BIOLOGICAL OVERVIEW

The Islet family of proteins is a distinct subfamily within the family of LIM homeodomain proteins. Prior to the discovery of Drosophila Islet, now properly termed Tailup, Apterous was the only known LIM homeodomain transcription factor in Drosophila. A Drosophila protein homologous to vertebrate Islet proteins was sought because of the intriguing distribution of LIM homeodomains in subsets of postmitotic neurons in the vertebrate spinal column. LIM homeodomain proteins, including two Islet and two other LIM proteins more closely related to Apterous, act in a combinatorial fashion to determine the fate of motor neurons that project to distinct muscle targets (Tsuchida, 1994).

While islet/tailup is expressed in sets of postmitotic motorneurons, apterous (Lundgren, 1995) is not expressed in motor neurons but in interneurons. islet is also expressed in sets of interneurons, but these are distinct from those expressing apterous. Both apterous and islet mutants show defects in axonal pathfinding. While mutation in either gene causes cell autonomous pathfinding defects (that is, defects in cells expressing the genes), defects in peripheral lateral bipolar dendrite neurons (LBDs) are cell non-autonomous. LBDs are defective in islet mutants, however these neurons do not normally express islet (Thor, 1997).

Of particular interest in Drosophila is the identity of the interneurons expressing islet. In the Drosophila embryonic ventral cord, a small number of neurons synthesize either dopamine or serotonin (Lundell and Hirsh, 1994) and can be identified by experimentally employing antibodies against tyrosine hydroxylase (an enzyme involved in dopamine synthesis), serotonin or Dopa decarboxylase (an enzyme involved in synthesis of both neurontransmitters).

The developmental origin of dopamine or serotonin synthesizing hormones is of particular interest to developmental biologists because of the importance of such neurons in vertebrates in addictive behavior and reward and punishment. In organisms like Drosophila, such neurons can be studied in a less complex context than in the human brain. There are three dopaminergic and four serotonergic cells per segment of the ventral cord. These include the unpaired midline and dorsal lateral dopamine neurons and the paired ventrolateral serotonin neurons (Lundell, 1994). All of the dopaminergic and serotonergic cells of the ventral cord express Islet protein and represent a subset of the islet expressing interneurons. islet mutants show no detectable tyrosine hydroxylase expression, and little or no expression of serotonin in the ventral cord. In addition, little or no expression of Dopa decarboxylase is found in islet mutants. Interestingly, a small subset of serotonergic and dopaminergic neurons in the fly's brain does not express islet, and both the timing and levels of tyrosine hydroxylase and serotonin expression in these cells are normal in islet mutants.

The serotonin neurons constitute a subset of islet interneurons and project axons across the midline in the posterior commissure to form a discrete fascicle with their partners on the contralateral side. The dopamine neurons also project within this fascicle; the lateral cells extend axons contralaterally while the unpaired ventral cell bifurcates at the midline within the fascicle. In islet mutants, these cells survive throughout embryogenesis and extend axons normally. However, in most segments, the serotonin-dopamine fascicle fails to form. In late embryos the segmentally repeated pattern of these fascicles is disrupted and the dopamine and serotonin neurons can be seen projecting axons abnormally within the commissures and connectives. Postmitotic expression of islet rescues tyrosine hydroxylase and serotonin expression in islet mutants (Thor, 1997).

Engrailed and Huckebein are also essential for development of serotonin neurons in the Drosophila CNS. en and hkb coexpress only in the serotonin neurons and in neuroblast 7-3 (NB7-3). In the grasshopper, the analogous serotonin neurons originate from the first ganglion mother cell produced from NB7-3. The corresponding NB7-3 in Drosophila can be identified by its time of birth, size, and relative position within each hemisegment. The serotonin neurons can be identified during late embryogenesis by the appearance of DOPA decarboxylase (DDC) immunoreactivity. The high selectivity of coexpression of these two gene products suggests that their combined activities may be important for the development of NB7-3 progeny. Serotonin neuron differentiation is abnormal in en and hkb mutants. Since NB 7-3 appears normal in hkb mutants, the effect of hkb on development of the serotonin cell lineage must be at a later stage of development, either at division of the neuroblast or ganglion mother cells, or on the identity of the GMC progeny (Lundell, 1996).

The finding of an islet requirement in serotonergic and dopaminergic cells suggests the existence of a combinatorial code that includes en, hkb, islet, and possibly other transcription factors (See Zn finger homeodomain 1) in the generation of serotonergic cells. The existence of a combinatorial code would explain why islet misexpression alone is insufficient to trigger ectopic serotonin expression. Dissection of the Dopa decarboxylase-promoter has implicated the POU proteins Drifter and I-POU in its regulation (Johnson, 1990 and Treacy, 1991). It is noteworthy that several studies have demonstrated direct interactions betweeen LIM homeodomain proteins and POU domain proteins; these interactions have been proposed as effecting a change in the specificity of DNA binding and/or as altering the regulatory activity of both proteins (Xue, 1993 and Bach, 1993).

Targeted disruption of islet-1 in the mouse leads to lack of motor neuronal differentation, as all mutant spinal motor neurons appear to undergo programmed cell death rapidly after their final mitotic division (Pfaff, 1996). A similar disappearance of motor neurons in Drosophila islet mutants is not apparent, and instead neurons in islet mutants appear to survive and elongate axons throughout embryogenesis (Thor, 1997). Whereas only a subset of Drosophila motor neurons express islet, all vertebrate motor neurons express islet-1, islet-2 or a combination of both. Thus only certain aspects of the function of the islet genes are common to vertebrates and flies.

Specification of Drosophila motoneuron identity by the combinatorial action of POU and LIM-HD factors

In both vertebrates and invertebrates, members of the LIM-homeodomain(LIM-HD) family of transcription factors act in combinatorial codes to specifymotoneuron subclass identities. In the developing Drosophila embryo,the LIM-HD factors Islet (Tailup) and Lim3, specify the set of motoneuronsubclasses that innervate ventral muscle targets. However, since severalsubclasses express both Islet and Lim3, this combinatorial code alone cannotexplain how these motoneuron groups are further differentiated. To identifyadditional factors that may act to refine this LIM-HD code, the expression of POU genes in the Drosophila embryonic nerve cord was analyzed. The class III POU protein, Drifter (Ventral veinless), isco-expressed with Islet and Lim3 specifically in the ISNb motoneuron subclass.Loss-of-function and misexpression studies demonstrate that the LIM-HDcombinatorial code requires Drifter to confer target specificity between theISNb and TN motoneuron subclasses. To begin to elucidate molecules downstreamof the LIM-HD code, the involvement of the Beaten path (Beat)family of immunoglobulin-containing cell-adhesion molecules was analyzed. beat Ic genetically interacts with islet and Lim3in the transverse nerve (TN) motoneuron subclass and can also rescue the TN fasciculation defectsobserved in islet and Lim3 mutants. These results suggestthat in the TN motoneuron context, Islet and Lim3 may specify axon targetselection through the actions of IgSF call-adhesion molecules (Certel, 2004).

In each abdominal hemisegment of the Drosophila embryo, the axonsof ~40 motoneurons exit the ventral nerve cord and specifically synapsewith 30 identified muscle fibers. The axons of these motoneurons form sixdiscrete fascicles and exit the VNC together, extending into the periphery toinnervate specific muscle groups (or fields). The ventral muscles areinnervated by the motoneurons of three subclasses: the transverse nerve (TN),intersegmental nerve b (ISNb) and intersegmental nerve d (ISNd). Inaddition, the segmental nerve c (SNc) innervates a set of externally locatedventral muscles. The two TN motoneurons contact ventral muscle 25 or theventral process of the lateral bipolar dendritic neuron (LBD). TheISNb motoneurons innervate the ventral muscles 6, 7, 12, 13, 14, 28 and 30,and the ISNd motoneurons muscles 15, 16 and 17. TheDrosophila LIM-HD genes, islet and Lim3, areco-expressed in a subset of CNS neurons, including several classes ofmotoneurons. Although Isl and Lim3 are required in these distinctmotoneurons to specific neuronal identity, this twomember 'LIM-code' cannot alone be responsible for the unique differentiationand function of each subclass. More specifically, since the TN and ISNbmotoneuron subclasses both express a combination of Isl and Lim3, it is likelythat other factors act to discriminate between these two subclasses (Certel, 2004).

To identify factors that act to further differentiate these motoneuronsubgroups, the expression pattern of previously characterizedtranscription factors was examined. Isl and Lim3 are co-expressed with theclass III POU factor, Drifter (Dfr), in a limited number of embryonic VNCneurons. Immunohistochemical experiments using double transgenic animals reporting on Isl and Lim3 expression and antibodies directed against Dfr reveal restrictedtriple expression specifically in the ISNb motoneuron subclass. Dfr, Isl and Lim3expression is clearly observed in the RP1, RP3, RP4 and RP5 motoneuronsthat project out the ISNb fascicle to innervate ventral muscles6, 7, 12 and 13.Dfr expression is not detected in the pair of Isl- and Lim3-expressing TNneurons. Toconfirm that the lateral co-expressing neurons belong to the ISNb subclass, embryos carrying the Lim3A-tau-myc transgene were double-labeled. Thisreporter construct drives expression in Isl-expressing motoneurons thatproject via the ISNb but not the ISNd fascicle. The lateral Dfr-expressing neurons do belong to the ISNb subclass since theyalso express the Tau-myc fusion protein. The co-expression of Dfr and Islwas further analyzed. In addition to theISNb neurons, Dfr and Islet co-expression is also observed in two serotonergicEW neurons (Certel, 2004).

To verify the lack of Dfr expression in the ISNd subgroup, focus was placed onthe well-characterized GW/7-3M motoneuron that innervates muscle 15 via theISNd pathway. To identify this ISNd motoneuron, the enhancer-trapline, eg^P289, was used that drives lacZ expression in the7-3M progeny. Although Dfr expression is seen in the EW interneurons, theISNd GW/7-3M motoneuron does not express Dfr. The resultsof these labeling experiments demonstrate that Dfr expression can be furtherused to subdivide the ISNb from the TN and ISNd neuronal classes (Certel, 2004).

The ISNb fascicle contains motor axons from at least eight motoneuronsinnervating ventral muscles 6, 7, 12, 13, 14, 28 and 30. Loss of isl or Lim3 function in ISNbmotoneurons affects axon targeting resulting in a reduction of target muscleinnervation. The most common phenotype in both isl andLim3 mutants is a failure to innervate the cleft between muscles 12and 13. In Lim3 mutants, muscles 12/13 were not innervated by motoraxons in 46% of hemisegments compared with 3% in wild-type hemisegments. Inisl mutants, the lack of muscle contacts was also coupled with theISNb motor axons, leaving the ventral muscle field and joining the TN (26% ofhemisegments in isl mutants versus 0% in wild type) (Certel, 2004).

Dfr is functionally required in a variety of tissues, including the midlineglia, trachea, wing veins, sensory neurons and antennal lobe projectionneurons. The early lethality of null dfr alleles,and the VNC perturbations caused by midline glia defects made analyzing therequirement for Dfr function in ISNb neurons difficult. To circumvent thisproblem, and to examine whether Isl, Lim3 and Dfr are required to specifysimilar aspects of motor axon targeting, the ISNb fasciclewas analyzed in trans-heterozygous combinations. Removing a single copy of isl,Lim3 and dfr results in motor axon targeting defectscharacterized by significant reductions in muscle innervation. The failure toinnervate muscles 12 and 13 observed in bothisl^37Aa/+;dfr^B129/+ andLim3^Bd1/+;dfr^B129/+ trans-heterozygotes iseasily quantifiable and observed in isl and Lim3 mutantembryos. Reducing the levels of Isl and Dfr also results in ISNb motor axons leaving the ventral muscle field and targeting the TN fascicle. Neither Dfr nor Isl is expressed in motoneurons that innervate the dorsal muscles or the externally located ventral muscles. In line with their restricted expression, no evidence was found of non-cell autonomous affects upon the targeting of the ISN and SNc motor axons in dfr, isl trans-heterozygotes (Certel, 2004).

In an attempt to address the specificity of the genetic interactionsbetween POU and LIM-HD genes, genetic interactions were tested betweendfr and another key regulator of ISNb identity, the Drosophilaexex/hb9 gene. However, no evidence of ISNb axon pathfindingdefects were found in embryos trans-heterozygotes for dfr and hb9(dfr^B129/hb9^KK30). Therefore,results from genetic interaction studies indicate that Dfr may be requiredspecifically in combination with Isl and Lim3 to specify the ISNb motoneuronsubclass (Certel, 2004).

isl and Lim3 mutants display axontargeting defects manifested by a reduction in muscle innervation. Results from thetrans-heterozygous genetic interaction studies suggest that Dfr is necessaryin combination with Isl and Lim3 to specify ISNb axon targeting. If thishypothesis is correct, then a loss of Dfr function in ISNb motoneurons shouldalso result in a reduction in muscle innervation. To address the possible roleof Dfr in ISNb specification, RNA interference was used to reduce or eliminatethe expression of Dfr protein specifically in neurons, without affecting itsexpression and function in midline glia. Transgenic flies were generatedexpressing double-stranded dfr RNA (UAS-dsdfr) under thecontrol of neuronal-specific Gal4 drivers. To further assist in theremoval of Dfr function, two independent UAS-dsdfr insertions wererecombined onto a chromosome containing a null dfr allele,dfr^B129. Although the protein produced by this allele isdetected by the Dfr antiserum, it is non-functional because of a prematurestop codon located before the DNA-binding POU domain. Several Gal4 drivers were used to analyze theeffectiveness of the UAS-dsdfr transgenes. Using both theC155(elav)-Gal4 and the Lim3B-Gal4 drivers, Dfr protein was reduced or eliminated in the majority of ISNbmotoneurons, including the RP motoneurons. As a control, theDfr-expressing midline glia showed no loss of Dfr protein and, accordingly, noloss of the midline glia-specific marker Slit. This shows that UAS-dsdfr could be used together with neuronal-specific Gal4 drivers to address how loss of Dfr function affects ISNb neuron specification (Certel, 2004).

Using both early (ftz_ng-Gal4.20) and post-mitotic(Lim3B-Gal4) drivers to reduce Dfr activity, two axonoutgrowth phenotypes were observed. (1) ISNb motor axons can now leave the ventral muscle field and contact or target the TN fascicle. This re-targetingsuggests that losing Dfr leaves the ISNb neurons in an Isl/Lim3-only specifiedstate generating a TN fate. (2) Reducing Dfr function causes an overalldecrease in muscle innervation by the ISNb motor axons similar to defectsobserved in isl mutants. Both of these axon targeting and innervation phenotypes indicate that Dfr plays an important role in ISNb neuronal specification (Certel, 2004).

If Dfr functions as part of a LIM/POU combinatorial code to specify ISNbmotoneurons, then ectopically expressing Dfr in the Isl/Lim3-expressing TNmotoneurons would be predicted to alter TN axon pathfinding toward anISNb-like behavior. To test this, Dfr was misexpressed in postmitoticIsl/Lim3-expressing TN neurons using the Lim3B-Gal4 driver. Inwild-type development, the transverse nerve forms from the fasciculation of asensory nerve axon (the lateral bipolar dendrite or LBD neuron) and the TMNpneuron. Atlate stage 15/16, these growth cones contact each other on the ventralinterior muscle surfaces. The TN fascicle is on a different focal plane and inwild-type embryos does not come in contact with the ISNb fascicle (Certel, 2004).

Misexpression of Dfr protein in Lim3B-Gal4;UAS-dfr doubletransgenic embryos did not have any effect on TN axons exiting the ventralnerve cord or lead to random innervation in the periphery. Instead, adding Dfrfunction to the TN neurons caused the motor axons to target the ISNb musclefield in a significant number of hemisegments (52%). TN motoraxons either send collaterals into ISNb muscle targets or in some caseseven innervated ISNb muscles. This change in motor axon targeting appears to be specific to the TN motoneurons. Misexpressing Dfr in most, if not all, motoneurons via the ftz_ng-Gal4.20 did not result in any targeting changes or defects in the SNc or ISN fascicles; it did, however, result in a similar percentage of TMNp targeting defects (Certel, 2004).

The ability of the TN motoneurons to be respecified by Dfrmisexpression was tested in isl mutant embryos. Without Isl function, theretargeting of TN motor axons did not occur, suggestingpossible cooperative actions between these transcription factors. These resultsindicate that it is the addition of Dfr specifically to the TN Isl/Lim3 LIM-HDcode that allows this motoneuron subclass to exhibit ISNb motoneuroncharacteristics (Certel, 2004).

In C. elegans touch receptor neurons, the POU protein UNC-86directly regulates the expression of the LIM-HD gene, MEC-3. And invertebrates, abLIM is a transcriptional target of the POU factor, Brn3.2. Todetermine whether Dfr, Isl and/or Lim3 are possible transcriptional targets ofone another, the individual expression patterns were examined in each mutantbackground. The results indicate that in Drosophila Dfr, Isl and Lim3 must function at the same hierarchical level in ISNb motoneuron specification (Certel, 2004).

Studies in various model systems have led to the identification of a numberof transcription factors with highly restricted expression. These factors areimportant for different aspects of neuronal differentiation, including axonpathfinding. By contrast, many other molecules, such as cell adhesionmolecules, receptor protein tyrosine phosphatases and semaphorins, also play acrucial role in axon guidance, yet these molecules are often, at least inDrosophila , more broadly expressed. This apparentdisjunction could indicate that the downstream genes regulated by highlyrestricted transcription factors such as Dfr and Isl are thus faruncharacterized molecules. Therefore, to identify possible targets of theLIM/POU or LIM-HD combinatorial codes, specific molecules were sought thatare differentially expressed between the ISNb and TN motoneuron subclasses. Restricted expression is exhibitied by 14Drosophila Beat-like members of the immunoglobulin superfamily (IgSF)of CAMs.Members of the Beat family in Drosophila and the Zig family in C.elegans contain two Ig domains and most members have been shown to behighly restricted in their expression pattern, largely confined to subsets ofneurons (Certel, 2004).

beat Ic is of particular interest because of itsexpression in a small number of embryonic neurons that include the TNmotoneurons but not the ISNb neurons. In situ hybridization and immunohistochemistry was used to verify the TN expression ofbeat Ic. beat Ic transcripts are expressed in lateral cell clustersthat contain the TN neurons but not in the ISNb RP neurons.In addition to restricted TN expression, removal of beat Icfunction through overlapping deficiencies results in TN phenotypesindistinguishable from those observed in isl and Lim3mutants. Embryos that lack beat Ic display errors in TNfasciculation. The TMNp axon fails to completely fasciculate with the LBDprojection, resulting in bifurcation and aberrant ventral muscle exploration. This TN fasciculation in beat Ic mutants is improved by elav-Gal4driven UAS-beat Ic expression. Inisl mutants, the TN axons either do not exit the VNC or fail tofasciculate with the LBD projection, also leading to aberrant ventral muscleexploration. Thisfailure of the TMNp and LBD axons to adhere is also observed in a significantnumber of Lim3 mutant hemisegments (Certel, 2004).

The similarity of TN mutant phenotypes suggests that Isl, Lim3 and Beat Icare required to specify analogous aspects of motor axon targeting. To testthis hypothesis, the TN fascicle was analyzed in trans-heterozygouscombinations. Mutations in beat Ic are not available, therefore, defineddeficiencies were used. Embryosthat lack one copy of isl, Lim3 and beat Ic display asignificant number of TN fasciculation defects. As in individualmutants, the TMNp and LBD axons in several trans-heterozygous combinationsfail to fasciculate, thus leading to bifurcation and/or aberrant axonoutgrowth. Twochromosomal deficiencies removing beat Ic, beat Ic^Df andbeat Ic^Df1 were used todemonstrate that other genes removed by an individual deficiency did notinfluence the TN phenotype. In addition, at least two different island Lim3 alleles were used to verify that the observed geneticinteractions were not due to specific chromosomes or alleles. Another Beatfamily member, beat Ib, is also expressed in the CNS; however, a lackof Beat Ib does not result in any defects in neuronal development. It was not possible to test for genetic interactions with any other Beat family members because neither overlapping deficiencies nor individual mutations are available forthese genes. However, to assess the specificity of these interactions amongIg domain-containing molecules, genetic interactions were examined betweenisl, Lim3 and the CAM Fas2, which contains five Ig-likedomains. No fascicle defects were detected in varioustrans-heterozygous combinations. These results raise thepossibility that in TN motoneurons Isl and Lim3 may specify axon targetselection through the actions of specific IgSF CAM members (Certel, 2004).

At least two possible explanations can be put forwards to explain the TNdefects observed in isl and Lim3 mutants. First, the defectsobserved are because the TMNp and the LBD axons cannot fasciculate or adhereto one another. A second hypothesis is that these two axons do not recognizeeach other and therefore do not grow close enough together to fasciculateproperly. If the defect lies in fasciculation, then increasing the levels ofBeat Ic, a promoter of motor axon adhesion, should reduce the TN defects inisl and Lim3 mutants. To increase Beat Ic levels, thepan-neuronal driver elav-Gal4 and UAS-beat Ic transgeneswere crossed into isl and Lim3 mutant backgrounds. As inprevious experiments, strong isl and Lim3 alleles werecrossed to isl^Df and lim^Dfdeficiencies to create embryos null for isl and Lim3,respectively (Certel, 2004).

Increasing the levels of Beat Ic through one copy of UAS-beat Icand the elav-Gal4 driver significantly reduces the percentage of TNdefects in isl mutants from 56% to 22%. In addition,the severity of TN adhesion defects decreased in the remaining 22% of affectedhemisegments. Likewise, increasing Beat Ic levels in Lim3 mutantsalso significantly decreased the occurrence of TN defects from 55% to 31%. These results suggest that the TN defects observed in isl and Lim3 mutants are a result of a decrease in the adhesive properties between the TN motor axon and the LBD projection. This suggests that Beat Ic may be a direct transcriptional target of the LIM code (Certel, 2004).

At this time, this hypothesis could not be tested because of theunavailability of a Beat Ic antibody. Attempts were made to analyze beat Ictranscript accumulation in isl and Lim3 double mutants butit was not possible to achieve cellular resolution in the mutant ventral nerve cords. However, DNA-binding site pattern searches using previously described LIM-HDbinding motifs indicate that there are significant clusters of LIM-HD sitessurrounding and within the beat Ic locus.The Isl1 consensus site (CTAATG) is found 15 times in the chromosomal regionencompassing the beat Ic locus; the Lhx3-bindingsite (AATTAATTA) is found nine times and 21 Isl 2.2 sites (YTAAGTG) have beenidentified. Although functional analyses will be needed to determine if thesesites are necessary and/or sufficient for beat Ic expression,searching the entire genome with the Isl 2.2 site places the beat Iclocus seventh out of the first 10 identified. Furthermore, studies in C.elegans indicate that two LIM-HD genes, the Lmx-class genelim-6 and the Lhx3-class gene, ceh-14, are requiredfor the expression of at least four IgSF zig genes (Certel, 2004).

Therefore beat Ic is transcribed in a small number of cells in the embryonicnerve cord, including the transverse motoneurons. Loss-of-function experiments indicate Beat Ic is required in apro-adhesive manner for the proper recognition/or fasciculation of the TMNpmotor axon and the LBD fascicle. These TN defects are identical to thefasciculation defects observed in isl and Lim3 mutants.Trans-heterozygous combinations reveal strong genetic interactions betweenisl, Lim3 and beat Ic, and furthermore, increasing Beat Ic expression in isl and Lim3 mutantssignificantly rescues these TN axon fasciculation defects (Certel, 2004).

LIM-HD proteins participate in a number of unique complexes throughprotein-protein interactions mediated by their LIM domains. For example, LIMdomains can interact with the widely expressed co-factor, NLI(Ldb1/CLIM-2) inmice or Chip in Drosophila. The NLI/CHIP proteins homodimerize and generate a bridge between two LIM-HD proteins, thereby leading to the formation of tetramericcomplexes. Although this complex is functional in vivo, it has beenfound that other proteins also participate to generate further tissuespecificity. In Drosophila, the newly identified Ssdp proteininteracts with Chip to modify the activity of complexes comprising Chip andthe LIM-HD protein Apterous in the wing. Invertebrates, the bHLH factors Ngn2 and NeuroM functionally interact with theNLI, Isl1 and Lhx3 complex to initiate motoneuron differentiation. Theseresults suggest that the formation of further specialized combinations couldbe used to confer not only tissue but also cellular specificity (Certel, 2004).

In Drosophila , Isl and Lim3 are co-expressed in a subset of CNS neurons including two neuron subclasses, the TN and ISNb motoneurons. Although Isl and Lim3 are required in these distinct motoneurons to specific motor axon pathway choice neuronal identity, these two factors alone cannot be responsible for the unique differentiation of each subclass. In this study, evidence has been provided that the class III POU domain protein Dfr functions in combination with this Isl/Lim3 LIM code, to specify the ISNb motoneuron class. Loss-of-function analyses indicate each of these transcription factors is required in the ISNb neurons for the specification of motor axon target selection. Without Dfr, Isl or Lim3, these motor axons fail to correctly innervate their designated muscle targets. In addition, genetic interaction studies suggest that this phenotype indicates a common aspect of motoneuron designation has been altered (Certel, 2004).

How might this LIM/POU code function in ISNb neurons? In C.elegans touch receptor neurons, the LIM-HD factor, MEC-3 and the POUprotein UNC-86, physically interact to control specification. In thepituitary, the LIM domain of Lhx3 (P-Lim) specifically interacts with the Pit1POU domain and is required for synergistic interactions with Pit1. Whether Dfr, Isl and or Lim3 physically interact to regulate ISNb motor axon target selection has not been determined. However, misexpression experimentsindicate that the re-specification of transverse motoneurons by the additionof Dfr does require functional Isl protein. Although, this result does notdistinguish between the possibilities of direct interactions between theseproteins or the binding of a common transcriptional target, it does indicatethat a functioning 'LIM code' is required for the re-specification of the TNneurons (Certel, 2004).

A second finding of the Dfr misexpression studies is that the target selection of postmitotic neurons can be robustly re-specified. TheLim3B-Gal4 line was used to add Dfr to the LIM-only transverse motoneurons.This Gal4 line does not activate reporter construct expression untilstage 14 -- a post-mitotic stage even for the late developing transversemotoneurons. At this stage, the TN motor axons have exited the CNS and arenavigating the periphery, although the TN motor axon and LBD fascicle have notcome into contact. Misexpressing Dfr even at this relatively late stage of TNmotoneuron differentiation can clearly alter axon pathfinding, and in asignificant percentage of hemisegments, TN motor axons actually appear toectopically innervate ISNb muscle targets. This result shows that thesemotoneurons remain plastic, even after becoming postmitotic and furtherindicate that the LIM/POU code may be acting directly on genes involved inaxon targeting (Certel, 2004).

The combinatorial expression of LIM-HD transcriptionfactors confers motoneuron subtypes with the ability to direct their axons toreach distinct muscle targets. If more than one subgroup of motoneurons use aLIM code, how does subtype-specific motor axon pathfinding occur? Presumably,it is the downstream targets of each LIM code that confer the ability ofindividual or groups of motor axons to find their correct innervation targets.What might be the target(s) of the LIM code in Drosophila transverseneurons? Studies in vertebrates and invertebrates have demonstrated that members of the IgSF class of CAMs play important roles in cell-cell recognition andcommunication -- processes that are crucial for nervous system wiring. The ability of Ig-domains to form linear rods when deployedin series, and their propensity to bind specifically to other proteins, hasmade these molecules ideal for functioning as cell-surface receptors and/orCAMs. Furthermore, IgSF molecules have dramatically increased the number ofcell-cell recognition molecules through family expansion, the generation ofmultiple variants through alternative splicing, receptor multimerization andcross-talking intracellular signaling pathways (Certel, 2004).

A recent genomic analysis indicates the Drosophila immunoglobulin superfamily repertoireconsists of about 150 proteins; in C. elegans, 80 immunoglobulin superfamily molecules arepredicted. Members of the Beat family in Drosophila and thezig family in C. elegans are located in gene clusters, haverestricted expression patterns and share the same domain architecture. Eachprotein is comprised exclusively of two Ig modules with either a transmembranedomain [beat Ic, beat Ib, beat IIa, beat VI (GPI), zig-1] or secretedsignals (10 Beat genes: zig-2 to zig-8). The Beatfamily members that have been functionally characterized appear to controlfasciculation through anti- and pro-adhesive properties.Furthermore, unlike many previously described CAMs, several members of thesefamilies are expressed in subsets of CNS neurons (Certel, 2004).

GENE STRUCTURE

Structural Domains

The predicted Islet protein contains two LIM domains and a C-terminal homeodomain, with extensive homology to the vertebrate Islet-1 and Islet-2 proteins. The homology is highest in the homeodomain (95% identity to Islet 1 and 2) and somewhat lower in the LIM domains (85%). Overall the Drosophila and vertebrate proteins show 57% identity. A highly conserved 16 amino acids stretch located C-terminal to the homeodomain denotes the Islet-specific domain, and is found in all members of the Islet subfamily, but not in other LIM-HD proteins. The Drosophila Islet homolog shows greater similarity to the vertebrate Islet proteins than to other Drosophila LIM-HD proteins. For example, within the homeodomain, Drosophila Islet shows only 38% amino acid identity to Apterous, which is 92% identical to its vertebrate homolog LH-2. Mutations affecting the isl locus completely abolish immunoreactivity with antibodies that recognize both vertebrate Islet-1 and Islet-2, suggesting that only a single islet homolog exists in Drosophila (Thor, 1997).

The LIM domain is a cysteine-rich domain composed of 2 special zinc fingers joined by a2-amino acid spacer. Some proteins are made up of only LIM domains, while others contain a varietyof different functional domains. LIM proteins form a diverse group that includes transcription factorsand cytoskeletal proteins. The primary role of LIM domains appears to be in protein-protein interaction,through the formation of dimers with identical or different LIM domains or by binding distinct proteins.In LIM homeodomain proteins, LIM domains seem to function as negative regulatory domains. LIMhomeodomain proteins are involved in the control of cell lineage determination and the regulation ofdifferentiation, and LIM-only proteins may have similar roles. LIM-only proteins are also implicated inthe control of cell proliferation since several genes encoding such proteins are associated withoncogenic chromosome translocations. In analyzing sequence relationships among various LIM domains it issuggested that they may be arranged into 5 groups that appear to correlate with the structural andfunctional properties of the proteins containing these domains. All N-terminal LIM domains (LIM1) are segregated into cluster A, whereas all LIM2 domains of the same proteins constitute cluster B. This relationship suggests that the putative duplication leading to the LIM A and B domains is ancient, preceding their association with different structural motifs (e.g., homeodomains, kinases). Furthermore, the sequence relationships between the LIM domains (LIM1 and LIM2) in the same protein may be conserved by functional constraints based on cooperation between LIMA and B domains. In contrast, the two type C LIM (another LIM domain cluster) domains of some of the LIM-only proteins like CRP are more similar to one another, implying the possibility of a more recent duplication. Cluster D is a rather divergent set of LIM domains that includes the cytoskeletal proteins Zyxin and Paxillin. The closest homologs of Apterous, for both LIM1 and LIM2 domains, are human and rat LH2. The Islet LIM1 and LIM2 domains define Islet as a cohesive subfamily of LIM proteins (Dawid, 1995).

date revised: 20 January 2005

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