A comparison of homeodomains implies that LIM homeodomain proteins fall into four groups, plus two genes with no close relatives (mec-3, lmx-1). Drosophila apterous and vertebrate LH-2/lhx2 are closely related and define a separate group well removed from the other three. C. elegans Ceh-14 and Drosophila BK64 fit within a second group, the lim/lhx3,4 group. C. elegans lin-11 belong to a third group, the lim/lhx1,5,6 genes. The islet group is distinct from the other three. While these sequence relationships are apparent, expression patterns are not obviously similar between the invertebrate and vertebrate species (Dawid, 1995). Drosophila Lin-1 belongs to the third group, the lim/lhx1,5,6 genes.
The lin-11 LIM homeobox gene of C. elegans (closest mammalian homolog: LIM-1) is expressed in nine classes of head, ventral cord, and tail neurons and functions at a late step in the development of a subset of these neurons. In a lin-11 null mutant, all lin-11-expressing neurons are generated. However, several of these neurons exhibit neuroanatomical as well as functional defects. In the lateral head ganglion, lin-11 functions in a neural network that regulates thermosensory behavior. It is expressed in the AIZ interneuron that processes high temperature input and is required for the function of AIZ in the thermoregulatory neural network. Another LIM homeobox gene, ttx-3 (closest fly homolog: apterous), functions in the antagonistic thermoregulatory interneuron AIY. Thus, distinct LIM genes specify the functions of functionally related antagonistic interneurons within a neural network dedicated to thermoregulatory processes. Both ttx-3 and lin-11 expression are maintained throughout adulthood, suggesting that these LIM homeobox genes play a role in the functional maintenance of this neural circuit. Particular LIM homeobox genes may specify the distinct features of functionally related neurons that generate patterned behaviors (Hobert, 1998).
Chemosensory neuron diversity in C. elegans arises from the action of transcription factors that specify different aspects of sensory neuron fate. In the AWB and AWA olfactory neurons, the LIM homeobox gene lim-4 and the nuclear hormone receptor gene odr-7 are required to confer AWB and AWA-specific characteristics respectively, and to repress an AWC olfactory neuron-like default fate. AWA neuron fate is also regulated by a member of the LIM homeobox gene family, lin-11. lin-11 regulates AWA olfactory neuron differentiation by initiating expression of odr-7, which then autoregulates to maintain expression. lin-11 also regulates the fate of the ASG chemosensory neurons, which are the lineal sisters of the AWA neurons. lin-11 is expressed dynamically in the AWA and ASG neurons, and that misexpression of lin-11 is sufficient to promote an ASG, but not an AWA fate, in a subset of neuron types. These results suggest that differential temporal regulation of lin-11, presumably together with its interaction with asymmetrically segregated factors, results in the generation of the distinct AWA and ASG sensory neuron types. It is proposed that a LIM code may be an important contributor to the generation of functional diversity in a subset of olfactory and chemosensory neurons in C. elegans (Sarafi-Reinach, 2001).
It is concluded that, in the AWA neurons, a temporally regulated cascade of transcription factors is required for fate specification. In C. elegans, expression of most LIM homeobox genes appears to be maintained throughout the postmitotic life of neurons. However, results from this work show that lin-11 is expressed only during a brief temporal window, shortly after the birth of the AWA neurons. This expression is sufficient to activate odr-7, which then autoregulates to maintain its expression and the expression of AWA-specific characteristics. The data suggest that forced prolonged expression of lin-11 in the AWA neurons may be partly sufficient to alter AWA fate, indicating that strict temporal control of lin-11 expression is critical for correct AWA fate specification. In vertebrates, the expression pattern of LIM homeobox genes is also regulated temporally, and is an important component in determining cell type identity. Isl1 is first expressed early in all motorneurons, while expression of other LIM homeobox genes follows in a temporally stereotyped manner. Expression of the Lhx3 and Lhx4 genes has also been shown to be dynamic. It will be interesting to investigate whether other LIM homeobox genes in C. elegans are also regulated dynamically (Sarafi-Reinach, 2001).
What are the mechanisms by which lin-11 expression is down-regulated in the AWA, but not in the ASG neurons? Maintenance of expression in one, but not the other sibling cell may arise from cell-cell interactions. Notch signaling between the two daughters of the pIIA sensory neuron progenitors in Drosophila has been shown to be required for the autorepressive and autoactivating functions of Suppressor of Hairless [Su(H)] in each of the two daughter cells, resulting in high levels of Su(H) in one, but not the other cell. A defect in specification of an AWA or ASG neuron does not result in a fate defect in the sibling neuron, suggesting that cell-cell signaling between the AWA and ASG neurons may not be the primary mediator of regulation of lin-11 expression. Instead, it is suggested that this difference in the temporal pattern of lin-11 expression arises from the asymmetric segregation of factors to the AWA and ASG neurons. The forkhead domain transcription factor UNC-130 plays an important role in the asymmetric division, giving rise to the AWA and ASG neurons. It is proposed that UNC-130 function in the AWA/ASG precursors is necessary for correct segregation of factors required for modulation of LIN-11 function in these two neurons. These factors could function to maintain lin-11 expression in the ASG neurons, or to repress lin-11 expression in the AWA neurons later in development. It is also possible that LIN-11 is autoregulatory, and functions along with these factors to maintain or repress its expression (Sarafi-Reinach, 2001).
It is also proposed that UNC-130 regulates the asymmetric segregation of factors that work together with LIN-11 to regulate either AWA- or ASG-specific gene expression. In unc-130 mutants, incorrect segregation of factors to the ASG neurons may result both in the downregulation of lin-11 expression and promotion of AWA-specific gene expression, thereby converting the ASG neurons to an AWA fate. These factors are likely to be different for each of the AWA and ASG cell types. The functions of LIM homeobox genes have been shown to be modified by interaction with a number of different proteins, including members of the paired-type homeodomain family, POU-homeodomain proteins and other LIM homeobox proteins. It is unlikely that lin-11 works with other LIM homeobox genes in the AWA and ASG neurons, since none of the six additional identified LIM homeobox genes in C. elegans is expressed in the AWA or ASG neurons, and mutations in these genes do not affect odr-7 expression (Sarafi-Reinach, 2001).
The C. elegans POU protein UNC-86, a homolog of Drosophila Acj6, specifies the HSN motor neurons, which are required for egg-laying, and six mechanosensory neurons. To investigate how UNC-86 controls neuronal specification, two unc-86 mutants have been characterized that do not respond to touch but show wild-type egg-laying behavior. Residues P145 and L195, which are altered by these mutations, are located in the POU-specific domain and abolish the physical interaction of UNC-86 with the LIM homeodomain protein, MEC-3 (most closely related to Drosophila Lim1). This results in a failure to maintain mec-3 expression and in loss of expression of the mechanosensory neuron-specific gene, mec-2. unc-86-dependent expression of genes in other neurons is not impaired. It is concluded that distinct residues in the POU domain of UNC-86 are involved in modulating UNC-86 activity during its specification of different neurons. A structural model of the UNC-86 POU domain, including base pairs and amino acid residues required for MEC-3 interaction, reveals that P145 and L195 are part of a hydrophobic pocket that is similar to the OCA-B-binding domain of the mammalian POU protein, Oct-1 (Rohrig, 2000).
The egg-laying system of Caenorhabditis elegans hermaphrodites requires development of the vulva and its precise connection with the uterus. This process is regulated by LET-23-mediated epidermal growth factor signaling and LIN-12-mediated lateral signaling pathways. Among the nuclear factors that act downstream of these pathways, the LIM homeobox gene lin-11 plays a major role. lin-11 mutant animals are egg-laying defective because of the abnormalities in vulval lineage and uterine seam-cell formation. However, the mechanisms providing specificity to lin-11 function are not understood. The regulation of lin-11 during development of the egg-laying system was examined. The tissue-specific expression of lin-11 is controlled by two distinct regulatory elements that function as independent modules and together specify a wild-type egg-laying system. A uterine pi lineage module depends on the LIN-12/Notch signaling, while a vulval module depends on the LIN-17-mediated Wnt signaling. These results provide a unique example of the tissue-specific regulation of a LIM homeobox gene by two evolutionarily conserved signaling pathways. Finally, evidence is provided that the regulation of lin-11 by LIN-12/Notch signaling is directly mediated by the Su(H)/CBF1 family member LAG-1 (Gupta, 2002).
LIM homeobox family members regulate a variety of cell fate choices during animal development. In C. elegans, mutations in the LIM homeobox gene lim-11 (most closely related to Drosophila Lim1) have been shown to alter the cell division pattern of a subset of the 2º lineage vulval cells. Multiple functions of lin-11 during vulval development have been demonstrated. The fate of vulval cells was examined in lin-11 mutant animals using five cellular markers: lin-11 is necessary for the patterning of both 1º and 2º lineage cells. In the absence of lin-11 function, vulval cells fail to acquire correct identity and inappropriately fuse with each other. The expression pattern of lin-11 reveals dynamic changes during development. Using a temporally controlled overexpression system, lin-11 is shown to be initially required in vulval cells for establishing the correct invagination pattern. This process involves asymmetric expression of lin-11 in the 2º lineage cells. Using a conditional RNAi approach, it has been shown that lin-11 regulates vulval morphogenesis. LDB-1, a NLI/Ldb1/CLIM2 family member, interacts physically with LIN-11, and is necessary for vulval morphogenesis. Together, these findings demonstrate that temporal regulation of lin-11 is crucial for the wild-type vulval patterning (Gupta, 2003).
One of the first intercellular signaling events in the vertebrate embryo leads to mesoderm formation and axis determination. In the mouse, a gene encoding a new member of the TGF-beta superfamily, nodal, is disrupted in a mutant deficient in mesoderm formation. nodal mRNA is found in prestreak mouse embryos, consistent with a role in the development of the dorsal axis. Injection of nodal mRNA into zebrafish embryos causes the formation of ectopic axes that include notochord and somites. Axis duplication is preceded by the generation of an apparent ectopic shield (organizer equivalent) in nodal-injected embryos, as indicated by the appearance of a region over-expressing gsc and lim1. These results suggest a role for a nodal-like factor in pattern formation in zebrafish (Toyama, 1995b).
A novel cysteine-rich motif, named LIM, has been identified in the homeo box genes lin-11, Isl-1, and mec-3; the mec-3 and lin-11 genes determine cell lineages in Caenorhabditis elegans. LIM class homeobox genes have been isolated from Xenopus laevis that are closely related to lin-11 and mec-3 in the LIM and homeo domains. This paper deals with one of these genes, Xlim-1. Xlim-1 mRNA is found in low abundance in the unfertilized egg; has a major expression phase at the gastrula stage; decreases, and rises again during the tadpole stage. In adult tissues the brain shows the highest abundance, by far, of Xlim-1 mRNA. The maternal and late expression phases of the Xlim-1 gene suggest that it has multiple functions at different stages of the Xenopus life cycle. In the gastrula embryo, Xlim-1 mRNA is localized in the dorsal lip and the dorsal mesoderm, that is, in the region of Spemann's organizer. Explant experiments have shown that Xlim-1 mRNA is induced by the mesoderm-inducer activin A and by retinoic acid, which is not a mesoderm inducer but affects patterning during Xenopus embryogenesis; application of activin A and retinoic acid together results in synergistic induction. The structure, inducibility, and localized expression in the organizer of the Xlim-1 gene suggest that it has a role in establishing body pattern during gastrulation (Tiara, 1992).
The LIM class homeobox gene Xlim-1 is expressed in Xenopus embryos in the lineages leading to (1) the notochord, (2) the pronephros, and (3) certain cells of the central nervous system (CNS). In its first expression phase, Xlim-1 mRNA arises in the Spemann organizer region, accumulates in prechordal mesoderm and notochord during gastrulation, and decays in these tissues during neurula stages, except that it persists in the posterior tip of the notochord. In the second phase, expression in lateral mesoderm begins at late gastrula, and converges to the pronephros at tailbud stages. Expression in a central location of the neural plate also initiates at late gastrula, expands anteriorly and posteriorly, and becomes established in the lateral regions of the spinal cord and hindbrain at tailbud stages. Thus Xlim-1 expression precedes morphogenesis, suggesting that it may be involved in cell specification in these lineages. Enhancement of Xlim-1 expression by retinoic acid (RA) is first detectable in the dorsal mesoderm at initial gastrula. During gastrulation and early neurulation, RA strongly enhances Xlim-1 expression in all three lineages and also expands its expressing domains; this overexpression correlates well with RA phenotypes, such as enlarged pronephros and hindbrain-like structure. Exogastrulation reduces Xlim-1 expression in the lateral mesoderm and ectoderm but not in the notochord, suggesting that the second phase of Xlim-1 expression requires mesoderm/ectoderm interactions. RA treatment of exogastrulae does not revert this reduction (Taira, 1994a).
Like all known LIM class homeobox genes, Xlim-1 encodes a protein with two tandemly repeated cysteine-rich LIM domains upstream of the homeodomain. In Xenopus laevis, Xlim-1 is specifically expressed in the Spemann organizer, whose major functions include neural induction and dorsalization of ventral mesoderm. From RNA injection experiments it has been concluded that: (1) the LIM domains behave as negative regulatory domains; (2) LIM domain mutants of Xlim-1 elicite neural differentiation in animal explants; (3) mutant, and to a lesser extent wild-type, Xlim-1 enhances muscle formation after coinjection with Xbra; (4) both of these activities are mediated by extracellular signals as seen in combined explant experiments; (5) Xlim-1 mutants activate goosecoid (gsc) expression in animal explants, but not expression of noggin or follistatin; (6) mutant Xlim-1 elicits formation of partial secondary axes, and cooperates with gsc in notochord formation. Thus Xlim-1 has latent activities, implicating it in organizer functions (Tiara, 1994b).
A new LIM-domain-binding factor, Ldb1, a novel protein, has been isolated on the basis of its ability to interact with the LIM-HD protein Lhx1 (Lim1). High-affinity binding by Ldb1 requires paired LIM domains and is restricted to the related subgroup of LIM domains found in LIM-HD and LMO proteins (see Drosophila Muscle LIM protein at 60A). The highly conserved Xenopus Ldb protein XLdb1, interacts with Xlim-1, the Xenopus ortholog of Lhx1. When injected into Xenopus embryos, XLdb1 (or Ldb1) can synergize with Xlim-1 in the formation of partial secondary axes and in activation of the genes encoding goosecoid, chordin, NCAM and XCG7, demonstrating a functional as well as a physical interaction between the two proteins (Agulnick, 1996).
Polyclonal antibodies to Xlim-1 homeodomain protein of Xenopus laevis were used to study the developmental expression pattern of this protein in Xenopus, rat and mouse. Western blotting of embryo extracts injected with different Xlim-1 constructs confirmed the specificity of the antibody. Beginning at the gastrula stage, Xlim-1 protein was detected in three cell lineages: (1) notochord, (2) pronephros and (3) certain regions of the central nervous system. In addition, Xlim-1 is expressed in the olfactory organ, retina, otic vesicle, dorsal root ganglia and adrenal gland. Similar expression patterns have been seen for the Lim-1 protein in frog and rodent tissues. These observations implicate the Xlim-1 gene in the specification of multiple cell lineages, particularly within the nervous system, and emphasize the conserved nature of the role of this gene in different vertebrate animals (Karavanov, 1996).
Anteroposterior patterning of neural tissue is thought to be directed by the axial mesoderm, which is functionally divided into head (or precordal) and trunk organizer (notochord). In Xenopus the homeobox genes goosecoid (Drosophila homolog: Goosecoid) and Otx2 (Drosophila homolog: Orthodenticle) are expressed in the pre-cordal mesoderm; the LIM class homeobox gene Xlim-1 is expressed in the entire axial mesoderm, whereas the distinct Brachyury related transcription factor Xbra (Drosophila homolog: T-related gene) is expressed in the notochord but not in the procordal mesoderm. Messenger RNA injection experiments show that Xenopus animal pole explants (caps) expressing an activated form of Xlim-1 (a LIM domain mutant named 3m) induce anterior neural markers, whereas caps coexpressing Xlim-1/3m and Xbra induce posterior neural markers. These data indicate that in terms of neural inducing ability, Xlim-1/3m-expressing caps correspond to the head organizer and Xlim-1/3m plus Xbra-coexpressing caps correspond to the trunk organizer. Thus the expression domains of Xlim-1 and Xbra correlate with, and possibly define, the functional domains of the organizer. In animal caps, Xlim-1/3m initiates expression of a neuralizing factor chordin (Drosophila homolog: Short gastrulation, which counteracts the antineurogenic effects of Decapentaplegic), whereas Xbra activates embryonic fibroblast growth factor (eFGF expression) (see Drosophila FGF homolog Branchless); these factors could mediate the neural inducing and patterning effects that are observed. A dominant-negative FGF receptor (XFD) inhibits posteriorization by Xbra in a dose-dependent manner, supporting the suggestion that eFGF or a related factor has posteriorizing influence. Retinoic acid, postulated to be a posteriorizing factor based on the observations that RA treatment of embryos leads to truncation of anterior structures in Xenopus, can posteriorize neural tissue generated by Xlim-1. RA strongly inhibits Otx2 expression and induces Krox-20 and beta2-tubulin expression, indicating that RA can act as a posteriorizing factor for neural tissue in the absence of mesoderm (Taira, 1997).
The Xlim-1 gene is activated in the late blastula stage of Xenopus embryogenesis in the mesoderm; its RNA product becomes concentrated in the Spemann organizer at early gastrula stage. A major regulator of early expression of Xlim-1 is activin or an activin-like signal. The 5' flanking region of Xlim-1 contains a constitutive promoter that is not activin responsive, whereas sequences in the first intron mediate repression of basal promoter activity and stimulation by activin. An intron-derived fragment of 212 nt is the smallest element that can mediate activin responsiveness. Nodal and act-Vg1, factors with signaling properties similar to activin, also stimulate Xlim-1 reporter constructs, whereas BMP-4 neither stimulates nor represses the constructs. The mechanism of activin regulation of Xlim-1 and the sequence of the response element are distinct from the activin response elements of other genes studied to date (Rebbert, 1997).
Recently, a model to explain the mechanism of Xenopus tail bud formation has been proposed. The NMC model proposes that three regions around the late blastopore lip are required to initiate tail formation. These are the posterior-most neural plate, fated to form tail somites (M); the neural plate (N), immediately anterior to M, and the underlying caudal notochord (C). To initiate tail formation, C must underlie (and presumably signal to) the junction of N and M, which subsequently forms the tip of the tail. During normal development, the NMC interaction leading to specification of the tail bud occurs at the end of gastrulation. Outgrowth of the tail bud commences much later, becoming clearly visible by stage 30 (Beck, 1998 and references).
Several domains of the Xenopus tail bud are defined by two phases of gene expression. The first group of genes are already expressed in the tail bud region before its determination at stage 13 and are subsequently restricted in the extending tail bud by stage 30. This group, the early genes, includes the Notch ligand X-delta-1, the lim domain homeobox factor Xlim1, the T-box factor Xbra, and the homeobox factor Xnot2 and Xcad2, a member of the caudal family. X-delta-1 is expressed specifically in the posterior wall of the neuroenteric canal but is excluded from the chordoneural hinge at stage 30, thus maintaining its earlier expression in the lateral and ventral blastopore lips. Xim1 is expressed in the notochord and dorsal blastopore lip at the end of gastrulation, and is maintained in the chordoneural hinge and posterior tip of the differentiated notochord in later stages. Xnot2 is expressed in the ventral neural tube and chordoneural hinge, but not in the posterior notochord. The posterior notochord therefore represents a novel tail bud region by stage 30, marked by Xlim but not Xnot transcripts, whereas the posterior ventral neural tube is marked by Xnot but not Xbra or Xlim1. Xbra is expressed in the chordoneural hinge and posterior wall. Xcad3 expression in the posterior neural plate is later maintained in the posterior wall and posterior dorsal neural tube. Xpo is expressed in all tissues of the tail bud with the exception of the chordoneural hinge, and is expressed in the fin and epidermis (Beck 1998).
Unlike the early genes, the regional expression of the second group of genes in the extended tail bud can not be traced back to the stage of tail bud initiation. These genes have a late onset of localized expression in the tail bud, corresponding to the beginning of tail outgrowth, although they may be expressed elsewhere in the embryo at stage 13. The dorsal roof domain of the tail bud is marked by expression of Xwnt3a and lunatic fringe. Xwnt5a expression is restricted to the tail bud roof. The distal tip of the tail, which comprises part of the posterior wall, is marked by expression of Xhox3, which marks the distal cells of the tail bud. Xhox3 is a vertebrate homolog of Drosophila evenskipped. Other late genes include BMP-4, X-serrate-1 and BMP-2 (Beck, 1998).
The existence of distinct domains in the positions predicted for C and M is proposed. The restriction of Xcad3 and Xlim1 transcripts to the posterior of the notochord in the early neurula demonstrates that the posterior part of the notochord differs from the crest, corresponding to the C region. Novel domains of the tail bud are proposed to express different combinations of genes. These domains include the dorsal roof of the tail bud, the distal tip of the tail, marked by Xhox3, the chordoneural hinge, the posterior tip of the chordoneural hinge, the posterior wall domain, the tip of the posterior wall, the posterior notochord, the posterior wall of the neuroenteric canal and the ventral neural tube (Beck, 1998).
Xlim-1, a LIM class homeobox gene expressed in Xenopus laevis, is one of the earliest known marker genes of pronephros development and is expressed in pronephros rudiment. The role of Xlim-1 in pronephros development has been examined. Temporal expression of Xlim-1 in explants was analyzed in a series of induction assays using RT-PCR analysis. Xlim-1 is expressed 9 to 15 h after activin/retinoic acid treatment, corresponding to pronephros differentiation in explants. The role of Xlim-1 was examined using a series of microinjection experiments. Presumptive pronephric anlagen of embryos were injected with various Xlim-1 mutants, and the effects of these Xlim-1 mutants on pronephrogenesis in embryos and in explants were analyzed by RT-PCR and immunohistochemistry. Dominant-negative Xlim-1 (Xlim-1-enR) inhibits differentiation of pronephros in activin/retinoic acid-treated animal caps. In embryos injected with a dominant-negative form of Xlim-1, development of pronephric tubules is inhibited at the late tail-bud stage. These results suggest that Xlim-1 may not initiate differentiation of the pronephros, but that it is necessary for growth and elongation in the development of pronephric tubules (Chan, 2000).
Kidney organogenesis requires the morphogenesis of epithelial tubules. Inductive interactions between the branching ureteric buds and the metanephric mesenchyme led to mesenchyme-to-epithelium transitions and tubular morphogenesis to form nephrons, the functional units of the kidney. The LIM-class homeobox gene Lim1 is expressed in the intermediate mesoderm, nephric duct, mesonephric tubules, ureteric bud, pretubular aggregates and their derivatives. Lim1-null mice lack kidneys because of a failure of nephric duct formation, precluding studies of the role of Lim1 at later stages of kidney development. This study shows that Lim1 functions in distinct tissue compartments of the developing metanephros for both proper development of the ureteric buds and the patterning of renal vesicles for nephron formation. These observations suggest that Lim1 has essential roles in multiple steps of epithelial tubular morphogenesis during kidney organogenesis. The nephric duct is essential for the elongation and maintenance of the adjacent Müllerian duct, the anlage of the female reproductive tract (Kobayashi, 2005).
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