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 specify motoneuron subclass identities. In the developing Drosophila embryo, the LIM-HD factors Islet (Tailup) and Lim3, specify the set of motoneuron subclasses that innervate ventral muscle targets. However, since several subclasses express both Islet and Lim3, this combinatorial code alone cannot explain how these motoneuron groups are further differentiated. To identify additional 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), is co-expressed with Islet and Lim3 specifically in the ISNb motoneuron subclass. Loss-of-function and misexpression studies demonstrate that the LIM-HD combinatorial code requires Drifter to confer target specificity between the ISNb and TN motoneuron subclasses. To begin to elucidate molecules downstream of 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 Lim3 in the transverse nerve (TN) motoneuron subclass and can also rescue the TN fasciculation defects observed in islet and Lim3 mutants. These results suggest that in the TN motoneuron context, Islet and Lim3 may specify axon target selection through the actions of IgSF call-adhesion molecules (Certel, 2004).

In each abdominal hemisegment of the Drosophila embryo, the axons of ~40 motoneurons exit the ventral nerve cord and specifically synapse with 30 identified muscle fibers. The axons of these motoneurons form six discrete fascicles and exit the VNC together, extending into the periphery to innervate specific muscle groups (or fields). The ventral muscles are innervated by the motoneurons of three subclasses: the transverse nerve (TN), intersegmental nerve b (ISNb) and intersegmental nerve d (ISNd). In addition, the segmental nerve c (SNc) innervates a set of externally located ventral muscles. The two TN motoneurons contact ventral muscle 25 or the ventral process of the lateral bipolar dendritic neuron (LBD). The ISNb motoneurons innervate the ventral muscles 6, 7, 12, 13, 14, 28 and 30, and the ISNd motoneurons muscles 15, 16 and 17. The Drosophila LIM-HD genes, islet and Lim3, are co-expressed in a subset of CNS neurons, including several classes of motoneurons. Although Isl and Lim3 are required in these distinct motoneurons to specific neuronal identity, this two member 'LIM-code' cannot alone be responsible for the unique differentiation and function of each subclass. More specifically, since the TN and ISNb motoneuron subclasses both express a combination of Isl and Lim3, it is likely that other factors act to discriminate between these two subclasses (Certel, 2004).

To identify factors that act to further differentiate these motoneuron subgroups, the expression pattern of previously characterized transcription factors was examined. Isl and Lim3 are co-expressed with the class III POU factor, Drifter (Dfr), in a limited number of embryonic VNC neurons. Immunohistochemical experiments using double transgenic animals reporting on Isl and Lim3 expression and antibodies directed against Dfr reveal restricted triple expression specifically in the ISNb motoneuron subclass. Dfr, Isl and Lim3 expression is clearly observed in the RP1, RP3, RP4 and RP5 motoneurons that project out the ISNb fascicle to innervate ventral muscles 6, 7, 12 and 13. Dfr expression is not detected in the pair of Isl- and Lim3-expressing TN neurons. To confirm that the lateral co-expressing neurons belong to the ISNb subclass, embryos carrying the Lim3A-tau-myc transgene were double-labeled. This reporter construct drives expression in Isl-expressing motoneurons that project via the ISNb but not the ISNd fascicle. The lateral Dfr-expressing neurons do belong to the ISNb subclass since they also express the Tau-myc fusion protein. The co-expression of Dfr and Isl was further analyzed. In addition to the ISNb neurons, Dfr and Islet co-expression is also observed in two serotonergic EW neurons (Certel, 2004).

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

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

Dfr is functionally required in a variety of tissues, including the midline glia, trachea, wing veins, sensory neurons and antennal lobe projection neurons. The early lethality of null dfr alleles, and the VNC perturbations caused by midline glia defects made analyzing the requirement for Dfr function in ISNb neurons difficult. To circumvent this problem, and to examine whether Isl, Lim3 and Dfr are required to specify similar aspects of motor axon targeting, the ISNb fascicle was analyzed in trans-heterozygous combinations. Removing a single copy of isl, Lim3 and dfr results in motor axon targeting defects characterized by significant reductions in muscle innervation. The failure to innervate muscles 12 and 13 observed in both isl^37Aa/+;dfr^B129/+ and Lim3^Bd1/+;dfr^B129/+ trans-heterozygotes is easily quantifiable and observed in isl and Lim3 mutant embryos. 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 interactions between POU and LIM-HD genes, genetic interactions were tested between dfr and another key regulator of ISNb identity, the Drosophila exex/hb9 gene. However, no evidence of ISNb axon pathfinding defects 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 required specifically in combination with Isl and Lim3 to specify the ISNb motoneuron subclass (Certel, 2004).

isl and Lim3 mutants display axon targeting defects manifested by a reduction in muscle innervation. Results from the trans-heterozygous genetic interaction studies suggest that Dfr is necessary in combination with Isl and Lim3 to specify ISNb axon targeting. If this hypothesis is correct, then a loss of Dfr function in ISNb motoneurons should also result in a reduction in muscle innervation. To address the possible role of Dfr in ISNb specification, RNA interference was used to reduce or eliminate the expression of Dfr protein specifically in neurons, without affecting its expression and function in midline glia. Transgenic flies were generated expressing double-stranded dfr RNA (UAS-dsdfr) under the control of neuronal-specific Gal4 drivers. To further assist in the removal of Dfr function, two independent UAS-dsdfr insertions were recombined onto a chromosome containing a null dfr allele, dfr^B129. Although the protein produced by this allele is detected by the Dfr antiserum, it is non-functional because of a premature stop codon located before the DNA-binding POU domain. Several Gal4 drivers were used to analyze the effectiveness of the UAS-dsdfr transgenes. Using both the C155(elav)-Gal4 and the Lim3B-Gal4 drivers, Dfr protein was reduced or eliminated in the majority of ISNb motoneurons, including the RP motoneurons. As a control, the Dfr-expressing midline glia showed no loss of Dfr protein and, accordingly, no loss 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 axon outgrowth phenotypes were observed. (1) ISNb motor axons can now leave the ventral muscle field and contact or target the TN fascicle. This re-targeting suggests that losing Dfr leaves the ISNb neurons in an Isl/Lim3-only specified state generating a TN fate. (2) Reducing Dfr function causes an overall decrease in muscle innervation by the ISNb motor axons similar to defects observed 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 ISNb motoneurons, then ectopically expressing Dfr in the Isl/Lim3-expressing TN motoneurons would be predicted to alter TN axon pathfinding toward an ISNb-like behavior. To test this, Dfr was misexpressed in postmitotic Isl/Lim3-expressing TN neurons using the Lim3B-Gal4 driver. In wild-type development, the transverse nerve forms from the fasciculation of a sensory nerve axon (the lateral bipolar dendrite or LBD neuron) and the TMNp neuron. At late stage 15/16, these growth cones contact each other on the ventral interior muscle surfaces. The TN fascicle is on a different focal plane and in wild-type embryos does not come in contact with the ISNb fascicle (Certel, 2004).

Misexpression of Dfr protein in Lim3B-Gal4;UAS-dfr double transgenic embryos did not have any effect on TN axons exiting the ventral nerve cord or lead to random innervation in the periphery. Instead, adding Dfr function to the TN neurons caused the motor axons to target the ISNb muscle field in a significant number of hemisegments (52%). TN motor axons either send collaterals into ISNb muscle targets or in some cases even 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 Dfr misexpression was tested in isl mutant embryos. Without Isl function, the retargeting of TN motor axons did not occur, suggesting possible cooperative actions between these transcription factors. These results indicate that it is the addition of Dfr specifically to the TN Isl/Lim3 LIM-HD code that allows this motoneuron subclass to exhibit ISNb motoneuron characteristics (Certel, 2004).

In C. elegans touch receptor neurons, the POU protein UNC-86 directly regulates the expression of the LIM-HD gene, MEC-3. And in vertebrates, abLIM is a transcriptional target of the POU factor, Brn3.2. To determine whether Dfr, Isl and/or Lim3 are possible transcriptional targets of one another, the individual expression patterns were examined in each mutant background. 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 number of transcription factors with highly restricted expression. These factors are important for different aspects of neuronal differentiation, including axon pathfinding. By contrast, many other molecules, such as cell adhesion molecules, receptor protein tyrosine phosphatases and semaphorins, also play a crucial role in axon guidance, yet these molecules are often, at least in Drosophila , more broadly expressed. This apparent disjunction could indicate that the downstream genes regulated by highly restricted transcription factors such as Dfr and Isl are thus far uncharacterized molecules. Therefore, to identify possible targets of the LIM/POU or LIM-HD combinatorial codes, specific molecules were sought that are differentially expressed between the ISNb and TN motoneuron subclasses. Restricted expression is exhibitied by 14 Drosophila 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 be highly restricted in their expression pattern, largely confined to subsets of neurons (Certel, 2004).

beat Ic is of particular interest because of its expression in a small number of embryonic neurons that include the TN motoneurons but not the ISNb neurons. In situ hybridization and immunohistochemistry was used to verify the TN expression of beat Ic. beat Ic transcripts are expressed in lateral cell clusters that contain the TN neurons but not in the ISNb RP neurons. In addition to restricted TN expression, removal of beat Ic function through overlapping deficiencies results in TN phenotypes indistinguishable from those observed in isl and Lim3 mutants. Embryos that lack beat Ic display errors in TN fasciculation. The TMNp axon fails to completely fasciculate with the LBD projection, resulting in bifurcation and aberrant ventral muscle exploration. This TN fasciculation in beat Ic mutants is improved by elav-Gal4 driven UAS-beat Ic expression. In isl mutants, the TN axons either do not exit the VNC or fail to fasciculate with the LBD projection, also leading to aberrant ventral muscle exploration. This failure of the TMNp and LBD axons to adhere is also observed in a significant number of Lim3 mutant hemisegments (Certel, 2004).

The similarity of TN mutant phenotypes suggests that Isl, Lim3 and Beat Ic are required to specify analogous aspects of motor axon targeting. To test this hypothesis, the TN fascicle was analyzed in trans-heterozygous combinations. Mutations in beat Ic are not available, therefore, defined deficiencies were used. Embryos that lack one copy of isl, Lim3 and beat Ic display a significant number of TN fasciculation defects. As in individual mutants, the TMNp and LBD axons in several trans-heterozygous combinations fail to fasciculate, thus leading to bifurcation and/or aberrant axon outgrowth. Two chromosomal deficiencies removing beat Ic, beat Ic^Df and beat Ic^Df1 were used to demonstrate that other genes removed by an individual deficiency did not influence the TN phenotype. In addition, at least two different isl and Lim3 alleles were used to verify that the observed genetic interactions were not due to specific chromosomes or alleles. Another Beat family member, beat Ib, is also expressed in the CNS; however, a lack of 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 for these genes. However, to assess the specificity of these interactions among Ig domain-containing molecules, genetic interactions were examined between isl, Lim3 and the CAM Fas2, which contains five Ig-like domains. No fascicle defects were detected in various trans-heterozygous combinations. These results raise the possibility that in TN motoneurons Isl and Lim3 may specify axon target selection through the actions of specific IgSF CAM members (Certel, 2004).

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

Increasing the levels of Beat Ic through one copy of UAS-beat Ic and the elav-Gal4 driver significantly reduces the percentage of TN defects in isl mutants from 56% to 22%. In addition, the severity of TN adhesion defects decreased in the remaining 22% of affected hemisegments. Likewise, increasing Beat Ic levels in Lim3 mutants also 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 the unavailability of a Beat Ic antibody. Attempts were made to analyze beat Ic transcript accumulation in isl and Lim3 double mutants but it was not possible to achieve cellular resolution in the mutant ventral nerve cords. However, DNA-binding site pattern searches using previously described LIM-HD binding motifs indicate that there are significant clusters of LIM-HD sites surrounding and within the beat Ic locus. The Isl1 consensus site (CTAATG) is found 15 times in the chromosomal region encompassing the beat Ic locus; the Lhx3-binding site (AATTAATTA) is found nine times and 21 Isl 2.2 sites (YTAAGTG) have been identified. Although functional analyses will be needed to determine if these sites are necessary and/or sufficient for beat Ic expression, searching the entire genome with the Isl 2.2 site places the beat Ic locus seventh out of the first 10 identified. Furthermore, studies in C. elegans indicate that two LIM-HD genes, the Lmx-class gene lim-6 and the Lhx3-class gene, ceh-14, are required for the expression of at least four IgSF zig genes (Certel, 2004).

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

LIM-HD proteins participate in a number of unique complexes through protein-protein interactions mediated by their LIM domains. For example, LIM domains can interact with the widely expressed co-factor, NLI(Ldb1/CLIM-2) in mice or Chip in Drosophila. The NLI/CHIP proteins homodimerize and generate a bridge between two LIM-HD proteins, thereby leading to the formation of tetrameric complexes. Although this complex is functional in vivo, it has been found that other proteins also participate to generate further tissue specificity. In Drosophila, the newly identified Ssdp protein interacts with Chip to modify the activity of complexes comprising Chip and the LIM-HD protein Apterous in the wing. In vertebrates, the bHLH factors Ngn2 and NeuroM functionally interact with the NLI, Isl1 and Lhx3 complex to initiate motoneuron differentiation. These results suggest that the formation of further specialized combinations could be 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 POU protein UNC-86, physically interact to control specification. In the pituitary, the LIM domain of Lhx3 (P-Lim) specifically interacts with the Pit1 POU 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 experiments indicate that the re-specification of transverse motoneurons by the addition of Dfr does require functional Isl protein. Although, this result does not distinguish between the possibilities of direct interactions between these proteins or the binding of a common transcriptional target, it does indicate that a functioning 'LIM code' is required for the re-specification of the TN neurons (Certel, 2004).

A second finding of the Dfr misexpression studies is that the target selection of postmitotic neurons can be robustly re-specified. The Lim3B-Gal4 line was used to add Dfr to the LIM-only transverse motoneurons. This Gal4 line does not activate reporter construct expression until stage 14 -- a post-mitotic stage even for the late developing transverse motoneurons. At this stage, the TN motor axons have exited the CNS and are navigating the periphery, although the TN motor axon and LBD fascicle have not come into contact. Misexpressing Dfr even at this relatively late stage of TN motoneuron differentiation can clearly alter axon pathfinding, and in a significant percentage of hemisegments, TN motor axons actually appear to ectopically innervate ISNb muscle targets. This result shows that these motoneurons remain plastic, even after becoming postmitotic and further indicate that the LIM/POU code may be acting directly on genes involved in axon targeting (Certel, 2004).

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

A recent genomic analysis indicates the Drosophila immunoglobulin superfamily repertoire consists of about 150 proteins; in C. elegans, 80 immunoglobulin superfamily molecules are predicted. Members of the Beat family in Drosophila and the zig family in C. elegans are located in gene clusters, have restricted expression patterns and share the same domain architecture. Each protein is comprised exclusively of two Ig modules with either a transmembrane domain [beat Ic, beat Ib, beat IIa, beat VI (GPI), zig-1] or secreted signals (10 Beat genes: zig-2 to zig-8). The Beat family members that have been functionally characterized appear to control fasciculation through anti- and pro-adhesive properties. Furthermore, unlike many previously described CAMs, several members of these families are expressed in subsets of CNS neurons (Certel, 2004).

Islet coordinately regulates motor axon guidance and dendrite targeting through the Frazzled/DCC receptor

Motor neuron axon targeting in the periphery is correlated with the positions of motor neuron inputs in the CNS, but how these processes are coordinated to form a myotopic map remains poorly understood. This study shows that the LIM homeodomain factor Islet (Isl) controls targeting of both axons and dendrites in Drosophila motor neurons through regulation of the Frazzled (Fra)/DCC receptor. Isl is required for fra expression in ventrally projecting motor neurons, and isl and fra mutants have similar axon guidance defects. Single-cell labeling indicates that isl and fra are also required for dendrite targeting in a subset of motor neurons. Finally, overexpression of Fra rescues axon and dendrite targeting defects in isl mutants. These results indicate that Fra acts downstream of Isl in both the periphery and the CNS, demonstrating how a single regulatory relationship is used in multiple cellular compartments to coordinate neural circuit wiring (Santiago, 2017).

The RP3 motor neurons innervate the NetrinB-expressing muscles 6 and 7 and are enriched for fra mRNA during the late stages of embryonic development, and it was reported previously that, in the absence of fra or Netrin, there are significant defects in the innervation of muscles 6 and 7. This phenotype is also detected in the absence of hb9/exex or isl/tailup, two transcription factors expressed in RP3 as well as in other ventrally projecting motor neurons, suggesting that hb9 or isl may regulate fra. Interestingly, Hb9, Isl, and the LIM homeodomain factor Lim3 were all recently shown to bind directly to the fra locus in vivo, as determined by a genome-wide DNA adenine methyltransferase identification (DAM-ID) analysis performed in Drosophila embryos. However, DAM-ID results do not provide information about the functional significance of the detected binding events or about the cell types in which they occur. To determine whether Hb9, Isl, or Lim3 regulate the expression of fra in embryonic motor neurons, situ hybridization experiments were performed and fra mRNA expression was analyzed with single-cell resolution in embryos mutant for these factors. Only isl is required for fra expression in the RP3 motor neurons at stage 15, when RP axons have reached the ventral muscle field but their final targets have not been selected. 80% of RP3 neurons in abdominal segments A2-A7 in isl/+ embryos are positive for fra mRNA versus 38% in isl mutant embryos. A significant difference was also observed in fra mRNA levels in RP3 neurons between isl mutants and heterozygotes when quantifying pixel intensity from the fra in situ, whereas no difference was detected in the signal of the isl-H-tau-myc transgene. No change was detected in the number or position of RP3 neurons in isl mutants, consistent with previous data demonstrating that Isl is not required for the generation or survival of Drosophila motor neurons. Importantly, no requirement was found for either hb9 or lim3 in regulating fra mRNA expression in any RP motor neurons, demonstrating that isl's effect on fra is specific and could not have been predicted simply from similarities in loss of function phenotypes or from transcription factor binding data (Santiago, 2017).

Hb9 has been shown to be required for robo2expression in RP3. Interestingly, just as hb9 is not required for fra expression in RP neurons, isl is not required for robo2expression. A previous study reported that isl; hb9 double mutants have a stronger intersegmental nerve b (ISNb) phenotype than either single mutant, but muscle 6/7 innervation defects were not quantified. This study scored motor axon guidance defects in isl; hb9 double mutants and found that the double mutants display significantly more muscle 6/7 innervation defects than either single mutant. Similarly, embryos mutant for both robo2and fra have a stronger motor axon phenotype than either robo2or fra single mutants. Note that, because robo2, fra double mutants have severe defects in midline crossing, motor axon phenotypes should be interpreted with caution . These results show that Hb9 and Isl act in parallel to regulate distinct downstream programs in RP3 neurons, demonstrating how combinations of transcription factors result in specific cell surface receptor profiles and axon trajectories (Santiago, 2017).

To determine whether isl and fra act in the same genetic pathway during RP3 guidance, embryos mutant for both genes were examined. In isl-null mutants, 20% of hemisegments lack muscle 6/7 innervation, whereas fra-null mutants have a significantly stronger phenotype (34% of hemisegments). isl, fra double mutants do not have more muscle 6/7 innervation defects than fra single mutants, consistent with isl and fra acting in the same pathway. If fra acts downstream of Isl during motor axon targeting, then it was reasoned that restoring Fra expression in isl mutant neurons might rescue muscle 6/7 innervation. Indeed, it was found that pan-neural overexpression of Fra in isl mutants partially but significantly rescues these defects. The difference between genotypes was most noticeable when hemisegments were counted in which a growth cone stalls at the 6/7 cleft as well as those in which it fails to reach it. In isl mutants, a growth cone stalls at or fails to reach the 6/7 cleft in 27% of hemisegments compared with 15% of hemisegments in sibling mutants overexpressing Fra. The data was also analyzed by comparing the number of embryos with 6/7 innervation defects. In isl mutants, 0% of embryos have no 6/7 innervation defects in A2-A6, 44% have one defect, and 56% have two or more defects. In contrast, in isl mutants overexpressing Fra, 29% of embryos have no innervation defects, 29% have one defect, and 41% have two or more defects. The incomplete rescue could be due to differences in the timing or levels of GAL4/UAS-mediated expression of Fra compared with its endogenous regulation or could indicate that Isl regulates additional downstream effectors important in this process. Nevertheless, these data strongly suggest that Fra is an essential downstream effector of Isl during the guidance of the RP3 axon to its target muscles (Santiago, 2017).

To further investigate the relationship between isl and fra, whether ectopic expression of isl is sufficient to induce fra expression was tested. These experiments used the apterous (ap) neurons. The axons from this subset of interneurons form a single fascicle on either side of the midline that are labeled by ap-Gal4. The ap neurons express low levels of fra, do not express isl, and do not cross the midline. Fra overexpression causes ectopic midline crossing of ap axons. Overexpression of Isl with ap-Gal4 produced high levels of midline crossing, phenocopying the effect of Fra overexpression. In stage 17 control embryos, ap axons cross the midline in 12% of segments, whereas in embryos overexpressing UAS-Isl with ap-Gal4, ap axons cross the midline in 60% of segments. This phenotype is dose-dependent because embryos with two copies of an UAS-Isl insertion display significantly more ectopic midline crossing than embryos with one insert (Santiago, 2017).

To determine whether Isl overexpression results in fra induction, the expression of fra mRNA in situ was examined in ap neurons. In stage 15 wild-type embryos, a low percentage of ap neurons express fra (25% of ventral ap clusters were fra+). In contrast, in embryos overexpressing isl from two UAS-Isl inserts, 37% of the ventral ap clusters were fra+. To test whether the ectopic crossing phenotype depends on fra function, Isl was overexpressed in embryos homozygous for a null allele of fra. Strikingly, removing fra completely suppresses the crossing phenotype, indicating that fra is required for Isl to produce its gain-of-function effect. Although it cannot be ruled out that Isl affects the expression of other genes in the Fra pathway to cause midline crossing, these results demonstrate that ectopically expressing Isl causes an increase in fra expression and a fra-dependent axon guidance phenotype and suggest that the functional relationship between isl and fra may be used in multiple contexts (Santiago, 2017).

Fra mutants have defects in RP axon midline crossing, as shown by retrograde labeling of single motor neurons. In addition, Netrin-Fra signaling controls the medio-lateral position of dendrites in several groups of motor neurons. Therefore, it was asked whether isl regulates midline crossing or RP3 dendrite development through fra. A genetic strategy was used to label single motor neurons by mosaic expression of a membrane-tethered GFP transgene under the control of lim3b-GAL4, which labels RP motor neurons, sensory neurons, and several other motor and interneuron populations. RP3 neurons were identified by the stereotyped position of the RP3 cell body and by the targeting of its axon to muscles 6 and 7. Because of the axon targeting defects observed in isl and fra mutants, cell body position was used to identify RP3 neurons in mutants. By this approach, significant midline crossing defects were detected in RP3 axons in fra mutants. Surprisingly, however, no defects were observed in RP axon midline crossing in isl mutants (Santiago, 2017).

Isl and fra expression both initiate earlier than stage 13, the time at which RP axons cross the midline. Therefore, whether isl is required for fra expression was examined during the early stages of commissural axon guidance. Interestingly, isl was not required for fra expression at stage 13 in any of the ventrally projecting RPs. In contrast, in stage 15 isl mutant embryos from the same collection, a decrease in was observed fra expression in RP1 and RP3. The temporal pattern of fra expression in RP motor neurons is dynamic, so that a larger proportion of RP1 and RP3 neurons express fra mRNA during late embryogenesis than during the stages of midline crossing. A requirement was detected for isl in regulating fra in RP1 and RP3 as early as stage 14, when the RP motor axons have exited the CNS. Taken together, these results suggest that isl is not essential for early fra expression but required for fra expression during the late stages of motor neuron differentiation. The stages at which a requirement was detected for isl in regulating fra correspond to when RP3 axons are exploring their ventral muscle targets, consistent with a model in which Isl instructs the final stages of RP3 axon targeting through Fra (Santiago, 2017).

Another essential feature of Drosophila larval motor neurons that is established late in embryogenesis is the morphogenesis and targeting of their dendrites in the ventral nerve cord. Motor neuron dendrites begin to form as extensions off the primary neurite at stage 15, a stage when a requirement is detected for isl in regulating fra. By early stage 17 (15 hr after egg laying, AEL), RP3 has assumed its stereotyped morphology, consisting of a small ipsilateral projection extending from the soma and a large dendritic arbor forming off the contralateral primary neurite (Santiago, 2017).

The FLP-out genetic labeling strategy was used to visualize individual late-stage RP motor neurons and analyze their dendrites. Focus was placed on the large contralateral arbor of the RP motor neurons that spans one side of the nerve cord in wild-type embryos and forms branches that extend into several medio-lateral zones. Analyses using isl-tau-myc and lim3a-tau-myc transgenes confirmed that the RP cell bodies retain their stereotyped positions in isl mutants and that the relative dorsal-ventral positions of RPs 1/4, 3, and 5 are preserved, allowing identification of distinct classes of RP motor neurons (Santiago, 2017).

Most RP3 neurons in late-stage isl/+ embryos neurons form contralateral dendritic arbors that send projections into the zone between the medial FasII+ axon pathways and the intermediate FasII+ pathways, hereafter referred to as the 'intermediate zone,' consistent with previously published images of RP3 neurons from wild-type embryos. Interestingly, the dendritic morphology of RP3 was distinct from that of a related neuron, RP5, that also expresses Isl and Lim3b-Gal4 and that can be unambiguously identified in both wild-type and mutant embryos because its cell body is found in a more ventral position than the other RP neurons. In wild-type embryos, the RP5 axon targets muscles 12 and 13 (VL1 and VL2) as well as other ventral muscles. Most RP5 neurons in isl/+ embryos exclusively target their dendrites to the lateral zone of the neuropile. Furthermore, the difference observed in the dendritic targeting of RP3 and RP5 neurons correlates with a difference in fra expression. Although fra expression in RP3 and RP5 neurons in control embryos is comparable when RP axons are crossing the midline, by stage 15, significantly fewer RP5 than RP3 neurons express fra. Interestingly, isl is not required for the low levels of fra expression in late-stage RP5 neurons, in contrast to its role in promoting high levels of fra in late-stage RP3 neurons (Santiago, 2017).

Finally, endogenous Netrin expression was monitored in late-stage nerve cords using a Myc-tagged NetB knockin allele, and significant enrichment of Netrin protein was detected in the area between the intermediate and medial FasII+ axon bundles. This area corresponds to the zone where contralateral dendritic projections from RP3 neurons were detected, suggesting that high levels of Fra in RP3 may instruct the formation of dendritic arbors in this region in response to Netrin (Santiago, 2017).

RP motor neuron dendrites were examined in isl mutant embryos to determine whether Isl regulates dendritic position or morphogenesis through Fra or other effectors. No significant difference in the morphology or medio-lateral position of RP5 dendrites was observed between heterozygous and mutant embryos. In striking contrast, many RP3 neurons in isl mutants fail to extend contralateral dendrites into the intermediate zone. Instead, the dendrites of these RP3 neurons remain fasciculated with the intermediate FasII+ axon pathways and do not send medial extensions toward the midline. To more quantitatively measure medio-lateral position and to address the possibility that defects in targeting are secondary to defects in outgrowth, RP3 neurons were traced using Imaris software and total contralateral dendrite lengths and the total number of dendrite tips were measured. Total length of contralateral dendrites was also measured in the intermediate zone of the neuropile, defined as the area between the medial FasII+ and the intermediate FasII+ axon pathways. Although RP3 neurons displayed increased variability in the size of their dendritic arbors in isl mutants, there was no significant difference in the total length or tip number of RP3 dendrites between isl mutants and heterozygotes, suggesting that targeting defects in isl mutants are not likely due to reduced outgrowth. However, the ratio of RP3 dendrites in the intermediate zone over total RP3 dendrite length was significantly reduced in isl mutants, confirming that isl mutant RP3 dendrites are shifted laterally relative to controls (Santiago, 2017).

The dendrites of RP3 neurons were examined in fra/+ and fra mutant embryos. As with isl mutants, cell body position was used to identify RP3 neurons, and neurons with ambiguous positions were excluded. In fra mutant RP3 neurons whose axons fail to cross the midline, a single dendritic arbor forms off the ipsilateral primary neurite, and this arbor was traced. A significant lateral shift was observed in the position of RP3 dendrites in fra mutants both by scoring for the presence of dendrites in the intermediate zone and by quantitative analysis of the dendrites of traced neurons. The lateral shift in fra mutants was more pronounced than in isl mutants, consistent with the observation that some RP3 neurons retain fra expression in the absence of isl. Of note, the lateral shift phenotype did not correlate with whether the RP3 axon had crossed the midline because it was detected at similar frequencies in both contralateral and ipsilateral arbors. Curiously, several RP3 contralateral dendritic arbors appeared reduced in size in fra mutants, whereas this phenotype was not seen in control embryos. However, as in isl mutants, there was no significant change in the total dendrite length or tip number in fra mutants compared with their sibling controls, although there was increased variability in the sizes of dendritic arbors in the mutants. These findings are consistent with previous reports that Netrin-Fra signaling does not play a major role in regulating the outgrowth of motor neuron dendrites in the nerve cord (Santiago, 2017).

A single-cell labeling method allows precise description of the axon targeting defects in isl and fra mutants and determine whether they correlate with defects in dendrite position. Axon and dendrite targeting occur at approximately the same developmental stage, and there is no evidence that one process depends on the other. Importantly, previous studies using retrograde labeling of motor neurons in mutant embryos were not able to address this question because they relied upon motor axons reaching the correct muscles to be visualized (Santiago, 2017).

To determine whether defects in dendrite position correlate with defects in axon targeting, both phenotypes were scored in single labeled RP3 neurons in embryos with muscles fully preserved following dissection. All of the RP3 axons that could be scored in isl heterozygous embryos innervated the muscle 6/7 cleft . In contrast, 18 of 26 isl mutant RP3 axons innervated muscles 6/7, and eight stalled at the 6/7 cleft or earlier along RP3's trajectory or bypassed the choice point. In fra mutant embryos, 10 of 22 RP3 neurons failed to innervate the muscle 6/7 cleft and stalled at or bypassed the choice point. This phenotype is stronger than the frequency at which a complete loss of muscle 6/7 innervation in isl or fra mutants was detected by scoring with anti-FasII. To determine whether this enhancement was due to the heat shock (H.S.) step that is required for genetic labeling, defects were scored using anti-FasII in embryos heat-shocked for either 5 min or 1 hr; it was found that the 1-hr H.S. mildly enhances muscle innervation defects in isl mutants (to 30.4%) whereas a 5-min H.S. does not (to 24.7%, data not shown). Importantly, the two H.S. protocols did not result in any difference in the frequency of dendrite targeting defects observed in isl mutants because 7 of 17 RP3 dendrites in isl mutants are shifted laterally in embryos treated with 1-hr H.S, and 9 of 16 dendrites are shifted after 5-min H.S (Santiago, 2017).

Surprisingly, no correlation was detected between axon and dendrite defects in isl mutants. Although 5 of 26 RP3 neurons displayed defects in both axons and dendrites in isl mutants, 12 of 26 neurons showed defects in one process but not the other). A similar analysis in fra mutants revealed that 8 of 22 RP3 neurons displayed defects in both muscle 6/7 innervation and dendrite position, whereas 8 of 22 displayed normal targeting in one process but not the other. These data suggest that axon and dendrite targeting can occur independently within an individual RP3 neuron and that the central targeting defects observed in isl mutants are not likely to be secondary to defects in muscle innervation (Santiago, 2017).

It was next asked whether isl and fra regulate dendrite development in other classes of motor neurons. RP1 and RP4 also express isl, fra, and lim3b-Gal4. A requirement was detected for isl in regulating fra expression in RP1, but not in RP4, at stage 15. Interestingly, most RP1 neurons, like RP3 neurons, retain high levels of fra at this stage, whereas few RP4 neurons express fra in late-stage control embryos. Previous descriptions of RP1 and RP4 neurons indicate that they form contralateral dendritic arbors of distinct morphologies; RP1's dendritic arbor is taller and found more medially. However, because the axons of RP1 and RP4 target adjacent muscles external to muscles 6 and 13, and their cell bodies are found close to the midline at a similar dorsal-ventral position, they could not nr unambiguously distinguished in single-cell labeling experiments. Nevertheless, when RP1 and RP4 neurons were scored together, a significant lateral shift was observed in the position of RP1 and RP4 dendrites in isl mutants compared with heterozygous siblings: 3 of 22 RP1 and RP4 dendritic arbors were excluded from the intermediate zone in isl heterozygous embryos (14%) compared with 16 of 22 in mutant embryos. A similar phenotype was detected in RP1 and RP4 dendrites in fra mutants. Specifically, 6 of 24 RP1 and RP4 dendritic arbors were excluded from the intermediate zone in heterozygotes (25%) compared with 17/19 in fra mutants. Although additional work will be necessary to determine whether the defects in dendrite position that were detect in RP1 and RP4 neurons in isl mutants correlate with changes in fra expression, these data demonstrate that Isl is required for high levels of fra expression in at least two classes of motor neurons (RP1 and RP3), both of which require isl and fra for dendritic targeting (Santiago, 2017).

To directly test whether isl regulates RP3 dendrite position through its effect on fra expression, a UAS-HA-Fra transgene was overexpressed using lim3b-GAL4 in isl mutants and the hsFLP technique was used to sparsely label RP motor neurons. Strikingly, in isl mutants overexpressing Fra, 0 of 21 RP3 contralateral dendritic arbors were excluded from the intermediate zone compared with 8 of 22 (36%) in sibling mutants lacking the UAS-Fra transgene. To quantitatively measure dendrite position, traces of RP3 dendrites were obtained. A robust rescue of the lateral shift phenotype was detected in isl mutants, as measured by the length of dendrites in the intermediate zone over the total dendrite length. Indeed, the ratio of dendrites in the intermediate zone in rescued mutants was higher than in heterozygous controls, perhaps reflecting a gain-of-function effect caused by artificially high levels of Fra from transgenic overexpression. Importantly, Fra overexpression did not have any effect on total dendritic arbor lengths or tip numbers, strongly arguing that the rescue that was observe is not caused by an increase in the total size of the arbors. Although it cannot be ruled out that Isl regulates dendrite position in part through additional effectors, the observation that cell-type-specific overexpression of Fra in isl mutants rescues dendrite targeting provides compelling support for the model that fra acts downstream of isl to control RP3 dendrite morphogenesis. Together with the demonstration that isl directs RP3 motor axon targeting through the regulation of fra, it is concluded that isl coordinately regulates the targeting of axons in the periphery and of dendrites in the CNS through a common downstream effector (Santiago, 2017).

In the vertebrate spinal cord, the position of motor neuron cell bodies correlates with the targeting of their axons in the periphery (Catela, 2015). This myotopic map may be established through the action of transcription factors that coordinately control cell migration and axon guidance. In particular, Lhx1 and Isl1 are expressed in limb-innervating lateral motor column (LMC) motor neurons and regulate the trajectory of their axons as well as the medio-lateral settling position of their cell bodies . Lhx1 and Isl1 regulate axon guidance through EphA4 and EphB receptors, respectively, and a recent study suggests that Lhx1 regulates cell body position through a distinct effector, the Reelin signaling protein Dab-1 (Santiago, 2017).

In Drosophila, unlike in vertebrates, the position of motor neuron cell bodies does not necessarily correlate with the targeting of their axons in the periphery because neurons that innervate adjacent muscles can be found far apart within a segment. Instead, recent studies have shown that both the larval and the adult Drosophila nervous systems use a myotopic map in which the position of motor neuron dendrites, rather than their cell bodies, correlates with the position of their target muscles (Brierley, 2009, Mauss, 2009). This may be a conserved feature of motor systems across phyla because the dendritic patterning of at least four motor neuron pools in the spinal cord correlates with muscle target identity in the mouse (Santiago, 2017).

Slit-Robo, Netrin-Fra, and Sema-Plexin signaling have been shown to control motor neuron dendrite targeting in Drosophila, and rescue experiments suggest that these guidance receptors act cell-autonomously in this process (Brierley, 2009, Mauss, 2009, Syed, 2016). In addition, the initial targeting of motor neuron dendrites in the embryo is largely unaffected by manipulations that affect the position or the activity of pre-synaptic axons or the presence of muscles, suggesting that this process is likely under the control of cell-autonomous factors, although these remain unidentified (Santiago, 2017).

This study has addressed several key questions about how motor neuron dendrite targeting is specified in Drosophila. First, it was shown that fra expression in two classes of motor neurons (RP3 and RP5) correlates with the medio-lateral position of their dendrites. Previous studies suggested that different classes of motor neurons express different levels of guidance receptors to direct the position of their dendrites, but this has not been demonstrated. Second, Isl, which was previously shown to regulate axon targeting in a subset-specific way, was also shown to regulate dendrite targeting. Third, it was found that Isl regulates both processes through fra. Surprisingly, no correlation was found between axon and dendrite phenotypes in isl mutants. The absence of a correlation suggests that the dendrite positioning defects are not secondary to defects in target selection, consistent with a previous study in which the general patterning of motor neuron dendrites was not disrupted in muscle-less embryos. However, additional experiments that disrupt axon targeting and monitor the medio-lateral position of dendrites will be necessary to confirm that the two occur independently (Santiago, 2017).

Future work will also be necessary to identify additional transcription factors that specify motor neuron dendrite development. A role has been identified for Hb9 in regulating robo2and robo3 expression, but it is not known whether these receptors regulate motor neuron dendrite development. No change was detected in robo1 mRNA levels in RP3 neurons in either hb9 or isl mutants. Robo signaling could be regulated post-transcriptionally. Comm is required for midline crossing of motor neuron dendrites and may endogenously regulate their medio-lateral position. The temporal pattern of comm expression does not support a role in dendrite targeting, however, because comm is not expressed in RP motor neurons at late stages of embryogenesis (Santiago, 2017).

The functional consequences of dendrite targeting defects remain to be explored. It is likely that shifting the position of motor neuron dendrites alters their connectivity, but testing this hypothesis will require identifying the pre-synaptic neurons that impinge on the RPs during locomotive behavior. Forcing a shift in the position of dendrites of dorsally projecting motor neurons does not abolish their connectivity with known pre-synaptic partners but does change the number of contacts established. In mice, the ETS factor Pea3/Etv4 is required for the dendritic patterning of a subset of motor neurons, and electrophysiological recordings reveal changes in connectivity in Pea3 mutant spinal cords. It will be of high interest to investigate whether analogous defects are detected in isl or fra mutant embryos (Santiago, 2017).

Drosophila Isl was initially described as a subset-specific regulator of axon guidance. More recently, Wolfram (2012) demonstrated that Isl also acts instructively to establish the electrophysiological properties of RP motor neurons through repression of the potassium ion channel Shaker. The curreng data show that, in addition to regulating the axonal trajectory and the electrophysiological properties of the RP3 neuron, Isl also establishes its dendritic position. Terminal selectors have been defined as transcription factors that coordinately regulate gene programs conferring multiple aspects of a neuron's identity, including its neurotransmitter phenotype, ion channel profile, and connectivity. Unlike the early-acting factors that function transiently to specify cell fate, terminal selectors are expressed throughout the life of an animal and are required for the maintenance of neural identity. Although there are several examples of transcription factors that act this way, it remains unclear how widespread a phenomenon it is. Does Isl fit the criteria for a terminal selector? Isl is not required for all aspects of RP3 identity because RP neurons retain expression of other motor neuron transcription factors in isl mutants, and their axons exit the nerve cord. Future work will be necessary to determine whether Isl is required throughout larval life for the maintenance of RP3's physiological and morphological features and to what extent Isl coordinately establishes multiple features of RP neuron identity (Santiago, 2017).

Co-expressed transcription factors could act synergistically to regulate specific downstream programs, in parallel through completely distinct effectors, or by some combination of the two mechanisms. Indeed, examples of all of these scenarios have been described. Both in vitro and in vivo studies demonstrate that, in vertebrate spinal motor neurons, Isl1 forms a complex with Lhx3 and that the Isl1-Lhx3 complex binds to and regulates different genes than Lhx3 alone or than a complex composed of Isl1 and Phox2b, a factor expressed in hindbrain motor neurons. In a subset of spinal commissural neurons, Lhx2 and Lhx9 act in parallel to promote midline crossing through upregulation of Rig-1/Robo3. In Drosophila dorsally projecting motor neurons, Eve, Zfh1, and Grain act in parallel to promote the expression of unc5, beat1a, and fas2, although Eve also regulates additional targets important for axon guidance that are not shared by Zfh1 or Grain (Santiago, 2017).

This study shows that Isl and Hb9 act in parallel through at least two distinct effectors and proposes that they regulate their targets by different mechanisms. Hb9 likely indirectly promotes robo2 expression by repressing one or multiple intermediate targets because its conserved Engrailed homology repressor domain is required for its function in motor axon guidance and for robo2 regulation. In vertebrate motor neurons, Isl1 forms a complex with Lhx3 to directly activate several of its known targets. A recent genome-wide DAM-ID analysis found that Isl binds to multiple regions within and near the fra locus in Drosophila embryos, suggesting that it may directly activate fra. The finding that lim3 is not required for fra expression in RP motor neurons, together with evidence that Isl can alter the electrical properties of muscle cells independently of Lim3, suggest that Drosophila Isl does not need to form a complex with Lim3 for all of its functions. Future research will be necessary to detect Isl binding events in embryonic motor neurons, although these experiments are challenging when binding occurs transiently or in a small number of cells. Interestingly, overexpression of Isl using ap-Gal4 or hb9-Gal4 induces fra only in certain subsets of these neurons, consistent with a model in which Isl binds to the fra locus in a cell-type-specific manner. The generation of many large-scale datasets for transcription factor binding sites presents the field with the task of reconciling these data with clearly defined genetic relationships during specific biological processes. This study and others have initiated this effort, but it will be important to investigate the functional significance of other putative transcription factor-effector relationships to achieve a better understanding of how transcriptional regulators control cell fate (Santiago, 2017).

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 a 2-amino acid spacer. Some proteins are made up of only LIM domains, while others contain a variety of different functional domains. LIM proteins form a diverse group that includes transcription factors and 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. LIM homeodomain proteins are involved in the control of cell lineage determination and the regulation of differentiation, and LIM-only proteins may have similar roles. LIM-only proteins are also implicated in the control of cell proliferation since several genes encoding such proteins are associated with oncogenic chromosome translocations. In analyzing sequence relationships among various LIM domains it is suggested that they may be arranged into 5 groups that appear to correlate with the structural and functional 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: 2 January 2023

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