Table of contents

LIM domain family members: General considerations

LIM domain family members, mec-3 and lin-11 determine cell lineages in C. elegans. Isl-1 and Xlim-1 may play similar roles in vertebrates (Bourgouin, 1992). LH-2 is a mouse LIM/Homeodomain involved in the differentiation of the immune system (Xu, 1992).

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, C. elegans ttx-3 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).

The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and the University of Geneva provides extensive documentation for the LIM domain signature.

LIM-homeodomain proteins direct cellular differentiation by activating transcription of cell-type-specific genes, but this activation requires cooperation with other nuclear factors. The LIM-homeodomain protein Lmx1 cooperates with the basic helix-loop-helix (bHLH) protein E47/Pan-1 (Drosophila homolog: Daughterless) to activate the insulin promoter in transfected fibroblasts. Two proteins originally called Lmx1 are the closely related products of two distinct vertebrate genes, Lmx1.1 and Lmx1.2. Yeast genetic systems were used to delineate the functional domains of the Lmx1 proteins and to characterize the physical interactions between Lmx1 proteins and E47/Pan-1 that produce synergistic transcriptional activation. The LIM domains of the Lmx1 proteins, and particularly the second LIM domain, mediate both specific physical interactions and transcriptional synergy with E47/Pan-1. The LIM domains of the LIM-homeodomain protein Isl-1, which cannot mediate transcriptional synergy with E47/Pan-1, do not interact with E47/Pan-1. In vitro studies demonstrate that the LIM2 domain of Lmx1.1 interacts specifically with the bHLH domain of E47/Pan-1. These studies provide the basis for a model of the assembly of LIM-homeodomain-containing complexes on DNA elements that direct cell-type-restricted transcription in differentiated tissues (Johnson, 1997).

Invertebrate Lim domain proteins

The POU-type homeodomain protein UNC-86 (See Drosophila I-POU) and the LIM-type homeodomain protein MEC-3, both of which specify neuronal cell fate in the nematode C. elegans, bind cooperatively as a heterodimer to the mec-3 promoter. Heterodimer formation increases DNA binding stability and, therefore, increases DNA binding specificity. The in vivo significance of this heterodimer formation in neuronal differentiation is suggested by (1) a loss-of-function mec-3 mutation whose product in vitro binds DNA well but forms heterodimers with UNC-86 poorly and (2) a mec-3 mutation with wild-type function, whose product binds DNA poorly but forms heterodimers well (Xue, 1993).

Neural pathways, which couple temperature-sensing neurons to motor and autonomic outputs, allow animals to navigate away from and adjust metabolism rates in response to the temperature extremes often encountered. ttx-3 is required for the specification of the AIY interneuron in the C. elegans neural pathway that mediates thermoregulation. ttx-3 null mutant animals exhibit the same thermotactic behavioral defect as that seen with laser ablation of AIY in wild type, suggesting that AIY does not signal in this mutant. ttx-3 encodes a LIM homeodomain protein, and is closely related to Drosophila Apterous. A ttx-3-GFP fusion gene is expressed specifically in the adult AIY interneuron pair, which connects to thermosensory neurons. In ttx-3 mutant animals, the AIY interneuron is generated but exhibits patterns of abnormal axonal outgrowth. Thus, the TTX-3 LIM homeodomain protein is likely to regulate the expression of target genes required late in AIY differentiation for the function of this interneuron in the thermoregulatory pathway. The ttx-3-dependent thermosensory pathway also couples to the temperature-modulated dauer neuroendocrine signaling pathway, showing that ttx-3 specifies AIY thermosensory information processing of both motor and autonomic outputs (Hobert, 1997).

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 for 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).

In C. elegans three pairs of neurons, AFD, AIY, and AIZ, play a key role in thermosensation. The LIM homeobox gene ceh-14, homolog of Drosophila Lim3, is expressed in the AFD thermosensory neurons. ceh-14 mutant animals display athermotactic behaviors, although the neurons are still present and differentiated. Two other LIM homeobox genes, ttx-3 and lin-11 (a Lim1 family protein), function in the two interneurons AIY and AIZ, respectively. Thus, the three key thermosensory neurons are specified by three different LIM homeobox genes. ceh-14 ttx-3 lin-11 triple mutant animals have a basic cryophilic thermotaxis behavior indicative of a second thermotaxis pathway. Misexpression of ceh-14 in chemosensory neurons can restore thermotactic behavior without impairing the chemosensory function. Thus, ceh-14 confers thermosensory function to neurons (Cassata, 2000).

The two LIM homeodomain genes ttx-3 and lin-11 have been shown to be essential for the function of the interneurons AIY and AIZ, respectively. These data together with the data on ceh-14 correlate well with cell ablation experiments. Thus, this simple three neuron thermosensory circuit is specified by a distinct three-LIM homeodomain factor system or code. The thermotaxis behavior of multiple mutants have been genetically analyzed using the three alleles ceh-14(ch3), ttx-3(ks5), and lin-11(n389). In ceh-14 ttx-3 double mutants a similar amount of animals are athermotactic compared to ceh-14, but none retain normal behavior. The remainder are either cryophilic or intermediate in these double mutants. In ceh-14 lin-11 double mutants most animals are thermophilic, as in lin-11, but the proportion of athermotactic animals has reached almost ceh-14(ch3) levels. In ttx-3 lin-11 double mutants, the phenotype is rather similar to ttx-3 alone; thus, ttx-3 can be considered to be epistatic to lin-11. Notably, like the laser ablation experiments, an unusually large proportion of the animals failed to move, possibly the consequence of conflicting downstream signals, which results in paralysis rather than athermotaxis. Similar to the ttx-3 lin-11 double mutants, the ceh-14 ttx-3 lin-11 triple mutants are mainly cryophilic, while the paralysis was partially rescued. This is surprising since one would have expected that impairment of AFD, AIY, and AIZ would result in athermotactic behavior. These experiments show that even if the key thermosensory circuitry is genetically removed, the animals tend to move to cold, although they do not perform isothermal tracking (Cassata, 2000).

The ancient origin of sleep is evidenced by deeply conserved signaling pathways regulating sleep-like behavior, such as signaling through the Epidermal growth factor receptor (EGFR). In Caenorhabditis elegans, a sleep-like state can be induced at any time during development or adulthood through conditional expression of LIN-3/EGF. The behavioral response to EGF is mediated by EGFR activity within a single cell, the ALA neuron, and mutations that impair ALA differentiation are expected to confer EGF-resistance. This study describes three such EGF-resistant mutants. One of these corresponds to the LIM class homeodomain (HD) protein CEH-14/Lhx3, and the other two correspond to Paired-like HD proteins CEH-10/Chx10 and CEH-17/Phox2. Whereas CEH-14 is required for ALA-specific gene expression throughout development, the Prd-like proteins display complementary temporal contributions to gene expression, with the requirement for CEH-10 decreasing as that of CEH-17 increases. Evidence is presented that CEH-17 participates in a positive autoregulatory loop with CEH-14 in ALA, and that CEH-10, in addition to its role in ALA differentiation, functions in the generation of the ALA neuron. Similarly to CEH-17, CEH-10 is required for the posterior migration of the ALA axons, but CEH-14 appears to regulate an aspect of ALA axon outgrowth that is distinct from that of the Prd-like proteins. These findings reveal partial modularity among the features of a neuronal differentiation program and their coordination by Prd and LIM class HD proteins (Van Buskirk, 2010).

The LIM homeobox genes are classified into five orthologous families (subclasses). lim-6 corresponds to the LMX subclass for which there is no known Drosophila homolog. The LMX3/4 subclass contains Xenopus Xlim-1, CEH-14 of C. elegans, and BK64 of Drosophila. The LMX1/5 subclass subclass contains C. elegans LIN-11 and BK87 of Drosophila. Drosophila Apterous, which typifies the Apterous subclass, is distantly related to the other subclasses and to the Islet subclass. Apterous corresponds to LHX2 of mouse and TTX-3 of C. elegans. Described here is the functional analysis of the C. elegans LIM homeobox gene lim-6, the ortholog of the mammalian Lmx-1a and b genes that regulate limb, CNS, kidney and eye development. lim-6 is expressed in a small number of sensory-, inter- and motorneurons, in epithelial cells of the uterus and in the excretory system. Loss of lim-6 function affects late events in the differentiation of two classes of GABAergic motorneurons, which control rhythmic enteric muscle contraction. lim-6 is required to specify the correct axon morphology of these neurons and also regulates expression of glutamic acid decarboxylase, the rate limiting enzyme of GABA synthesis in these neurons. Moreover, lim-6 gene activity and GABA signaling regulate neuroendocrine outputs of the nervous system. In the chemosensory system lim-6 regulates the asymmetric expression of a probable chemosensory receptor. lim-6 is also required in epithelial cells for uterine morphogenesis. The function of lim-6 is compared to those of other LIM homeobox genes in C. elegans and it is suggested that LIM homeobox genes share the common theme of controlling terminal neural differentiation steps, which, when disrupted, lead to specific neuroanatomical and neural function defects (Hobert, 1999).

The most prominent sites of expression of lim-6 in the nervous system are GABAergic neurons; five of the nine lim-6- expressing neurons are GABAergic. The complete GABAergic nervous system of C. elegans consists of 26 neurons. They can be functionally subdivided into the D-motorneurons of the ventral nerve cord, which are required for locomotion, the AVL/DVB motorneurons required for enteric muscle contraction; the RME motorneurons, required for foraging, and the RIS interneuron, of unknown function. The unc-30 homeobox gene is exclusively expressed in and required by one specific subset of the GABAergic neurons, the D-type motorneurons, for the acquisition of the GABAergic neural cell fate. Interestingly, the expression pattern and function of lim-6 is almost entirely complementary to the the expression pattern and function of unc-30 in the GABAergic nervous system; lim-6 is not expressed in the D-type motorneurons, but is expressed in all other GABAergic neurons (with the exception of two dorsal and ventral types of the fourfold symmetric RME motorneurons). However, lim-6 does not appear to be the functional counterpart of unc-30 in these neurons, since unlike unc-30, the lim-6 gene is not as tightly required for the expression of all GABAergic-specific cell specializations. For example, the expression of the unc-47 GABA transporter in non-D-type GABAergic neurons is largely unaffected and the expression of the unc-25/GAD gene is only partially affected by the lim-6 null mutation. Presumably other transcription factors can also regulate unc-25 and unc-47 expression in the non D-type motorneurons, suggesting that the genes that define GABAergic neural identity are regulated by different regulatory mechanisms in different subtypes of GABAergic cells (Hobert, 1999).

The functional and neuroanatomical defects of the AVL and DVB neurons in the lim-6 null mutant strongly suggest that the function of these neurons is defective. The neuroanatomical defects which manifest as sprouting defects or the inability of the main axonal projection to follow correct paths could either represent an inability of the neuron to correctly engage its axonal outgrowth machinery, or alternatively, could represent a secondary consequence of neural activity defects. Neural activity defects have been shown before to cause axonal misrouting in both C. elegans and vertebrates. lim-6 could be required for neural activity of GABAergic neurons by regulating the expression of genes directly involved in generating electric activity or regulating genes required to generate the synaptic connectivity required to receive and transmit electrical signals. The defects in non-D-type GABAergic function of the lim-6 mutant can account for the defects in defecation cycle that the animals show. Defecation behavior requires the activation of the defecation motor program. This program represents a rhythmic behavior composed of three stereotyped muscle contractions; one of these three rhythmic contractions, the enteric muscle contraction, is controlled by two GABAergic motorneurons, AVL and DVB. Mutations in GABAergic signaling, for example in the unc-47 GABA transporter or the unc-25 GABA synthesizing enzyme, cause very similar enteric muscle contraction defects. Similarly, GABAergic neurons are also part of the neural circuit that controls gut function in vertebrates. Enteric GABA has been shown to regulate the peristaltic effects in the vertebrate enteric system. Moreover, muscle contractions of the enteric system are also rhythmically active in vertebrates. This rhythmicity is regulated by the electric pacemaker activity of the interstitial cells of Cajal; these cells function as intermediaries between enteric nerves and smooth muscle cells. It will be interesting to see if human patients with Nail Patella syndrome, a haploinsufficency of Lmx-1b, or mice carrying Lmx-1a or Lmx-1b knockout mutations, also show defects in enteric motor function, perhaps due to a common GABAergic input to enteric muscles (Hobert, 1999).

The data favors an output from the GABAergic neurons to the neuroendocrine regulation of dauer formation, which depends on lim-6 activity. lim-6 loss of function strongly enhances the neuroendocrine signaling defects caused by a daf-7/TGF beta mutation. This neuroendocrine function of lim-6 is likely to be due to its function in GABAergic neurons, since unc-25 mutant animals with defects in GABA synthesis show the same phenotype. Because animals with defective D-type motor neurons do not have these neuroendocrine defects, these data argue that it is the lim-6-regulated GABAergic neurons that regulate C. elegans endocrine signaling. Although the suggestion cannot be excluded that lim-6 affects dauer arrest through other, as yet unidentified sites of expression, the simplest model is that lim-6 specifies functional aspects of the non-D-type GABAergic neurons, which in turn regulate dauer arrest via GABAergic signaling (Hobert, 1999).

The expression of lim-6 in the GABAergic neuroendocrine system also reveals an interesting similarity to the expression of its vertebrate homlog Lmx-1a. Vertebrate Lmx-1a participates in the control of insulin gene expression in the b cells of the pancreas, which also contain high levels of the neurotransmitter GABA. GABA regulates insulin secretion from the beta cells via an autocrine loop. Both lim-6 and non D-type GABAergic neurons in C. elegans regulate dauer arrest, which is regulated by an insulin-like neuroendocrine pathway. It is possible that lim-6 regulates the development of the particular GABAergic neurons, which in turn regulate insulin expression and secretion in C. elegans. For example, these GABAergic neurons may couple temperature or food or other sensory inputs to the endocrine output of dauer regulatory proteins such as the many insulin-like proteins that may converge on the single insulin-like receptor that clearly regulates dauer arrest. The synergizing effects of mutations in lim-6 and GABA signaling on the daf-7/TGF beta control of dauer formation indeed mirror the synergism between insulin signaling and daf-7/TGF beta signaling. However, the complexity of the insulin family in C. elegans complicates the matter significantly (Hobert, 1999 and references).

The major head ganglia of C. elegans display an obvious overall bilateral symmetry. The majority of neural cell types have both a left and right representative that share a comparable lineage history and morphology and make similar synaptic connections. In those cases tested, both left and right neurons are required for a full functional response of, for example, a given pair a chemosensory neurons. The asymmetric expression of lim-6 in ASEL (the left neuron of a chemosensory pair) but not in ASER thus represents a surprising observation. It may suggest a potential diversification of apparently symmetric sensory neurons, possibly leading to a diversification in sensory function. This notion is supported by the asymmetric expression of several receptor-type guanylyl cyclases in the ASER and ASEL neurons, one of which is under control of lim-6 activity. Since the function of these receptor-type guanylyl cyclases are as yet unknown, the physiological consequences of ectopic activation of one of these receptors cannot be assessed. Gross defects in ASE sensory functions are not obvious in lim-6 mutant animals (Hobert, 1999 and references).

Homeobox genes have been shown to act at a variety of different stages of neurogenesis, including such early steps as the determination of neural identity and later steps, such as neural differentiation. The analysis of four of the seven LIM homeobox genes in C. elegans demonstrates that they are required for the terminal differentiation of the neurons that express them. In many respects the neural defects caused by mutations in these genes are similar. Mutations in lim-6, like mutations in the ttx-3 and lin-11 LIM homeobox genes do not affect the generation of the neurons that express them, nor do they affect several aspects of neurotransmitter choice. However, the lim-6-, ttx-3- and lin-11- expressing neurons are functionally as well as structurally defective. Similarly, mec-3 is required for the structural integrity of mechanosensory neurons. These similarities in LIM homeobox gene function suggest a common theme in the action of this class of transcriptional regulators. It is speculated that each gene is required in its respective neuron to make a specific target choice. In the absence of intact signaling partners, retrograde signaling events induce the neuron to find a signaling partner by sprouting additional processes or by inducing abnormal turns of the main axonal process. Alternatively, each gene might be required within its given neuron to directly regulate its axon outgrowth machinery. The finding that defects in the GABA neurotransmitter synthesis do not cause axonal sprouting and a report that GABAergic neurons and their postsynaptic partners show no defects in synaptic connectivity in unc-25 mutants indicate that defects in synaptic outputs are unlikely to feedback on neurite outgrowth functions. However, defects in other synaptic outputs, such as signaling by neuropeptides might be feeding back into axonal sprouting or, alternatively, defects in synaptic inputs to the GABAergic neurons (caused, for example, by connectivity defects) could induce axonal sprouting (Hobert, 1999 and references).

Another common feature of all C. elegans LIM homeobox genes studied to date is their onset of expression in postmitotic neurons and the maintenance of their expression throughout adulthood. These observations are unlikely to be a reporter gene artifact, since the expression in other tissues, such as lim-6 in the uterus or lin-11 in the vulva is dynamic and transient. Maintained expression of a regulatory gene throughout the life of a given cell suggests, but does not prove, an involvement of the particular gene in maintenance of the differentiated features of the given cell. In the case of LIM homeobox genes it is possible that they are required for the maintenance of such neural features as synaptic connectivity. Lastly, all of the C. elegans LIM homeobox genes described so far are expressed in a largely non-overlapping subsets of neurons. The most prominent non-neural tissue that expresses C. elegans LIM homeobox genes is the epithelium of the somatic gonad and the vulva. Intriguingly, the LIM homeobox genes are also expressed in a complementary, non-overlapping pattern in this epithelium. lin-11 is expressed in the vulva and cells that connect the uterus to the vulva; lim-6 is expressed in uterine toroid cells and in spermathecal junction cells; ceh-14 is expressed in the epithelial cells of the spermatheca, and another LIM homeobox gene, lim-7, is expressed in the gonadal sheath cells. The unifying feature of all these cells is their highly polarized morphology and their engagement in complex morphogenetic events for which specific cell-cell contacts are required. These morphological features are shared by neurons as well. Moreover, epithelial cells and neurons utilize similar sorting and targeting mechanisms for specific cell surface molecules. It is conceivable that by regulating the expression of specific cell surface proteins, LIM homeodomain proteins in C. elegans are generally involved in determining the specificity of cell-cell recognition and attachment to neighboring cells both in the nervous system and in epithelial cells. Indeed, the structural defects of uterine cells in lim-6 mutants point to a role for lim-6 at the very least in cell recognition and adhesion (Hobert, 1999 and references).

The C. elegans AWA, AWB, and AWC olfactory neurons are each required for the recognition of a specific subset of volatile odorants. lim-4 mutants express an AWC reporter gene inappropriately in the AWB olfactory neurons and fail to express an AWB reporter gene. The AWB cells are morphologically transformed toward an AWC fate in lim-4 mutants, adopting cilia and axon morphologies characteristic of AWC. AWB function is also transformed in these mutants: rather than mediating the repulsive behavioral responses appropriate for AWB, the AWB neurons mediate attractive responses, as do AWC neurons. LIM-4 is a predicted LIM homeobox gene that is expressed in AWB and a few other head neurons. Ectopic expression of LIM-4 in the AWC neuron pair is sufficient to force those cells to adopt an AWB fate. The AWA nuclear hormone receptor transcription factor ODR-7 also represses AWC genes, as well as inducing AWA genes. It is proposed that the LIM-4 and ODR-7 transcription factors function to diversify C. elegans olfactory neuron identities, driving them from an AWC-like state into alternative fates (Sagasti, 1999).

These results suggest a model for the specification of olfactory neuron identities in the C. elegans amphid. lim-4 and odr-7 mutants reveal a common AWC-like developmental potential in the olfactory neurons AWA and AWB, which are not closely related, either to AWC or to one another by lineage. This potential could be specified by an olfactory neuron fate determinant that is generated through the cell lineage, or induced by the embryonic environment, in three separate cells: AWA, AWB, and AWC. Without further modification, all of these cells can take on some of the characteristics of AWC. Modification of the AWC-like state in AWA and AWB is achieved by the transcription factors ODR-7 and LIM-4, either by themselves or in cooperation with other unidentified factors. It is these factors that allow the three olfactory neurons to establish their unique patterns of gene expression, their cell morphologies, and perhaps their synaptic connectivities, ultimately determining the behavioral outputs mediated by each cell. It is speculated that the AWC-like fate may serve as an olfactory ground state on which the other neuronal fates can be elaborated. If this is true, the AWC-like fate could be a basic blueprint for making an olfactory cell, to which evolution can make alterations that diversify the animal's repertoire of responses to volatile chemicals. A possible role for AWC as a cellular module is likely just one example of a general strategy used during evolution to transform a simple organ with a few cell types into a complex multifunctional organ (Sagasti, 1999).

The development of the nervous system requires the coordinated activity of a variety of regulatory factors that define the individual properties of specific neuronal subtypes. A regulatory cascade composed of three homeodomain proteins is described that acts to define the properties of a specific interneuron class in the nematode C. elegans. A set of differentiation markers characteristic for the AIY interneuron class is described. The ceh-10 paired-type (Drosophila homolog: CG4136) and ttx-3 LIM-type homeobox (homolog of Apterous) genes function to regulate all known subtype-specific features of the AIY interneurons. ceh-10 regulates, either directly or indirectly, both ttx-3 and ceh-23 expression. In contrast, the acquisition of several pan-neuronal features is unaffected in ceh-10 and ttx-3 mutants, suggesting that the activity of these homeobox genes separates pan-neuronal from subtype-specific differentiation programs. The LIM homeobox gene ttx-3 appears to play a central role in regulation of AIY differentiation. Not only are all AIY subtype characteristics lost in ttx-3 mutants, but ectopic misexpression of ttx-3 is also sufficient to induce AIY-like features in a restricted set of neurons. One of the targets of ceh-10 and ttx-3 is a novel type of homeobox gene, ceh-23 (Drosophila homolog: Vnd) ceh-23 is not required for the initial adoption of AIY differentiation characteristics, but instead is required to maintain the expression of one defined AIY differentiation feature. The regulatory relationship between ceh-10, ttx-3 and ceh-23 is only partially conserved in other neurons in the nervous system. These findings illustrate the complexity of transcriptional regulation in the nervous system and provide an example for the intricate interdependence of transcription factor action (Altun-Gultekin, 2001).

Gene batteries are sets of coregulated genes with common cis-regulatory elements that define the differentiated state of a cell. The nature of gene batteries for individual neuronal cellular subtypes and their linked cis-regulatory elements is poorly defined. Through molecular dissection of the highly modular cis-regulatory architecture of individual neuronally expressed genes, a conserved 16 bp cis-regulatory motif has been identified that drives gene expression in a single interneuron subtype, termed AIY, in the nematode C. elegans. This motif is bound and activated by the Paired- and LIM-type homeodomain proteins CEH-10 and TTX-3 (Drosophila homolog: Apterous). Using genome-wide phylogenetic footprinting, the location, distribution, and evolution of AIY-specific cis-regulatory elements throughout the genome were delineated: this defined a large battery of AIY-expressed genes, all of which represent direct Paired/LIM homeodomain target genes. The identity of these homeodomain targets provides novel insights into the biology of the AIY interneuron (Wenick, 2004).

This study provides insights into the genomic cis-regulatory architecture controlling cell-type specification and demonstrates its phylogenetic conservation. (1) The phylogenetic footprinting that was employed to analyze cis-regulatory architecture, pioneers the use of the entire C. briggsae genome data set and illustrates the usefulness of interspecies comparisons. (2) The phylogenetic comparisons allowed documentation in several cases of 'evolution at work,' that is, changes in cis-regulatory architecture between C. elegans and C. briggsae. (3) The analysis can be viewed from the standpoint of transcription factor target isolation. Given the preponderance of transcription factors in all genomes, it is of pivotal importance to employ methods that will help in the understanding of how they function on a genome-wide level, that is, through what target genes they act to affect cellular differentiation and function. Such network construction is a particularly pressing issue for the developmentally important homeodomain transcription factors for which few downstream target genes are known to date. (4) This analysis provides new insights into the biology of the AIY interneurons and offers a starting point for the functional analysis of previously unknown AIY-expressed genes (Wenick, 2004).

The modular architecture of cis-regulatory elements allows for easy evolvability of gene expression control. In spite of overall patterns of conservation, phylogenetic comparison of the C. elegans and C. briggsae cis-regulatory architecture indeed permits glimpses into evolution at work, notably the differential loss, gain, or modification of cis-regulatory elements. One example is the ser-2a gene, expressed in AIY, RID, head muscles, and other neurons in C. elegans. In C. briggsae, the gene is in the same cells except AIY; this difference is due to the absence of the AIY motif in the C. briggsae 5'-regulatory region. Another example is the sra-11 gene. Both the C. elegans and C. briggsae genes contain the AIY motif and are expressed in AIY, yet the C. briggsae 5?-regulatory region contains elements that drive expression in additional sets of neurons. Evolutionary divergence can be also observed when comparing paralogous genes within one species. One such example is provided by the sra-type of orphan seven transmembrane receptors (7TMR), and, specifically, the adjacent C. elegans sra-12 and sra-11 genes which are paralogous genes originating from a gene duplication event. It was asked whether the ancient gene duplication event also encompassed a duplication of its presumptive 5'-regulatory regions. A reporter gene fusion to the sra-11 gene is expressed in AIY, as expected by the presence of an AIY motif and a set of other neurons, including RIF or RIG, PVT, and ventral cord neurons, while a reporter gene fusion to the sra-12 gene is also expressed in RIF/RIG and PVT but lacks expression in AIY and also does not contain an AIY motif. The AIY motif has either been recently acquired by the sra-11 locus or has been lost in the sra-12 locus (Wenick, 2004).

The experimentally verified description of direct targets of homeodomain proteins provides an understanding of the molecular mechanisms of homeodomain protein function. The strict cooperativity of CEH-10/TTX-3 binding to their target sequence illustrates why individual homeodomain proteins have in the past shown limited in vitro DNA binding affinity and specificity. The findings also demonstrate the strict cellular context dependency of homeodomain protein function. Although each is expressed in several neuronal types, CEH-10 and TTX-3 are only coexpressed in AIY where they jointly trigger activation of an AIY-specific gene battery. In other cellular contexts, in which TTX-3 and CEH-10 expression does not overlap, these proteins presumably interact with other factors to determine target gene expression. This is well illustrated in the example of the CAN cells; upon delineation of a 315 bp regulatory element in the ceh-23 locus that drives ceh-10-dependent expression in the CAN neurons, the presence of a motif in this element, TAATTGGCT, was noted that shares a half-site of the AIY motif. This motif was mutated and loss of expression in CAN was observed, thus confirming its functional relevance. A ceh-10-dependent cis-regulatory element has thus diverged to either coexist with a sequence that allows for cooperative TTX-3 binding, hence creating the AIY motif, or to coexist with another neighboring sequence that binds a different CEH-10 partner in the context of the CAN neuron to allow expression of target genes such as ceh-23. This partner protein is unlikely a homeodomain protein given that the neighboring sequence contains no TAAN core. Nevertheless, the relatively modest nature of changes required to generate new sites of expression illustrates the 'evolvability' of cis-regulatory elements to create novel expression profilesser-2a (Wenick, 2004).

In sum, the trigger determining expression of the AIY-specific gene battery lies in the cis-regulatory elements of the ceh-10 and ttx-3 genes; these elements determine gene coexpression exclusively in AIY. The cis-regulatory dissection of the ceh-10 and ttx-3 loci revealed cis-elements required for AIY expression. Since these cis-elements are themselves CEH-10/TTX-3 responsive, they are likely to provide an elegant way to self-stabilize and maintain their own expression. At this point, it is not clear what other factors provide the initial trigger for CEH-10/TTX-3 expression, yet the self-stabilizing feedback loop illustrates that this input needs only to be transient (Wenick, 2004).

Most CEH-10/TTX-3 target genes only contain a single AIY motif in their cis-regulatory region. While other transcription factors have binding sites that often occur in multiple copies in the cis-regulatory regions of target genes (Davidson, 2001), two other cell-type-defining transcription factors, the RFX transcription factor DAF-19 and the MEC-3 LIM homeodomain protein, also target single binding sites in their sensory neuron-specific target genes (Swoboda et al., 2000; Zhang et al., 2002). Since the dissection of cis-regulatory architecture on a genome-wide level is still in its infancy, it is difficult to assess how common gene is regulation through single binding sites (Wenick, 2004).

This analysis provides the molecular basis for understanding why genetic removal of ttx-3 causes the same diverse range of behavioral effects seen upon microsurgical removal of AIY. Providing solid support for a previous hypothesis, the AIY interneuron fails to adopt its normal identity in ttx-3 null mutant animals, as measured by the presumptive lack of expression of all tested members of the AIY-expressed gene battery that has been defined in this study. Orthologs of CEH-10 and TTX-3 (Chx10 and Lhx2/Apterous) affect neuronal differentiation in other species, and it will be interesting to determine whether in mature retinal bipolar interneurons, in which Chx10 and Lhx2 are exclusively coexpressed, these two factors may¬ólike CEH-10/TTX-3¬ócooperate to affect a whole interneuron-specific, terminally functioning gene battery (Wenick, 2004).

Although this analysis does not allow for the prediction of the absolute number of AIY-expressed genes, the nature of the coding sequences adjacent to the highest-scoring AIY motifs provides interesting glimpses into the function of the AIY interneuron. Clearly, the most striking feature of the AIY gene expression battery is its highly significant enrichment of putative neurotransmitter receptors. Almost one-quarter of all C. elegans G protein-coupled neuropeptide receptors are likely to be expressed in AIY. Moreover, AIY is predicted to express at least six ion channels, gated by four different ligands (acetylcholine, GABA, glutamate, and serotonin), two biogenic amine receptors, and two metabotropic receptors (acetylcholine and glutamate). These observations suggest that AIY is capable of sampling an impressive array of neurochemical signaling inputs. This is consistent with the microscopically determined synaptic connectivity diagram demonstrating that AIY is a highly interconnected neuron, postsynaptic to many classes of sensory neurons and presynaptic to various interneurons from which it may obtain nonsynaptically released feedback signals. Since peptidergic and aminergic neurotransmission does not require a direct synaptic connection between signaling and receiving cells, AIY may sample an even larger number of inputs than anticipated by the connectivity diagram. It will be interesting to determine how these different neurochemical inputs can shape the electric activity state of the AIY interneurons (Wenick, 2004).

A previous functional analysis of the AIY interneurons underscores the significant functional diversity of this neuron to which a possible molecular basis in the form of a diverse set of neuronal signaling molecules expressed in AIY can now be added. The identification of proteins, both of known and unknown molecular activity, provides an entry point for a further functional dissection of these proteins and will also yield further insights into the mechanisms of AIY interneuron development and function (Wenick, 2004).

Hrlim is a LIM class homeobox gene that was first isolated from the ascidian Halocynthia roretzi (phylum Urochordata). Transcription of Hrlim is activated at the 32-cell stage specifically in the endoderm lineage. Hrlim is also transiently expressed in all notochord precursor cells. Expression in the endoderm lineage continues through to the middle of gastrulation. After gastrulation, Hrlim is expressed in certain lineages that give rise to subsets of cells in the brain and spinal cord (Wada, 1995).

Two hypotheses have been proposed to explain the origin of insect wings. One holds that wings evolved by modification of limb branches already present in multibranched ancestral appendages that probably first functioned as gills. The second hypothesis proposes that wings arose as novel outgrowths of the body wall, not directly related to any pre-existing limbs. If wings derive from dorsal structures of multibranched appendages, it is expected that some of their distinctive features have been built on genetic functions that were already present in the structural progenitors of insect wings, and in homologous structures of other arthropod limbs. Crustacean homologs have been isolated for two genes that have wing-specific functions in insects: pdm and apterous. Their expression patterns support the hypothesis that insect wings evolved from gill-like appendages that were already present in the aquatic ancestors of both crustaceans and insects. Artemia franciscana PDM and Apterous are specifically expressed in cells of the distal epipodite before these acquire their characteristic differentiated morphology (large nuclei, large intercellular spaces). These expression patterns contrast markedly with that of Distal-less which is expressed in all outgrowing appendages (including insect legs and wings, and all crustacean limb branches). Artemia pdm and apterous are associated specifically with a distal epipodite of crustacean limbs. Crustacean epipodites are dorsally located limb branches with respiratory and osmoregulatory functions, precisely the type of structures that would have given rise to insect wings, according to some hypotheses. An alternative interpretation might be that wings did not derive from epipodites but have nevertheless independently coopted a number of gene functions that already existed in epipodites (Averof, 1997).

Table of contents

apterous: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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