Ladybird late Precomputed BLAST | Entrez Gene |
Fly GRID: Lbl genetic and physical interactions

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 90ƒ 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.

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).

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).

date revised: 6 Jan 97 

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