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

Using nuclear extracts derived from C. elegans embryos, the POU homeodomain protein (UNC-86) alone is able to activate transcription of the mec-3 promoter in vitro, while the LIM homeodomain protein MEC-3 fails to bind DNA or activate transcription on its own. However, in the presence of both UNC-86 and MEC-3, cooperative binding to the mec-3 promoter and synergistic activation of transcription in vitro is observed. Protein-protein interaction assays reveal that UNC-86 can bind directly to MEC-3, and in vitro transcription studies indicate that both proteins contain a functional activation domain. Thus, formation of a heteromeric complex containing two activation domains results in a highly potent activator. These studies provide direct functional evidence for coordinated transcriptional activation by two C. elegans DNA binding proteins that have been defined genetically as regulators of gene expression during embryogenesis (Lichtsteiner, 1994).

The POU homeobox gene unc-86 specifies many neuroblast and neural fates in the developing C. elegans nervous system. Genes regulated by unc-86 are mostly unknown. A genetic strategy was devised for the identification of downstream pathways regulated by unc-86. UNC-86 transcription activity was activated by inserting the VP16 activation domain into an unc-86 genomic clone that bears all regulatory sequences necessary for normal expression in C. elegans. unc-86/VP16 complements unc-86 mutations in the specification of neuroblast and neural cell fates, but displays novel genetic activities: it can suppress non-null mutations in the downstream genes mec-3 and mec-7 (coding for a mecanosensory neuron-specific ß-tubulin) that are both necessary for mechanosensory neuron differentiation and function. Note: for additional information on tubulin functions, see ß1 tubulin. These data suggest that UNC-86/VP16 increases the expression of mec-3 and mec-7 to compensate for the decreased activities of mutant MEC-3 or MEC-7 proteins. The suppression of mutations in downstream genes by an activated upstream transcription factor should be a general strategy for the identification of genes in transcriptional cascades. unc-86/VP16 also causes neural migration and pathfinding defects and novel behavioral defects. Thus, increased or unregulated expression of genes downstream of unc-86 can confer novel neural phenotypes suggestive of roles for unc-86-regulated genes in neural pathfinding and function. Genetic suppression of these unc-86/VP16 phenotypes may identify the unc-86 downstream genes that mediate these events in neurogenesis (Sze, 1997).

The presence of the LIM domain of Isl-1 inhibits binding of the homeodomain to its DNA target. This in vitro inhibition can be released either by denaturation/renaturation of the protein or by truncation of the LIM domains. A similar inhibition is observed in vivo using reporter constructs. LIM domains in a chimeric protein can inhibit binding of the Ultrabithorax homeodomain to its target. The ability of LIM domains to inhibit DNA binding by the homeodomain provides a possible basis for negative regulation of LIM-homeodomain proteins in vivo (Sanchez-Garcia, 1993).

Described here is the cloning and expression pattern of Ci-isl, a homolog of vertebrate islet genes, in the ascidian Ciona intestinalis (phylum Urochordata). Early in development, Ci-isl expression occurs in the primordia of palps and brain vesicle; then, in the tailbud embryo, it is transiently extended to the notochord cells. At larva stage, the expression is down-regulated in the notochord, and it persists predominantly in the compartments of the nervous system. These observations indicate that also in invertebrates, islet genes show an expression pattern during differentiation of the nervous system (Giuliano, 1998).

The vertebrate embryonic hindbrain is segmented into rhombomeres. Gene expression studies suggest that amphioxus, the closest invertebrate relative of vertebrates, has a hindbrain homolog. However, this region is not overtly segmented in amphioxus, raising the question of how hindbrain segmentation arose in chordate evolution. Vertebrate hindbrain segmentation includes the patterning of cranial motor neurons, which can be identified by their expression of the LIM-homeodomain transcription factor islet1. To learn if the amphioxus hindbrain homolog is cryptically segmented, an amphioxus gene closely related to islet1 was cloned, and named islet. Amphioxus islet expression includes a domain of segmentally arranged cells in the ventral hindbrain homolog. It is hypothesized that these cells are developing motor neurons and reveal a form of hindbrain segmentation in amphioxus. Hence, vertebrate rhombomeres may derive from a cryptically segmented brain present in the amphioxus/vertebrate ancestor. Other islet expression domains provide evidence for amphioxus homologs of the pineal gland, adenohypophysis, and endocrine pancreas. Surprisingly, homologs of vertebrate islet1-expressing spinal motor neurons and Rohon-Beard sensory neurons appear to be absent (Jackman, 2000).

Sensory neuron fates are distinguished by a transcriptional switch that regulates dendrite branch stabilization

Sensory neurons adopt distinct morphologies and functional modalities to mediate responses to specific stimuli. Transcription factors and their downstream effectors orchestrate this outcome but are incompletely defined. This study shows that different classes of mechanosensory neurons in C. elegans are distinguished by the combined action of the transcription factors LIM-type homeodomain protein MEC-3, bHLH PAS domain protein AHR-1, and Zn finger/homeodomain factor ZAG-1. Low levels of MEC-3 specify the elaborate branching pattern of PVD nociceptors, whereas high MEC-3 is correlated with the simple morphology of AVM and PVM touch neurons. AHR-1 specifies AVM touch neuron fate by elevating MEC-3 while simultaneously blocking expression of nociceptive genes such as the MEC-3 target, the claudin-like membrane protein HPO-30, that promotes the complex dendritic branching pattern of PVD. ZAG-1 exercises a parallel role to prevent PVM from adopting the PVD fate. The conserved dendritic branching function of the Drosophila AHR-1 homolog, Spineless, argues for similar pathways in mammals (Smith, 2013).

Sensory neurons display a wide range of morphological motifs and functional modalities that serve to transduce diverse types of external stimuli into specific physiological responses. Transcription factors define both the identity and number of each type of sensory neuron and thus are critical determinants of organismal behavior. The downstream pathways that distinguish the architectural and functional properties of different sensory neuron classes are largely unknown, however. This study shows that the conserved transcription factors MEC-3, AHR-1 and ZAG-1, function together to define distinct sensory neuron fates in C. elegans and identify downstream targets that are necessary for these roles (Smith, 2013).

The MEC-3 LIM homeodomain protein is expressed in both touch receptor neurons (TRNs) and in PVD but is responsible for distinctly different sets of characteristics displayed by these separate classes of mechanosensory neurons. In PVD neurons, MEC-3 promotes the creation of a highly branched dendritic arbor and nociceptive responses to harsh stimuli, whereas in the TRNs, MEC-3 is necessary for light touch sensitivity and for the adoption of a simple, unbranched morphology. Genetic ablation of mec-3 or its upstream regulator, the POU domain protein UNC-86, disrupts the function and morphological differentiation of both of these types of mechanosensory neurons. How are these different MEC-3-dependent traits produced? The results suggest that low levels of MEC-3 are sufficient to specify the PVD fate, whereas elevated MEC-3 drives TRN differentiation. The existence of this threshold effect is also supported by the finding that overexpression of MEC-3 induces TRN-specific gene expression in the PVD-like FLP neuron. This simple model is not sufficient, however, to explain why PVD nociceptor genes, which are turned on by low levels of MEC-3, are actually repressed in the TRNs as MEC-3 expression is elevated. The current findings now provide a mechanism for this effect. In the light touch AVM neuron, AHR-1 elevates MEC-3 expression while simultaneously blocking downstream MEC-3 targets that drive PVD branching and nociceptor function. It is suggested that ZAG-1 may exercise a similar role in PVM. This mechanism is robust because each of these TRNs is effectively transformed into a functional PVD-like neuron when either ahr-1 or zag-1 is genetically eliminated. Thus, this work has revealed the logic of alternative genetic regulatory pathways in which a single type of transcription factor (e.g., MEC-3) can specify the differentiation of two distinct classes of mechanosensory neurons. A related mechanism accounts in part for the dose-dependent effects of the homeodomain transcription factor Cut on the branching complexity of larval sensory neurons in Drosophila. The transcription factor Knot/Collier is selectively deployed in Type IV da neurons to antagonize expression of Cut targets that produce the dendritic spikes that are characteristic of Type III da neurons. In this case, however, Knot does not regulate Cut expression but functions in a parallel pathway. The finding that the Zinc-finger transcription factor ZAG-1 is required to prevent the PVM touch neuron from adopting a PVD nociceptor fate mirrors the recent observation that genetic ablation of the mammalian ZAG-1 homolog Zfhx1b (Sip1, Zeb2) results in cortical interneurons adopting the fate of striatal GABAerigic cells (McKinsey, 2013). The current results are suggestive of a potentially complex regulatory mechanism in which AHR-1 and ZAG-1 inhibit expression of nociceptor genes (e.g., hpo-30) whereas MEC-3 activates transcription of these targets. Additional upstream regulators of mec-3, UNC- 86, and ALR-1, are also likely involved in this pathway (Smith, 2013).

Although transcription factors are well-established determinants of sensory neuron fate, the downstream pathways that they regulate are largely unknown. As a solution to this problem for MEC-3, a cell-specific profiling strategy was used to detect mec-3-regulated transcripts in the PVD neuron. A combination of RNAi and mutant analysis was used to identify the subset of targets that affect PVD branching morphogenesis. Additional experiments with one of these hits, the claudin-like protein HPO-30, revealed a key role in the generation of PVD branches. It is noted that HPO-30 is expressed in the FLP neuron, where it also mediates the higher order branching morphology shared by FLP and PVD. Time-lapse imaging has revealed that PVD lateral or 2 branches may adopt either of two different modes of outgrowth along the inside surface of the epidermis: (1) fasciculation with existing motor neuron commissures or (2) independent extension as noncommissural or 'pioneer' dendrites. The results show that the principle role of HPO-30 is to stabilize pioneer 2 branches and, thus, that additional unknown factors may drive fasciculation with motor neuron commissures. Because claudins serve as key constituents of junctions between adjacent cells, it seems likely that HPO-30 functions in this case to link growing 2 dendrites with the nematode epidermis. It is noted that an additional membrane component, the LRR protein DMA-1, displays a mutant PVD branching phenotype strongly resembling that of Hpo-30 and therefore could also function in this pathway. The intimate association of topical sensory arbors with the skin and the broad conservation of junctional proteins across species point to the likelihood that homologs of HPO-30/Claudin and similar components could be widely utilized to pattern sensory neuron morphogenesis (Smith, 2013).

ahr-1 encodes a member of the bHLH-PAS family of transcription factors and is the nematode homolog of the aryl hydrocarbon receptor (AHR) protein. In mammals, AHR is activated by the xenobiotic compound dioxin to trigger a wide range of pathological effects. Invertebrate AHR proteins are not activated by dioxin, which suggests that this toxin-binding function represents an evolutionary adaptation unique to vertebrates. An ancestral role for AHR is suggested by AHR mutants in C. elegans and Drosophila that display distinct developmental defects in which a given cell type or tissue adopts an alternative fate. For example, stochastic expression of the Drosophila AHR homolog, Spineless, promotes the adoption of one specific photoreceptor sensory neuron identity at the expense of another (Smith, 2013).

The current results parallel those findings with the demonstration that AHR-1 function is required in C. elegans to distinguish between alternative types of mechanosensory neurons; in ahr-1 mutants, the unbranched light touch neuron, AVM, is transformed into a functional homolog of the highly branched PVD nociceptor. This role for ahr-1 in C. elegans is particularly notable because the AHR-1 homolog, Spineless, also regulates branching complexity in Drosophila. In spineless (Ss) mutants, Class I and II sensory neurons, which normally display simple branching patterns, adopt more complex dendritic arbors. This phenotype resembles the current finding in C. elegans that the simple morphology of the AVM neuron is transformed into the highly branched architecture of the PVD nociceptor in ahr-1 mutants. Ss mutants in Drosophila also show the opposite phenotype of more complex class III and class IV da neurons assuming simpler branching patterns, which could therefore reflect an additional role for spineless in this context of promoting the creation of dendritic branches. On the basis of these results, it is suggested that the striking conservation of the shared role of AHR homologs in regulating sensory neuron fate and branching complexity in nematodes and insects argues that this function is evolutionarily ancient and, thus, that the downstream effectors that have been identified in C. elegans may also pattern the dendritic architecture of vertebrate sensory neurons (Smith, 2013).

A LIM-homeobox gene is required for differentiation of Wnt-expressing cells at the posterior end of the planarian body

Planarians have high regenerative ability, which is dependent on pluripotent adult somatic stem cells called neoblasts. Recently, canonical Wnt/β-catenin signaling was shown to be required for posterior specification, and Hedgehog signaling was shown to control anterior-posterior polarity via activation of the Djwnt1/P-1 gene at the posterior end of planarians. Thus, various signaling molecules play an important role in planarian stem cell regulation. However, the molecular mechanisms directly involved in stem cell differentiation have remained unclear. This study demonstrates that one of the planarian LIM-homeobox genes, Djislet, is required for the differentiation of Djwnt1/P-1-expressing cells from stem cells at the posterior end. RNA interference (RNAi)-treated planarians of Djislet [Djislet(RNAi)] show a tail-less phenotype. Thus, it is speculated that Djislet might be involved in activation of the Wnt signaling pathway in the posterior blastema. When the expression pattern of Djwnt1/P-1 was carefully examined by quantitative real-time PCR during posterior regeneration, two phases of Djwnt1/P-1 expression were found: the first phase was detected in the differentiated cells in the old tissue in the early stage of regeneration and then a second phase was observed in the cells derived from stem cells in the posterior blastema. Interestingly, Djislet is expressed in stem cell-derived DjPiwiA- and Djwnt1/P-1-expressing cells, and Djislet(RNAi) only perturbed the second phase. Thus, it is proposed that Djislet might act to trigger the differentiation of cells expressing Djwnt1/P-1 from stem cells (Hayashi, 2011).

Transcriptional regulation of islet homologs: dissection of vertebrate islet cis-regulatory region

Islet-1 (Isl1) is a member of the Isl1 family of LIM-homeodomain transcription factors (LIM-HD) that is expressed in a defined subset of motor and sensory neurons during vertebrate embryogenesis. To investigate how this specific expression of isl1 is regulated, enhancers of the isl1 gene were sought that are conserved in vertebrate evolution. Initially, two enhancer elements, CREST1 (conserved regulatory element for isleti) and CREST2, were identified downstream of the isl1 locus in the genomes of fugu, chick, mouse, and human by BLAST searching for highly similar elements to those originally identified as motor and sensory neuron-specific enhancers in the zebrafish genome. The combined action of these elements is sufficient for completely recapitulating the subtype-specific expression of the isl1 gene in motor neurons of the mouse spinal cord. Furthermore, by direct comparison of the upstream flanking regions of the zebrafish and human isl1 genes, another highly conserved noncoding element, CREST3 was identified, and subsequently C3R, a similar element to CREST3 with two CDP CR1 recognition motifs was identified, in the upstream regions of all other isl1 family members. In mouse and human, CRESTs are located as far as more than 300 kb away from the isl1 locus, while they are much closer to the isl1 locus in zebrafish. Although all of zebrafish CREST2, CREST3, and C3R activate gene expression in the sensory neurons of zebrafish, CREST2 of mouse and human does not have the sequence necessary for sensory neuron-specific expression. These results reveal both a remarkable conservation of the regulatory elements regulating subtype-specific gene expression in motor and sensory neurons and the dynamic process of reorganization of these elements whereby each element increases the level of cell-type specificity by losing redundant functions with the other elements during vertebrate evolution (Uemura, 2005).

Sequence comparison and TRANSFAC analysis has revealed that there are putative recognition motifs for multiple transcription factors in the evolutionarily conserved region of CREST1. Among them, homeodomain proteins such as MNR2, Hb9, and Phox2b are known to be expressed in premature motor neurons and act as key regulators for motor neuron differentiation. Moreover, motor neurons are generated from the progenitor domain for motor neurons (pMN) delineated by the specific expression of homeodomain proteins Nkx6.1 and Pax6. Thus, focus was placed on the three homeodomain recognition motifs ATTA or TAAT of zCREST1. Each of them was replaced with GGGG. One mutated zCREST1 termed zCREST1m, which had such nucleotide conversion in one of these motifs, was completely inactive as assessed by transient transgene expression assay in zebrafish. This region was predicted as an Nkx protein recognition motif by TRANSFAC analysis. This region partly overlapped with a putative v-Maf recognition motif; however, nucleotide conversion of this motif to GGGGG never reduced the expression level of GFP in a transient transgene expression assay. Taken together, the homeodomain protein might target CREST1 via TAAT sequence (Uemura, 2005).

Sequence alignment and TRANSFAC analysis has revealed that the highly conserved region between CREST3 and C3R contains one POU recognition motif and two CDP CR1 (Cut Repeat1 of CDP) recognition motifs. The CDP family constitutes a characteristic group of homeodomain proteins, containing one to three repeats of Cut repeat(s). Cut repeat itself is known to bind to specific DNA sequence. One such gene D-onecut, a member of Drosophila Cut domain proteins, is thought to be a transcriptional activator and implicated in the regulation of neural differentiation in the eye. Moreover, zonecut, a zebrafish ortholog of D-onecut, is expressed in primary neurons including trigeminal ganglion neurons and Rohon-Beard neurons in zebrafish embryos at the stage just prior to the expression of isl1 begins. Thus, the expression of isl1 in primary sensory neurons might be directly regulated by Cut domain protein(s). To confirm this hypothesis, either or both of the core recognition motifs of CDP CR1 were replaced with GGGGG and their enhancer activities were examined. Double mutations in zCREST3 alone caused remarkable reduction in the enhancer activity of zCREST3 as assessed by the number of embryos expressing GFP in primary sensory neurons among all injected embryos. These data indicate that Cut domain proteins may play important roles for the regulation of isl1 gene expression in primary sensory neurons (Uemura, 2005).

Timing of the retinoid-signalling pathway determines the expression of islet neural progenitor cells

By culturing neural progenitor cells in the presence of retinoid receptor agonists, the components of the retinoid-signalling pathway that are important for the birth and maintenance of neuronal cells have been defined. Evidence is provided that depending on the order and combination of retinoid receptors activated, different neuronal cells are obtained. Astrocytes and oligodendrocytes are predominantly formed in the presence of activated retinoic acid receptor (RAR) alpha, whereas motoneurons are formed when RARß is activated. The regulation of islet-1 and islet-2, which are involved in neuronal development, was examined. Activated RARß up-regulates islet-1 expression, whereas activation of RARalpha can either act in combination with RARß signalling to maintain islet-1 expression or induce islet-2 expression in the absence of activated RARß. RARgamma cannot directly regulate islet-1/2 but can down-regulate RARbeta expression, which results in loss of islet-1 expression. Activated RARalpha is one of the final steps required for a mature motoneuron phenotype (Goncalves, 2005).

Protein interactions of Islet homologs

Islet-2 is a LIM/homeodomain-type transcription factor of the Islet-1 family expressed in embryonic zebrafish. Two Islet-2 molecules bind to the LIM domain binding protein (Ldb) dimers. Overexpression of the LIM domains of either Islet-2 or the LIM-interacting domain of Ldb proteins prevents binding of Islet-2 to Ldb proteins in vitro and causes similar in vivo defects in positioning, peripheral axonal outgrowth, and neurotransmitter expression by the Islet-2-positive primary sensory and motor neurons. Inhibition of Islet-2 translation with antisense morpholino oligonucleotide against Islet-2 mRNA reproduces the defects caused by overexpression of LIM domains of Isl-2. These and other experiments, i.e., mosaic analysis, coexpression of full-length Islet-2, and overexpression of the chimeric LIM domains, derived from two different Islet-1 family members, demonstrate that Islet-2 regulates neuronal differentiation by forming a complex with Ldb dimers and possibly with some other Islet-2-specific cofactors (Segawa, 2001).

Repression of Islet-2 function either by overexpressing the LIM domains of Isl-2 or injecting antisense against Islet-2 mRNA impairs outgrowth of the peripheral axons from the primary sensory neurons, while keeping their central axons intact. In vitro study using the explants of the chick dorsal root ganglion (DRG) has demonstrated that the DRG neurons extend their axons into the peripheral tissue only in the presence of the nerve growth factor (NGF), while they extend the axons into the CNS tissue irrespective of the presence of NGF. This suggests that NGF may selectively promote the peripheral outgrowth of the DRG neurons. Recently, double mutant mice for the genes encoding the proapoptotic BCL-2 homolog BAX and NGF/TrkA were generated. All DRG neurons (which would normally die in the absence of the NGF/TrkA signaling) survive if BAX is also eliminated. In BAX-/-;NGF-/- or BAX-/-;TrkA-/- mice, only the peripheral axons of the DRG neurons are lost, while their central axons remain intact. Therefore, the NGF/TrkA signaling regulates outgrowth of the peripheral axons from the DRG neurons. In view of this, it would be intriguing to examine whether Islet-2 is involved in the NGF/TrkA signaling (Segawa, 2001).

Islet and early specification of ascidian larval motor neurons

In the tadpole larvae of the ascidian Halocynthia roretzi, six motor neurons, Moto-A, -B, and -C (a pair of each), are localized proximal to the caudal neural tube and show distinct morphology and innervation patterns. To gain insights into early mechanisms underlying differentiation of individual motor neurons, an ascidian homologue of Islet, a LIM type homeobox gene, has been isolated. Earliest expression of Islet was detected in a pair of bilateral blastomeres on the dorsal edge of the late gastrula. At the neurula stage, this expression begins to disappear and more posterior cells start to express Islet. Compared to expression of a series of motor neuron genes, it was confirmed that early Islet-positive blastomeres are the common precursors of Moto-A and -B, and late Islet-positive cells in the posterior neural tube are the precursors of Moto-C. Overexpression of Islet induces ectopic expression of motor neuron markers, suggesting that Islet is capable of regulating motor neuron differentiation. Since early expression of Islet colocalizes with that of HrBMPb, the ascidian homologue of BMP2/4, a role for BMP in specification of the motor neuron fate was tested. Overexpression of HrBMPb leads to expansion of Lim and Islet expression toward the central area of the neural plate, and microinjection of mRNA coding for a dominant-negative BMP receptor weakens the expression of these genes. These results suggest that determination of the ascidian motor neuron fate takes place at late gastrula stage and local BMP signaling may play a role in this step (Katsuyama, 2005).

Expression and function of Islet Homologs in Zebrafish

Hours before the neural tube is formed in zebrafish, the expression of Isl-1 is initiated in Rohon-Beard cells, primary motor neurons, interneurons and cranial ganglia. The expression is initiated simultaneously in the Rohon-Beard cells and the primary motor neurons, at the axial level of the presumptive first somite. The Isl-1-expressing motor neurons appear on either side of the ventral midline whereas the interneurons and Rohon-Beard cells initiate expression while located at the edge of the germinal shield. Isl-1 expression is initiated in these cells before the formation of a differentiated notochord. Isl-1 is expressed in the various functional classes of primary neurons at 24 hours postfertilization. This selective expression of a homeodomain protein in the primary neurons implies that these neurons share a common program of early development and that they have evolved and been selected for as a coordinated system. One of the functions of the primary neurons is to send long axons that pioneer the major axon tracts in the zebrafish embryo. This suggests an evolutionary conserved functional role for Isl-1 in the expression of the pioneering phenotype of the primary neurons (Korzh, 1993).

Zebrafish Isl-1 mRNA first appears immediately after gastrulation in the polster, the cranial ganglia, and in Rohon-Beard neurons and ventromedial cells of the spinal cord. The expression by the ventromedial cells is segmentally repeated and becomes restricted to the one or two cells slightly anterior to the segment borders. Double staining by in situ hybridization and an antibody that stains most axons suggests that these segmentally distributed cells may be either the rostral primary motoneuron (RoP) or middle primary motoneuron (MiP). This raises a possibility that Isl-1 may be involved during determination of subtype identities of the primary motoneurons. Furthermore, the specific Isl-1 mRNA expression in the spinal cord is under the control of the somites, since mutant embryos with defective somites fail to maintain this pattern (Inoue, 1994).

Zebrafish embryos have three or four identifiable primary motoneurons per hemisegment. While several ventral cells initially express the zebrafish Islet-1 (Isl-1) gene, a member of the LIM/homeobox gene family, the expression of this gene becomes restricted by 16 hr after fertilization to a single cell or a pair of cells slightly anterior to each segment border. Double staining by in situ hybridization and immunohistochemistry strongly suggests that these cells are mainly rostral primary motoneurons. There are two more Isl-1 family genes, termed zfIsl-2 and zfIsl-3. zfIsl-2 mRNA starts to be expressed in the ventral midsegmental cells around 15 hr. Double labeling experiments have shown that these midsegmental cells are the caudal primary motoneuron (CaP) and its variant equivalence pair. These results reveal the heterogeneity in the expressed genes among primary motoneurons before the fates of the primary motoneurons are irreversibly determined, and further suggest the involvement of the Isl-1 and zfIsl-2 genes in the determination of cellular identities by primary motoneurons in embryonic zebrafish. zfIsl-3 mRNA is not expressed in motoneurons but is expressed at 17 hr, mainly in the ventral myotomes. This suggests that zfIsl-3 may be involved in the regional specification of the myotome and also in target recognition by CaP. zfIsl-2 is also expressed throughout the developing eye and tectal region of the midbrain, the target for the retinal axons. In the ventral spinal cord of the spadetail mutant embryo, which has defects in the somites, the cells expressing zfIsl-2 mRNA are significantly decreased in number, in contrast to the increase in cells expressing Isl-1 mRNA, suggesting the influence of the somites on the expression of both genes (Tokumoto, 1995).

In zebrafish, individual primary motoneurons can be uniquely identified by their characteristic cell body positions and axonal projection patterns. The fate of individual primary motoneurons remains plastic until just prior to axogenesis when they become committed to particular identities. Distinct primary motoneurons express particular combinations of LIM homeobox genes. Expression precedes axogenesis as well as commitment, suggesting that LIM homeobox genes may contribute to the specification of motoneuronal fates. By transplanting them to new spinal cord positions, it has been demonstrated that primary motoneurons can initiate a new program of LIM homeobox gene expression, as well as the morphological features appropriate for the new position. It is concluded that the patterned distribution of different primary motoneuronal types within the zebrafish spinal cord follows the patterned expression of LIM homeobox genes, and that this reflects a highly resolved system of positional information controlling gene transcription (Appel, 1995).

The first site at which neurogenesis occurs in the roof of the zebrafish forebrain is the epiphysial region of the dorsal diencephalon. There are two classes of neurons in the zebrafish epiphysis (or pineal organ): photoreceptors and projection neurons. Analysis of Pax6 (Drosophila homolog eyeless) distribution in the CNS reveals that it is expressed in projection neurons. In contrast to Pax6, the islet-1 gene is found to be expressed by photoreceptors and projection neurons. Zash-1a, a zebrafish homolog of Drosophila achaete-scute proneural genes is also expressed in the epiphysis. Islet-1 positive cells are produced within the Zash-1 expression domain. The homeobox gene floating head is required for neurogenesis to proceed in the epiphysis. In flh mutant embryos the first few epiphysial neurons are generated, but beyond the 18 somite stage neuronal production ceases. Islet-1 positive cells and mature neurons are reduced in the epiphysis of floating head mutants. In contrast, in masterblind mutant embryos, epiphysial neurons are generated throughout the dorsal forebrain. Thus mbl acts negatively, preventing the expression of flh in dorsal forebrain cells rostral to the epiphysis. Furthermore, epiphysial neurons are not ectopically induced in mbl/flh double mutant embryos, demonstrating that the epiphysial phenotype of mbl mutants is mediated by ectopic Flh activity. It is thought that Zash-1 is downstream of flh, and that Pax6 acts even further downstream, differentiating projection neurons from photoreceptors. A role for Flh is proposed in linking the signaling pathways that regulate regional patterning to the signaling pathways that regulate neurogenesis (Masai, 1997)

Overexpression of the LIM-domains-only truncated form of the Islet-3 LIM/homeodomain protein of zebrafish induces ocular and cerebellar defects. Islet-3 is expressed specifically in the eyes and the presumptive tectum of the zebrafish. Overexpression of Islet-3 LIM-domains-only protein prevents formation of the optic vesicles, causes abnormal termination of the expression of wnt1, engrailed2 and pax2 in the mesencephalic and metencephalic region between 14 hours and 20 hours posterfertilization, and severely impairs morphogenetic movement in this region between 20 and 26 hours, which should normally lead to formation of the cerebellar primordium. Such defects were all rescued by simultaneous overexpression of Islet-3, suggesting that the overexpressed LIM domains act as a specific dominant-negative variant of Islet-3. The phenotypic defects of cells overexpressing Islet-3 LIM-domains-only protein can be rescued by forming mosaic embryos (transplanting normal cells in the mesencephalic and metencephalic region). Normal embryos made mosaic by injecting cells overexpressing LIM-domains-only protein show a complete exclusion of overexpressing cells from the optic vesicles, but incorporation of cells in the mes met region. The results show that neighboring normal cells can rescue the phenotypic defect of cells overexpressing the LIM-domains-only protein, suggesting a possibility that the Isl-3 LIM-domains-only protein may interfere with levels of expression of a secreted signaling molecule that is essential for normal differention of these regions but not impair the reception of this signal. Initiation and maintenance of expression of wnt1, pax2 and eng2 is essential for normal development of the mesencephalon and metencephalon regions. The zebrafish embryos overexpressing Isl-3 LIM-domains-only protein phenotypically resembles mouse embryos carrying a mild Wnt1 mutant allele that selectively deletes the cerebellum while leaving the midbrain relatively intact in morphology. It is thought that in zebrafish Islet-3 is required for the Wnt1 signal. These data suggest that Islet-3 functions to promote evagination of the optic vesicles and to maintain reciprocal interaction between the mesencephalon and the mesencephalic-metencephalic boundary essential for normal development of this region (Kikuchi, 1997)

Development of the tectum and the cerebellum is induced by a reciprocal inductive signaling between their respective primordia, the midbrain and the midbrain/hindbrain boundary (MHB). It is of interest to identify molecules that function in and downstream of this reciprocal signaling. Overexpression of LIM domain of the transcription factor Islet-3 (LIM Isl-3) leads to inhibition of this reciprocal signaling and to resultant defects in tectal and cerebellar development. Genes were sought that may be either up- or down-regulated by overexpression of LIMIsl-3 by comparing the gene expression profiles in the midbrain and the MHB of normal embryos and embryos in which Islet-3 function is repressed, using a combination of ordered differential display and whole-mount in situ hybridization. Among genes identified in this search, two cDNA fragments encode Wnt1 and FGF8, which are already known to be essential for the reciprocal signaling between the midbrain and the MHB, confirming the effectiveness of this strategy. Four other partial cDNA clones were identified that were specifically expressed around the MHB, ten cDNAs specifically expressed in the tectum, and three cDNAs expressed in neural crest cells, including those derived from the midbrain level. The ephrin-A3 gene is specifically expressed in posterior tectum in a gradient that decreases anteriorly. Although ephrin-A2 and ephrin-A5 have been reported to be expressed in the corresponding region in mouse embryos (the superior/inferior colliculi), mouse ephrin-A3 is not expressed prominently in this region, suggesting that the role of ephrin-A3 in brain development may have been altered in the process of brain evolution (Hirate, 2001).

In Drosophila, Slit acts as a repulsive cue for the growth cones of the commissural axons which express a Robo, thus preventing the commissural axons from crossing the midline multiple times. Experiments using explant culture have shown that vertebrate Slit homologs also act repulsively for growth cone navigation and neural migration, and promote branching and elongation of sensory axons. Overexpression of Slit2 in vivo in transgenic zebrafish embryos severely affects the behavior of the commissural reticulospinal neurons (Mauthner neurons), promotes branching of the peripheral axons of the trigeminal sensory ganglion neurons, and induces defasciculation of the medial longitudinal fascicles. In addition, Slit2 overexpression causes defasciculation and deflection of the central axons of the trigeminal sensory ganglion neurons from the hindbrain entry point. The central projection is restored by either functional repression or mutation of Robo2, supporting its role as a receptor mediating the Slit signaling in vertebrate neurons. Furthermore, Islet-2, a LIM/homeodomain-type transcription factor, is essential for Slit2 to induce axonal branching of the trigeminal sensory ganglion neurons, suggesting that factors functioning downstream of Islet-2 are essential for mediating the Slit signaling for promotion of axonal branching (Yeo, 2004).

In Drosophila, Slit acts as a repulsive cue for the growth cones of the commissural axons which express a Robo, thus preventing the commissural axons from crossing the midline multiple times. Experiments using explant culture have shown that vertebrate Slit homologs also act repulsively for growth cone navigation and neural migration, and promote branching and elongation of sensory axons. Overexpression of Slit2 in vivo in transgenic zebrafish embryos severely affects the behavior of the commissural reticulospinal neurons (Mauthner neurons), promotes branching of the peripheral axons of the trigeminal sensory ganglion neurons, and induces defasciculation of the medial longitudinal fascicles. In addition, Slit2 overexpression causes defasciculation and deflection of the central axons of the trigeminal sensory ganglion neurons from the hindbrain entry point. The central projection is restored by either functional repression or mutation of Robo2, supporting its role as a receptor mediating the Slit signaling in vertebrate neurons. Furthermore, Islet-2, a LIM/homeodomain-type transcription factor, is essential for Slit2 to induce axonal branching of the trigeminal sensory ganglion neurons, suggesting that factors functioning downstream of Islet-2 are essential for mediating the Slit signaling for promotion of axonal branching (Yeo, 2004).

The expression of LIM homeobox genes islet1 and islet2 is tightly regulated during development of zebrafish primary motoneurons. All primary motoneurons express islet1 around the time they exit the cell cycle. By the time primary motoneurons undergo axogenesis, specific subtypes express islet1, whereas other subtypes express islet2, suggesting that these two genes have different functions. Islet1 is shown to be required for formation of zebrafish primary motoneurons; in the absence of Islet1, primary motoneurons are missing and there is an apparent increase in some types of ventral interneurons. Evidence is provided that Islet2 can substitute for Islet1 during primary motoneuron formation. Surprisingly, the results demonstrate that despite the motoneuron subtype-specific expression patterns of Islet1 and Islet2, the differences between the Islet1 and Islet2 proteins are not important for specification of the different primary motoneuron subtypes. Thus, primary motoneuron subtypes are likely to be specified by factors that act in parallel to or upstream of islet1 and islet2 (Hutchinson, 2006; full text of article).

The ability of animals to carry out their normal behavioral repertoires requires exquisitely precise matching between specific motoneuron subtypes and the muscles they innervate. However, the molecular mechanisms that regulate motoneuron subtype specification remain unclear. This study used individually identified zebrafish primary motoneurons to describe a novel role for Nkx6 and Islet1 proteins in the specification of vertebrate motoneuron subtypes. Zebrafish primary motoneurons express two related Nkx6 transcription factors. In the absence of both Nkx6 proteins, the CaP motoneuron subtype develops normally, whereas the MiP motoneuron subtype develops a more interneuron-like morphology. In the absence of Nkx6 function, MiPs exhibit normal early expression of islet1, which is required for motoneuron formation; however, they fail to maintain islet1 expression. Misexpression of islet1 RNA can compensate for loss of Nkx6 function, providing evidence that Islet1 acts downstream of Nkx6. It is suggested that Nkx6 proteins regulate MiP development at least in part by maintaining the islet1 expression that is required both to promote the MiP subtype and to suppress interneuron development (Hutchinson, 2007).

Expression of Islet Homologs in Chickens and Mammals

Continued: islet Evolutionary Homologs part 2/3 | part 3/3

islet/tailup: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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