The segmental subdivision of the head is one of the more dramatic examples of how genes work to define specific strucures. Each segment of the head is determined by a set of genes whose transcription is directed to that segment.

ocelliless, often called orthodenticle (otd) is essential for defining the antennal segment, which determines both the eye and the antenna, as well as parts of the brain. empty spiracles, buttonhead and sloppy paired are also required to define the antennal segment. otd can be considered a gap gene since it is activated by bicoid and torso in a very narrow band in the developing head, and mutation of otd specifically eliminate certain adult head structures. In the eye antennal disc, otd is expressed in a very specific manner to define areas of the dorsal head [Images], including ocelli, eye and bristles. otd expression areas that define ocelli and bristles are neither in the eye or antennal anlagen of the eye-antennal disc, but rather in a third area, a circumferential segment of the eye disc (Royet, 1995).

hedgehog and wingless have an role in specifying adult head structures. Reduction of hedgehog activity results in flies completely lacking medial head structures, while loss of wingless results in deletion of lateral (orbital) and mediolateral (frons) head structures. Ectopic expression of hh results in the induction of ectopic ocelli at more lateral locations, while ectopic wg results in an invasion of mediolateral frons cuticle into the ocellar region. In otd mutants, specifically ocelliless regulatory mutations, wg expression fails to disappear from the medial region and instead persists across the entire primordium of the head vertex. At the same time, hh expression in lost. In a complementary fashion, hh also seems to have a positive effect on otd expression; ectopic hh activates otd suggesting that otd expression in the head vertex primordium may be activated by hh during normal eye-imaginal disc development. Thus otd is required for regional head development, and has a critical role in regulating wg and hh expression (Royet, 1996).

The effect of otd on bristle structures is the basis for its name, which might be loosely translated as "correctly toothed (bristled)." Otd's effects on neural tissues are not confined to the head, since it is also involved in specifying particular neurons in the ventral midline of the central nervous system.

The mechanisms of action of cephalic gap genes remain poorly understood. orthodenticle (otd), which establishes a specific region of the anterior head, has been proposed to act in a combinatorial fashion with the cephalic gap genes empty spiracles (ems) and buttonhead (btd) to assign segmental identities in this region. To test this model, a heat-inducible transgene was used to generate pulses of ubiquitous otd expression during embryonic development. Ectopic otd expression causes significant defects in head formation, including the duplication of sensory structures derived from otd-dependent segments. However, these defects do not appear to result from the transformation of head segment identities predicted by the combinatorial model. To determine if the combinatorial model is correct, focus was placed on the dorsomedial papilla (dmp), antennal sense organ (anso), and dorsolateral papilla (dlp). The epidermal portions of these structures serve as markers of the ocular, antennal, and intercalary segments, respectively. According to the combinatorial model, ubiquitous otd expression should cause a transformation of the intercalary segment to a second antennal segment, without affecting the identity of the more anterior ocular segment. This would be indicated by duplication of the anso, loss of the dlp, and no change in the dmp. In a significant fraction of the cuticles that developed after an early pulse of otd expression, the anso is indeed duplicated. Significantly, however, the dlp is generallly not lost in these embryos, indicating that at least part of the intercalary segment is still present. These results do not indicate a transformtion of segmental identity, but rather the specific duplication of otd-dependent sensory structures (Gallitano-Mendel, 1998).

It is likely that misexpression of otd causes intrasegmental transformations rather than intersegmental transformations. The results correlate with specific regulatory effects of otd on the expression of the segment polarity genes engrailed (en) and wingless (wg). In wild-type embryos, en is expressed in each of the head segments. In the anterior head, en expression first appears during germ band extension in the antennal primordium, as a stripe 1-2 cells in width. Expression subsequently appears in the ocular segment (a small spot), the intercalary segment (a small stripe), and eventually in the clypeolabral region. Early induction of otd causes a broadening of the en antennal stripe to a width of as many as eight cells. In wild-type embryos, wg expression first apppears in the forgut primordium at the blastoderm stage. Subsequently, a broad anterior cephalic stripe forms. Following germ-band extension, wg is activated in a discrete stripe or spot in each head segment, anterior and adjacent to the engrailed counterpart. Ubiquitous otd expression causes the reduction or loss of wg in the antennal, intercalary, gnathal, and trunk segments. More anterior wg expression in the forgut, clypeolabral region, and ocular segment is not reduced (Gallitano-Mendel, 1998).

Mutant embryonic cuticles were examined for en or wg: en mutant embryos lack ansos; wg mutant embryos exhibit severe disruptions in head formation. However, unlike in en embryos, the anso is not missing but instead is frequently duplicated. These results indicate that en is required for anso formation. They also suggest that wg plays an inhibitory role in the specification of this sensory strucure. In double en;wg mutant embryos, the anso is absent, indicating that, although the absence of wg permits the formation of multiple ansos, this requires en activity. Although ubiquitous otd represses wg expression in the antennal segment and all segments posterior to it, otd induction has the opposite effect in the ocular segment, positively regulating wg expression. It is concluded that cephalic gap genes define head morphology through the direct modulation of segment polarity gene expression (Gallitano-Mendel, 1998).

Ectopic otd expression also causes the loss of head structures derived from the maxillary segment, which lies posterior to the otd domain. Ubiquitous otd affects the cirri and maxillary sense organ. This effect is associated with otd repression of the homeotic selector gene Deformed (Dfd). While Dfd mutants lack mouth hooks, the maxillary sense organ, cirri, and the ventral organ, it has been shown that otd induction reduces the number of cirri and maxillary sense organ papillae, but does not eliminate them altogether (Gallitano-Mendel, 1998).

The elbow/no ocelli (elb/noc) complex of Drosophila melanogaster encodes two paralogs of the evolutionarily conserved NET family of zinc finger proteins. These transcriptional repressors share a conserved domain structure, including a single atypical C2H2 zinc finger. In flies, Elb and Noc are important for the development of legs, eyes and tracheae. Vertebrate NET proteins play an important role in the developing nervous system, and mutations in the homolog ZNF703 human promote luminal breast cancer. However, their interaction with transcriptional regulators is incompletely understood. This study shows that loss of both Elb and Noc causes mis-specification of polarization-sensitive photoreceptors in the 'dorsal rim area' (DRA) of the fly retina. This phenotype is identical to the loss of the homeodomain transcription factor Homothorax (Hth)/dMeis. Development of DRA ommatidia and expression of Hth are induced by the Wingless/Wnt pathway. The current data suggest that Elb/Noc genetically interact with Hth, and two conserved domains crucial for this function were identified. Furthermore, Elb/Noc specifically interact with the transcription factor Orthodenticle (Otd)/Otx, a crucial regulator of rhodopsin gene transcription. Interestingly, different Elb/Noc domains are required to antagonize Otd functions in transcriptional activation, versus transcriptional repression. It is proposed that similar interactions between vertebrate NET proteins and Meis and Otx factors might play a role in development and disease (Wernet, 2014; PubMed: 24625735).

The transcription factors Homothorax (Hth) and Extradenticle (Exd) have been well characterized as co-factors for Hox genes. Hth/Exd can also act as co-factors for non-Hox transcription factors, like for Engrailed. This study showed that loss of both Elb and Noc phenocopies the loss of Hth at the dorsal rim of the retina. All markers of DRA ommatidia are lost in elb,noc double mutants: Rh3 expression and Sens repression in DRA R8, as well as the DRA-specific inner photoreceptor rhabdomere morphology in DRA R7 and DRA R8. The data shows that Elb/noc act downstream of Hth in the specification of DRA cell fates. Elb and Noc are expressed strongly in DRA R7 and R8. This expression is expanded to all R7 and R8 by ectopic Hth (but never into outer photoreceptors R1-6), while Hth expression is not affected in elb,noc double mutants. One possibility is that Elb/Noc serve as cofactors for Hth/Exd, since Hth loses its potential to induce the DRA fate in a double mutant retina. The vertebrate homologs of Elb and Noc function as repressors of transcription (Nakamura, 2008). Therefore, aspects of the Hth/Exd and Elb/Noc loss-of-function phenotypes could be due to a direct failure of their complex to repress common target genes. For instance, the de-repression of the R8 marker Sens by dominant-negative hth^HM, as well as in elb,noc double mutants could be explained by loss of a repressor complex containing all four proteins. Interestingly, functional antagonism between the Hox/Hth/Exd complex and Sens have been described in the Drosophila embryo. However, in this case the factors were shown to compete for overlapping binding sites in the promoter of the common target gene rhomboid. Gene expression profiling data revealed that the Hox gene Abd-B also directly represses Sens in the embryo using Hth/Exd as cofactors. Elb and Noc might therefore provide a missing link for transcriptional repression of Sens by Hth/Exd (Wernet, 2014).

Much work on NET family proteins has focused on functional characterization of their evolutionarily conserved domains. The C-terminus of NET proteins is required for nuclear localization (Pereira-Castro, 2013; Runko, 2004), as well as for self-association of the zebrafish ortholog Nlz1, although neither self-association nor heterodimerization with Nlz2 was found to be necessary for wild type function (Runko ). 'buttonhead box' , a conserved 7-10 amino acid motif which was not investigated in this study, may be required for transcriptional activation (Athanikar, 1997). Deletion of the 'buttonhead box' in zebrafish Nlz proteins transformed them into dominant-negatives, an effect that was proposed to be due to reduced affinity to co-repressor Groucho and histone de-acetylases. Interestingly, deletion of N-terminal sequences, including the Sp/SPLALLA motif also leads to dominant negative proteins. These data are consistent with findings that a protein with a mutated Sp/SPLALLA motif has a dominant-negative effect on DRA specification. The Sp motif was proposed to mediate transcriptional repression by directly binding to cofactors. It should be noted that both N-terminal Sp/SPLALLA deletion and the VP16 fusions have the same dominant-negative effect for zebrafish Nlz1. While this is consistent with a pure repressor function of the zebrafish protein, the differences between Sp/SPLALLA mutation and VP16-fusion (as well as the observation of a phenotype for the Engrailed fusion) reported in this study hint towards a more complex role of Elb and Noc in transcriptional regulation (Wernet, 2014).

This study has shown that mutation of the conserved zinc finger of Elbow also transforms this protein into a dominant-negative. Usually, multiple zinc fingers are required for DNA binding, suggesting that the NET family zinc finger is a protein-protein interaction domain. Deletion of the zinc finger from zebrafish Nlz proteins leads to a loss of nuclear localization, and the Nlz1 zinc finger is necessary for transcriptional repression. Although the possibility that Elb and Noc bind DNA through their zinc finger cannot be excluded, it is likely that mutation of the zinc finger either leads to an inactive complex by sequestration of another co-repressor, or that such complex could be trapped in the cytoplasm. Given that mutation of either Sp/SPLALLA motif or zinc finger both lead to a dominant-negative effect raises the possibility that protein binding to both motifs could be necessary for in vivo function, possibly through the formation of higher order transcriptional complexes (Wernet, 2014).

Loss of both elb and noc does not result in Rhodopsin phenotypes outside the DRA. However, over-expression of different forms of Elb or Noc recapitulates all Rhodopsin phenotypes observed in otd^UVI mutants. This phenotype might therefore arise from forcing a direct interaction between over-expressed Elb protein and Otd. Little is known about the regulatory relationship between Elb/Noc and Otd. However, the overlapping expression patterns and similar phenotypes for certain alleles of otd named ocelliless, and for no ocelli (noc) at the anterior pole of the fly embryo, as well as their common requirement in the morphogenesis of ocelli suggests that these proteins also interact positively outside of the retina. The antagonism that was observed might therefore be a dominant-negative effect resulting from sequestration of the Otd protein by over-expressed Elb. Alternatively, different combinations of transcriptional cofactors present between tissues (for instance DRA versus non-DRA R8 cells) might decide whether Elb and Noc act in concert with Otd, or as antagonists (Wernet, 2014).

In the retina, Otd acts in a 'coherent feedforward loop' with Spalt to directly activate transcription of rh3 and rh5. As a consequence, Rh3 and Rh5 are lost in otd mutants. Furthermore, Otd activates transcription of the repressor Dve, forming an 'incoherent feedforward loop', resulting in repression of rh3 and rh5 in outer photoreceptors. Since rh6 is activated by a distinct factor, Pph13, loss of Otd leads to a specific de-repression of rh6 into outer photoreceptors. This study shows that different domains of Elb specifically interfere with different aspects of Otd function in these feedforward loops. Mutation of the Groucho-binding motif FKPY only abolishes the ability of over-expressed Elbow protein to antagonize Otd function in repressing rh6 in outer photoreceptors, while mutation of the Sp/SPLALLA motif specifically antagonizes Otd function in activating both rh3 and rh5, without affecting repression of rh6 in outer photoreceptors (mediated by induction of Dve). Furthermore, while the Elb zinc finger is also required for antagonizing the function of Otd in outer photoreceptors, it is also necessary for antagonizing activation of rh3 by Otd, but not rh5. Hence, these two activator functions of Otd could be separated by mutating the zinc finger (Wernet, 2014).

The different Rhodopsin phenotypes caused by loss of Otd can be mapped to different protein domains. The current data therefore reveal specific genetic interactions between the protein domains of Elb/Noc and Otd. Such interactions could be direct or be mediated through additional proteins. For instance, the Otd C-terminus mediates the repression of rh6 in outer photoreceptors, making it a possible interaction domain for Groucho binding to the Elb/Noc FKPY motif. The N-terminus of Otd is necessary for most activation potential on rh3, while activation of rh5 predominantly maps to the C-terminus. This correlates well with the Rhodopsin-specific phenotypes seen after mutation of Sp/SPLALLA (affecting rh3 and rh5), or the zinc finger (affecting rh3 and rh6) motifs. Finally, the results using VP16- and en[R]-fusions of Noc show that potentially direct transcriptional effects on rhodopsin genes can only be induced in R8 cells. Both fusion proteins specifically regulate expression of rh5, while all other rhodopsins remain unaffected. Elb and Noc are both expressed strongly in R8 cells outside of the DRA where they may contribute the repression of Rh5. The absence of a non-DRA R8 rhodopsin phenotype in elb,noc double mutants, as well as the R8-specific action of VP16:noc could therefore be due to the existence of redundant, R8-specific factors required for Elb/Noc function there, but not for DRA specification. These factors remain unknown, since it was found that expression of elb and noc is not altered in homozygous mutants affecting p/y cell fate decisions in R8 cells (melt and wts (Wernet, 2014).

Mutations in the human Elb/Noc homolog ZNF703 promote metastasis (Slorach, 2011). This study has shown that over-expression of both human NET family proteins UAS-ZNF503 and UAS-ZNF703 in the Drosophila retina result in weak co-expression of Rh5 and Rh6, resembling over-expression of a VP16:noc protein. It is therefore possible that the genetic interaction of NET family proteins with Otd/Otx proteins is evolutionarily conserved, especially since a central domain of Otd was previously shown to mediate mutual exclusion of Rh5 and Rh6 (McDonald, 2010). This study presents a new role for Drosophila NET proteins in retinal patterning. Both zebrafish homologs of Elb/Noc, Nlz1 and Nlz2 are also required for optic fissure closure during eye development (Brown, 2009). Furthermore, expression of the Elb/Noc mouse homologue znf503 suggests that NET family genes are involved in the development of mammalian limbs(McGlinn, 2008). Given previous reports from Drosophila on the proximo-distal specification of leg segments, it appears that NET family members act in similar processes across species. This raises the possibility that NET proteins serve as evolutionarily conserved modules that have been re-utilized for analogous processes during evolution. Based on the current data, their conserved domain structure might be crucial for interacting with transcription factor networks involving conserved families of factors like Otx or Meis. Given their medical relevance in breast cancer, a better understanding of the role NET proteins play in the transcriptional control of tissue patterning will be of great importance (Wernet, 2014).

The Drosophila medulla, part of the visual processing center of the brain, contains more than 70 neural types generated by medulla neuroblasts which sequentially express several temporal transcription factors (TTFs), including Homothorax (Hth), eyeless (Ey), Sloppy paired 1 and 2 (Slp), Dichaete (D) and Tailless (Tll). However, it is not clear how such a small number of TTFs could give rise to diverse combinations of neuronal transcription factors that specify a large number of medulla neuron types. This study reports how temporal patterning specifies one neural type, the T1 neuron. The T1 neuron is the only medulla neuron type that expresses the combination of three transcription factors Ocelliless (Oc or Otd), Sox102F and Ets65A. Using CRISPR-Cas9 system, this study shows that each transcription factor is required for the correct morphogenesis of T1 neurons. Interestingly, Oc, Sox102F and Ets65A initiate expression in neurons beginning at different temporal stages and last in a few subsequent temporal stages. Oc expressing neurons are generated in the Ey, Slp and D stages; Sox102F expressing neurons are produced in the Slp and D stages; while Ets65A is expressed in subsets of medulla neurons born in the D and later stages. The TTF Ey, Slp or D is required to initiate the expression of Oc, Sox102F or Ets65A in neurons, respectively. Thus, the neurons expressing all three transcription factors are born in the D stage and become T1 neurons. In neurons where the three transcription factors do not overlap, each of the three transcription factors can act in combination with other neuronal transcription factors to specify different neural fates. This study shows that this way of expression regulation of neuronal transcription factors by temporal patterning can generate more possible combinations of transcription factors in neural progeny to diversify neural fates (Naidu, 2020).

T1 neurons are a class of mysterious neurons that connect the lamina and the medulla part of the optic lobe. They are uni-columnar neurons with one in each of the 800 columns of the medulla. The cell body of the T1 neuron is found in the medulla cortex, and its axon branches in a characteristic 'T' shape on the distal surface of the medulla. One branch projects through the outer optic chiasm back to the lamina and then forms a basket like structure of processes surrounding each lamina cartridge. The other branch arborizes in the M2 layer of the medulla with a dense bush like structure. T1 neuron is post-synaptic to amc (lamina amacrine cells), and the amc/T1 pathway was shown to enhance the lamina neuron L1 motion detection pathway at intermediate contrast. Depolarizing T1 neurons affected the flight steering responses to visual stimuli (Naidu, 2020).

Through screening antibodies and GFP fusion lines, this study found that Ocelliless (oc), Sox102F and Ets65A are expressed in T1 neurons, and the combination of these three TFs can distinguish T1 neurons from all other medulla neurons. Using CRISPR-Cas9 system, bi-allelic somatic mutations of each of the three TF genes were generated in T1 neurons; knock-down of each one affected different aspects of the T1 neuron morphology. Next, how the expression of each TF is controlled by temporal patterning to generate the combination code was examined. Oc expression in neurons starts in the Ey temporal stage, and continues in the Slp and D temporal stages, and Ey is required for the initiation of Oc expression in neurons; while Sox102F expression in neurons starts in the Slp temporal stage, and continues in the D temporal stage, and Slp is required for initiating the expression of Sox102F in neurons; finally, Ets65A is expressed in subsets of medulla neurons born in the D and later temporal stages, and D is required for the expression of Ets65A. Thus, the three TFs that control T1 neuron morphology initiate their expression in neurons beginning at different temporal stages controlled by different TTFs, but each of them spans a few temporal stages, and the neurons expressing all three TFs are born in the D stage and become T1 neurons. In neurons where the three transcription factors do not overlap, each of the three TFs could also act with other neuronal TFs to specify different neural fates. In this way, more combinations of TFs can be generated through temporal patterning (Naidu, 2020).

This study identified a combination of three transcription factors that control T1 neuron morphology, and examined how the expression of these three transcription factors are controlled by temporal patterning of medulla neuroblasts. Oc is turned on in neurons starting in the late Ey stage, and Oc expressing neurons continue to be generated in the Slp and D stages, although the fates will be different, possibly dependent on the co-expression with other neuronal TFs. Sox102F expressing neurons start to be generated in the Slp stage and continue in the D stage. Ets65A expressing neurons are generated in the D and later temporal stages. Thus, the three TFs that control T1 neuron morphology start their expression in neurons born at different temporal stages, and require the corresponding TTF for initiation of their expression, and each neuronal TF is expressed in neurons spanning a few temporal stages. One advantage for such temporal control of neuronal TFs is that more combinations of TFs can be generated to specify different fates. For example, Toy is expressed in the N-on neuronal progeny born from the Slp and D stages, and also in some N-off progeny born from the late Ey stage neuroblasts in some regions of the medulla. Results from this study and others suggest that the subset of Sox102F neurons that do not express Oc, express Toy and Ap instead, and they are specified as Tm5 neurons. In addition, the neurons that express both Toy and Oc in the N-off progeny of some late Ey stage neuroblasts could determine another unknown neural type. Although it remains to be determined whether these TF combinations are indeed required for the corresponding neural fates, these examples do suggest that different combinations of neuronal TFs can be created that might determine different fates (Naidu, 2020).

Mutation of each of the three TFs expressed in T1 caused a certain morphological defect, similar to the morphology TFs that act in combinations to determine motor neuron morphology. For oc and Ets65A mutant neurons, it appeared that they still maintained the T1 fate, but the morphology was abnormal. Some Sox102F mutant neurons resembled medulla intrinsic neurons, but without functional assay, it was not clear whether they were fully transformed to a normal Mi neuron fate, or they still maintained some T1 neuron charateristics but underwent dramatic morphological changes. One question is whether the combination of TFs regulate neuron morphology by simple addition (each TF determines one feature, and the simple addition of these features determines one neural type), or in a synergistic way (three TFs together can determine features not determined by either TF alone). In the case of T1, when Sox102F was removed from T1 neurons, the driver used (T1-LexA) was still expressed in the mutant neurons, but the neurons became more like medulla intrinsic neurons, and some neurons lost the projection back to the lamina. However, Sox102F is not expressed in other neurons that project back to the lamina like lamina wide field neurons (lawf 1/2) which express Hth and Eya. Instead, Sox102F is also expressed in a Transmedulla neural type (Tm5) which do not resemble T1 neurons. Thus, these results favor the synergistic action model of neuronal TFs to control neuron morphology (Naidu, 2020).

The results are consistent with the principle that integration between temporal/spatial patterning of neuroblasts and the Notch-dependent binary neuron fate choice further diversifies neural fates. This study found that T1 neurons are derived from the Notch-off hemilineage of D stage neuroblasts. In addition, although T1 neurons are uni-columnar neurons that are generated throughout the main medulla region, there is a spatial component that regulates Oc expression and neural fate specification. Neurons that co-express Oc and Forkhead are only localized in the Dpp domains. Through analyzing the sequencing data published for all medulla neurons, the neurons expressing both Oc and Fkh should become the Dm12 neuron, a multi-columnar neuron with arborizations spanning several columns. Thus, these results support the conclusion that uni-columnar neurons are generated throughout the medulla main region, while multi-columnar neurons are generated in special spatial domains determined by spatial patterning (Naidu, 2020).

In summary, this study of T1 neuron specification illustrated an example how temporal patterning of neuroblasts sequentially turns on the expression of three TFs in neuronal progeny, and generates different combinational codes to determine neural fates. In the future it will be interesting to examine how TTFs in neuroblasts regulate the expression of neuronal TFs in neurons that often span a few temporal stages. Only a subset of neurons maintain the expression of TTFs, while other neurons do not. Thus the TTFs should determine the expression of neuronal TFs already in neuroblasts. It is possible that the TTF promotes epigenetic modifications in the neuronal TF gene locus, so that the TF will be turned on in its progeny as well as in neurons born in subsequent temporal stages. It is also possible that the expression of the same neuronal TF in two subsequent temporal stages are controlled by two separate enhancers that respond to different TTFs. Addressing these questions will further advance understanding of the link between neuroblast temporal patterning and neural fate specification (Naidu, 2020).

Bases in 5' UTR - 777

Exons - 4

Bases in 3' UTR - 1018

Structural Domains

The homeodomain is paired-group-like, but OTD lacks a paired domain (Finkelstein, 1990).