via an as-yet-unknown factor to modulate sls expression in the SBM (Junion, 2007).

The reported here Lb targets fit into three main categories: (1) genes encoding transcription factors contributing to cell-type-specific identity gene code; (2) genes controlling cell shapes, adhesion, and cell motility; and (3) late-acting genes required for functional properties of cardiac and muscle cells. Importantly, Lb is able to bind to enhancers of genes from all these categories. Also, the functional properties of identified Lb targets strongly suggest that Lb-dependent multistep acquisition of cell identity is executed in a similar way in cardiac and in muscle cells. With respect to the Lb targets encoding transcription factors bab2 was identified as a new component of cardiac cell identity code. Considering the regulation of cell shape, cell adhesion, and motility, a large number of Lb targets code for proteins involved in remodeling of the actin cytoskeleton, for the ECM components and for integrins. Down-regulation of the selected microarray candidates from this category leads to dramatic muscle phenotypes affecting founder migration, myoblast fusion or muscle fiber attachment. Among the late-acting genes, expression and function of a direct, ChEST-identified Lb target gene, CG8698, and demonstrated that it was found to be specifically expressed in differentiated muscles and required for muscle contraction. Thus, it is concluded that the Lb-governed acquisition of cell identity is a long-term process (1) initiated by the spatially restricted expression of a set of transcription factors and (2) executed by precise regulation of genes determining the morphological, dynamic and functional properties specific to a given muscle or heart cell type (Junion, 2007).


GENE STRUCTURE

The 93D/E homeobox gene cluster is composed of the two Drosophila genes (lady-bird-late [previously named nkch4] and lady-bird-early) as well as tinman/NK4, bagpipe/NK3, S59/NK1 and 93Ba1 (Jagla, 1994). lbe maps distally to lbl and both genes are transcribed from the opposite DNA strand compared to tin, bap and NK1/S59. The proximal most gene is tinman, followed by bagpipe and the two ladybird genes. The Drosophila homolog of the insulin receptor (inr) intervenes between the ladybird genes and NK1/S59 (Jagla, 1997a).

Bases in 5' UTR - 227 (lbe) and about 150 (lbl)

Exons - 2 (lbe) and 5 (lbl)

Bases in 3' UTR - 378 lbe


PROTEIN STRUCTURE

Amino Acids - 479 for Lbe and 342, 372, and 411 for Lbl

Structural Domains

LBE has a Prd-like repeat (not the same as a paired domain) in the N-terminal region. The homeodomain (Jagla, 1993) is in the C-terminal region followed by a poly-Ala stretch. There is a second poly-Ala stretch between the Prd-like repeat and the homeodomain. LBL is homologous to the C-terminal 4/5ths of LBE, and lacks a Prd-like repeat and poly-Ala stetches. The homology extends beyond the homeodomains, which are 97% homologous, suggesting a relatively recent origin for the duplication (Jagla, 1994). LBL is alternatively spliced with cDNAs having been isolated for three proteins. One of the three has a different C-terminal sequence. lbl genomic and cDNA sequences reveal a large intron ( 20 kb long) upstream of the exon coding for the homeodomain (Jagla, 1997a).


EVOLUTIONARY HOMOLOGS

Two novel homeobox genes are the mouse (Lbx1) and human (LBX1) homologs of the Drosophila ladybird genes. They are highly related not only within the coding region but also in 5' and 3' untranslated regions. Several amino acid residues inside and around the homeodomain, have been conserved between the mammalian Lbx genes and their Drosophila counterparts. The mouse Lbx1 gene is located on chromosome 19 (region D) and the human LBX1 gene maps to the related q24 region of chromosome 10, known as a breakpoint region in translocations t(7;10) and t(10;14) involved in T-cell leukemias. Thus, LBX1 and the protooncogene HOX11 map to a common chromosomal region, as do their Drosophila counterparts, the ladybird and 93Bal genes. The mouse Lbx1 gene is specifically expressed during embryogenesis. From 10.5 days of gestation, Lbx1 expression is detected in the central nervous system and some developing muscles. In the CNS, Lbx1 transcripts are expressed in the dorsal part of the mantle layer of the spinal cord and hindbrain, up to a sharp boundary within the developing metencephalon. Thus, Lbx1 may be involved in spinal cord and hindbrain differentiation and/or patterning; its restricted expression pattern could depend upon evolutionarily conserved inductive signals involving some mammalian Wnt and Pax genes, as is the case for Drosophila ladybird genes and wingless or gooseberry (Jagla, 1995).

In vertebrates all skeletal muscles of trunk and limbs are derived from condensations of the paraxial mesoderm, that is from the somites. Limb muscle precursor cells migrate during embryogenesis from somites to limb buds, where migration stops and differentiation occurs. lbx1 homeobox genes (related to the Drosophila ladybird genes) have been characterized in chicken and mice. These genes are expressed in migrating limb muscle precursor cells in both species. Analysis of splotch mutant mice shows that lbx1 and c-met are differently affected by the lack of Pax-3 (Drosophila homolog: Paired). Limb buds of splotch (Pax-3 mutant) mice are devoid of lbx1 transcripts, while expression of c-met is still detectable at a low level. The presence of c-met-positive cells in splotch mice entering the limbs indicates that migration of cells from somites to limbs is not entirely dependent on Pax-3. Induction of epithelial to mesenchymal transition of Pax-3-positive cells by SF/HGF is not sufficient to induce ectopic lbx-1 expression at the inter-limb level, while ectopic limb formation is able to activate lbx1 expression. It is postulated that Pax-3 is necessary for lbx1 expression in the lateral tips of somites but additional, yet unknown signals derived from limb buds are needed to initiate lbx1 expression. The role of limb bud-derived signals involved in targeted muscle precursor cell migration, and lbx1 activation was further confirmed by analysis of explanted somite/limb bud co-cultures in collagen gels (Mennerich, 1998).

Myogenic differentiation can be initiated by a limited number of molecules. Overexpression of Lbx1 in vivo and in vitro leads to a strong activation of various muscle markers. Cell proliferation, which is strongly stimulated by Lbx1 and Pax3, is required for Lbx1- or Pax3-dependent myogenic activation. Inhibition of cell proliferation prevents expression of muscle differentiation markers, while the activation of other putative downstream targets of Pax3 and Lbx1 is not affected. These findings imply that a critical function of Pax3 and Lbx1 during muscle cell formation is the enlargement of muscle cell populations. The growth of the muscle precursor cell population may increase the bias for myogenic differentiation and thus enable myogenic cells to respond to environmental cues. These results might provide an explanation of how Pax3 and Lbx1 induce myogenesis by amplification of the myogenic precursor cell pool with an increasing potential for myogenic differentiation, and emphasize the critical role of the size of cell populations biased for differentiation (Mennerich, 2001).

Lbx1 staining resides within the expression domain of the lateral marker Sim1, a basic helix-loop-helix transcription factor, indicating that Lbx1 belongs to the lateral program of the somite. However, Sim1 staining extends throughout the lateral half of the dermomyotome and, at lower levels, encompasses the lateral sclerotome. Similar to Sim1, the putative VEGF receptor Quek1 and the recently cloned divergent homeobox gene Meis2 also occupy a wide lateral territory. Thus, Lbx1 marks a sub-population of lateral somite cells only. Within the lateral somite, expression of Lbx1 co-localizes with elevated signals for the paired and homeobox containing transcription factor Pax3, and with signals for the receptor tyrosine kinase c-Met, both thought to be indicative of prospective hypaxial myoblasts. However, Pax3 expression commences throughout the dorsoventral perimeter of the anterior segmental plate, and continues in the medial and lateral dermomyotome before the up-regulation in the lateral dermomyotomal lip takes place. Likewise, c-Met displays an additional expression domain in the medial dermomyotomal lip. Therefore, while the mechanisms leading to the dorsally restricted expression pattern of Sim1, the lateral up-regulation of Pax3 and the lateral activation of c-Met may be related to those stimulating Lbx1, Lbx1 is the only known marker that is exclusive for the lateral dermomyotomal lip. Unlike Sim1, Pax3 and c-Met, Lbx1 expression is excluded from flank regions, where the lateral dermomyotomal lips remain epithelial, and hypaxial muscle precursors are added to the lateral myotome during demomyotomal elongation. In such flank regions myoblasts begin to express muscle-determining genes as soon as they enter the lateral myotome. In contrast, where Lbx1 is expressed, the lateral dermomyotomal lips de-epithelialise, and activation of muscle determining genes appears delayed. Thus, the expression pattern of Lbx1 along the anteroposterior axis prefigures the different fates of lateral dermomyotomal lips, before they become morphologically evident: the gene labels lateral dermomyotomal lips bound to de-epithelialise and to generate late differentiating hypaxial muscle precursors. Occipitally and at the level of the limbs, the dispersing lateral dermomyotomal lips release migratory muscle precursors that head toward branchial arches and limbs to provide the intrinsic tongue and limb musculature, respectively. During the course of their migration, these cells continue to express Lbx1. In mammals, cervical cells populating the septum transversum to form the muscular diaphragm are also Lbx1 positive. In chick and mouse, Lbx1 is down-regulated when the migrating cells settle at their target sites and initiate expression of the muscle determining genes. Thus, Lbx1 serves as a bona fide marker for muscle precursors capable of long range migration (Dietrich, 1998 and references).

During development of the amniote embryo, the dorsolateral territory of the somite is destined to give rise to the hypaxial skeletal musculature. The lateral mesoderm has been suggested to emit signals required for somite lateralization. In line with these studies, separation of paraxial mesoderm from intermediate and lateral mesoderm prevents expression of Lbx1 and upregulation of Pax3. Similarly, when an additional strip of paraxial mesoderm is inserted parallel to the endogenous one, only the strip in contact with the lateral structures expresses the lateral markers. Lastly, when strips of paraxial mesoderm are grafted on top of the notochord after neural tube removal, the lateral markers are lacking in the graft, while the medial markers are found in their normal dorsoventrally restricted domains. This emphasizes that the formation of the hypaxial musculature belongs to the lateral developmental pathway, since it depends on appositional signaling by the lateral structures (Dietrich, 1998 and references).

To study the mechanisms that lead to the formation of this musculature, Lbx1 was used as a marker for the anatomical structures that produce the signals necessary for the specification of the hypaxial musculature. These signals have been characterized by ablating muscles or transplanting them to ectopic locations in the chick embryo. In addition, BMP4 soaked beads were inserted medial to the somite. The data suggest that lateralizing signals from intermediate and lateral mesoderm have to synergize with dorsalizing signals from the surface ectoderm to induce the formation of the hypaxial musculature. However, the lateralizing function of the lateral mesoderm can only in part be mimicked by BMP4. The following model is proposed for the induction of hypaxial musculature: in the paraxial mesoderm, lateral identities are induced and medial identities are repressed by intermediate and lateral mesoderm, involving BMP4 as a general lateralizing signal, and possibly additional factors involved in the specification of the hypaxial musculature. Within the lateral somite half, dorsal identities are induced by the surface ectoderm, possibly via WNT signalling. The dorsalizing signals may be antagonized by signals released by the underlying aorta or endoderm. However, where the lateralizing and dorsalizing signals synergize, formation of the hypaxial musculature is induced, as monitored by the upregulation of Pax3, and at occipital, cervical and limb levels, by the expression of the novel marker Lbx1 (Dietrich, 1998).

During vertebrate embryogenesis, myogenic precursor cells of limb muscles delaminate from the ventro-lateral edge of the somitic dermomyotome and migrate to the limb buds, where they congregate into dorsal and ventral muscle masses. It has been proposed that the surrounding connective tissue controls muscle pattern formation in limbs. Regulatory molecules such as receptor tyrosine kinases like c-Met and those encoded by homeobox-containing genes, including c-Met, Tbx1, Mox2, Six1 and Six2, Pitx2, Pax3 and Lbx1h (a homolog of Drosophila Ladybird genes), are expressed in migrating limb precursor cells. The role of these genes in the patterning of limb muscles is unknown, although mutation of Pax3 or Met causes disruption of limb muscle development at an initial step, disturbing the epithelial-to-mesenchymal transition of the somitic epithelium. No limb muscle cells form in these mutants, and the early loss of myogenic precursor cells prevents an analysis of later functions of these genes during limb muscle development. Based on quail-chick chimaera studies, it was assumed that a cell-autonomous contribution of myogenic cells to the formation of individual limb muscles is negligible, and that an instructive role of limb mesenchyme is critical in this process. Lbx1h determines migratory routes of muscle precursor cells in a cell-autonomous manner, thereby leading to the formation of distinct limb muscle patterns. Inactivation of Lbx1h, which is specifically expressed in migrating muscle precursor cells, leads to a lack of extensor muscles in forelimbs and an absence of muscles in hindlimbs. The defect is caused by the failure of all muscle precursor cells of hindlimbs and of precursor cells of extensor muscles of forelimbs to migrate to their corresponding muscle anlagen. These results demonstrate that Lbx1h is a key regulator of muscle precursor cell migration and is required for the acquisition of dorsal identities of forelimb muscles (Schafer, 1999).

Described here is the cloning, expression pattern, and genomic organization of Lbx2, a murine homolog of the Drosophila and mammalian ladybird genes. Lbx2 includes a homeodomain motif most closely related to those of Lbx1 and the Drosophila Ladybird proteins. Lbx2 transcripts are first detected at E10.5 when they are located in the gonadal component of the urogenital ridge. Expression of Lbx2 dramatically increases by E11.5 in the urogenital ridges, and in the cranial surface ectoderm. At later stages, Lbx2 transcripts are expressed in the brain and organs derived from the urogenital ridge, including the gonadal tubercle, kidneys, and adrenal glands. From E14.5 to birth, Lbx2 expression is evident in the developing retinal neuroepithelium and the vibrissa (Chen, 1999).

The homeobox gene Lbx1 is expressed in migrating hypaxial muscle precursor cells during development. These precursors delaminate from the lateral edge of the dermomyotome and form distinct streams that migrate over large distances, using characteristic paths. The targets of migration are limbs, septum transversum and the floor of the first branchial arch where the cells form skeletal muscle of limbs and shoulders, diaphragm and hypoglossal cord, respectively. Gene targeting was used to analyse the function of Lbx1 in the mouse. Myogenic precursor cells delaminate from the dermomyotome in Lbx1 mutants, but migrate in an aberrant manner. Most critically affected are migrating cells that move to the limbs. Precursor cells that reach the dorsal limb field are absent. In the ventral limb, precursors are present but distributed in an abnormal manner. As a consequence, at birth some muscles in the forelimbs are completely lacking (extensor muscles) or reduced in size (flexor muscles). Hindlimb muscles are affected strongly, and distal limb muscles are more affected than proximal ones. Other migrating precursor cells heading towards the floor of the first branchial arch move along the appropriate path in Lbx1 mutants. However, these cells migrate less efficiently and reduced numbers of precursors reach their distal target. At birth, the internal lingual muscle is therefore reduced in size. It is suggested that Lbx1 controls the expression of genes that are essential for the recognition or interpretation of cues that guide migrating muscle precursors and maintain their migratory potential (Brohmann, 2000).

The somitic mesoderm gives rise to multiple tissues in the developing embryo including bone, connective tissue and muscle. Somites form as an epithelial ball of cells that later segregate into sclerotome and dermomyotome in response to patterning signals that arise from adjacent tissues. Cells in the ventral half of the nascent somite undergo a transition from epithelium to mesenchyme, forming the sclerotome. Subsequently, sclerotome differentiates further to give rise to the axial skeleton. Cells in the dorsolateral half of the somite retain their epithelial morphology, forming the dermomyotome, which contains precursors for both the dermis and for skeletal muscles. Dermomyotome derived muscle precursors not only generate the epaxial muscles that attach to the vertebral column, but also the hypaxial musculature of the limb, tongue, diaphragm and ventral body wall. Whereas the epaxial muscles arise from cells in the medial dermomyotome, hypaxial muscles are derived from precursors in the lateral half of the dermomyotome. The precursors for hypaxial muscles exhibit markedly different morphogenetic behaviours at different axial levels of the embryo. Cells in the ventral dermomyotome at limb and cervical levels delaminate and undergo long range migration to form diaphragm, tongue muscles and appendicular muscles. In contrast, hypaxial muscle precursors at interlimb levels do not migrate. Instead, they retain their epithelial morphology, forming a bud that extends ventrally toward the midline to give rise to ventral body wall muscles (Gross, 2000).

Previous studies have shown that Pax3, together with the c-Met receptor tyrosine kinase and its ligand Scatter Factor (SF) are necessary for the migration of hypaxial muscle precursors in mice. Lbx1 and Pax3 are co-expressed in all migrating hypaxial muscle precursors, raising the possibility that Lbx1 regulates their migration. To examine the function of Lbx1 in muscle development, the Lbx1 gene was inactivated by homologous recombination. Mice lacking Lbx1 exhibit an extensive loss of limb muscles, although some forelimb and hindlimb muscles are still present. The pattern of muscle loss suggests that Lbx1 is not required for the specification of particular limb muscles, and the muscle defects that occur in Lbx1 mutant mice can be solely attributed to changes in muscle precursor migration. c-Met is expressed in Lbx1 mutant mice and limb muscle precursors delaminate from the ventral dermomyotome but fail to migrate laterally into the limb. Muscle precursors still migrate ventrally and give rise to tongue, diaphragm and some limb muscles, demonstrating that Lbx1 is necessary for the lateral, but not ventral, migration of hypaxial muscle precursors. These results suggest that Lbx1 regulates responsiveness to a lateral migration signal that emanates from the developing limb (Gross, 2000).

Association and relay neurons that are generated in the dorsal spinal cord play essential roles in transducing somatosensory information. During development, these two major neuronal classes are delineated by the expression of the homeodomain transcription factor Lbx1. Lbx1 is expressed in and required for the correct specification of three early dorsal interneuron populations and late-born neurons that form the substantia gelatinosa. In mice lacking Lbx1, cells types that arise in the ventral alar plate acquire more dorsal identities. This results in the loss of dorsal horn association interneurons, excess production of commissural neurons, and disrupted sensory afferent innervation of the dorsal horn. Therefore, Lbx1 plays a critical role in the development of sensory pathways in the spinal cord that relay pain and touch (Gross, 2002).

It is generally held that vertebrate muscle precursors depend totally on environmental cues for their development. Instead, it is shown that somites are predisposed toward a particular myogenic program. This predisposition depends on the somite's axial identity: when flank somites are transformed into limb-level somites, either by shifting somitic boundaries with FGF8 or by overexpressing posterior Hox genes, they readily activate the program typical for migratory limb muscle precursors. The intrinsic control over myogenic programs can only be overridden by FGF4 signals provided by the apical ectodermal ridge of a developing limb (Alvares, 2003).

Since the competence to start a particular program of hypaxial myogenesis is linked to the position of the somites along the anteroposterior body axis, it was reasoned that this competence may be a consequence of the somite's axial identity. Indeed, when the ability was exploited of FGF8 to move the somitic boundaries, thereby changing the axial identity of somites in the flank into the identity of hindlimb-level somites, these somites gained the ability to produce Lbx1-expressing migratory muscle precursors (MMPs). This suggests that the positional values intrinsic to the somites determine their competence to generate either migratory or nonmigratory hypaxial muscle precursors (Alvares, 2003).

It is established that the axial identity of somites is controlled by the overlapping expression of Hox/HOM genes. In particular, a code of Hox gene expression is conserved for crucial anatomical landmarks such as the neck-thorax transition. Significantly, conserved Hox gene expression boundaries also demarcate the transition of limb-flank somites. Moreover, Hox gene expression patterns are maintained when somites are heterotopically grafted, as is the ability to express the MMP marker Lbx1. It was therefore reasoned that Hox genes may control the somitic competence to initiate a particular myogenic program. To test this possibility, expression constructs were engineered for HoxD9 and HoxA10, both normally present in hindlimb-level somites but not in the flank. Upon electroporation of either of the constructs into flank somites, these somites readily expressed Lbx1, while control constructs were unable to evoke expression of the Lbx1 gene. Thus, Lbx1 expression-directly or indirectly-is under the control of Hox genes (Alvares, 2003).

Evolving enhancer-promoter interactions within the tinman complex of the flour beetle, Tribolium castaneum

Modifications of cis-regulatory DNAs, particularly enhancers, underlie changes in gene expression during animal evolution. This study presents evidence for a distinct mechanism of regulatory evolution, whereby a novel pattern of gene expression arises from altered gene targeting of a conserved enhancer. The tinman gene complex (Tin-C) controls the patterning of dorsal mesodermal tissues, including the dorsal vessel or heart in Drosophila. In Drosophila, all members of the Tin-C are involved in muscle cell differentiation, and many of the mesodermal patterning functions of Tin-C genes are conserved between flies, annelids and vertebrates. For example, the founding member, tinman (also known as NK4), is expressed in the cardiac mesoderm of all three major animal groups. Moreover, bagpipe (NK3) is involved in patterning both fly and vertebrate visceral mesoderm, whereas ladybird/Lbx, slouch/Nk1, C15/Txl and Msh (Dr)/Msx regulate the patterning of somatic muscle precursors in both flies and annelids. Despite broad conservation of Tin-C gene expression patterns in the flour beetle (Tribolium castaneum), the honeybee (Apis mellifera) and the fruit fly (Drosophila melanogaster), the expression of a key pericardial determinant, ladybird, is absent from the dorsal mesoderm of Tribolium embryos. Evidence is presented that this loss in expression is replaced by expression of C15, the neighboring gene in the complex. This switch in expression from ladybird to C15 appears to arise from an inversion within the tinman complex, which redirects a conserved ladybird 3' enhancer to regulate C15. In Drosophila, this enhancer fails to activate C15 expression owing to the activity of an insulator at the intervening ladybird early promoter. By contrast, a chromosomal inversion allows the cardiac enhancer to bypass the ladybird insulator in Tribolium. Given the high frequency of genome rearrangements in insects, it is possible that such enhancer switching might be widely used in the diversification of the arthropods (Cande, 2009).

The homeodomain protein Ladybird late regulates synthesis of milk proteins during pregnancy in the Tsetse fly (Glossina morsitans)

Regulation of tissue and development specific gene expression patterns underlies the functional specialization of organs in multi-cellular organisms. In the viviparous tsetse fly (Glossina), the female accessory gland is specialized to generate nutrients in the form of a milk-like secretion to support growth of intrauterine larva. Multiple milk protein genes are expressed specifically in the female accessory gland and are tightly linked with larval development. Disruption of milk protein synthesis deprives developing larvae of nutrients and results in extended larval development and/or in abortion. The ability to cause such a disruption could be utilized as a tsetse control strategy. This study identified and delineated the regulatory sequence of a major milk protein gene (milk gland protein 1:mgp1) by utilizing a combination of molecular techniques in tsetse, Drosophila transgenics, transcriptomics and in silico sequence analyses. The function of this promoter is conserved between tsetse and Drosophila. In transgenic Drosophila the mgp1 promoter directs reporter gene expression in a tissue and stage specific manner orthologous to that of Glossina. Analysis of the minimal required regulatory region of mgp1, and the regulatory regions of other Glossina milk proteins identified putative homeodomain protein binding sites as the sole common feature. Annotation and expression analysis of Glossina homeodomain proteins identified ladybird late (lbl) as being accessory gland/fat body specific and differentially expressed between lactating/non-lactating flies. Knockdown of lbl in tsetse resulted in a significant reduction in transcript abundance of multiple milk protein genes and in a significant loss of fecundity. The role of Lbl in adult reproductive physiology is previously unknown. These results suggest that Lbl is part of a conserved reproductive regulatory system that could have implications beyond tsetse to other vector insects such as mosquitoes. This system is critical for tsetse fecundity and provides a potential target for development of a reproductive inhibitor (Attardo, 2014).


ladybird early and ladybird late: Regulation | Developmental Biology | Effects of Mutation | References

date revised: 6 Jan 97 

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