functions of lim homeobox gene

LIM-homeodomain (LIM-HD) proteins are encoded bya subfamily of homeobox-containing genes1,2. The

homeodomains (HD) of LIM-HD proteins are signifi-cantly more similar to each other than to those of otherHD proteins, suggesting that all LIM-HD proteins origi-nated from one common ancestor, and that LIM-HD pro-teins have acquired a distinctive DNA-binding specificity.Like other homeobox genes, this subfamily of transcrip-tion factors has been well preserved throughout evolution.Twelve mammalian and five Drosophila genes that encodeLIM-HD proteins have been described to date (Fig. 1a).The complete genome sequence of the nematodeCaenorhabditis elegans reveals seven different LIM-HD-encoding genes. A phylogenetic analysis of individualLIM-HD proteins shows that they can be subdivided intosix subgroups based on conserved features within thehomeodomain (Fig. 1a). In general, every group usuallycontains a single C. elegans and Drosophila representativeand two representatives within a mammalian species(Fig. 1a). Owing to extra genome duplications, Zebrafishis usually represented with three group members. Thestereotypical appearance of two mammalian genes pergroup suggests that most, if not all, mammalian LIM-HDgenes have been identified. The absence of groups that arespecific for vertebrates or invertebrates indicates that, inthe course of speciation, a strong selective pressure haspreserved members from each individual group.

The characteristic features of LIM-HD proteins are twospecialized zinc fingers, called LIM domains, that arelocated N-terminally of the homeodomain (Fig. 1b). Basedon their structure and protein-binding specificities, theLIM domains of LIM-HD proteins can be distinguishedfrom the LIM domains of primarily cytoplasmic proteins,such as zyxin, cysteine-rich protein (CRP) or muscle LIMprotein (MLP)3,4. The LIM domain is recognized by anumber of co-factors that mediate LIM-HD function, aswe discuss in detail below. The presence of the LIMdomain protein-binding interface sets LIM-HD proteinsapart from other transcription factors of the homeo-

domain superfamily. With the exception of homeobox(HOX) cluster proteins, which contain a short protein-interaction motif that allows for co-factor binding5, mosthomeodomain proteins do not contain additional con-served domains outside the HD; in proteins that do, suchas POU-HD or paired homeobox (PAX) proteins, the sec-ond domain represents a DNA- but not a protein-bindingdomain2. Thus, by means of their LIM domains, LIM-HDproteins might have acquired a unique potential to combi-natorially interact with other transcriptional regulators ina homomeric or heteromeric fashion. The formation ofhigher-order transcriptional-regulator complexes thatregulate transcription in a tissue-specific manner couldallow LIM-HD proteins to participate in a wide range ofdevelopmental events.

LIN-11 groupInvertebrate members of this group have been detected inDrosophila and in C. elegans. The Drosophila gene bk87was identified in a DNA-target-binding screen6 and hasnot been functionally characterized to date. The C. elegansgenes lin-11 and mec-3 are founding members of the LIM-HD gene family (the ‘L’ and the ‘M’ in the LIMacronym)7,8. Whereas lin-11 is closely related to vertebratemembers of this group, the similarity of mec-3 to membersof this group is more remote, yet significant (Fig. 1a).Whether mec-3 defines a novel family of LIM homeoboxgenes for which vertebrate homologs have yet to be identi-fied, or whether it represents a nematode-specific genethat originated by duplication and subsequent diversionfrom an ancestor common to this group, remains an openquestion. lin-11 and mec-3 are both required for the ter-minal differentiation of a subset of specific, non-overlap-ping sensory, motor neurons and interneurons8–10. Themec-3 gene is required for the terminal differentiation oftouch-receptor neurons8 and directly regulates the expres-sion of the mec-7 gene encoding b-tubulin and the mec-4gene encoding an ion channel11. The lin-11 gene of C. ele-gans was originally identified based on its patterning role

LIM-homebox gene function

TIG February 2000, volume 16, No. 20168-9525/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(99)01883-1

Oliver [email protected]

Heiner Westphal*[email protected]

Columbia University,College of Physicians andSurgeons, Department ofBiochemistry andMolecular Biophysics,701 W.168th Street, NewYork, NY 10032, USA.*Laboratory ofMammalian Genes andDevelopment, NICHD,NIH, Bethesda, MD20892-2790, USA.

75

Homeobox genes play fundamental roles in development. They can be subdivided into several subfamilies, oneof which is the LIM-homeobox subfamily. The primary structure of LIM-homeobox genes has been remarkablyconserved through evolution. Have their functions similarly been conserved? A host of new data has beenderived from mutational analysis in diverse organisms, such as nematodes, flies and vertebrates. These studieshave revealed a prominent involvement of LIM-homeodomain proteins in tissue patterning and differentiation,and their function in neural patterning is evident in all organisms studied to date. Here, we summarize therecent findings on LIM-homeobox gene function, compare the function of these genes from different organismsand describe specific co-factor requirements.

Functions of LIM-homeobox genes

Reviews

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FIGURE 1. LIM homeodomain proteins

(a) Classification of LIM-HD proteins. The phylogenetic tree of the homeodomains of the LIM-HD proteins was constructed using the neighbor-joining method.Invertebrate members are labeled in blue, vertebrate members in red. LIM-HD group names were chosen based on the first identified member of each group. Genes forwhich mutant phenotypes have been characterized (see text) are underlined. Other major classes of HD proteins are schematically depicted in order to illustrate thatmembers from distinct HD classes are not merely defined by the presence of additional domains, but by distinctive sequence features within their homeodomains thatmake them cluster in a separate branch of a phylogenetic tree of homeodomain proteins. A detailed phylogenetic analysis of other homeodomain classes can be foundelsewhere2. Species designations are: C, chicken; CE, Caenorhabditis elegans; DM, Drosophila melanogaster; H, human; HR, Halocynthia roretzi; M, mouse; MA,hamster; PC, butterfly; R, rat; X, Xenopus; ZF, Zebrafish. Alternative gene names can be found at: http://www.informatics.jax.org/. (b) Schematic structure of LIM-HDproteins. The structure and function of LIM domains have been discussed elsewhere1,4. In vitro DNA-binding abilities of LIM homeodomain proteins have beencharacterized using DNA-binding site selection76,77 and direct promoter-binding assays59,68.

in the developing vulva12. Within the nervous system,neurons do not need lin-11 to acquire neuron identity, butin lin-11 null-mutant animals at least one class of interneu-rons is dysfunctional and displays neurite sproutingdefects10. Moreover, ventral nerve cord (VNC) motor neur-ons show axon fasciculation defects in these mutants10.

The Lhx1 and Lhx5 proteins (also called Lim1 andLim5) are the two known vertebrate members of the LIN-11 group. Gain-of-function experiments using Xenopusembryos strongly suggest that the Lhx1 gene functions inneural induction13. In mouse mutants that lack Lhx1 func-tion, head formation is severely compromised14. However,because Lhx1 null mutant embryos die at mid-gestation, ananalysis of Lhx1 function at later stages of neural develop-ment has been precluded. The closely related mouse Lhx5gene is similarly expressed during gastrulation. Thereafter,its expression becomes rapidly restricted to an anteriorregion of the developing neural plate and later to most ofthe presumptive diencephalon15. Within the diencephalon,Lhx5 expression can be detected in the hypothalamus,which contains the main thermoregulatory processing cen-ters of the brain. This observation might be significant inthe light of the expression and function of the C. eleganshomolog lin-11 in thermoprocessing interneurons of the C.elegans brain10. Lhx5 expression extends to restrictedregions of the midbrain, hindbrain and spinal cord andshows significant overlaps but also differences with theexpression of Lhx1 (Ref. 15). A knockout analysis of Lhx5in the mouse has brought to light a crucial function in thedevelopment of the hippocampus16. Lhx5-null mutants failto form normal structures of Ammon’s horn and the den-tate gyrus (Fig. 2). Precursor cells for the hippocampal anla-gen are specified and proliferate in the Lhx52/2 embryo, butmany fail to exit the cell cycle. Those that do are unable tomigrate to their appropriate target positions. Thus, Lhx5 isessential for the regulation of neural-precursor cell prolifer-ation and migration during hippocampus formation. It isinteresting to note that the invertebrate LIN-11 group mem-bers, lin-11 and mec-3, also affect neural migration; in mec-3-mutant animals the position of a class of mec-3-express-ing touch sensory neuron is altered8, whereas in lin-11mutants, several lin-11-expressing interneurons appear tobe slightly misplaced10.

Apterous groupThe invertebrate members of this group include theDrosophila apterous gene, the founding member of thissubclass, and the C. elegans ttx-3 gene. Both genes act inthe development of the nervous system17–19. Whereas thefunction of the ttx-3 gene appears to be largely restricted tothe nervous system, apterous functions in a wide variety ofdevelopmental events, such as wing development, muscledevelopment, axon guidance and determination of neuro-transmitter choice. In the developing Drosophila wing,dorsally expressed apterous controls dorso–ventral polar-ity20,21 and plays a prominent role in defining the expres-sion domains of PS1 and PS2 integrins (Ref. 22), fringe(Ref. 23) and serrate (Ref. 23). With regard to mesodermalderivatives, apterous gene activity is both necessary andsufficient for the generation of a subset of embryonic mus-cles20,21. It will be interesting to explore whether apterous-expressing muscles are innervated by apterous-expressingmotor neurons, which would suggest that apterous mightregulate the expression of homophilic adhesion moleculesrequired for motor neuron target recognition21. Such a role

for apterous in selective cell recognition might also be envi-sioned for interneurons of the central nervous system(CNS) that require apterous for correct fasciculation18. Theregulation of integrin expression by apterous in the wingfurther supports the involvement of LIM-HD-encodinggenes in determining cell adhesion. apterous plays anadditional role in neurons as a direct regulator of theexpression of the neuropeptide FMRF-amide17.

The well-defined functions of apterous in nervous sys-tem and wing formation led O’Keefe et al.24 to address thehighly relevant question of whether distinct LIM-HD fac-tors fulfill similar functions that are distinguished byrecruitments into distinct cellular contexts, as shown forPax genes by Li and Noll25, or whether there are intrinsicdeterminants that are specific for individual members of thisgene family. By swapping the LIM and homeodomains ofapterous and lim3 (see below), LIM domains were found tobe interchangeable for the function of apterous in wing, butnot in nervous system, development. These results demon-strate that individual LIM domains from distinct LIM-HDproteins specify LIM-HD protein function; they also revealthat LIM-HD proteins act differently in distinct tissues.

In C. elegans, the post-embryonic expression of theapterous-related ttx-3 gene is required for one class ofinterneurons to process thermosensory information19. Inthe absence of ttx-3, these interneurons are generated butthey are dysfunctional, and display axonal sprouting

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FIGURE 2. Neural defects caused by loss-of-function mutations inLIM-HD genes

(a) Axonal defects in Caenorhabditis elegans ttx-3 mutant animals19. The white arrow points to aberrantneurite sprouts of the AIY interneuron. The main axon (white triangles) projects correctly into the nervering, the major axon bundle of the worm. (b) Targeting defects of ISNb motor neurons in Drosophila lim-3 mutant animals; ISNb motor neurons fail to innervate target muscles 6, 7, 12 and 13 (blackarrowheads)38. (c) Defects in the morphogenesis and cell proliferation in the developing hippocampus(hi) of Lhx5 mutant mice16.

defects that might represent a secondary consequence ofsignaling defects of the interneurons19 (Fig. 2). Notably,lin-11 and ttx-3, members of distinct groups of LIM-HD-encoding genes, are expressed in two different classes of C.elegans interneurons that control the thermoregulatorycircuit in a ‘Yin-and-Yang’ fashion26. They seem to affectthe respective interneurons in similar ways, suggestingthat both genes fulfill similar functions in these neurons10,19.

The Apterous group of LIM-HD proteins contains twovertebrate members, Lhx2 (also called LH-2 or LH2A)and Lhx9 (LH2B)27–29. The phenotype of the mouse Lhx2gene knockout showed the gene’s function in brain andeye development and in hematopoiesis30. The embryoswere anophthalmic because of a developmental arrest ofthe eye anlagen, prior to the formation of the optic cup. Inaddition, deficient cell proliferation in the forebrainresulted in hypoplasia of the neocortex and aplasia of thehippocampal anlagen. Lhx2-null-mutant embryos died inutero, possibly because of a cell non-autonomous defect ofdefinitive erythropoiesis that caused severe anemia. A rolefor Lhx2 in the control of cell fate decision and/or prolif-eration in the hematopoietic system is also revealed by theforced expression of Lhx2 in embryonic stem cells, whichgives rise to multipotent hematopoietic precursor cells thatrequire Steel factor for growth31.

The structurally closely related Lhx9 is expressed in theD1 interneurons, a specific class of interneurons in thedorsal spinal cord of chicken29,32. Interestingly, Lhx9expression completely overlaps with the sites of expres-sion of the highly related Lhx2 gene at early embryonicstages; however, between E10.5 and E12 this interneuronpool diversifies in response to a GDF7 signal (a secretedbone morphogenic protein-like signaling molecule) toyield the D1A and D1B pools, which express differentcombinations of Lhx2 and Lhx9 genes29. D1A and D1Binterneurons migrate to distinct sites of the spinal cord, aprocess that might be governed by Lhx2 and Lhx9,respectively. In the mouse, in situ hybridization exper-iments revealed high levels of expression of Lhx2 andLhx9 in the developing limbs. Explant experiments pointto the involvement of Lhx2 and Lhx9 in proximal–distaland anterior–posterior positioning during chick limbdevelopment28. The functions of Lhx2 and Lhx9 mightoverlap during this stage of limb formation because mouseembryos that are homozygous for a Lhx2 null mutationdisplay no apparent defects in early limb development30.By contrast, expression of a dominant-negative form ofchicken Lhx2 does interfere with limb outgrowth33.Bearing in mind the caveats of dominant-negativeapproaches and the low penetrance of effects observed,these results suggest that dominant-negative Lhx2 inter-feres with the function of endogenous Lhx2, as well asLhx9. A double knockout will be needed to clarify theextent of overlap, if any, of Lhx2 and Lhx9 function inlimb development. Lhx9 expression was also reported inspecific areas of the developing brain, some distinct, oth-ers overlapping with Lhx2 (Refs 34, 35).

Comparisons of expression and function reveal severalinteresting features that are conserved between apterousand its vertebrate homologs. apterous and Lhx2 areexpressed in the VNC of flies and the spinal cord of verte-brates, respectively, as well as in the eye, the olfactoryorgan and the limbs of the respective species. Theinterneurons that express apterous and Lhx2 in the nerve

cords of Drosophila and vertebrates, respectively, displaycomparable axonal projections18,36. The sites of expressionof both genes differ in the limb, with apterous being dor-sally expressed and Lhx2 displaying no polarity in expres-sion; it is possible that Lhx9 reveals more similarities toapterous expression. However, not only can humanLHX2 rescue the fly apterous mutant phenotype, butdominant-negative Lhx2, presumably through the inhibi-tion of both Lhx2 and Lhx9, can also inhibit limb out-growth, which is one of the two functions of apterous inwing development33,36. Human LHX2 and apterous alsoinduce the expression of similar downstream marker geneswhen ectopically expressed in the fly wing. These impres-sive similarities reveal how highly divergent organisms usehomologous sets of proteins to control basic developmen-tal processes. But they also reveal that distinct organismsrecruit regulatory factors into new contexts, as shown bythe role of apterous in the dorsalization of the wing.

Lim-3 groupInvertebrate homologs of this group have been identifiedin C. elegans37, in Drosophila38 and in the ascidianHalocynthia roretzi39. Expression of the Drosophila lim3gene is largely confined to postmitotic neurons38. Withinspecific motor neurons of the CNS, lim3 participates in acombinatorial fashion with the Drosophila islet LIM-HD-encoding gene to define the patterns of axonal projectionsof subtypes of specific motor neurons38 (Fig. 2).Interestingly, the expression of lim3 in interneurons doesnot seem to overlap with the expression of other LIM-HD-encoding genes, suggesting that some neurons requirecombinatorial expression by distinct members of the LIM-HD gene family, whereas others do not. The sites of lim3,islet and apterous expression also reveal striking similari-ties to the expression of their vertebrate counterparts inthe spinal cord; in both systems the LIM-HD-encodinggenes are expressed in a mutually exclusive manner in anon-overlapping set of interneurons, whereas they arecombinatorially expressed in distinct sets of motor neuroncolumns32,38,40.

Knockout studies brought to light essential functionsof the vertebrate Lhx3 and Lhx4 genes during pituitaryorganogenesis and motor neuron development41–44. Bothgenes act in concert to determine the formation of thepituitary gland and its primordium, Rathke’s pouch. Thepouch is formed in two steps, first as a rudiment, andlater as a definitive pouch. Lhx3 and Lhx4 control theformation of a definitive pouch in a redundant fashion.After Rathke’s pouch is formed, Lhx3 non-redundantlycontrols a critical step of pituitary fate commitment.Later, Lhx3, as well as Lhx4, regulates the proliferationand differentiation of pituitary-specific cell lineages.Thus, Lhx3 and Lhx4 dictate pituitary organ identity bycontrolling developmental decisions at multiple stages oforganogenesis. Rathke’s pouch forms in mice that lackLhx3 and Lhx4 function and, thus, a different set ofgenes controls the earliest stages of pituitary develop-ment45. The homeodomains of Lhx3 and Lhx4 are 95%identical, and the genes can functionally replace eachother in some, but not all developmental contexts.Whereas this differential activity might be owing to smallbut significant differences in timing and/or levels ofexpression of each of the two homologs, it is also possiblethat each of the genes uses a distinct set of co-factorsand/or regulates distinct sets of target genes.



Members of the Lim-3 group also function prominentlyin vertebrate motor neuron development. Here, the timingof Lhx3 and Lhx4 expression is strictly controlled; bothgenes are transiently expressed in motor neurons that pro-ject their axons ventrally from the neural tube44. Mousemutants that lack the function of both genes developmotor neurons whose axons acquire properties of dorsallyprojected axons from a different class of motor neurons.Conversely, misexpression of Lhx3 reorients dorsally ori-ented axonal projections of motor neurons ventrally44.Therefore, Lhx3 and Lhx4 are engaged in the specifi-cation of dorsal versus ventral motor-axon projection andthus contribute to the diversification of motor neuronaxonal projections.

Islet groupThe Drosophila islet gene is expressed in mesodermally

derived cell types and in a discrete subset of motor andinterneurons of the CNS. In islet null-mutant animals, dis-crete aspects of neural identity are altered46. The axons ofseveral motor and interneurons of the VNC are incorrectlytargeted. Also, markers for the dopaminergic and seroton-ergic cell fate fail to be expressed. Drosophila islet partici-pates in a combinatorial coding mechanism together withthe lim3 gene to determine motor neuron axonal out-growth and targeting choices38. The defects in neurotrans-mitter expression can be rescued by islet expression inpostmitotic neurons, suggesting that islet does not affectthe lineage of these neurons, but instead their terminal dif-ferentiation features46. In addition, other islet-expressingtissues, such as heart and alary muscles, exhibit morpho-logical defects in islet null-mutant animals.

Islet homologs have also been identified, although notyet functionally characterized, in other invertebratespecies, such as the ascidian Ciona intestinalis47 and thenematode C. elegans (O. Hobert, unpublished). In bothspecies, the respective Islet homolog is expressed in severalcell types, including neural cells and endothelial cells.

Rat Isl-1 was one of the first Lhx genes identified (the‘I’ in the LIM acronym)48. Loss-of-function studies in themouse have revealed crucial roles for Isl1, one of two Isletgroup genes in mice, in motor neuron and pancreas devel-opment. In Isl1-null mutants, motor neurons cannot bedetected; cells that are destined to undergo motor neurondifferentiation are removed by apoptosis49. Thus, itappears that cell-intrinsic mechanisms exist that allow theremoval of cell populations that lack specific cell-fatedeterminants. The motor neuron defect seems to affectsome populations of interneurons that fail to differentiatein the absence of Isl1-expressing motor neurons49.Pancreatic development is also severely affected in theIsl1-null mutant embryo. Here, Isl1 appears to be specifi-cally required for the induction of the pancreas and subse-quently for the generation of endocrine islet cells50. TheIsl2 gene, although not functionally studied to date, mightbe involved in defining motor neuron pool identity in thespinal cord together with other LIM-HD-encodinggenes40. A third Islet group member present in fish, Isl3, isrequired for eye development51.

Drosophila islet and vertebrate Isl1 both function inmotor neuron differentiation, yet their loss-of-functionphenotypes reveal that Drosophila islet is required foraxonal targeting of motor neurons, whereas the absence ofmouse Isl1 leads to a lack of motor neurons. As previouslysuggested by Thor and Thomas46, there are several ways to

view these ostensible discrepancies between vertebrate andinvertebrate gene functions. Most simply, vertebrate Isl2,whose function has not been reported to date, could be thetrue functional ortholog of Drosophila islet. If so, verte-brate Isl1 expression and function has diverged after agene-duplication event that generated Isl1 and Isl2.Conversely, it is also possible that Drosophila islet andvertebrate Isl1 are, indeed, functionally related, but thateach organism deals differently with the loss of the respec-tive gene function46. Whereas defects in the specificationof neurotransmitter identities and axonal trajectoriesmight be tolerated in Drosophila, the same defects mightlead to the removal of these neurons in vertebrates49.Whichever view is taken, the combinatorial expression ofIslet class and Lim-3 class genes in motor neurons of bothflies and vertebrates and the recently established func-tional relevance of this combinatorial expression arguesfor an impressive degree of conservation of LIM-HD-encoding gene function in vertebrates and invertebrates.

Lhx6/Lhx8 groupThis group represents the most divergent class of LIM-HD-encoding genes. Features of the homeodomain sup-port the notion that the genes in this group are derivedfrom a common ancestor, yet their divergence is muchgreater than that among individual members of otherLIM-HD groups (Fig. 1a). Moreover, the LIM domain ofone representative of this group, the Drosophila arrow-head gene reveals highly unusual sequence features52.arrowhead is expressed in the nervous system but its onlyreported function involves the establishment of the propernumbers of cells in a subset of imaginal disc precursor tis-sues52,53. Given the specialized nature of Drosophilametamorphosis, the role of arrowhead is difficult to relate to the function of Lhx6/Lhx8 group genes in otherorganisms.

lim-4, the C. elegans member of this group, is expressedexclusively in a set of sensory, motor and interneurons inthe C. elegans brain. Recent loss-of-function studiesrevealed its involvement in the specification of odorsen-sory neural fate; in lim-4 null mutant animals, a certainclass of odorsensory neurons acquire a distinct cell fate54.lim-4 is also required to determine the correct axon mor-phology of a class of motor neurons.

The two vertebrate genes included in this group, Lhx6and Lhx8, are expressed in areas of the first branchial archand in the forebrain55,56. The expression of Lhx8 (alsotermed L3 or Lhx7) in defined regions of the embryonicmouse brain is mutually exclusive of the expression ofLhx1 and Lhx2 and suggests a function for these genes inregion-specific differentiation events57. The phenotype ofLhx8 null mutants, an isolated cleft palate78, reflects a common human congenital disorder and is thus of obvious medical importance.

Lmx groupThe only invertebrate member of the LMX group describedto date is the C. elegans lim-6 gene, which is expressed inseveral classes of postmitotic neurons, endothelial cells ofthe uterus and in parts of the excretory system58. Althoughthe execution of several aspects of GABAergic cell fates arenot altered in lim-6 mutant animals, lim-6 acts in a subsetof GABAergic neurons to determine their correct axonaltrajectory and also impacts on the regulation of the enzymerequired for synthesis of the neurotransmitter GABA



(g aminobutyric acid)58. These defects result in the dysfunc-tioning of enteric muscle contractions, which are con-trolled by a set of GABAergic motor neurons.

The founding member of this group, currently termedLmx-1a, was first cloned in the hamster as one of themany proteins that can bind to the promoter of the geneencoding insulin59. Its interaction with other promoter-binding proteins, for example with basic helix–loop–helix(bHLH) proteins, has been demonstrated60; however, thephysiological relevance of Lmx-1a binding to the insulin-gene promoter is still unclear. Lmx-1b, which is identicalto Lmx-1a in its homeodomain, is involved in a variety ofdevelopmental events. In the chick, it is responsible for thedorsal–ventral patterning of limbs, where it specifies dor-sal fate under the control of Wnt7a (Refs 61, 62). TheLmx-1b gene is also involved in patterning of calvarialbones63, and in early patterning of the otic vesicle64.Interestingly, Lmx-1b, like lim-6 of C. elegans, is alsoexpressed in the excretory system, suggesting some func-tional conservation during evolution. Loss of a single copyof the human LMX-1b gene causes Nail Patella Syndrome,a disorder that is characterized by limb-patterning defectsand kidney malfunctions65,66. Like other LIM-HD pro-teins, the mouse Lmx-1b gene is a marker for specific sub-types of interneurons in the spinal cord67.

Co-factorsLIM-HD proteins appear to harbor a significant discrimi-natory power of DNA binding in vitro. For example,

rat Isl-1, mouse Lhx2 and hamster Lmx1a were cloned as sequence-specific enhancer binding proteins48,59,68.Nevertheless, it is likely that in vivo DNA-binding speci-ficity is modulated by the interaction with distinct tran-scriptional regulators. With the exception of the POU pro-tein UNC-86 and the LIM-HD protein MEC-3, whoseinteraction is not dependent on LIM domains69, all LIM-HD interactions reported so far depend on the LIMdomains of LIM-HD proteins. LIM domain-dependentinteractions with cofactors can be classified into severaltypes (Fig. 3), including direct interactions between twodifferent transcription factors (Fig. 3a) or indirect interac-tions that are mediated by members of the Ldb cofactorfamily (Fig. 3b,c). The intrinsic dimerization capacity ofLdb allows LIM-HD proteins to interact with distinct tran-scriptional regulatory proteins, such as Otx proteins70 andallows any given LIM-HD to form homomeric and het-eromeric complexes with other LIM-HD proteins71. It ispossible that the combinatorial function of LIM-HD pro-teins in determining motor neuron identity might be owingto distinct sets of heteromeric and homomeric LIM-HDcomplexes that bind to distinct sets of identity-determiningtarget genes. Given that there is little, if any, overlap ofLIM-HD gene expression in C. elegans, the formation ofheteromeric LIM-HD protein complexes might not be anevolutionarily conserved feature of LIM-HD proteins. A C.elegans homolog of Ldb identified by the genome-sequenc-ing project might serve to form homodimers of LIM-HDproteins or to allow association with distinct types of tran-scriptional regulators, such as Otx proteins. However,genetic studies on the Drosophila Ldb homolog CHIP sug-gest that Ldb function might not be restricted to LIM-HDprotein-related functions72; instead, Ldb serves as a tran-scriptional co-activator for other homeodomain proteins aswell, such as Bicoid (D. Dorsett, pers. commun.).

Are LIM domains providing more than just an interac-tion surface with other proteins? Based on LIM domain-deletion studies, it was initially suggested that LIMdomains confer an autoinhibitory role within a givenLIM-HD protein, and that this negative autoregulation isrelieved by association of the LIM domain with cofactorsof the Ldb family73. However, LIM domain deletion stud-ies in Drosophila failed to show a negative regulatory roleof the LIM domains. Instead, they are essential for apter-ous LIM-HD protein function24. Moreover, LIM domainswap experiments of Drosophila apterous and lim3 clearlyshowed that different LIM domains can confer distinct,tissue-specific functional outputs24, also arguing against asimple role of LIM domains in autoinhibition.

The versatility in LIM domain interactions was recentlyunderscored by the identification of RLIM (for RING fingerLIM domain-binding protein), a widely expressed cofactorthat binds to the LIM domain of all LIM-HD protein testedand that recruits the Sin3A/histone acetylase transcriptionalrepressor complex74. These findings raise some intriguingquestions: For example, is there a dynamic balance betweenLIM-HD activator (Fig. 3a,b,c) and repressor (Fig. 3d) com-plexes? If so, how is this balance regulated? Do the activatorand repressor complexes assemble on all LIM-HD targetsor are they promoter specific? Remarkably, the C. elegansgenome sequence reveals no RLIM-like protein, suggestingthat RLIM-mediated regulatory mechanisms represent anadditional layer of transcriptional regulatory complexity,added later in evolution to allow the diversity and tight regulation of LIM-HD function.



trends in Genetics

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FIGURE 3. LIM-HD protein interactions

Different types of cofactor complexes in which LIM-HD proteins have been foundto be involved69–71,74. Complex formation shown in (a–c) presumably leads totranscriptional activation, whereas the interactions shown in (d) will causetranscriptional repression.

Common themes of LIM-HD gene functionThe data reviewed here demonstrate that LIM-HD genesact in a wide variety of developmental contexts and dis-play functional similarities in organisms that are as dis-tantly related as nematodes, arthropods and vertebrates.All LIM-HD genes have expression domains in the nerv-ous system, and the analysis of LIM-HD mutant animalshas revealed a neural function for each individual LIM-HD gene examined (arrowhead function in the nervoussystem has not yet been examined; Fig. 2). These func-tional studies indicate that LIM-HD genes confer subtype-specific cell identities in the developing embryo. There isno indication that they are involved in the determinationof pan-neural features. Rather, most LIM-HD genesappear to be active at a step in neural development whencells leave the cell cycle and begin to express determinantsthat convey specific identities. This is possibly best demon-strated in C. elegans. Based on complete genome sequencedata, C. elegans encodes a total of seven LIM-HD genes,six of which have been functionally characterized. Each ofthese fulfills a neural regulatory role and each is expressedin postmitotic neurons. Moreover, LIM-HD gene functionis required to determine the correct axonal arrangement ofdiverse groups of LIM-HD-expressing neurons, such assensory, motor and interneurons. These observations sug-gest that individual LIM-HD group members in C. elegansshare overlapping functions in distinct cell types andmight regulate common sets of downstream target genes.Conversely, the regulation of touch-neuron-specific genesby mec-3 (Ref. 11), or a GABAergic marker and a sensory-neuron-specific receptor by lim-6, also point to highly spe-cialized functions of LIM-HD genes in C. elegans58. It isremarkable that, in the absence of LIM homeobox genes,such as ttx-3, lin-11 or lim-6, no dramatic changes in cellfate occur. Only very specific aspects of terminal differen-tiation appear to be affected. A notable exception appearsto be the lim-4 LIM homeobox gene, whose loss of func-tion leads to a complete neural cell fate change54. Becauseof the absence of appropriate cell-fate markers, it has beendifficult to assess the extent of cell-fate switches that occurin mec-3 mutant animals.

We propose that the role of LIM-HD-encoding genes indetermining terminal differentiation features, such as thepattern of axonal projections, is a feature of LIM-HD genefunction that is conserved in evolution. In more-complexnervous systems, such as those of flies and vertebrates,LIM-HD-encoding genes appear to affect terminal differ-entiation at a more fundamental level because manipu-lations of LIM-HD gene expression convey distinct identi-ties to neurons. This has been well illustrated by studiesthat examined the role of members of the Lim-3 and Isletgroup of LIM-HD-encoding genes in motor neuron devel-opment. These neurons are assembled in columns that runat fixed positions along the anterior–posterior axis of thegrowing embryo. Cells within each column project axonsto specific target fields that they innervate. The identitiesof cells from each column and thus the direction that cellstake as they leave the ventricular zone, and the target tra-jectory that their axons follow, appear to be under controlof the LIM-HD-encoding genes expressed in individualmotor neuron columns. Taken together with the timingand the dynamics of LIM-HD gene expression, theseresults suggest that LIM-HD-encoding genes are requiredin a relatively short time window to affect neural subtypeidentities in a rather global way.

LIM-HD-encoding genes in C. elegans reveal few, ifany, spatial overlaps in expression. Although this conclu-sion is based mainly on the analysis of reporter genes, thephenotypic characterization of LIM-HD gene mutants isconsistent with LIM-HD-encoding genes functioning in adistinct set of cells. Thus, combinatorial coding of LIM-HD-encoding genes to determine distinct cell fates appearsto be a mechanism to create a higher complexity of neuralcell types in animals that are more complex than C. ele-gans. This can be best exemplified by comparing the func-tions of LIM-HD in the VNC of C. elegans, Drosophilaand in the spinal cord of vertebrates. In C. elegans, onlyone of the eight classes of VNC motor neurons expresses aLIM-HD-encoding gene, lin-11, which is required for cor-rect axon fasciculation. Because there is also no evidenceyet that the C. elegans Islet homolog lim-7 functions inVNC motor neurons (O. Hobert, unpublished), it appearsthat LIM-HD-encoding genes in C. elegans fulfill no majorfunction in determining VNC motor neuron identity. Thismight be explained by the low number and limited net-work complexity of VNC motor neurons in C. elegans;instead of extending axonal processes to their muscle tar-gets, these cells receive innervation from body-wall mus-cles through thin muscle arms that extend into the VNC(Ref. 75). By contrast, Drosophila and vertebrates containcolumns of motor neurons in their VNCs that are signifi-cantly more complex and that are distinguishable by a dis-tinct set of axonal projections to peripheral muscle targets.A combinatorial code of LIM-HD expression has presum-ably been selected to create neural diversity in the courseof evolution. A similar level of conservation appears toexist in interneurons, which express non-overlapping setsof LIM-HD proteins in vertebrates and invertebrates.Human LHX2 (Ref. 36) appears to rescue the mutant phe-notypes of Drosophila apterous completely, suggesting aconservation of LIM-HD function in interneurons as well.

The similarities between defects of terminal neural differentiation caused by LIM-HD loss-of-functionmutations in diverse organisms suggest that some ances-tral LIM-HD-encoding gene (LIM-HD ‘ur-gene’) fulfilleda crucial role in determining neural identity, axonal target-ing or correct cell positioning in a common ancestor ofworms, flies and vertebrates. Although such an ancestor islong extinct, it should be interesting to examine whetherextant animals of early metazoan origin with a very simplenervous system, such as hydra with its nerve net, containsuch an ancestral LIM-HD-encoding gene. Such a genemight have regulated the expression of cell-surface mol-ecules that confer a specific identity to a neuron requiredfor its axonal outgrowth and targeting properties. Duringthe course of evolution, LIM-HD gene duplication led tothe diversification not only of the coding region of theduplicated genes but also, and most notably, of the regula-tory sequences surrounding these genes. For example, thefunction of the vertebrate Lhx2 gene in erythroid develop-ment serves as a co-option in a new context, as C. elegansdoes not contain a hematopoietic system. Also, the role ofthe vertebrate Lhx1 gene during embryonic inductions atpre-gastrulation stages of development might represent anovel recruitment because C. elegans uses different modesof embryonic patterning. Further examples of functionsadded during evolution include LIM-HD-encoding generegulation during the assembly of pituitary gland, eye andpancreas, organs that were presumably not present in thecommon ancestor of C. elegans and vertebrates.



The key question that needs to be addressed in the futureregards the nature of the target genes of LIM-HD proteins.Are there overlaps among the arrays of downstream genesbeing activated by individual LIM-HD-encoding genes in dis-tinct regions of the developing organism? How does a LIM-HD-encoding gene exert a seemingly diverse array of func-tions in the same developing organism? For example, does agene product such as Lhx2 complex with sets of proteins thatdiffer in nature and/or composition in the developing brain,limb or hematopoietic system? These are only some of the

important questions that need to be answered in an effort toilluminate the mechanism of LIM-HD protein action.

AcknowledgementsWe thank D. Dorsett for communicating unpublishedresults, Z. Altun-Gultekin, S. Pfaff, S. Thor, T. Jessell andanonymous reviewers for helpful comments and suggestionson the manuscript. Because of severe space constraints someof the earlier work on LIM-HD-encoding genes could notbe cited but can be found in earlier reviews.



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Much of the charm in the recent study of the ADAMproteins has been in discovering their novel and

unexpected functions in particular biological systems. Asecond (sometimes philosophical) pleasure is to speculateon how these functions arise from the organization of theADAMs’ signature domains. An ADAM is a multi-domainprotein, ~750 amino acids long, and includes multipledomains: the pro-domain, and the metalloprotease, disin-tegrin, cysteine-rich, epidermal growth factor (EGF)-like,transmembrane and cytoplasmic tail domains (Fig. 1).Currently, there is evidence that each of these domains(with the exception of the transmembrane domain) has afunctional, and not just structural, role in at least oneADAM (Refs 1–12; see Table 1).

The emerging properties of the ADAM gene family havebeen the subject of several recent reviews13–17 (see Table 2).Among the 29 known ADAM cDNAs to date, 17 have ametalloprotease active site with the correct amino acidsequence (deduced from the cDNA) to be a functional pro-tease. In the other 12 ADAMs, the amino acid sequence(deduced from the cDNA sequence) has one or moreresidues in the active-site region that are incompatible withmetalloprotease activity. Thus, these ADAMs are believedto lack protease activity17 (see Table 2). Expression studiesof the known ADAM genes have shown that they have asurprising tissue distribution. A total of 14 ADAM genesare expressed in a wide variety of somatic tissues, the other15 are expressed exclusively (12 ADAMs) or very predom-inantly (3 ADAMs) in the testis. This suggests a specialrelationship between ADAM function and the processes ofspermatogenesis and fertilization, or it might be the result

of more intense searching through testis cDNA libraries forADAM family members. Whichever the reason, it is daunt-ing to imagine how 12 testis-specific ADAMs and 3 moretestis-predominant ADAMs might be required in sper-matogenesis and fertilization. Current research efforts sug-gest that more ADAM family members will be revealed inthe next year or two.

Some ADAM proteases are sheddasesDuring development and in the adult, cells have the abilityto modify their surface to regulate various kinds of functions. For example, the extracellular domain of.40 plasma membrane-anchored cytokines, growth factors,

0168-9525/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(99)01926-5

An ADAM is a transmembrane protein that contains a disintegrin and metalloprotease domain and, therefore, itpotentially has both cell adhesion and protease activities. Currently, the ADAM gene family has 29 members,although the function of most ADAM gene products is unknown. We discuss the ADAM gene products withknown functions that act in a highly diverse set of biological processes, including fertilization, neurogenesis,myogenesis, embryonic TGF-a release and the inflammatory response.

The ADAM gene familysurface proteins with adhesion and proteaseactivity

ReviewsLIM-homeobox gene function


transcription factors. J. Biol. Chem. 273, 3152–315772 Morcillo, P. et al. (1997) Chip, a widely expressed

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74 Bach, I. et al. (1999) RLIM inhibits functional activity of LIMhomeodomain transcription factors via recruitment of thehistone deacetylase complex. Nat. Genet. 22, 394–399

75 White, J.G. et al. (1976) The structure of the ventral nerve cordof Caenorhabditis elegans. Philos. Trans. R. Soc. London Ser.B Biol. Sci. 275, 327–348

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Biochemistry 33, 15149–1515877 Sanchez-Garcia, I. et al. (1993) The cysteine-rich LIM domains

inhibit DNA binding by the associated homeodomain in Isl-1.EMBO J. 12, 4243–4250

Reference added in proof78 Zhao, Y. et al. (1999) Isolated cleft palate in mice with a

targeted mutation of the LIM homeobox gene Lhx8. Proc. Natl.Acad. Sci. U. S. A. 96, 1500–1506

trends in Genetics

N 1

P M D C E TCy

200 400 600 800 C

FIGURE 1. The general domain structure of ADAM proteins

The documented functions of the domains are: the pro-domain (P) blocks protease activity; themetalloprotease domain (M) has protease activity; the disintegrin domain (D) has adhesion activity; thecysteine-rich domain (C) has adhesion activity; the EGF-like domain (E) stimulates membrane fusion;the cytoplasmic tail (Cy) is phosphorylated and regulates other ADAM activities. Various other functionshave been proposed, for example a membrane-fusion activity in the cysteine-rich domain and active SH3domains in the cytoplasmic domain; but, so far, evidence is lacking that these specific domains actuallyhave these functions. The scale bar represents amino acid number.

Paul [email protected]

Diana G. Myles*[email protected]

Department of CellBiology and Anatomy,School of Medicine and*Section of Molecularand Cellular Biology,University of CaliforniaDavis, CA 95616, USA.

functions of lim homeobox gene

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