InteractiveFly: GeneBrief

Lim1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The Drosophila lim1 cDNA has been isolated by polymerase chain reaction (PCR) using degenerate primers designed from the conserved sequences within the Xenopus Xlim-1 and murine Lim-1 (Lhx1) homeoboxes. The sequence and expression of dLim1 is highly related to its vertebrate homologs. Within the Drosophila embryo, Lim1 is expressed in the head primordia, the brain lobes, and in distinct sets of motorneurons and interneurons within the ventral nerve cord. For comparison, in vertebrates the lim homeodomain proteins [Lim-1 along with Lim-3 (Lhx3), Gsh-4 (Lhx4), Isl-1 and Isl-2] are expressed in developing motorneurons along the spinal column, where their overlapping expression suggests a role lim proteins in the establishment of specific motorneuron subtypes. Drosophila Lim1 is absent from all cells expressing Islet, Lim3, and Apterous (Ap), an indication that these proteins function independently within the Drosophila embryo. Drosophila lim1 is an essential gene that when mutated results in lethality near the larval-pupal boundary. Mice mutant for Lim-1 fail to form anterior structures, resulting in the complete absence of a fore- and mid-brain in mutant embryos (Shawlot, 1995). In contrast to this, Drosophila lim1 has no apparent role in anterior patterning of the Drosophila embryo. Thus Drosophila lim1 has been evolutionarily conserved, however the Drosophila lim1 gene exhibits unique properties that distinguish it from its vertebrate homologs (Lilly, 1999).

While the vertebrate Lim-1 gene is expressed in the head folds of the young embryo, Drosophila Lim1 is expressed as a stripe within the head primordia just after cellularization. At later stages both genes are found in subsets of motorneurons and interneurons within their respective nerve cords. The similarities in sequence and expression between the vertebrate and Drosophila orthologs strongly suggest that their function has also been conserved. Consistent with this, the molecular capabilities of the Drosophila Lim1 protein have been compared with those of vertebrates. Just as the vertebrate proteins, Xlim-1 and Xldb-1 (NLI, CLIM-2) interact in vitro, Drosophila Lim1 and Chip exhibit a comparable association. Thus, within each species these factors have retained common characteristics that are important to the function of these proteins. However, within each organism the Lim-1 proteins have adapted species-specific functions that reflect divergent specialization (Lilly, 1999).

Lim1 is expressed in many cells along the ventral nerve cord of the Drosophila embryo. Lim1 is found in neural cells but not glial cells within the ventral nerve cord. Expression is observed in interneurons and motorneurons, specifically RP2, aCC and the U motorneurons, all of which innervate the dorsal muscles of the embryo. Because of the combinatorial expression observed with LIM homeodomain members in the chick spinal column and the Drosophila ventral nerve cord, it was of interest to assess the relative expression of the LIM homeodomain proteins in Drosophila. In the vertebrate spinal column the overlapping expression of Lim-1, Lim-3, Isl-1 and Isl-2 demarcates regions of motorneurons with common targets. In Drosophila, the LIM homeodomain genes (Lim1, Lim3, Isl, and Ap) are expressed in select subtypes of neurons within the ventral nerve cord. Drosophila Lim1 is expressed in motoneurons that innervate dorsal muscles, while Isl-expressing motoneurons innervate ventral muscles. Additionally, the pools of interneurons in which Lim1 and Isl are expressed do not overlap. Moreover, the results show that Lim1 and Lim3 have mutually exclusive expression patterns. The significance of this exclusive expression has yet to be determined, however based on the vertebrate model and the combinatorial relationship observed for isl and lim3 in Drosophila, it seems feasible that Lim1 may provide pathfinding identity to subclasses of neurons. As in the chick spinal column, the overlapping or exclusive expression of these proteins provides instructional information to subsets of neurons. This is supported by the pathfinding defects observed in the ap, lim3 and isl mutants and the combinatorial relationship observed with isl and lim3. The subtlety of the pathfinding defects seen with ap, isl, and lim3, and the apparent lack of any defects in Lim1 mutants, supports the idea that there are multiple factors involved in regulating this process (Lilly, 1999).

In an effort to elucidate the role of Lim1 in Drosophila, loss-of-function alleles were generated in the Lim1 locus. As a function of the screening strategy, all Lim1 mutations recovered were lethal. These mutants survive to the larval and pupal stages, but never eclose to produce viable adult flies. Morphologically, the Lim1 mutants appear normal, however at the third instar stage, larvae began to exhibit coordination defects and are unable to crawl in a wild-type fashion. Several nervous system markers were examined to assess the cause of lethality. The cells are specified correctly and they appear to extend their axon projections normally. The general behavior of the larvae suggest that these mutants have motor-coordination defects. What these defects are at a morphological and molecular level remains to be determined. What is clear is that the Drosophila Lim1 mutants die during their development, demonstrating that Lim1 is an essential gene. Clonal analysis and characterization of the Lim1 locus may unveil the specific nature of the Lim1 phenotype (Lilly, 1999).

Notch-dependent epithelial fold determines boundary formation between developmental fields in the Drosophila antenna

Compartment boundary formation plays an important role in development by separating adjacent developmental fields. Drosophila imaginal discs have proven valuable for studying the mechanisms of boundary formation. This study examined the boundary separating the proximal A1 segment and the distal segments, defined respectively by Lim1 and Dll expression in the eye-antenna disc. Sharp segregation of the Lim1 and Dll expression domains precedes activation of Notch at the Dll/Lim1 interface. By repressing bantam miRNA and elevating the actin regulator Enabled, Notch signaling then induces actomyosin-dependent apical constriction and epithelial fold. Disruption of Notch signaling or the actomyosin network reduces apical constriction and epithelial fold, so that Dll and Lim1 cells become intermingled. These results demonstrate a new mechanism of boundary formation by actomyosin-dependent tissue folding, which provides a physical barrier to prevent mixing of cells from adjacent developmental fields (Ku, 2017).

This study attempted to unravel the molecular and cellular mechanisms of boundary formation in the Drosophila head. Focus was placed on the antennal A1 fold that separates the A1 and A2-Ar segments. The results showed that the expression of the selector genes Lim1 and Dll, which are expressed in A1 and A2-Ar, respectively, was sharply segregated. This step was followed by differential expression of Dl, Ser and Fng, as well as activation of N signaling at the interface between A1 and A2. N signaling then induced apical constriction and epithelial fold, possibly through repression of bantam to allow levels of the bantam target Ena to become elevated, with this latter inducing the actomyosin network. The actomyosin-dependent epithelial fold then provided a mechanical force to prevent cell mixing. When N signaling or actomyosin was disrupted, or when bantam was overexpressed, the epithelial fold was disrupted and Dll and Lim1 cells become mixed. Thus this study describes a clear temporal and causal sequence of events leading from selector gene expression to the establishment of a lineage-restricting boundary (Ku, 2017).

Sharp segregation of Dll/Lim1 expressions began before formation of the A1 fold, suggesting that fold formation is not the driving force for segregation of Dll/Lim1 expression. Instead, the fold functions to safeguard the segregated lineages from mixing. Whether Dll/Lim1 segregated expression is due to direct or indirect antagonism between the two proteins is not known (Ku, 2017).

Actomyosin-dependent apical constriction is an important mechanism for tissue morphogenesis in diverse developmental processes, e.g., gastrulation in vertebrates, neural closure and Drosophila gastrulation, as well as dorsal closure and formation of the ventral furrow and segmental groove in embryos. This study describes a new function of actomyosin, i.e., the formation of lineage-restricting boundaries via apical constriction during development (Ku, 2017).

This actomyosin-dependent epithelial fold provides a mechanism distinctly different from other known types of boundary formation. The cells at the A1 fold still undergo mitosis, suggesting that mitotic quiescence is not involved. Perhaps epithelial fold as a lineage barrier is needed in situations in which mitotic quiescence does not happen. Mechanically and physically, epithelial folds could serve as stronger barriers than intercellular cables when mitotic activity is not suppressed. The drastic and sustained morphological changes, including reduced apical area and cell volume, may be accompanied by increased cortical tension of cells along the A1 fold, with such high interfacial tension then preventing cell intermingling and ensuring Dll and Lim1 cell segregation. Although similar to actomyosin boundaries, the epithelial fold in the A1 boundary is distinctly different from the supracellular actomyosin cable structure in fly parasegmental borders, the wing D/V border, and the interrhombomeric boundaries of vertebrates. The adherens junction protein Echinoid, which is known to promote the formation of supracellular actomyosin cables, is not involved in A1 fold formation. Although actomyosin is enriched in a ring of cells in the A1 fold, it does not exert a centripetal force to close the ring, unlike the circumferential cable described in dorsal closure and wound healing (see review. In the A1 fold, the constricting cells become smaller in both their apical and basolateral domains, thus differing from ventral furrow cells where cell volume remains constant (Ku, 2017).

A tissue fold probably provides a strong physical or mechanical barrier to prevent cell mixing. In addition, whereas in a flat tissue where the boundary involves only one to two rows of cells, the tissue fold involves more cells engaging in cell-cell communication. The close apposition of cells within the fold may allow efficient signaling within a small volume. This may be an evolutionarily conserved mechanism for boundary formation that corresponds to stable morphological constrictions such as the joints in the antennae and leg segments (Ku, 2017).

Although N signaling has been reported to be involved in many developmental processes, a role in inducing actomyosin-dependent apical constriction and epithelial fold is a novel described function for N. For the A1 boundary, N activity is possibly mediated through repression of bantam and consequent upregulation of Ena. In the wing D/V boundary, N signaling is also mediated through bantam and Ena, but the outcome is formation of actomyosin cables, i.e., without apical constriction and epithelial fold [19]. Thus, the N/bantam/Ena pathway for tissue morphological changes is apparently context-dependent (Ku, 2017).

Tissue constriction also occurs later in joint formation of the legs and antennae. N activation also occurs in the joints of the leg disc and is required for joint formation. This role is conserved from holometabolous insects like the fruitfly Drosophila melanogaster and the red flour beetle Tribolium castaneum to the hemimetabolous cricket Gryllus bimaculatus. It is possible that for segmented structures that telescope out in the P/D axis, like the antennae, legs, proboscis and genitalia, N signaling is used to demarcate the boundaries between segments, which are characterized by tissue constriction. N-dependent epithelial fold morphogenesis has also been reported in mice cilia body development without affecting cell fate, suggesting that such N-dependent regulation in morphogenesis is evolutionarily-conserved (Ku, 2017).

It is proposed that N signaling is important in all boundaries that involve stable tissue morphogenesis. For those boundaries corresponding to stable morphological constrictions, e.g., the joints in insect appendages, N acts via actomyosin-mediated epithelial fold. The wing D/V boundary represents a different type of stable tissue morphogenesis. It becomes bent into the wing margin and involves N signaling via actomyosin cables, rather than apical constriction. In contrast, actomyosin-dependent apical constrictions do not involved N signaling and are involved in transient tissue morphogenesis, such as gastrulation in vertebrates, neural closure, Drosophila gastrulation, dorsal closure, as well as formation of the ventral furrow, eye disc morphogenetic furrow, and segmental groove in embryos (Ku, 2017).

N signaling is also involved in the boundary between new bud and the parent body of Hydra, where it is required for sharpening of the gene expression boundary and tissue constriction at the base of the bud [78]. Whether the role of N in these tissue constrictions is due to actomyosin-dependent apical constriction and epithelial fold is not known (Ku, 2017).

Boundaries may be established early in development. As the tissue grows in size through cell divisions and growth, boundary maintenance become essential. This study found that N activity is maintained by actomyosin, suggesting feedback regulation to stably maintain the boundary. Mechanical tension generated by actomyosin networks has been suggested to enhance actomyosin assembly in a feedback manner. Interestingly, the N-mediated wing A/P and D/V boundaries, which form actomyosin cables rather than tissue folds, did not exhibit such positive feedback regulation. Instead, the stability of the Drosophila wing D/V boundary is maintained by a complex gene regulatory network involving N, Wg, N ligands and Cut. Perhaps this is necessary for a boundary not involving tissue morphogenesis (Ku, 2017).

The segmented appendages of arthropods (antennae, legs, mouth parts) are homologous structures of common evolutionary origin. It has been proposed that the generalized arthropod appendage is composed of a proximal segment called the coxopodite and a distal segment called the telopodite, either of which can further develop into more segments. The coxopodite is believed to be an extension of the body wall, whereas the telopodite represents the true limb, and thus represents an evolutionary addition. Dll mutants lack all distal segments except for the coxa in legs and the A1 segment in antennae. Lineage tracing studies have shown that Dll-expressing cells contributed to all parts of the legs except the coxa. These results indicate that the leg coxa and antenna A1 segment correspond to the Dll-independent coxopodite, and that Dll is the selector gene for the telopodite. Therefore, the antennal A1 fold is the boundary between the coxopodite and telopodite. It is postulated that the same N-mediated epithelial fold mechanism also operates in the coxopodite/telopodite boundary of legs and other appendages (Ku, 2017).

REGULATION

Transcriptional Regulation

The expression of Lim1 is very similar to that of the PD gene aristaless. al is expressed in the most distal part of the leg discs, and in two rings in the peripheral and medial regions. Confocal immunofluorescence staining has shown that the expression of Lim1 and al are coincident in the most distal parts of the leg and antenna disc. Furthermore, there are similarities between the al and Lim1 mutant phenotypes, since in al mutants the claw organ, the sternopleural bristles in the leg and the arista are missing or reduced. A possible functional relationship between the two genes was explored. Since al is expressed earlier than Lim1, one possibility is that Lim1 expression is regulated by al. The expression of Lim1 was examined in al mutants, and Lim1 was found to be lost in the presumptive tip of the leg where both genes are co-expressed. In contrast, al expression is normal in Lim1 mutants. These results suggest that the Lim1 locus is regulated by the al gene, one possibility being that the Lim1 gene is a direct downstream target of the homeodomain Al protein (Pueyo, 2000).

In both al and Lim1 mutants, the strongest pupal lethal alleles eliminate the claw and reduced the rest of the pretarsal organs, but do not eliminate the whole pretarsus in all legs. This could be due to a functional cooperation between al and Lim1, such that the presence of any one product in the absence of the other would still provide enough function for some organs of the pretarsus to occasionally develop. Alternatively, the remnant pretarsal organs observed in al and Lim1 mutants could be due to hypomorphy of the mutants available and no functional relationship needs to be implied between the two genes. It was reasoned that if a functional relationship does exist, a double mutant should show an enhanced phenotype: a double mutant Lim1^R12.4;al^ice was generated, and found to be embryonic lethal. Therefore, al and Lim1 double mutants show a synergistic effect that might betray a functional relationship. This synergistic relationship is also shown in ectopic expression experiments. Ectopic expression of either Lim1 or al driven by the 30AGal4 line produces only small defects in the joint between tarsus four and five. However, simultaneous expression of both al and Lim1 in 30AGal4;UASLim1;UASal flies produces stronger defects, including partial or complete fusion of tarsus four and five (Pueyo, 2000).

The distal region of the Drosophila leg, the tarsus, is divided into five segments (ta I-V) and terminates in the pretarsus, which is characterized by a pair of claws. Several homeobox genes are expressed in distinct regions of the tarsus, including aristaless (al) and lim1 in the pretarsus, Bar (B) in ta IV and V, and apterous (ap) in ta IV. This pattern is governed by regulatory interactions between these genes; for example, Al and Bar are mutually antagonistic, resulting in exclusion of Bar expression from the pretarsus. Although Al is necessary, it is not sufficient to repress Bar, indicating another factor is required. This factor has been identified as the product of the C15 gene, also termed clawless (cll), a homeodomain protein that is a homolog of the human Hox11 oncogene. C15 is expressed in the same cells as al -- together, C15 and Al appear to directly repress Bar and possibly to activate Lim1. C15/Al also act indirectly to repress ap in ta V, i.e., in surrounding cells. To do this, C15/Al autonomously repress expression of the gene encoding the Notch ligand Delta (Dl) in the pretarsus, restricting Dl to ta V and creating a Dl+/Dl− border at the interface between ta V and the pretarsus. This results in upregulation of Notch signaling, which induces expression of the bowl gene, the product of which represses ap. Similar to aristaless, the maximal expression of C15 requires Lim1 and its co-factor, Chip. Bar attenuates aristaless and C15 expression through Lim1 repression. Aristaless and C15 proteins form a complex capable of binding to specific DNA targets, which cannot be well recognized solely by Aristaless or Clawless (Campbell, 2005; Kojima, 2005).

To investigate regulatory interactions between C15, Al, and Lim1, each was misexpressed in the leg and expression of the other two was examined. This was achieved initially with a UAS-C15 line and by generating Gal4 expressing clones using the FLPout technique and Tub-Gal4; the clones were monitored with UAS-GFP. This revealed that ectopic C15 could, in fact, induce ectopic expression of both Al and Lim1, although this was somewhat random with Al and Lim1 being expressed only in some cells ectopically expressing C15. Ectopic C15 can also repress Bar and loss of Bar results in expansion of the Al expression domains, but only in the cells immediately surrounding their normal domains. Repression of Bar does not appear to account for the ectopic Al and Lim1 expression induced by C15, because, Al and Lim1 can be induced some distance from their endogenous domains. In contrast, misexpression of al or lim1 in Tub-Gal4 clones has no effect on expression of the other genes. It has been shown that driving higher levels of lim1 can induce ectopic expression of al and this was confirmed using dpp-Gal4. However, there was no ectopic C15 in the UAS-lim1; dpp-Gal4 discs. Similarly, driving higher levels of al with dpp-Gal4 does not induce ectopic expression of C15 (Campbell, 2005).

Therefore, although Al is still expressed in C15 mutants, and vice versa, indicating that both are probably activated independently by EGFR signaling, C15 can induce expression of al and lim1. This may act as a feedback mechanism to ensure all three are expressed in the same cells. Since expression of Lim1 is completely lost in the center of discs from C15 and al mutants, it may simply be a direct target of either or both and may not be directly activated by EGFR signaling (Campbell, 2005).

Since al is still expressed, albeit in a much smaller domain, in C15 mutants and C15 is still expressed in al mutants, it appears possible that each may play an additional, redundant role, in patterning the leg. This was ruled out by examining al^ice, C15² double mutants (both alleles are either null or very close to being null), which have legs and antennae that are indistinguishable from either single mutant, indicating that, in the absence of the other, neither Al nor C15 provides any function during leg development (Campbell, 2005).

That Lim1 expression becomes discernible in the future pretarsus shortly after the appearance of Al and Cll signals may imply that Lim1 is positively regulated by al and/or cll. However, it is difficult to directly determine whether al and cll are required for Lim1 expression, since Bar, serving as a repressor for Lim1, is de-repressed in the pretarsus of al or cll mutants. Thus, the effect was examined of cll or al misexpression on Lim1-lacZ, whose expression is essentially identical to that of Lim1. cll-misexpressing flip-out clones were generated in first-second instar, and Lim1-lacZ signals were detected in late third instar. Lim1-lacZ misexpression occurred in some cll-misexpressing cells, indicating that Cll can serve as a positive regulator of Lim1 (Kojima, 2005).

A previous experiment showed the sole misexpression of al is incapable of inducing Lim1 misexpression. However, this does not necessarily mean that al is not involved in Lim1 regulation. Rather the notion is preferred that Lim1 is positively regulated by a concerted action of al and cll, since Lim1-LacZ signals are detected in all cll-misexpressing cells, only in those cells in which al expression is simultaneously observable (Kojima, 2005).

Targets of Activity

Lim1 expression becomes discernible slightly later than expression al, clawless (cll/C15) and Bar, and maximal al expression in late third instar depends on Lim1 function. Cll expression is also significantly reduced in Lim1^7B2 (a null allele) clones in late third instar discs, indicating that not only al but also cll is positively regulated by Lim1 in the late third instar. Chi encodes a LIM domain binding protein and has been suggested to act as a co-factor for Lim1. Cll and Al signals are significantly reduced in clones of Chi^e5.5, a null allele of Chi. The concerted action of Lim1 and Chi is thus shown to be required for the maximal expression of cll and al in the late-third-instar pretarsus region (Kojima, 2005).

When Lim1 is misexpressed using blk-GAL4, the expression of al but not cll is induced. Thus, unlike al, cll may require an additional component for its maximal expression. Alternatively, cll may be less sensitive to activation by Lim1 than al (Kojima, 2005).

Protein Interactions

At the sequence level, Lim1 is highly related to its vertebrate homologs. Given their conservation, it was of interest to see if this sequence homology translates into functional similarities at a molecular level. In Xenopus, Xlim-1 and the LIM-domain-binding protein (Xldb-1/NLI/CLIM- 2) interact in vitro, and cooperate in vivo to induce secondary axis structures (Agulnick, 1996). As the name implies, this association takes place through the LIM domains. More recently, the Drosophila homolog of Xldb1/NLI/CLIM-2, Chip has been cloned and shown to interact with the Apterous protein (Morcillo, 1997; Fernandez-Funez, 1998). To determine if Lim1 and Chip interact in vitro, co-immunoprecipitation experiments were carried out. Using the Lim1 antibody, the ability of Chip to be immunoprecipitated by full-length and truncated Lim1 proteins was carried out. The results show that Chip can be immunoprecipitated in the presence of full-length Lim1, and a truncated Lim1 protein that contains the LIM domains (LIM-Lim1). HD-Lim1, which lacks the LIM domains and includes the homeodomain fails to coimmunoprecipitate Chip. Additionally, Chip by itself is not immunopreciptated by the Lim1 antibody. These results show that Lim1 has the capacity to interact with the LIM-domain-binding protein, Chip. This interaction requires the LIM domains of Lim1 and is independent of the Lim1 homeodomain. Similar to its vertebrate counterparts, and Apterous in Drosophila, Lim1 and Chip may cooperate in vivo to modulate the transcriptional activity of its downstream target genes. Chip is ubiquitously expressed and therefore is present in all Lim1-expressing cells suggesting that an in vivo interaction is possible (Lilly, 1999).

Chip-mediated partnerships of the homeodomain proteins Bar and Aristaless with the LIM-HOM proteins Apterous and Lim1 regulate distal leg development

Proximodistal patterning in Drosophila requires division of the developing leg into increasingly smaller, discrete domains of gene function. The LIM-HOM transcription factors apterous (ap) and Lim1 (also known as dlim1), and the homeobox genes Bar and aristaless (al) are part of the gene battery required for the development of specific leg segments. Genetic results show that there are posttranslational interactions between Ap, Bar and the LIM-domain binding protein Chip in tarsus four, and between Al, Lim1 and Chip in the pretarsus, and that these interactions depend on the presence of balanced amounts of such proteins. In vitro protein binding is observed between Bar and Chip, Bar and Ap, Lim1 and Chip, and Al and Chip. Together with evidence for interactions between Ap and Chip, these results suggest that these transcription factors form protein complexes during leg development. It is proposed that the different developmental outcomes of LIM-HOM function are due to the precise identity and dosage of the interacting partners present in a given cell (Pueyo, 2004).

Biochemical studies in vitro have shown that LIM-HOM transcription factors confer little transcriptional activation of target genes on their own. LIM-HOM proteins interact with a variety of proteins, including members of the bHLH family, the POU family and also other LIM family members, to control specific developmental processes. It has been suggested that these protein interactions confer specificity and modulate LIM-HOM activity. For example, Dlmo proteins reduce LIM-HOM activity, and Lbd proteins such as Chip modulate LIM-HOM activity by acting as a bridge between LIM-HOM proteins and Chip-binding cofactors, thus leading to the formation of heteromeric complexes. LIM-HOM protein activity functions in different contexts is the development of Drosophila (Pueyo, 2004).

Bar and ap genes are expressed in the fourth tarsal segment and are required for its proper development, whereas the al and Lim1 genes are expressed and required in the pretarsus. All of these genes encode putative transcription factors and display canonical regulatory relationships. Thus, al activates lim1 expression and then both genes cooperate to repress Bar expression in the pretarsus. Reciprocally, Bar represses al and lim1 expression while activating the expression of ap in tarsus four. After the refinement of their gene expression domains by these regulatory interactions, Bar directs tarsus five development, whereas cooperation between al and lim1 directs pretarsus development, and cooperation between Bar and ap directs tarsus four. The results of this study offer more evidence for the existence of this regulatory network, but also suggest an interesting role for direct protein interactions in its mechanism (Pueyo, 2004).

The cooperation between Bar and Ap on the one hand, and Al and Lim1 on the other, is likely to be carried out by transcriptional complexes involving Chip. The Chip protein is required for development of the tarsus four, five and pretarsus, and Gst (Glutathione-S-transferase-Chip fusion construct) experiments reveal Chip's ability to bind Ap, Bar, Lim1 and Al. However, the results also show that modulation of LIM-HOM protein activity by Chip alone does not explain distal leg development. For example, Ap function is not modulated primarily by Chip and Dlmo. The relative amount of Chip and Ap has to be grossly unbalanced before a phenotype is obtained in the leg, and dlmo is not expressed or required in leg development. Furthermore, the interaction between Ap and Chip does not confer the developmental specificity that allows LIM-HOM proteins to produce different outcomes in different parts of the leg. (1) Ap and Chip also interact in the wing and the CNS. (2) A chimaeric Lim3-Ap protein containing the LIM domains of Lim3 and the HOM domain of Ap does not behave as a dominant negative when expressed in tarsus four, and is even able to fulfil Ap function and rescue ap mutants. In the distal leg, developmental specificity seems to be achieved at the level of DNA binding and the transcriptional control of targets genes, mediated by partnerships between LIM-HOM and HOM proteins (Pueyo, 2004).

The evidence for this is presented first by dosage interactions between LIM-HOM and HOM proteins. Whereas there seems to be a relative abundance of endogenous Ap in tarsus four, an excess of Bar or Chip leads to a mutant phenotype, which is rescued by restoring the normal balance between Ap, Bar and Chip proteins in co-expression experiments. The effects observed could be explained simply by independent competition and the binding of Bar and Ap to Chip, leading, for example, to an excess of Bar-Chip complexes and a reduction of the pool of Chip available for Ap-Chip complexes. However, this hypothesis alone does not explain the additional dominant-negative effects of ectopic LIM-HOM and HOM proteins in tarsus four (Lim3, Islet and Al), which are also not mediated by transcriptional regulation but are nonetheless rescued by co-expression of appropriate endogenous proteins. For example, ectopic expression of UAS-islet or UAS-Lim3 in the ap domain produces loss of tarsus four without affecting Ap or Bar expression, and simultaneous co-expression of UAS-Bar partially suppresses this phenotype. If the sole effect of both UAS-Bar and UAS-Lim3 or UAS-islet were to quench Chip away from Ap, then simultaneous co-expression of Bar and Lim3 or Islet should worsen the phenotype, not correct it as observed. Moreover, ectopic expression of Islet or Lim3 proteins is not corrected by simultaneous co-expression of either UAS-Chip or UAS-ap. Altogether these results show instead that UAS-islet and UAS-Lim3 must interfere posttranslationally with Bar. The most direct explanation is that Islet and Lim3 have the ability to quench Bar protein into a non-functional state. Interestingly, the hybrid UAS-Lim3:ap does not behave as dominant negative but as an endogenous Ap protein in these experiments, since it does not produce a mutant phenotype on its own and it rescues UAS-Bar overexpression. This suggests that the LIM domains are not very specific when it comes to interaction with Bar, and points to the involvement of a common LIM-binding intermediary such as Chip. These results suggest that a protein complex involving Ap, Chip and Bar is the correct functional state of these proteins in tarsus four, and deviations from this situation into separate Bar-Chip, Ap-Chip, or Bar-Chip-Lim3 or Bar-Chip-Islet complexes leads to a mutant phenotype (Pueyo, 2004).

The notion of a protein complex involving Ap, Chip and Bar together is also supported by the Gst pull-down assays. The domain of Chip involved in Ap binding, the LIM interaction domain (LID), is not involved in Bar binding. However, the LID and the dimerisation domains of Chip are necessary to rescue the dominant-negative effect of UAS-Bar on tarsus four, suggesting a requirement for the formation of a complex with a LIM-HOM protein such as Ap. In agreement with this view, the Ap protein, and the LIM domains of Ap alone, are able to retain Bar protein in a Gst assay (Pueyo, 2004).

In the pretarsus, Al and Lim1 are possibly engaged in a partnership with Chip similar to that suggested for Ap, Chip and Bar. Synergistic cooperation between Al and Lim1 is required to direct pretarsus development and to repress Bar expression and function. Their cooperation entails a close functional relationship because a proper balance of Al, Lim1 and Chip is required, as is shown by the loss of pretarsal structures in UAS-Chip or UAS-Lim1 flies. Ectopic expression of LIM-HOM proteins in the pretarsus also disrupts pretarsal development without affecting Lim1 and Al expression. The possibility of direct protein interactions between Al, Lim1 and Chip is also suggested by the reciprocal ability of Al to interfere posttranscriptionally with Bar and Ap in tarsus four, and by the binding of Chip to Lim1 and to Al in in vitro experiments (Pueyo, 2004).

Comparison of tarsal development with other developmental processes illustrates how LIM-HOM proteins are versatile factors to regulate developmental processes. It had been observed that the outcome of LIM-HOM activity depends on their developmental context. This context can now be analysed as being composed of the presence, concentration and relative affinities of other LIM-HOM proteins, Ldb adaptors, and other cofactors such as LMO proteins and HOM proteins. It is proposed that the different developmental outcomes of LIM-HOM protein function could be due to the precise identity and dosage of cofactors available locally (Pueyo, 2004).

Ectopic expression experiments distort these contexts and lead to non-functional or misplaced LIM-HOM activities. In the wing, a finely balanced amount of functional Ap protein is modulated by Dlmo and Chip. Over-abundance of Chip stops the formation of functional tetramers in the wing but not in the CNS, where the relative amount of Ap, which is not modulated by Dlmo, is limiting for the formation of Ap-Chip functional complexes. In tarsus four, the Ap-Chip-Bar partnership is affected by experimentally induced over-abundance of Chip, presumably also because ectopic Ap-Chip tetramers typical of the CNS and the wing, and Bar-Chip complexes typical of tarsus five, are produced. Similarly, an excess of Bar might be interpreted by the cells as being a wrong developmental outcome, since high levels of Bar in the absence of Ap direct tarsus five development. Overexpression of Ap rescues this Bar dominant-negative effect, by restoring the relative amounts of Bar and Ap, which are determinant and limiting for tarsus four development. Finally, the dominant-negative effects produced by overexpression of either Chip or Lim1 in the pretarsus could either prevent the formation of Al-Chip-Lim1 complexes, or could favor the existence of Lim1-Chip complexes typical of the CNS (Pueyo, 2004).

The wing and the CNS models have postulated that Ap function is carried out by an Ap-Chip tetramer; however, the molecular scenario might be more complex. A new component of Ap-Chip complexes, named Ssdp, has been identified and is required for the nuclear localisation of the complex. Thus it is possible that an Ap-Chip tetramer also contains two molecules of Ssdp. In addition, different types of Chip-mediated transcriptional complexes and different regulators have been identified in other developmental contexts, such as in sensory organ development and thorax closure, in which the GATA factor Pannier forms a complex with Chip and with the bHLH protein Daughterless. Heterodimers of this complex are negatively regulated by a protein interaction with Osa. Thus, although the current results indicate that in different segments of the leg there exist specific interactions between LIM-HOM, Chip and HOM proteins, the involvement of further elements in these multiprotein complexes is not excluded (Pueyo, 2004).

The results support a partnership between HOM and LIM-HOM proteins in the specification of distinct segments of the leg, and the results are compatible with Ap-Chip-Bar, Bar-Chip and Lim1-Chip-Al forming transcriptional complexes. Although the characterisation of the target sequences, followed by further biochemical and molecular assays, is necessary to study the transcriptional mechanism of these interactions, it has been shown that LIM-HOM proteins can interact specifically and directly with other transcription factors to regulate particular genes. For instance, mouse Lim1 (Lhx1) interacts directly with the HOM protein Otx2. In addition, the bHLH E47 transcription factor interacts with Lmx1, and both synergistically activate the insulin gene. This interaction is specific to Lmx1, since E47 is unable to interact with other LIM-HOM proteins such as Islet. Moreover, Chip is able to bind to other Prd-HOM proteins, such as Otd, Bcd and Fz, to activate downstream genes. Chip also complexes with Lhx3 and the HOM protein P-Otx, increasing their transcriptional activity. The current results reinforce the notion of Chip as a multifunctional transcriptional adaptor that has specific domains involved in each interaction (Pueyo, 2004).

Experiments in Drosophila have demonstrated a conservation of LIM-HOM activity at the functional and developmental level in the CNS between Drosophila and vertebrates. In addition, xenorescue experiments have shown that the mechanism of action of Ap and its vertebrate homolog Lhx2 is very conserved in Drosophila wings, whereas ectopic expression of dominant-negative forms of chick Lim1, Chip, Ap and Lhx2 mimic both Ap and Lhx2 loss-of-function phenotypes. The developmental role of Ap, Bar and Al in the fly leg, and their putative molecular interactions may also have been conserved because their vertebrate homologs Lhx2, Barx and Al4 are also co-expressed in the limb bud. It is expected that the interactions between the LIM-HOM and Prd-HOM proteins shown here represent a conserved mechanism to specify different cellular fates during animal development (Pueyo, 2004).

DEVELOPMENTAL BIOLOGY

Lim1 protein, detected using a specific antiserum, is first detected at the cellular blastoderm stage in a circumferential stripe just anterior to the cephalic furrow. Prior to this, no expression is evident within the syncytium, indicating a lack of maternal contribution. Zygotic expression persists in the procephalic lobes as gastrulation proceeds and becomes compartmentalized to defined regions within the head segments. At maximum germ band extension, transcripts are detected in clusters of cells in the thoracic and abdominal segments. Expression is confined to the cells at the neuroectoderm border where the neuroblasts are segregating from the ectoderm. In later stages of embryogenesis, high levels of Lim1 are present in the head segments, particularly in the clypeolabrum, the mandible, and maxilla. High levels of expression are also observed in the cells of the ventral nerve cord, as well as the embryonic brain lobes. In addition, the Lim1 protein is present in small groups of cells in the peripheral nervous system and cells of the ventral epidermis. The expression of Lim1 RNA was analyzed by whole mount in situ hybridization. In comparing the expression of RNA and protein no obvious differences were observed between their individual patterns (Lilly, 1999).

To further define the Lim1 expressing cells within the nervous system, double labeling experiments were carried out with the Lim1 antibody and cell specific markers. Using an antibody against the Repo protein, which is expressed exclusively in glial cells, no overlap was observed between Lim1 and Repo expression. Double stainings for Lim1 and the Elav protein, which is expressed in all neural cells, shows that Lim1 is confined to a subset of neuronal cells within the ventral nerve cord. To help identify the neurons within the nerve cord that are positive for Lim1, double labelling using an antibody that recognizes the Evenskipped (Eve) protein has been carried out. Lim1 was localized to motoneurons, RP2, aCC, and the U neurons. All of these motorneurons innervate the dorsal-most muscle groups within each segment. Interestingly, the motorneurons in which Isl is expressed innervate only ventral muscle groups. Lim1 is localized to a subset of interneurons that express the Derailed protein. Thus, within the ventral nerve cord Lim1 is expressed in both motorneurons and interneurons, and is absent from the glial cell population (Lilly, 1999).

Additionally, the expression of Lim1 was analyzed in dissected larvae by immunostaining. Lim1 protein is present in discrete cells of the larval nerve cord, and in the lobes of the brain. There is particularly strong expression in the cells of brain from which the optic stalk emerges to attach to the eye disc. Lim1 immunoreactivity is detected in the antennal disc and the leg disc. No protein was observed in the eye disc, or the wing and haltere discs. Lim1 in the leg and antennal disc are expressed in concentric rings, corresponding to the major segmental folds in the disc epithelium. The center spot in each disc represents the distal-most segment of what will become the adult leg and antennae. The outer rings correspond to the progenitors of more proximal segments. In addition, Lim1 expression is detected in the labial disc. All discs that express Lim1 are derived from imaginal progenitors that originate from the ventral region within the embryo. Thus, as in the embryo, Lim1 shows a very defined pattern of expression within subsets of imaginal discs and adult progenitors (Lilly, 1999).

The unique expression of Lim1 in a subset of motoneurons and interneurons led to an examination of whether this expression overlaps with other LIM homeodomain members. Characterization of the expression of a group of vertebrate LIM homeodomain genes (Isl-1, Isl-2, Lim-1 and Lim-3) along the chick spinal column has demonstrated that these genes overlap with one another in very distinct patterns (Tsuchida, 1994). Their spatial overlap demarcates regions of motorneuron subclasses, suggesting that the LIM homeodomain genes confer an identity to pools of motorneurons by Lim protein combinatorial expression. More recently, a combinatorial code for motorneuron pathway selection has been demonstrated for isl and lim3 in Drosophila (Thor, 1999). In order to evaluate the expression of Lim1 with respect to its LIM homeodomain relatives, a series of double labeling experiments were carried out using late stage embryos. These embryos carried enhancers from either islet (Thor, 1997), lim3 (Thor, 1999) or apterous (O'Keefe, 1998) that recapitulated their expression using a tau-LacZ or tau-c-myc fusion construct as a reporter. By double staining for enhancer expression and the Lim1 protein, no overlap of expression was observed between Lim1 and Isl, Ap or Lim3. All neurons that stain positively for Lim1 in the nuclei lack enhancer expression within their cell bodies. Similar to what is observed in vertebrates, the LIM homeodomain genes that were analyzed in Drosophila are expressed in distinct subclasses of neurons within the ventral nerve cord. The expression of Lim1 is confined to a subset of motorneurons and interneurons that are lacking the other LIM homeodomain genes tested. To assess the possibility that the absence of Lim1 in cells expressing other LIM homeodomain proteins was due to repression by these family members, the expression of Lim1 was analyzed in ap and lim3 mutant embryos. In embryos with a null mutation in ap, the expression of Lim1 remains unchanged. Likewise, analysis of the Lim3-expressing RP neurons in lim3 mutants shows no upregulation of Lim1. These results indicate that the exclusion of Lim1 from cells expressing other LIM homeodomain proteins is not a result of repression by these LIM homeodomain family members (Lilly, 1999).

In addition to the exclusive expression of Lim1, Ap and Isl do not overlap (Thor, 1997), while Lim3 fails to overlap with Ap, but is found in a subset of Isl positive cells (Thor, 1999). Thus, as in vertebrates, the expression of these genes in the Drosophila nerve cord may provide instructional cues for proper pathfinding and target identity in the embryo (Lilly, 1999).

Intrinsic control of precise dendritic targeting by an ensemble of transcription factors

Proper information processing in neural circuits requires establishment of specific connections between pre- and postsynaptic neurons. Targeting specificity of neurons is instructed by cell-surface receptors on the growth cones of axons and dendrites, which confer responses to external guidance cues. Expression of cell-surface receptors is in turn regulated by neuron-intrinsic transcriptional programs. In the Drosophila olfactory system, each projection neuron (PN) achieves precise dendritic targeting to one of 50 glomeruli in the antennal lobe. PN dendritic targeting is specified by lineage and birth order, and their initial targeting occurs prior to contact with axons of their presynaptic partners, olfactory receptor neurons. A search was performed for transcription factors (TFs) that control PN-intrinsic mechanisms of dendritic targeting. Two POU-domain TFs, acj6 and drifter have been identified as essential players. After testing 13 additional candidates, four TFs were identified, (LIM-homeodomain TFs islet and lim1, the homeodomain TF cut, and the zinc-finger TF squeeze) and the LIM cofactor Chip, that are required for PN dendritic targeting. These results begin to provide insights into the global strategy of how an ensemble of TFs regulates wiring specificity of a large number of neurons constituting a neural circuit (Komiyama, 2007).

For technical simplicity, larval born GH146-Gal4-positive PNs, originating from three neuroblast lineages, anterodorsal (adPNs), lateral (lPNs), and ventral (vPNs), were studied. Out of ~25 classes defined by their glomerular targets, focus was placed on 17 classes whose target glomeruli are reliably recognized across different animals. The MARCM technique allows visualization and genetic manipulation of PNs in neuroblast and single-cell clones in otherwise heterozygous animals, so PN-intrinsic programs can be studied for dendritic targeting. GH146 is expressed only in postmitotic PNs (Komiyama, 2007).

acj6 and drifter have been identified as lineage-specific regulators of PN dendritic targeting. To identify additional transcription factors (TFs) that regulate dendritic targeting of different PN classes, candidates were tested that have been shown to regulate neuronal subtype specification and targeting specificity and have available loss-of-function mutants. The following was tested; (1) the expression of candidate genes in PNs at 18 hr after puparium formation (APF) when PN dendrites are in the process of completing their initial targeting, and/or (2) their requirement in PNs by examining dendritic targeting in homozygous mutant MARCM clones (Komiyama, 2007).

In addition to the eight genes described below, five other TFs were examined that were not pursued because of the lack of expression in GH146-PNs at 18 hr APF (aristaless and pdm-1) or the lack of targeting defects in homozygous mutant PNs (abrupt [ab^k02807], kruppel [Kr¹], and Dichaete [Dichaete⁸⁷]) (Komiyama, 2007).

LIM-HD factors and PN targeting: LIM-homeodomain (LIM-HD) TFs are involved in multiple events during neuronal development. Most functions of LIM-HD factors require the LIM domain-binding cofactor, which is represented in Drosophila by ubiquitously expressed Chip. Chip antibody revealed ubiquitous expression of Chip in cells around the antennal lobe (AL) including all GH146-PNs at 18 hr APF (Komiyama, 2007).

The requirement of Chip in PN dendritic targeting was tested. Wild-type adPNs, lPNs, and vPNs target stereotyped sets of glomeruli. PNs homozygous for a Chip null allele (Chip^e5.5) failed to target most of the correct glomeruli and occupied inappropriate glomeruli. Most adPN and lPN clones (12/13) also mistargeted a fraction of dendrites to the structure ventral to the AL, the suboesophaegeal ganglion (SOG). Thus, Chip is required for targeting specificity of most, if not all, PN classes studied here, and Chip-interacting proteins including LIM-HD factors likely play important roles in PN dendritic targeting (Komiyama, 2007).

Five LIM-HD factors have been characterized in Drosophila: apterous, arrowhead, islet, lim1, and lim3. apterous, arrowhead, or lim3 were not pursued because they are not expressed in GH146-PNs at 18 hr APF (apterous) or they do not have targeting defects in PNs homozygous for null alleles (lim3^37Bd6 and awh¹⁶) (Komiyama, 2007).

Islet antibody detected Islet expression in ~50% adPNs and most lPNs but not in vPNs at 18 hr APF and adult. isl^−/− adPNs failed to target many (but not all) of the normal target glomeruli, including VA1lm, VA3, and VM7. In addition, DA1, a lPN target, was often specifically mistargeted. Defects of isl^−/− lPNs were very similar to Chip^−/− lPN defects. A fraction of dendrites often mistargeted to the SOG. Within the AL, dendrites were diffusely spread, although DA1 and DL3 were always correctly innervated. Targeting of isl^−/− vPNs was normal, consistent with their lack of Islet expression (Komiyama, 2007).

Lim1 antibody revealed Lim1 expression in most or all vPNs, but not in adPNs or lPNs in adults. The expression pattern appears similar at 18 hr APF, although vPNs are difficult to identify unambiguously at early stages. lim1^−/− adPNs showed no defects, consistent with the lack of Lim1 expression. lim1^−/− lPNs rarely showed a cell number decrease, but in clones in which the cell number was normal, lim1^−/− lPNs targeted correct glomeruli. In contrast, lim1^−/− vPNs showed a specific targeting defect. Wild-type vPNs innervate DA1 and VA1lm densely because of the single vPNs that specifically innervate these glomeruli, in addition to the diffuse innervation all over the AL contributed by the pan-AL vPN. In lim1^−/− vPNs, DA1 innervation was greatly reduced and sometimes undetectable. Therefore, lim1 is required for dendritic targeting by a single vPN class, vDA1, despite its general expression in vPNs. lim1 might be redundant with other factors in non-DA1 vPNs. It was note that phenotypes of islet and lim1 combined are only a subset of the Chip phenotype. Additional Chip phenotype may be explained by non-Lim-HD molecules interacting with Chip (Komiyama, 2007).

cut is required for targeting of several lPN and all vPN classes: cut encodes a homeodomain TF that regulates sensory organ identity and dendritic morphogenesis in Drosophila peripheral nervous system. A monoclonal antibody detected Cut in subsets of adPNs and lPNs (~8 for each) and in all vPNs. The expression pattern appeared similar at 18 hr APF. Costaining with Mz19-Gal4 and various single-cell clones with GH146-Gal4 further narrowed down Cut-expressing PNs; Cut-positive adPNs are likely embryonically born and thus not included in the functional analysis, while DM1 and DM2 lPNs express Cut, but DA1, DL3, and DM5 lPNs do not (Komiyama, 2007).

cut^−/− adPNs targeted all their normal glomeruli correctly, consistent with their lack of expression. cut^−/− lPNs failed to target DM1, DM2, and VA5. cut^−/− vPNs were severely affected, with their cell numbers reduced from 4–6 in wild-type to 2–3 in cut^−/− clones. cut^−/− vPNs failed to elaborate their dendrites correctly in the AL and mistargeted the SOG. In summary, cut is required by a specific subset of lPNs and all vPNs that express Cut (Komiyama, 2007).

cut appears to control global targeting of PNs along mediolateral axis, as indicated by the fact that loss and gain of cut in lPNs causes a lateral and medial shift of dendrites, respectively. adPNs do not show a cut loss-of-function defect, consistent with the lack of expression. Nevertheless, cut misexpression in adPNs shifted their dendrites medially. Interestingly, adPNs misexpressing cut usually avoid DM1 and DM2, suggesting that cut controls global targeting, rather than simply promoting innervation of these glomeruli (Komiyama, 2007).

Postmitotic expression of a cut transgene only in labeled cut^−/− lPNs completely rescued targeting of DM1, DM2, and VA5. There were also gain-of-function phenotypes, and DA1 and DL3 innervation was often lacking in these clones. Thus, cut postmitotically rescues dendritic targeting defects of lPNs that normally express cut, whereas postmitotic misexpression in other lPNs disrupts their targeting fidelity (Komiyama, 2007).

The vPN rescue phenotype was more complex. The cell number decrease was not rescued by postmitotic cut expression. However, the targeting defect was partially rescued. 71% of vPN rescue clones examined sent some dendrites to the AL (the rest completely failed to innervate the AL), and 68% innervated VA1lm. This is markedly better than cut^−/−, in which only 51% entered the AL and 23% innervated VA1lm. DA1 targeting was not rescued, raising the possibility that the DA1 vPN was never born or correctly specified in these animals (Komiyama, 2007).

Relationship of cut and lim1 in vPNs: The lim1 phenotype in vPNs is a subset of the cut phenotype. Lim1 immunoreactivity in cut^−/− vPNs was either absent or greatly reduced compared to wild-type. Therefore, Cut directly or indirectly controls Lim1 expression (Komiyama, 2007).

If a major function of Cut in vPNs is to upregulate Lim1, then transgenic lim1 expression in cut^−/− vPNs might suppress part of the cut^−/− phenotype. In cut^−/− vPNs expressing a lim1 transgene, the reduction of cell number was not suppressed. However, 67% clones innervated the AL (compared to 51% in cut^−/−). VA1lm innervation was also mildly improved (36% in UAS-lim1 versus 23% in cut^−/−). Thus, UAS-lim1 expression partially suppresses cut^−/− targeting defects, although not quite as well as UAS-cut. In contrast, UAS-lim1 expression in cut^−/− lPNs, which normally do not express Lim1, did not suppress the cut^−/− targeting defects. Therefore, Cut and Lim1 are not simply interchangeable, and the partial suppression of cut^−/− defects by lim1 is specific to vPNs (Komiyama, 2007).

Although postmitotic expression of cut partially rescued the cut^−/− vPN phenotypes, it failed to rescue Lim1 expression. In addition, postmitotic misexpression of cut in adPNs or lPNs did not lead to an ectopic expression of Lim1. Therefore, cut is not sufficient to upregulate Lim1 expression in postmitotic neurons. It is proposed that cut functions at two distinct stages of vPN development. First, cut controls the proliferation and/or fate specification of the vPN neuroblast, including Lim1 expression. Second, cut controls dendritic targeting by postmitotic VA1lm vPNs, partially redundantly with lim1. This partial redundancy may explain the observation that lim1^−/− vPNs target VA1lm normally. These pre- and postmitotic functions of cut in the same neuronal lineage are reminiscent of its function in peripheral nervous system development (Komiyama, 2007).

EFFECTS OF MUTATION

Lim1 mutation is lethal. Lim1 mutant embryos hatch from their egg case and can develop to the 3rd instar larval stage. In good culture conditions most progress to the 3rd instar stage, and some will pupate. Two alleles appear to be hypomorphs, because 90% of the mutants pupate, compared to only 10%-20% for null allele E9 and allele E4. Heteorallelic combinations of the Lim1 mutations with each other and with Df(1)lz-90b24 give similar results. The mutant larvae exhibit no developmental delay: they molt and reach the wandering stage with no obvious defects. Movement of larvae appears typical until the 3rd instar wandering stage. At this stage the mutants appear sluggish and fail to wander normally. This causes them to either arrest or pupate on or near their food source. From analysis of dissected pupal cases, a few mutants are seen to develop into pharate adults, but are incapable of eclosion and survival beyond this point. These behavioral defects suggest that the Lim1 mutants have abnormal motor coordination, leaving them unable to wander properly and eclose into adult flies. Molecular analysis of these mutants has not revealed any striking defects. By using an array of molecular markers to analyze both embryos and larvae, the overall structure of the nervous system has been found to be normal. The mutants were analyzed with several nervous system markers including 22C10 (see Futsch), FasII BP102, and BP104; no structural defects or misguided axon projections were detected. In particular, the projections of the RP2 and aCC neurons were analyzed in dissected embryos and larvae and they were found to be morphologically normal. For each marker, approximately 5 individual hemisegments from 50 Lim1 mutant embryos were analyzed and compared to wild-type embryos using whole mount immunocytochemistry. For the dissected preparations, more than 20 mutant embryos and 20 mutant larvae were dissected, from which 5 to 6 hemisegments where analyzed in detail and compared in parallel to wild-type preparations (Lilly, 1999).

Although it is quite possible that Lim1 mutants have subtle pathfinding defects, as is the case with ap and isl, they could not be identified. Lim1 is not found in serotonin- and dopamine-producing neurons, and is absent from the ring gland in larva, suggesting that these defects are not due to disruptions in these neurosecretory pathways. Thus a clear explanation for the cause of lethality for the Lim1 mutants remains to be elucidated. Additionally the neuronal phenotype of Lim1 null mutants in embryos heterozygous for chip-/+ was examined. However, as with the Lim1 mutation alone, no phenotype was observed in the Lim1-/- null, chip-/+ heterozygous background (Lilly, 1999).

During Drosophila leg development, the distal-most compartment (pretarsus) and its immediate neighbor (tarsal segment 5) are specified by a pretarsus-specific homeobox gene, aristaless, and tarsal-segment-specific Bar homeobox genes, respectively; the pretarsus/tarsal-segment boundary is formed by antagonistic interactions between Bar and pretarsus-specific genes that include aristaless. Drosophila Lim1 is involved in pretarsus specification and boundary formation through its activation of aristaless. Ectopic expression of Lim1 causes aristaless misexpression, while aristaless expression is significantly reduced in Lim1-null mutant clones. Pretarsus Lim1 expression is negatively regulated by Bar and is abolished in leg discs lacking aristaless activity, which is associated with strong Bar misexpression in the presumptive pretarsus. No Lim1 misexpression occurred upon aristaless misexpression. The concerted function of both Lim1 and aristaless is required to maintain Fasciclin 2 expression in border cells and form a smooth pretarsus/tarsal-segment boundary. Lim1 is also required for femur, coxa and antennal development (Tsuji, 2000).

To isolate genes that possibly act with al in pretarsus specification, a search was made for genes expressed in the pretarsus but not the segment immediately adjacent to it at late third instar stages. P0092 is an enhancer trap line, in which lacZ expression in leg and antennal discs was found to be similar to al expression in these tissues. lacZ is coexpressed in virtually all Al-positive cells in the pretarsus, tibia, femur and possibly coxa in leg discs, and the arista and first antennal segment in antennal discs. Although Al expression is restricted to ventral cells in the tibia and dorsal cells in the femur, coxa and first antennal segment, lacZ expression has been noted in both ventral and dorsal cells uniformly, which gives rise to complete circular expression. In wing and haltere discs, in which al is also expressed, no appreciable expression of P0092-lacZ is observed. Nucleotide sequence analysis indicates that the putative P0092 gene encodes a LIM-homeodomain protein identical in amino acid sequence to Lim1 (Tsuji, 2000).

Flies neither homozygous nor hemizygous for the P0092 P insertion show any obvious morphological defects. Thus, Lim1 loss-of-function mutants generated by imprecise P-element excision and six independent larval or pupal lethal mutant lines were obtained. These frequently produce pharate adults with apparent defects in mouth parts, leg and antennal morphology, making it possible to examine the roles of Lim1 in leg and antennal development (Tsuji, 2000).

In legs and antenna completely lacking al activity, all pretarsus structures and arista are lost, respectively. In moderate hypomorphic al mutants, claws are frequently lost without loss of other pretarsus structures such as pulvilli and empodia, while in weak hypomorphic mutants, claws and aristae are not lost but only reduced in size. Lim1 minus mutants are very similar in leg phenotype to moderate al hypomorphic mutants. In about half of all cases, the antenna are absent from the Lim1 minus half head. When antennae is present, arista is deformed and reduced in size. That is, Lim1 minus arista are morphologically similar to those of weak hypomorphic al mutants. These findings indicate that Lim1 is essential for proper development of pretarsus and arista as well as al, although Lim1 mutant phenotypes are much less severe than a1 minus mutant phenotypes. In Lim1 minus legs that are simultaneously homozygous for al, not only claws but also empodia and pulvilli are frequently lost. The concerted function of Lim1 and al would thus appear to be required for normal pretarsus/aristal development (Tsuji, 2000).

Apart from the future pretarsus, Lim1 is expressed circularly in proximal segments such as the coxa, femur and tibia. In Lim1 minus flies, the femur is extensively reduced in size and the coxa is missing for the most part or present only as a small bulb-like structure, suggesting the requirement of Lim1 for proper development of the femur and coxa. Although the tibia is bent and fused with the femur, morphological analysis indicates the presence of essentially normal characteristic structures of the tibia, such as transverse rows of bristles, preapical bristles, tibial sense organs and tibial sensilla trichodea; tibial sense organs and tibial sensilla trichodea are structures situated near the proximal tibial end. The tibial phenotype may thus possibly derive from secondary effects of the femoral deformation. In late third instar, Dll expression is evident in the central region spanning from the most distal tip to distal half of the tibia and in the future trochanter. Consistent with shortening of the femur, appreciable reduction in mass has already taken place in the region flanked by the central Dll domain and the proximal Dll ring at late third instar. In Lim1 minus leg and antennal discs, Al expression in the proximal region, such as in the femur, coxa and first antennal segment, is virtually absent. In Lim1 mosaic clones in the femur or coxa, Al expression is abolished cell autonomously. Tibial Al expression remains in Lim1 discs but mosaic analysis clearly indicates substantial reduction in Al expression in Lim1 clones. al is expressed considerably prior to Lim1 and thus Lim1 may thus be involved in maintenance of pretarsus al expression. But loss of Al expression would not completely explain the femoral and coxal defects, since al is dispensable for normal development of the femur and coxa (Tsuji, 2000).

Mutually antagonistic interactions between al and Bar are essential for the strict separation of Al and Bar domains, leading to localized Fas2 induction by Bar in border cells. Although the absence of Lim1 shows little Bar misexpression in the pretarsus, increased Bar misexpression in Lim1;al leg discs could indicate the involvement of Lim1 in the repression of Bar expression in the pretarsus. Remarkable decrease in Fas2 expression in putative Lim1;al mutant border cells indicates that Fas2 expression requires al and Lim1 functions, in addition to cell non-autonomous functions of Bar. Lim1 may be involved in pretarsus specification and boundary formation only through its activation of al. Low al expression in Lim1 single mutants may still be sufficient for maintaining the normal expression of Bar and Fas2, but with further reduction in al expression in Lim1;al double mutants, Bar misexpression and loss of Fas2 expression may result. Alternatively, Lim1 may act independently of al, and simultaneous reduction in al and Lim1 expression may cause Bar misexpression and reduction of Fas2 expression in the double mutants. These considerations are not mutually exclusive (Tsuji, 2000).

Proximal-distal leg development in Drosophila involves a battery of genes expressed and required in specific proximal-distal (PD) domains of the appendage. apterous is required for PD leg development, and the functional interactions between ap, Lim1 and other PD genes during leg development have been explored. A regulatory network formed by ap and Lim1 plus the homeobox genes aristaless and Bar specifies distal leg cell fates in Drosophila (Pueyo, 2000).

Lim homeobox (Lhx) genes have been shown to interact functionally in the nervous systems of Drosophila and vertebrates. It has been suggested that different combinations of Lhx proteins shunt cells into different cell fates, and this model predicts that Lhx proteins can act combinatorially, possibly forming complexes to activate target genes. In appropriate experiments, ectopic generation of a given combination of Lhx proteins shunts cells into an ectopic, but coherent and predictable, cell fate. This study has explored the possibility of similar interactions between Lim1 and other Lhx genes in the appendages of Drosophila. A computer search of the Drosophila genome has identified four other Lhx genes. The Lhx genes Lim3 and Islet (tailup) have been characterized previously in Drosophila, but their mutant phenotypes and patterns of expression do not involve the appendages. A search has identified a new putative Lhx gene, homologous to vertebrate Lmx1, which is not expressed in legs either. The only other Lhx gene identified is the apterous (ap) gene, which is homologous to vertebrate Lhx2. ap is expressed in the leg in the presumptive tarsal segment four, near the tip of the leg close to where Lim1 is expressed. A mutant phenotype for ap in legs has not been described, but using allelic mutant combinations that produce extreme loss of function of ap the following phenotypes were observed: either fusion of tarsus four and five, reduction and deformities in tarsus four, or complete loss of tarsus four, the latter producing legs with only four tarsi but looking otherwise normal. Lim1 and ap expression was combined using UAS constructs to express ap and Lim1 ectopically in legs (Pueyo, 2000).

Expression of UASap over the presumptive claw region using several different Gal4 lines produces no discernible phenotype. In contrast, expression of UASLim1 driven by apGal4, which faithfully reproduces ap expression, produces complete absence of tarsus four, thus mimicking extreme loss of function of ap. This loss of tarsus four fates is specific, since it is also accomplished by ectopic expression of Lim3, a close sequence paralogue of Lim1, but not by other, unrelated proteins, and it is accompanied by the loss of ap expression. However, this apparent dominant negative effect of Lim1 on ap was not rescued by simultaneous co-expression of extra ap in apGal4;UASLim1;UASap flies, as would be expected if the phenotype of UASLim1 were due to either loss of ap expression or competition with the Ap protein. Furthermore, mild tarsal fusions produced by expressing UASLim1 under the control of the weak line 30AGal4 were not made worse by simultaneous reduction of endogenous ap function in ap minus 30AGal4;UASLim1 flies. Altogether these results suggest that, although there exists an effect of ectopic Lim1 on ap expression, the Lim1 and Ap proteins are not interfering directly with each other. Rather, Lim1 must interact with another element involved in tarsus four development and related to ap function (Pueyo, 2000).

The Drosophila Chip gene has been shown to encode a ubiquitous transcriptional cofactor. Chip proteins bind to the Lim domains of Ap and the ap and Chip genes have to be present in similar doses to ensure normal wing development. Interestingly, Chip has been shown to bind the Lim domains of other Lhx proteins, among them Lim1. However, no such dose relationships were found, between chip and Lim1 or between chip and ap in the leg, or in flies expressing UASChip and UASLim1. Furthermore, intermediate ap or Lim1 mutants are not rescued by UASChip, and co-expression of UASChip together with UASLim1 in the ap domain does not rescue the dominant-negative effect of UASLim1 on ap. Therefore, it is concluded that Chip is either not required for Lhx protein function in leg development, or not present in either limited amounts or stoichiometric doses. Thus Chip is unlikely to be the putative ap partner affected by Lim1 (Pueyo, 2000).

Ectopic expression of Lim1 leads to loss of ap expression and of tarsus four, but a direct regulatory relationship between ap and Lim1 in the wild type does not need to exist, since they are never expressed in the same cells. Furthermore, expression of ap in Lim1 mutants, and of Lim1 in ap mutants is normal, which indicates the absence of long-range regulatory cell signals between these genes. However, expression of Bar in the presumptive tarsus abuts that of Lim1 and al. Bar encodes two redundant Hox proteins expressed in tarsus four and five, which are required for the development of these structures and for the expression of ap in tarsus four. When Lim1 is ectopically expressed in the Bar territory, a reduction of Bar expression occurs. This loss of Bar expression could explain the loss of ap expression seen in apGal4;UASLim1 flies and suggests that in the wild type, an important regulatory role of Lim1 is to restrict Bar expression to the presumptive tarsus five. The apparent paradox that apGal4;UASLim1;UASap flies still show a mutant phenotype can be understood if Bar also has a direct requirement for tarsus four development, one beyond simply activating ap expression (Pueyo, 2000).

PD patterning in Drosophila legs seems to proceed stepwise after it is initiated by the Wg- and Dpp-mediated activation of Dll, dac and al. Later on, these genes interact among themselves and with Hth to activate, in a Wg- and Dpp-independent phase, the expression of further PD genes in new domains of expression. Similar interactions of this kind must lead to the eventual allocation of all different PD fates. The genes downstream of the initial PD genes are still to be identified but Lim1 and ap may serve downstream functions. In early third instar, shortly after 72 hours AEL, al expression at the presumptive leg tip is possibly initiated by a combination of Wg and Dpp signaling, with a requirement for Dll. Around mid-third instar, al-expressing cells in the presumptive tip of the leg activate the expression of Lim1. At this time, the expression of Bar is present in a ring in the presumptive distal tarsal region, partially overlapping that of al and Lim1. This overlap then resolves into an abutment by late third instar. This refinement is important for proper development of the claw organ and tarsus five, and could be based on direct repressory action between the Hox transcription factors Bar and al. However, whereas ectopic Bar expression represses al expression, in the reciprocal experiment ectopic al does not repress Bar. Interestingly, ectopic expression of lim1 results in a reduction of Bar expression. It is concluded that al and Bar do have a mutual repressory relationship that involves Lim1 (Pueyo, 2000).

Whereas Bar might repress al expression directly, the repressory effect of al on Bar is mediated by Lim1. This regulatory circuit between Bar, al and Lim1 establishes the abutting fields of tarsus five cells expressing Bar, and claw organ cells expressing al and Lim1. This circuit also explains why although al mutants lead to an expansion of Bar expression, ectopic al does not reduce Bar expression. Whereas loss of al produces loss of Lim1 and hence leads to ectopic Bar expression, ectopic al on its own is not able to repress Bar. The final element in the determination of distal leg fates is the expression of ap, which is activated in the presumptive tarsus four around mid-third instar. Although ap expression is reduced by ectopic Lim1, this is probably an indirect consequence of the loss of Bar, because appropriate levels of Bar are responsible for the activation of ap. Whereas low levels of Bar are needed for ap expression in tarsus four, high levels of Bar in tarsus five prevent it. Thus, the tip of the leg gets divided into its three final domains during the second half of the third instar: the presumptive claw organ or pretarsus, defined by the expression of al and Lim1; the presumptive tarsus five, defined by the expression of high levels of Bar; and the presumptive tarsus four, defined by the expression of ap and low levels of Bar. During the subsequent pupal metamorphosis into an adult fly, these transcription factors must control the expression of appropriate downstream genes, leading to the differentiation of appropriate structures in each of these presumptive leg segments (Pueyo, 2000).

The transcriptional co-factor Chip acts with LIM-homeodomain proteins to set the boundary of the eye field in Drosophila

Development involves the establishment of boundaries between fields specified to differentiate into distinct tissues. The Drosophila larval eye-antennal imaginal disc must be subdivided into regions that differentiate into the adult eye, antenna and head cuticle. The transcriptional co-factor Chip is required for cells at the ventral eye-antennal disc border to take on a head cuticle fate; clones of Chip mutant cells in this region instead form outgrowths that differentiate into ectopic eye tissue. Chip acts independently of the transcription factor Homothorax, which was previously shown to promote head cuticle development in the same region. Chip and its vertebrate CLIM homologues have been shown to form complexes with LIM-homeodomain transcription factors, and the domain of Chip that mediates these interactions is required for its ability to suppress the eye fate. Two LIM-homeodomain proteins, Arrowhead and Lim1, are shown to be expressed in the region of the eye-antennal disc affected in Chip mutants, and both require Chip for their ability to suppress photoreceptor differentiation when misexpressed in the eye field. Loss-of-function studies support the model that Arrowhead and Lim1 act redundantly, using Chip as a co-factor, to prevent retinal differentiation in regions of the eye disc destined to become ventral head tissue (Roignant, 2009).

Regionalization of the eye-antennal disc is a progressive process in which selector genes and signaling pathways specify the fates of different head structures. Clones of eye-antennal disc cells induced during the second larval instar can contribute to multiple organs, indicating that these cells retain developmental plasticity at this stage. The anteroposterior boundary of the wing disc is established much earlier; expression of the selector gene engrailed (en) specifically in the posterior cells during embryogenesis generates an affinity border that keeps the two compartments clonally separated. By contrast, the eye selector gene ey is uniformly expressed throughout the early eye-antennal disc, and only retracts to the eye field in the second instar. It was initially proposed that localized Notch signaling controls this retraction, as expression of dominant-negative forms of Notch in the eye disc abolishes ey expression and leads to antennal duplications. However, a later study demonstrated that loss of Notch function does not affect ey expression directly, but reduces cell proliferation in the retinal field, preventing the initiation of eya expression. This study shows that Chip and Lim1 are both necessary to repress ey expression in the anterior of the antennal disc. Additional factors probably help to restrict ey expression to the eye disc, because ey expression does not extend throughout the normal Lim1 expression domain in Lim1 or Chip mutant clones in the antennal disc (Roignant, 2009).

Since Lim1 mutant clones always misexpress Ey, but rarely misexpress Eya and never differentiate ectopic photoreceptors, additional proteins must interact with Chip to repress retinal differentiation. Awh is a good candidate because it is expressed at the ventral margin of the eye-antennal disc, its misexpression in the retina represses photoreceptor differentiation in a Chip-dependent manner, and loss of both Lim1 and Awh leads to ectopic photoreceptor differentiation in the ventral eye-antennal disc. Since ectopic photoreceptors differentiate only in the absence of both Lim1 and Awh, whereas Ey expansion is observed in Lim1 single mutants, Awh must control the expression of target genes other than ey. It may negatively regulate other genes involved in retinal determination, such as eya, or positively regulate genes important for head capsule development, such as Deformed and odd-paired (Roignant, 2009).

Like Chip, Hth is required to prevent retinal differentiation at the ventral eye-antennal disc boundary. Investigation of the relationship between Chip and Hth indicates that Chip is not required for Hth expression or activity. The ability of Hth to repress photoreceptor differentiation in Chip mutant clones rules out the possibility that Chip acts as a co-factor for Hth or an essential downstream mediator of its effects. The normal expression of Hth and its target gene wg in Chip mutant clones also make it unlikely that Chip controls the expression of Hth or its co-factor Exd. However, the possibility that Hth and Chip act in parallel poses the paradox that misexpressed Hth is sufficient to repress photoreceptor development in the eye field in the absence of Chip, but endogenous Hth is insufficient to do so in the head field. It is possible that Hth expression levels in the head field early in development are too low to repress the eye fate in the absence of Chip. Consistent with this hypothesis, it was found that overexpression of Hth in Chip mutant cells prevents ectopic photoreceptor differentiation. Similarly, overexpression of Awh or Lim1 prevents ectopic photoreceptor differentiation in hth mutant cells, suggesting that endogenous levels of these LIM-HD proteins are not sufficient to compensate for the absence of Hth. The two classes of transcription factors may normally act on different sets of target genes, but show some cross-regulatory ability when overexpressed (Roignant, 2009).

The boundary between the eye and the dorsal head appears to be established differently from the boundary in the ventral region. The LIM-HD gene tup is expressed at the dorsal eye-antennal disc boundary, in a pattern resembling the mirror image of the Awh pattern, and is capable of repressing photoreceptor development in a Chip-dependent manner. However, loss of Chip in this region does not lead to ectopic eye formation, although it can cause overgrowth and mispatterning of the head. In the absence of Chip, the GATA transcription factor Pannier (Pnr) and its target gene wg may be sufficient to maintain dorsal head fate. The ventral margin of the eye-antennal disc may be particularly susceptible to ectopic photoreceptor differentiation because of the high level of Dpp signaling there. A 5' enhancer element has been shown to direct dpp expression specifically in the ventral marginal peripodial epithelium of the eye-antennal disc. The ability of Dpp and Ey to synergize to drive retinal differentiation therefore makes it critical to repress Ey in this region, which is fated to form head capsule (Roignant, 2009).

In addition, this domain of Dpp overlaps with Wg present at the anterior lateral margin of the eye disc; the combination of these two growth factors induces proximodistal growth of the leg. One function of Chip and its partner proteins might thus be to repress the outgrowth that would otherwise be triggered by the combination of Dpp and Wg. Unlike growth of the wild-type eye disc, growth of Chip mutant regions appears to be Notch-independent, as they do not contain a fng expression boundary and do not show activation of the Notch target genes E(spl)mβ or eyg. Notch has been thought to trigger growth by inducing the expression of the JAK/STAT ligand Unpaired (Upd); however, a recent report describes an earlier function for Upd upstream of Notch, raising the possibility that upd expression is activated independently of Notch in Chip mutant clones. As hth mutant clones, or clones lacking the Odd skipped family member Bowl, frequently show ectopic ventral photoreceptor differentiation but rarely induce outgrowths like those seen in Chip mutants, the functions of Chip in growth and differentiation are likely to be separable (Roignant, 2009).

LIM-HD proteins also set developmental boundaries in other imaginal discs, acting in concert with other classes of transcription factors. In the wing disc, Tup specifies the notum in collaboration with homeodomain transcription factors of the Iroquois complex, and Ap specifies the dorsal compartment. Ap interacts with the homeodomain protein Bar and Lim1 with Aristaless to establish specific tarsal segments within the leg disc. LIM-HD proteins have also been implicated in vertebrate eye development, although those that have been studied appear to play positive roles. The Ap homologue Lhx2 is expressed within the mouse retinal field at the neural plate stage, and contributes to the expression of Pax6, Six3 and Rx. Lmx1b, the homologue of CG32105, is required for the development of anterior eye structures such as the cornea and iris, and is mutated in human patients with nail-patella syndrome, often characterized by glaucoma. Within the retina, loss of Lim1 results in mispositioning of horizontal cells within the amacrine cell laye. Drosophila Lim3 shows photoreceptor-specific expression, and might therefore have a positive function in eye development (Roignant, 2009).

In the central nervous system, LIM-HD proteins act combinatorially to specify different neuronal cell fates. In both Drosophila and vertebrates, combinations of Islet and Lhx3/4/Lim3 proteins regulate motoneuron specification and pathfinding. The ability of Chip to interact with LIM-HD proteins and other transcription factors as well as to dimerize enables it to form heteromeric transcription factor complexes. In the wing disc, the active complex is a tetramer containing two subunits each of Chip and Ap, whereas in motoneuron development the Chip homologue NLI can form either a tetramer with Lhx3 or a hexamer containing both Isl1 and Lhx3. The finding that Lim1 and Awh act redundantly to prevent eye development in the ventral head primordium, whereas Chip is absolutely required, seems most consistent with regulation of distinct subsets of target genes by independent Chip-Awh and Chip-Lim1 complexes; however, a contribution from a complex containing all three proteins, or even additional transcription factors, cannot be ruled out. The role of the Chip co-factor may be to coordinate multiple transcriptional regulatory complexes to restrict developmental fates within the eye-antennal imaginal disc, allowing it to give rise to the head cuticle as well as distinct external sensory structures (Roignant, 2009).

EVOLUTIONARY HOMOLOGS

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 Lim-1 belongs to the third group, the lim/lhx1,5,6 genes.

Lim-1 homologs in C. elegans

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).

The LIM and POU homeobox genes ttx-3 and unc-86 act as terminal selectors in distinct cholinergic and serotonergic neuron types

Transcription factors that drive neuron type-specific terminal differentiation programs in the developing nervous system are often expressed in several distinct neuronal cell types, but to what extent they have similar or distinct activities in individual neuronal cell types is generally not well explored. This problem was investigated using, as a starting point, the C. elegans LIM homeodomain transcription factor ttx-3, which acts as a terminal selector to drive the terminal differentiation program of the cholinergic AIY interneuron class. Using a panel of different terminal differentiation markers, including neurotransmitter synthesizing enzymes, neurotransmitter receptors and neuropeptides, it was shown that ttx-3 also controls the terminal differentiation program of two additional, distinct neuron types, namely the cholinergic AIA interneurons and the serotonergic NSM neurons. The type of differentiation program that is controlled by ttx-3 in different neuron types is specified by a distinct set of collaborating transcription factors. One of the collaborating transcription factors is the POU homeobox gene unc-86, which collaborates with ttx-3 to determine the identity of the serotonergic NSM neurons. unc-86 in turn operates independently of ttx-3 in the anterior ganglion where it collaborates with the ARID-type transcription factor cfi-1 to determine the cholinergic identity of the IL2 sensory and URA motor neurons. In conclusion, transcription factors operate as terminal selectors in distinct combinations in different neuron types, defining neuron type-specific identity features (Zhang, 2014).

Vertebrate Lim-1 homologs

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).

Cloning and genomic organization of Lim1

This paper reports on cloning, sequence analysis, and developmental expression pattern of lim1, a member of the LIM class homeobox gene family in the mouse. lim1 cDNA encodes a predicted 406 amino acid protein that is 93% identical with the product of the Xenopus LIM class homeobox gene Xlim1. lim1 expression from day 8.5 post coitum onward has been characterized. Northern blot analysis of RNA transcripts indicate that lim1 is expressed both during embryogenesis and in the adult brain. Analysis by whole-mount and section in situ hybridization shows lim1 expression in the central nervous system from the telencephalon through the spinal cord and in the developing excretory system including the pronephric region, mesonephros, nephric duct, and metanephros. In the metanephros, lim1 is strongly expressed in renal vesicles and S-shaped bodies, and transcripts are also detected in the ureteric branches (Fujii, 1999).

The sequence and genomic organization of the mouse Lim1 gene have been determined. The mouse Lim1 gene has five coding exons. The Lim1 transcription initiation start site was determined by 5' RACE. Results indicate that the first exon encodes the translation initiation codon and a 1360-bp 5' untranslated region. Sequence analysis of the 450-bp upstream of the transcription start site reveals the presence of a CATTAA motif at -32 bp and a CAATT box located in reverse orientation at -68 bp. HNF3 beta and Pbx1 binding sites have also been identified. Like most LIM domain encoding genes, the LIM domains of Lim1 are each encoded on separate and adjacent exons. Knowledge of the sequence and structure of the mouse Lim1 gene provides important information for the genetic manipulation of the Lim1 locus (Li, 1999).

Evolutionary origins of blastoporal expression and organizer activity of the vertebrate gastrula organizer gene lhx1 and its ancient metazoan paralog lhx3

Expression of the LIM homeobox gene lhx1 (lim1) is specific to the vertebrate gastrula organizer. Lhx1 functions as a transcriptional regulatory core protein to exert 'organizer' activity in Xenopus embryos. Its ancient paralog, lhx3 (lim3), is expressed around the blastopore in amphioxus and ascidian, but not vertebrate, gastrulae. These two genes are thus implicated in organizer evolution, and this study addressed the evolutionary origins of their blastoporal expression and organizer activity. Gene expression analysis of organisms ranging from cnidarians to chordates suggests that blastoporal expression has its evolutionary root in or before the ancestral eumetazoan for lhx1, but possibly in the ancestral chordate for lhx3, and that in the ascidian lineage, blastoporal expression of lhx1 ceased, whereas endodermal expression of lhx3 has persisted. Analysis of organizer activity using Xenopus embryos suggests that a co-factor of LIM homeodomain proteins, Ldb, has a conserved function in eumetazoans to activate Lhx1, but that Lhx1 acquired organizer activity in the bilaterian lineage, Lhx3 acquired organizer activity in the deuterostome lineage and ascidian Lhx3 acquired a specific transactivation domain to confer organizer activity on this molecule. Knockdown analysis using cnidarian embryos suggests that Lhx1 is required for chordin expression in the blastoporal region. These data suggest that Lhx1 has been playing fundamental roles in the blastoporal region since the ancestral eumetazoan arose, that it contributed as an 'original organizer gene' to the evolution of the vertebrate gastrula organizer, and that Lhx3 could be involved in the establishment of organizer gene networks (Yasuoka, 2009).

Lim1: function during gastrulation and in the head organizer of vertebrates

To investigate Lim1 function during gastrulation, transcript depletion through DEED antisense oligonucleotides was used in Xenopus and cell transplantation was used in mice. Xenopus embryos depleted of Lim1 lack anterior head structures and fail to form a proper axis as a result of a failure of gastrulation movements, even though mesodermal cell identities are specified. Similar disruption of cell movements in the mesoderm is also observed in Lim1-/- mice. Paraxial protocadherin (PAPC) expression is lost in the nascent mesoderm of Lim1-/- mouse embryos and in the organizer of Lim1-depleted Xenopus embryos; the latter can be rescued to a considerable extent by supplying PAPC exogenously. It is concluded that a primary function of Lim1 in the early embryo is to enable proper cell movements during gastrulation (Hukriede, 2003).

Lim1 is a homeobox gene expressed in the organizer region of mouse embryos. To investigate the role of Lim1 during embryogenesis, a targeted deletion of the Lim1 gene was generated in embryonic stem cells. Embryos homozygous for the null allele lack anterior head structures but the remaining body axis develops normally. A partial secondary axis develops anteriorly in some mutant embryos. Lim1 is thus an essential regulator of the vertebrate head organizer (Shawlot, 1995).

Recent experiments have implicated the visceral endoderm in anterior neural induction in mouse. The visceral endoderm is an extraembryonic tissue that surrounds the epiblast of the egg cylinder stage embryo. During gastrulation the visceral endoderm is replaced by definitive endoderm that derives from the anterior portion of the primitive streak. Although no morphological asymmetries are apparent in the visceral endoderm, molecular studies have shown that a distinct anterior-posterior pattern exists in the visceral endoderm prior to the formation of the primitive streak. These studies reveal that the VE-1 antigen and the homeobox genes Hesx1/Rpx and Hex are expressed in the anterior visceral endoderm that underlies the ectoderm fated to form the anterior portion of the neural plate. Ablation experiments have shown that, if the anterior visceral endoderm is removed at the early streak stage, expression of Hesx1/Rpx in the anterior neuroectoderm in late streak/headfold stage embryos is absent or greatly reduced (Shawlot, 1999 and references therein).

Lim1 is a homeobox gene expressed in the extraembryonic anterior visceral endoderm and in primitive streak-derived tissues of early mouse embryos. Mice homozygous for a targeted mutation of Lim1 lack head structures anterior to rhombomere 3 in the hindbrain. To determine in which tissues Lim1 is required for head formation and its mode of action, chimeric mouse embryos were generated and tissue layer recombination explant assays were performed. In chimeric embryos in which the visceral endoderm is composed of predominantly wild-type cells, Lim1 -/- cells are able to contribute to the anterior mesendoderm of embryonic day 7.5 chimeric embryos but embryonic day 9.5 chimeric embryos display a range of head defects. In addition, early somite stage chimeras generated by injecting Lim1 -/- embryonic stem cells into wild-type tetraploid blastocysts lack forebrain and midbrain neural tissue. Furthermore, in explant recombination assays, anterior mesendoderm from Lim1 -/- embryos is unable to maintain the expression of the anterior neural marker gene Otx2 in wild-type ectoderm. In complementary experiments, embryonic day 9.5 chimeric embryos in which the visceral endoderm is composed of predominantly Lim1 -/- cells and the embryo proper of largely wild-type cells, also phenocopies the Lim1 -/- headless phenotype. These results indicate that Lim1 is required in both primitive streak-derived tissues and visceral endoderm for head formation and that its inactivation in these tissues produces cell non-autonomous defects. A double assurance model in which Lim1 regulates sequential signaling events required for head formation in the mouse is discussed (Shawlot, 1999).

Recent embryological and genetic experiments have suggested that the anterior visceral endoderm and the anterior primitive streak of the early mouse gastrula function as head- and trunk-organizing centers, respectively. HNF3beta and Lim1 are coexpressed in both organizing centers suggesting synergistic roles for these genes in regulating organizer functions and hence axis development in the mouse embryo. To investigate this possibility, compound HNF3beta and Lim1 mutant embryos were generated. An enlarged primitive streak and a lack of axis formation were observed in double mutant but not in single homozygous mutant embryos. Chimera experiments indicate that the primary defect in these double homozygous mutants is due to loss of activity of HNF3beta and Lim1 in the visceral endoderm. Altogether, these data provide evidence that these genes function synergistically to regulate organizer activity of the anterior visceral endoderm. Moreover, double mutant embryos also exhibit defects in mesoderm patterning that are likely due to lack of specification of anterior primitive streak cells (Perea-Gomez, 1999).

The first morphological sign of A-P pattern in the epiblast of the mouse embryo is the site of formation of the primitive streak at the posterior end of the embryo. The genetic pathway that initiates primitive streak formation remains to be elucidated, but expression of T on one side of the epiblast at the onset of gastrulation marks posterior primitive streak cells. In HNF3beta,Lim1 double mutant embryos, T expression in the epiblast is no longer restricted posteriorly, but is instead expressed throughout the epiblast by the mid-streak stage. Thus, A-P polarity of the epiblast is abnormal in HNF3beta,Lim1 embryos and widespread expression of T strongly suggests that mutant epiblast cells are transformed into primitive streak cells. The loss of epiblast cells is confirmed by the absence and reduction of expression of Otx2 and Oct4, respectively. In addition, mid-streak-stage embryos also show ectopic mesoderm formation as demonstrated by the expression of MesP1 and Lefty2. As a consequence of these early patterning defects, ectoderm and neurectoderm cells that are derived from distal and anterior epiblast cells are missing in these embryos at 7.5-7.75 d.p.c. These epiblast defects are not observed in single homozygous HNF3beta and Lim1 mutants. Altogether, these data demonstrate that HNF3beta and Lim1 function synergistically to establish A-P patterning of the epiblast and to restrict primitive streak formation to the posterior side of mouse embryos (Perea-Gomez, 1999).

Investigation of the developmental fates of cells in the endodermal layer of the early bud stage mouse embryo has revealed a regionalized pattern of distribution of the progenitor cells of the yolk sac endoderm and the embryonic gut. By tracing the site of origin of cells that are allocated to specific regions of the embryonic gut, it was found that by late gastrulation, the respective endodermal progenitors are already spatially organized in anticipation of the prospective mediolateral and anteriorposterior destinations. The fate-mapping data further showed that the endoderm in the embryonic compartment of the early bud stage gastrula still contains cells that will colonize the anterior and lateral parts of the extraembryonic yolk sac. In the Lhx1(Lim1)-null mutant embryo, the progenitors of the embryonic gut are confined to the posterior part of the endoderm. In particular, the prospective anterior endoderm is sequestered to a much smaller distal domain, suggesting that there may be fewer progenitor cells for the anterior gut that is poorly formed in the mutant embryo. The deficiency of gut endoderm is not caused by any restriction in endodermal potency of the mutant epiblast cells but more likely the inadequate allocation of the definitive endoderm. The inefficient movement of the anterior endoderm, and the abnormal differentiation highlighted by the lack of Sox17 and Foxa2 expression, may underpin the malformation of the head of Lhx1 mutant embryos (Tam, 2004).

How multiple developmental cues are integrated on cis-regulatory modules (CRMs) for cell fate decisions remains uncertain. The Spemann-Mangold organizer in Xenopus embryos expresses the transcription factors Lim1/Lhx1, Otx2, Mix1, Siamois (Sia) and VegT. Reporter analyses using sperm nuclear transplantation and DNA injection showed that cerberus (cer) and goosecoid (gsc) are activated by the aforementioned transcription factors through CRMs conserved between X. laevis and X. tropicalis. ChIP-qPCR analysis for the five transcription factors revealed that cer and gsc CRMs are initially bound by both Sia and VegT at the late blastula stage, and subsequently bound by all five factors at the gastrula stage. At the neurula stage, only binding of Lim1 and Otx2 to the gsc CRM, among others, persists, which corresponds to their co-expression in the prechordal plate. Based on these data, together with detailed expression pattern analysis, a new model of stepwise formation of the organizer is proposed, in which (1) maternal VegT and Wnt-induced Sia first bind to CRMs at the blastula stage; then (2) Nodal-inducible Lim1, Otx2, Mix1 and zygotic VegT are bound to CRMs in the dorsal endodermal and mesodermal regions where all these genes are co-expressed; and (3) these two regions are combined at the gastrula stage to form the organizer. Thus, the in vivo dynamics of multiple transcription factors highlight their roles in the initiation and maintenance of gene expression, and also reveal the stepwise integration of maternal, Nodal and Wnt signaling on CRMs of organizer genes to generate the organizer (Sudou, 2012).

Lhx1 controls terminal differentiation and circadian function of the suprachiasmatic nucleus

Vertebrate circadian rhythms are organized by the hypothalamic suprachiasmatic nucleus (SCN). Despite its physiological importance, SCN development is poorly understood. This study shows that Lim homeodomain transcription factor 1 (Lhx1) is essential for terminal differentiation and function of the SCN. Deletion of Lhx1 in the developing SCN results in loss of SCN-enriched neuropeptides involved in synchronization and coupling to downstream oscillators, among other aspects of circadian function. Intact, albeit damped, clock gene expression rhythms persist in Lhx1-deficient SCN; however, circadian activity rhythms are highly disorganized and susceptible to surprising changes in period, phase, and consolidation following neuropeptide infusion. These results identify a factor required for SCN terminal differentiation. In addition, this in vivo study of combinatorial SCN neuropeptide disruption uncovered synergies among SCN-enriched neuropeptides in regulating normal circadian function. These animals provide a platform for studying the central oscillator's role in physiology and cognition (Bedont, 2014).

An LHX1-regulated transcriptional network controls sleep/wake coupling and thermal resistance of the central circadian clockworks

The suprachiasmatic nucleus (SCN) is the central circadian clock in mammals. It is entrained by light but resistant to temperature shifts that entrain peripheral clocks. The SCN expresses many functionally important neuropeptides, including vasoactive intestinal peptide (VIP), which drives light entrainment, synchrony, and amplitude of SCN cellular clocks and organizes circadian behavior. The transcription factor LHX1 (see Drosophila Lim1) drives SCN Vip expression, and cellular desynchrony in Lhx1-deficient SCN largely results from Vip loss. LHX1 regulates many genes other than Vip, yet activity rhythms in Lhx1-deficient mice are similar to Vip-/- mice under light-dark cycles and only somewhat worse in constant conditions. It is thought that LHX1 targets other than Vip have circadian functions overlooked in previous studies. This study compared circadian sleep and temperature rhythms of Lhx1- and Vip-deficient mice and found loss of acute light control of sleep in Lhx1 but not Vip mutants. Loss of circadian resistance to fever was also found in Lhx1 but not Vip mice that was partially recapitulated by heat application to cultured Lhx1-deficient SCN. Having identified VIP-independent functions of LHX1, the VIP-independent transcriptional network downstream of LHX1 and a largely separable VIP-dependent transcriptional network were also mapped. The VIP-independent network does not affect core clock amplitude and synchrony, unlike the VIP-dependent network. These studies identify Lhx1 as the first gene required for temperature resistance of the SCN clockworks and demonstrate that acute light control of sleep is routed through the SCN and its immediate output regions (Bedont, 2016).

Control of roof plate formation by Lmx1a (LIM-1) in the developing spinal cord

Numerous studies have identified the roof plate as an embryonic signaling center critical for dorsal central nervous system patterning, but little is known about mechanisms that control its formation and its separation from clonally related neural crest cells and dI1 sensory interneurons. The LIM homeodomain transcription factor, Lmx1a, mutated in the dreher mouse, acts to withdraw dorsal spinal cord progenitors from the cell cycle and simultaneously direct their differentiation into functional roof plate cells. Lmx1a cell-autonomously represses the dI1 progenitor fate, distinguishing the roof plate and dI1 interneuron programs, two major developmental programs of the dorsal neural tube. Lmx1a is not directly involved in neural crest development. Bmp signaling from epidermal ectoderm is necessary and sufficient for inducing Lmx1a and other co-factors that also regulate the extent of roof plate induction. It is concluded that Lmx1a controls multiple aspects of dorsal midline patterning and is a major mediator of early Bmp signaling in the developing spinal cord (Chizhikov, 2004).

Lim-1 and motor neurons: a LIM domain protein combinatorial code

Motor neurons located at different positions in the embryonic spinal cord innervate distinct targets in the periphery, establishing a topographic neural map. The topographic organization of motor projections depends on the generation of subclasses of motor neurons that select specific paths to their targets. A family of LIM homeobox genes has been cloned in the chick. The combinatorial expression of four of these genes, Islet-1, Islet-2 (homologs of Drosophila Islet), Lim-1, and Lim-3, defines subclasses of motor neurons that segregate into columns in the spinal cord and select distinct axonal pathways. Thus the combination of LIM domain proteins serve to code motor neuron identity in the spinal cord (Tsuchida, 1994).

These LIM homeodomain proteins are expressed prior to the formation of distinct motor axon pathways and before motor columns appear. Depending on their arrangement in columns and eventual synaptic targets, motor neurons of the chick brain stem are designated as belonging to somatic motor (sm) visceral motor (vm), or branchiomotor (bm) classes or to the ipsilateral or contralateral vestibuloacoustic effect neuronal population. Sm neurons innervate muscle derived from the paraxial mesoderm and prechordal plate mesoderm. Bm, vm and vestibuloacoustic axons extend dorsolaterally for some distance through the neuroepithelium before converging on large single exit points within the dorsal neural tube (alar plate). Bm neurons innervate muscle derived from paraxial mesoderm within the branchial arches, while vm neurons innervate parasympathetic ganglia associated with lacrimal and salivary glands or neuronal plexuses that innervate smooth muscle; vestibuloacoustic efferent neurons innervate the hair cells of the inner ear. Subpopulations of spinal motor neurons within specific locations in the spinal cord and distinct targets in the periphery express different combinations of LIM homeobox genes. Sm neurons of the medial division of the median motor column express Islet-1, Isl-2, and Lim-3, while those of the lateral division of the median motor column and the medial division of the later motor column express Isl-1 and Isl-2. Sm neurons of the lateral division of the lateral motor column express Lim-1 and Isl-2. Since the lateral motor column is present only at limb levels, Lim-1 expression is restricted to these levels of the neuraxis. At early stages, visceral motor neurons express both Isl-1 and Isl-2, but after their migration to form the column of Terni, only a subset of these neurons continues to express Isl-1. These genes are good candidates to confer target specificity upon motor neuron classes, since they are expressed at times before the motor columns have fully segregated and before axons have reached their targets (Tsuchida, 1994).

The generation of distinct classes of motor neurons is an early step in the control of vertebrate motor behavior. To study the interactions that control the generation of motor neuron subclasses in the developing avian spinal cord, in vivo grafting studies were performed in which either the neural tube or flanking mesoderm was displaced between thoracic and brachial levels. The positional identity of neural tube cells and motor neuron (MN) subtype identity was assessed by Hox and LIM homeodomain protein expression. Brachial (B) levels of the median motor column (MMC) are organized into three columns: neurons of the medial MMC (MMCM) co-express Isl1, Isl2 and Lim3; neurons of the medial lateral motor column (LMCM) co-express Isl1 and Isl2, and motoneurons of the lateral LMC (LMCL) coexpress Isl2 and Lim1. At thoracic (T) levels motoneurons are also organized into three columns: MMCM neurons; lateral MMC neurons that coexpress Isl1 and Isl2 but not Lim3, and dorsomedially positioned Column of Terni (CT) neurons that express only Isl1. Grafts of 13-15 segment quail T neural tube were placed rostrally at the B level of 12-15 segment chick hosts. Marker and morphological analysis reveals that grafted neural cells divert their normal T fates and their neuronal progeny acquire the molecular properties of B MNs. These changes in the neural tube are restricted to a limited time frame. The rostrocaudal identity of neural cells is plastic at the time of neural tube closure and is sensitive to positionally restricted signals from the paraxial mesoderm. Such paraxial mesodermal signals appear to control the rostrocaudal identity of neural tube cells and the columnar subtype identity of motor neurons. Analysis of neural Hoxc8 expression provides evidence that the change in cell identity after neural tube displacement is not restricted to the MNs; the change occurs in a graded manner along the rostrocaudal axis of the spinal cord, and is associated with both a rostral and caudal respecification in cell fate. In contrast, neural tube grafts between B and T levels do not change the pattern of Hoxc8 expression in the flanking paraxial mesodem. These results suggest that the generation of motor neuron subtypes in the developing spinal cord involves the integration of distinct rostrocaudal and dorsoventral patterning signals that derive, respectively, from paraxial and axial mesodermal cell groups (Ensini, 1998).

The diversification of neuronal cell types in the vertebrate central nervous system depends on inductive signals provided by local organizing cell groups of both neural and nonneural origin. The link between neuronal birth date, migratory pattern, and identity is also evident in the generation of motor neurons in the spinal cord. These conserved features are particularly apparent for motor neurons of the lateral motor column (LMC). This class of motor neurons is generated selectively at brachial and lumbar levels of the spinal cord, and their axons innervate target muscles in the limb. Within the LMC, motor neurons can be further divided into two subclasses: medial LMC neurons that project to ventrally derived limb muscles, and lateral LMC neurons that project to dorsally derived limb muscles. Motor neurons destined to form the medial LMC leave the cell cycle before lateral LMC neurons; as a consequence, prospective lateral LMC neurons emerge from the ventricular zone and migrate past medial LMC neurons to their final position. The time of generation and the distinct migratory environment represent two prominent differences between the development of lateral LMC neurons and other motor neurons. In addition, the total number of motor neurons generated at limb levels of the spinal cord is greater than that at nonlimb levels, presumably to accommodate the formation of the LMC (Sockanathan, 1998 and references).

All somatic motor neurons initially express Isl1 and Isl2, and most maintain the expression of these genes. Lateral LMC neurons, however, extinguish Isl1 and initiate Lim1 expression as they begin to migrate past medial LMC neurons, thus acquiring a unique LIM homeobox gene code. Studies of LIM homeobox gene function in vertebrates and invertebrates have provided evidence that this gene family has a role in motor neuron differentiation and axon pathfinding. The diversification of motor neuron subtypes is initiated by inductive signals from the axial and paraxial mesoderm that operate along the dorsoventral and rostrocaudal axes of the neural tube. However, medial and lateral LMC motor neurons are generated from progenitor cells that occupy the same dorsoventral and rostrocaudal positions, and thus it is unlikely that mesodermal signals impose this distinction. The late birth date of lateral LMC neurons and their migration past early-born LMC neurons prompted a consideration of whether the fate of lateral LMC neurons might be directed by signals provided by early-born LMC neurons. This hypothesis invokes the idea that LMC motor neurons generated at early stages express a local but non-cell-autonomous signal that induces the lateral LMC phenotype in late-born LMC neurons. A retinoid-mediated signal provided by one subset of early-born spinal motor neurons (the medial) imposes a local variation in the number of motor neurons generated at different axial levels and also specifies the identity of a later-born subset of motor neurons (the lateral). Thus, in the vertebrate central nervous system the distinct fates of late-born neurons may be acquired in response to signals provided by early-born neurons (Sockanathan, 1998).

Zebrafish Lmx1b.1 and Lmx1b.2 are required for maintenance of the isthmic organizer

The mesencephalic and metencephalic region (MMR) of the vertebrate central nervous system develops in response to signals produced by the isthmic organizer (IsO). The LIM homeobox transcription factor Lmx1b is expressed within the chick IsO, where it is sufficient to maintain expression of the secreted factor wnt1. This paper shows that zebrafish express two Lmx1b orthologs, lmx1b.1 and lmx1b.2, in the rostral IsO; these genes are necessary for key aspects of MMR development. Simultaneous knockdown of Lmx1b.1 and Lmx1b.2 using morpholino antisense oligos results in a loss of wnt1, wnt3a, wnt10b, pax8 and fgf8 expression at the IsO, leading ultimately to programmed cell death and the loss of the isthmic constriction and cerebellum. Single morpholino knockdown of either Lmx1b.1 or Lmx1b.2 has no discernible effect on MMR development. Maintenance of lmx1b.1 and lmx1b.2 expression at the isthmus requires the function of no isthmus/pax2.1, as well as Fgf signaling. Transient misexpression of Lmx1b.1 or Lmx1b.2 during early MMR development induces ectopic wnt1 and fgf8 expression in the MMR, as well as throughout much of the embryo. It is proposed that Lmx1b.1- and Lmx1b.2-mediated regulation of wnt1, wnt3a, wnt10b, pax8 and fgf8 maintains cell survival in the isthmocerebellar region (O'Hara, 2005).

Motor neurons and retinoid signaling

To begin to define the contribution of retinoid signaling to motor neuron differentiation, the pattern of expression of retinaldehyde dehydrogenase 2 (RALDH2) in the developing spinal cord was examined. At brachial levels, RALDH2 expression is first detected at stage 19, and at this and subsequent stages, expression in the ventral spinal cord appears to be restricted to motor neurons. By stage 27, when the medial motor column (MMC) and LMC have segregated, expression of RALDH2 is restricted to LMC neurons. Within the LMC, RALDH2 is expressed by both medial and lateral LMC neurons. A similar LMC-specific pattern of RALDH2 expression is detected at lumbar levels. Consistent with the restriction of RALDH2 expression to LMC neurons, no expression of the gene is detected in motor neurons at thoracic levels. The expression of RALDH2 in motor neurons at brachial and lumbar levels persists until at least stage 35, although from stage 29 onward, expression gradually becomes restricted to specific motor neuron pools. The only other site of RALDH2 expression in the spinal cord is in the roof plate, both at limb and nonlimb levels. These selective results show that (1) RALDH2 expression is initiated during the early phase of motor neuron generation at brachial levels of the spinal cord; (2) RALDH2 expression distinguishes developing LMC neurons from other somatic or visceral motor neurons, and (3) RALDH2 expression precedes the appearance of lateral LMC neurons marked by Isl2 and Lim1 expression (Sockanathan, 1998).

The number of Isl+ motor neurons was counted in brachial ventral/floor plate (vf) explants grown either alone or with retinol (Rol), a metabolic precursor of retinoic acid, or with all-trans retinoic acid (RA). The number of Isl+ motor neurons in [vf] explants grown in the presence of either Rol or RA is increased by 60%. The detection of an increase in motor neuron number with Rol, as well as with RA, indicates that explants grown in medium with no added retinoid are deprived of the metabolic substrate required for synthesis of RA by RALDH2. To examine further the involvement of RALDH2 activity in the control of motor neuron number, thoracic [vf] explants, which do not express RALDH2, were exposed to Rol or RA and the number of Isl+ motor neurons measured. In contrast to results obtained with brachial level explants, exposure of thoracic [vf] explants to Rol does not increase motor neuron number, whereas RA similarly induces a 60% increase in Isl+ motor neurons. Taken together, these results provide evidence that (1) retinoids increase the number of motor neurons; (2) the increase in motor neuron number detected after exposure of brachial [vf] explants to Rol is correlated with the synthesis of active retinoids by RALDH2 activity, and (3) the apparent requirement for RALDH2-generated retinoids can be overcome by exogenous RA. The retinoid-induced increase in motor neuron number at brachial levels appears to result from an increase in the number of progenitor cells. These experiments suggest that, at limb levels, a RALDH2-generated LMC source of retinoids acts non-cell-autonomously to increase the number of motor neuron progenitors and, consequently, postmitotic motor neurons. Studies using an RAR antagonist show that retinoid receptor activation is required for the generation of lateral LMC neurons and for the control of motor neuron number. Maintenance of the lateral LMC phenotype appears to require ongoing retinoid signaling over the period that these neurons are migrating to their lateral position (Sockanathan, 1998).

RALDH2-dependent induction of lateral LMC neurons requires non-autonomous RA signaling. The onset of RALDH2 and Lim1 expression by lateral LMC neurons was examined. At stage 23, many Isl2+, Lim1+ lateral LMC neurons are still located medial to Isl1+, Isl2+ medial LMC neurons. These Isl2+, Lim1+ neurons do not express RALDH2, suggesting that their lateral LMC phenotype has not been acquired through cell-intrinsic RALDH2 activity. Many of the motor neurons that are located in an even more medial position, distant from RALDH2+ neurons, will populate the lateral LMC, but at this stage these neurons express Isl1/2 but not Lim1. These observations support the idea that the lateral phenotype of LMC neurons is acquired by virtue of the proximity of the neurons to a RALDH2-dependent signal provided by earlier-born LMC neurons. The late birth date of lateral LMC neurons requires that they migrate past early-born neurons to reach their final position. What role might this inside-out program of neuronal migration have in the establishment of the lateral LMC phenotype? The detection of late-born Isl2+, Lim1+ lateral LMC neurons in positions adjacent but medial to early-born RALDH2+ medial LMC neurons provides evidence that proximity to early-born neurons is sufficient to achieve a lateral LMC identity. The failure of late-born LMC neurons to migrate past medial LMC neurons might, however, have the consequence that some LMC neurons fail to be exposed to retinoid signals before they lose competence to respond. In this view, the migration of prospective lateral LMC neurons through early-born LMC neurons would achieve a rapid intermixing of inductive and responsive neurons and ensure that the entire population of late born LMC neurons efficiently encounters a local source of retinoid signals (Sockanathan, 1998).

Interaction of LIM-homeodomain proteins with LBd1, a LIM Domain-binding protein

LIM domains mediate protein-protein interactions. Within LIM-homeodomain proteins, the LIM domains act as negative regulators of the transcriptional activation function of the protein. The recently described protein Ldb1 (also known as NLI, or LIM domain-binding protein) binds LIM domains in vitro and synergizes with the LIM-homeodomain protein Xlim-1 in frog embryo microinjection experiments. The transcriptional activation domain of Xlim-1 has been localized to its carboxyl-terminal region; the interactions of the amino-terminally located LIM domains with Ldb1 have been characterized. Ldb1 binds LIM domains through its carboxyl-terminal region, and can form homodimers via its amino-terminal region. Optimal binding to Ldb1 requires tandem LIM domains, while single domains can bind with lower, yet clearly measurable efficiencies. In animal explant experiments, synergism of Ldb1 with Xlim-1 in the activation of downstream genes requires both the region containing the dimerization domain of Ldb1 and the region containing the LIM-binding domain. The role of Ldb1 may be to recruit other transcriptional activators depending on the promoter context and LIM-homeodomain partner involved (Breen, 1998).

The transcriptional activity of LIM-homeodomain (LIM-HD) proteins is regulated by their interactions with various factors that bind to the LIM domain. Reduced expression of single-stranded DNA-binding protein 1 (Ssdp1), which encodes a co-factor of LIM domain interacting protein 1 (Ldb1), in the mouse mutant headshrinker (hsk) disrupts anterior head development by partially mimicking Lim1 mutants. Although the anterior visceral endoderm and the anterior definitive endoderm, which together comprise the head organizer, are able to form normally in Ssdp1^hsk/hsk mutants, development of the prechordal plate was compromised. Head development is partially initiated in Ssdp1^hsk/hsk mutants, but neuroectoderm tissue anterior to the midbrain-hindbrain boundary is lost, without a concomitant increase in apoptosis. Cell proliferation is globally reduced in Ssdp1^hsk/hsk mutants, and approximately half also exhibit smaller body size, similar to the phenotype observed in Lim1 and Ldb1 mutants. Ssdp1 contains an activation domain and is able to enhance transcriptional activation through a Lim1-Ldb1 complex in transfected cells, and Ssdp1 interacts genetically with Lim1 and Ldb1 in both head development and body growth. These results suggest that Ssdp1 regulates the development of late head organizer tissues and body growth by functioning as an essential activator component of a Lim1 complex through interaction with Ldb1 (Nishioka, 2005).

Ssdp1 mutants exhibit a global reduction in cell proliferation after E8.5 and an increase in apoptosis in somites at E9.0. These changes may be at the root of the abnormalities such as growth retardation and kinked neural tube that were observed in Ssdp1 mutants. Although the mechanism by which Ssdp1 regulates cell proliferation is unknown at present, growth retardation of Ssdp1^+/hsk;Lim1^+/- and Ssdp1^+/hsk;Ldb1^+/- compound mutants suggests involvement of a Ssdp1-Lim1-Ldb1 complex in this process. A shortened body axis was also observed in embryos lacking either Ldb1 or Lim1, supporting this hypothesis. However, if the Lim1 complex plays a major role in the regulation of cell proliferation and cell death, it must be through an indirect mechanism, since Lim1 is not expressed in all of the affected cells. It is conceivable that defective gastrulation movements or the inability of cells with reduced Lim1 complex activity to induce lateral plate mesoderm genes secondarily affects the proliferation and survival of surrounding cells. Furthermore, it is possible that Ssdp1 may also function independently of Lim1, in which case the Ldb1-Ssdp1 complex may regulate cell proliferation in a cell-autonomous manner by controlling the activities of transcription factors involved in cell cycle regulation and cell survival. Alternatively, Ssdp1 might play a direct role in the DNA replication process as a single stranded DNA-binding protein (Nishioka, 2005).

Analysis of hsk mutants shows that disruption of the Ssdp1 gene and the resulting reduction in Ssdp1 expression causes defects in the prechordal plate development and anterior truncations, with some mutants also exhibiting smaller body size. In vitro data have demonstrated that Ssdp1 acts as a coactivator that enhances transcriptional activation by the Lim1-Ldb1 complex. Moreover, genetic interactions between Ssdp1 and Lim1 or Ldb1 suggest that the phenotypes observed in Ssdp1 mutants very probably reflect reduced activity of a Lim1 complex. Together, these data demonstrate that Ssdp1 acts as an essential activator component of a Ssdp1-Lim1-Ldb1 complex in the development of the prechordal plate and body growth (Nishioka, 2005).

LIM homeobox transcription factors integrate signaling events that control three-dimensional limb patterning and growth

Vertebrate limb development is controlled by three signaling centers that regulate limb patterning and growth along the proximodistal (PD), anteroposterior (AP) and dorsoventral (DV) limb axes. Coordination of limb development along these three axes is achieved by interactions and feedback loops involving the secreted signaling molecules that mediate the activities of these signaling centers. However, it is unknown how these signaling interactions are processed in the responding cells. This study found that distinct LIM homeodomain transcription factors, encoded by the LIM homeobox (LIM-HD) genes Lhx2, Lhx9 and Lmx1b integrate the signaling events that link limb patterning and outgrowth along all three axes. Simultaneous loss of Lhx2 and Lhx9 function resulted in patterning and growth defects along the AP and the PD limb axes. Similar, but more severe, phenotypes were observed when the activities of all three factors, Lmx1b, Lhx2 and Lhx9, were significantly reduced by removing their obligatory co-factor Ldb1. This reveals that the dorsal limb-specific factor Lmx1b can partially compensate for the function of Lhx2 and Lhx9 in regulating AP and PD limb patterning and outgrowth. It was further shown that Lhx2 and Lhx9 can fully substitute for each other, and that Lmx1b is partially redundant, in controlling the production of output signals in mesenchymal cells in response to Fgf8 and Shh signaling. These results indicate that several distinct LIM-HD transcription factors in conjunction with their Ldb1 co-factor serve as common central integrators of distinct signaling interactions and feedback loops to coordinate limb patterning and outgrowth along the PD, AP and DV axes after limb bud formation (Tzchori, 2009).

Requirement of Lim1 for female reproductive tract development

Lim1 encodes a LIM-class homeodomain transcription factor that is essential for head and kidney development. In the developing urogenital system, Lim1 expression has been documented in the Wolffian (mesonephric) duct, the mesonephros, metanephros and fetal gonads. Using a Lim1 lacZ knock-in allele in mice. a previously unreported urogenital tissue for Lim1 expression was identified, the epithelium of the developing Müllerian duct that gives rise to the oviduct, uterus and upper region of the vagina of the female reproductive tract. Lim1 expression in the Müllerian duct is dynamic, corresponding to its formation and differentiation in females and regression in males. Although female Lim1-null neonates have ovaries they lack a uterus and oviducts. A novel female mouse chimera assay was developed and revealed that Lim1 is required cell autonomously for Müllerian duct epithelium formation. These studies demonstrate an essential role for Lim1 in female reproductive tract development (Kobayashi, 2003).

Inactivation of FGF8 in early mesoderm reveals an essential role in kidney development: FGF and Lim1 expression

To bypass the essential gastrulation function of Fgf8 and study its role in lineages of the primitive streak, a new mouse line, T-Cre, was used to generate mouse embryos with pan-mesodermal loss of Fgf8 expression. Surprisingly, despite previous models in which Fgf8 has been assigned a pivotal role in segmentation/somite differentiation, Fgf8 is not required for these processes. However, mutant neonates display severe renal hypoplasia with deficient nephron formation. In mutant kidneys, aberrant cell death occurs within the metanephric mesenchyme (MM), particularly in the cortical nephrogenic zone, which provides the progenitors for recurring rounds of nephron formation. Prior to mutant morphological changes, Wnt4 and Lim1 expression, which is essential for nephrogenesis, is absent in MM. Furthermore, comparative analysis of Wnt4-null homozygotes reveals concomitant downregulation of Lim1 and diminished tubule formation. These data support a model whereby FGF8 and WNT4 function in concert to induce the expression of Lim1 for MM survival and tubulogenesis (Berantoni, 2005).

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date revised: 22 January 2017

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