BIOLOGICAL OVERVIEW

It is possible to artificially direct eye morphogenesis to any appendage of the body. Genetically engineered sleight of hand has been used to express the eyeless gene in various tissues throughout the body of the fly in order to clarify an understanding of development.

Directed gene expression in such experiments is carried out using Enhancer Trap GAL4 lines. GAL4 is a gene activator. It turns genes on and is easily manipulated using genetic engineering techniques to modify the original genome. A genetic vector carrying the yeast GAL4 gene is randomly integrated into various sites along the genome. Some GAL4 vectors insert next to enhancers. These are promoter-like regions that regulate the induction of specific genes. The GAL4 vector comes under the control of enhancers expressed in various tissues of the fly. These modified genomes and the animals that carry them are termed enhancer trap GAL4 lines. Different enhancer trap GAL4 lines express GAL4 in different tissues depending on the activity of the enhancer into which the vector has inserted.

Other lines have been genetically engineered by introduction of a vector carrying the eyeless gene. The gene is regulated by a promoter region, termed an upstream activating sequence (UAS), activated by GAL4. By placing the eyeless gene under the regulation of GAL4, the gene can be activated wherever gene activator GAL4 is expressed. When these two lines are cross bred and the progeny examined, eyeless appears to be activated in the various tissues expressing the GAL4 transcription factor.

In this manner, by targeting expression of the eyeless complementary DNA in various imaginal disc primordia, ectopic eye structures have been induced on Drosophila wings, legs, and antennae. The ectopic eyes appear morphologically normal, consisting of groups of fully differentiated ommatidia with a complete set of photoreceptor cells. These experiments form the basis for considering eyeless to be the master control gene for eye morphogenesis (Halder, 1995).

Because homologous genes are present in vertebrates, ascidians (sea squirts), insects, cephalopods (squids and octopus), and nemerteans (worms), eyeless may function as a master control gene throughout the metazoa (Halder, 1995). eyeless is not the only homeodomain functioning early in eye morphgenesis. sine oculis is expressed in the optic primordium during stage 5 (the cellular blastoderm). optimotor blind, a brachyury homolog, is expressed in the optic placode from stage 12 onward (Cheyette, 1994).

The model for eyeless function suggests eyeless is required for the determination of cell fate in the eye (see Specification of the eye disc primordium and establishment of dorsal/ventral asymmetry). The embyonic and larval expression patterns of ey are consistent with the early requirement of this gene: it is expressed in the eye primordia in the embryo and before the morphogenetic furrow at the time of photoreceptor determination in the third instar larva (Halder, 1995). A subsequent study shows that ey is not only required at earlier developmental stages for the initiation of photoreceptor development, but also in later stages when terminal photoreceptor-specific genes are expressed. A novel form of ey is expressed in the adult stage. It contains both the first and the second exons. Exon 2 is in frame with exon 1 and its 5' boundary is 15 base pairs in front of the first methionine reported in the D1 transcript. Late ey expression begins in the late pupal stage and coincides with rhodopsin expression. The transition from the patterning of the eye disc to the expression of terminal photoreceptor-specific genes occurs during late pupal development. There is an initial decrease of ey expression in the first half of pupal life, but ey transcripts start to accumulate again starting at 2/3 of the way through pupal development (Sheng, 1997).

Eyeless directly regulates rhodopsin 1 (rh1) expression in photoreceptor cells. rh1 is expressed specifically in photoreceptor cells R1 to R6. eyeless is expressed in both larval and adult terminally differentiated photoreceptor cells. The homeodomain of Eyeless binds to a palindromic homeodomain binding site P3/RCS1 in the rh1 proximal promoter, which is essential for rh1 expression. These results suggest that Pax-6/Ey directly regulates rh1 gene expression by binding to the conserved P3RCS1 element in the promoter (Sheng, 1997).

eyeless is expressed both early and late in Bolwig's organ cells, which serve as the larval photoreceptor. The ey expression in Bolwig's organ occurs during embryonic development at the end of stage 12. Krüppel expression can also be detected in all 12 Bolwig's organ precursors, but whether ey and Krüppel are coexpressed in all precursors is unknown. ey is down-regulated and absence during most phases of Bolwig's organ development, which includes morphogenetic movement and axonal growth, elongation and projection (Sheng, 1997).

Drosophila contains a second Pax-6 gene, twin of eyeless (toy), due to a duplication during insect evolution. Toy is more similar to vertebrate Pax-6 proteins than Ey, with regard to overall sequence conservation, DNA-binding function, and early expression in the embryo. toy and ey share a similar expression pattern in the developing visual system, and targeted expression of Toy, like Ey, induces the formation of ectopic eyes. Genetic and biochemical evidence indicates, however, that Toy functions upstream of ey by directly regulating the eye-specific enhancer of ey. Toy is therefore required for initiation of ey expression in the embryo and acts through Ey to activate the eye developmental program (Czerny, 1999).

The predicted Toy protein with its 543 amino acids is considerably shorter (by 295 amino acids) than Ey and thus in terms of its size, much more resembles the known Pax-6 proteins of other animals. Ey and Toy share the same sequence identity (90%) in the homeodomain with vertebrate Pax-6 proteins, while Ey is more closely related in the paired domain to vertebrate Pax-6 proteins than Toy (95% vs. 91% sequence identity). In contrast to Ey, Toy shows significant sequence conservation in the C-terminal region with other Pax-6 proteins, Hence, the Drosophila Toy protein has more features (length, conserved sequence motifs) in common with Pax-6 proteins of other animal phyla than with its previously characterized Drosophila Pax-6 paralog, Eyeless. Two Pax-6 genes are present in D. melanogaster and in the closely related species D. virilis and in a more distantly related holometabolous insect, the silkmoth Bombyx mori. However, a single Pax-6 gene could be isolated from squid, sea urchin, mouse, and human, which represents three different animal phyla. The absence of a second Pax-6 gene in the springtail and grasshopper further suggests that the duplication of the Pax-6 gene occurred during insect evolution, in agreement with the observed conservation of intron positions in toy and ey (Czerny, 1999).

During embryogenesis, transcripts of the toy gene are first detected at the cellular blastoderm stage in the posterior procephalic region, including the optic lobe area. During subsequent development, the toy expression domain in the dorsolateral head ectoderm gives rise to the brain and to most, if not all, parts of the visual system, including the optic lobe, the larval eyes (Bolwig's organ), and the eye imaginal discs from which the adult compound eyes and the three ocelli develop. Throughout gastrulation, toy expression is confined to the head region anterior to the cephalic furrow. After germband retraction, toy expression is detected in the optic lobe primordia in a broad region of the brain, and in the anlagen of the eye-imaginal discs. At this stage, toy expression is also observed in a segmentally reiterated pattern in the ventral nerve cord. toy transcripts could not be detected in the differentiating photoreceptor cells of Bolwig's organ, which originates from the posterior procephalic head region. In third instar larvae, toy expression is observed in defined regions of the brain and in the eye-antennal imaginal discs, but not in the leg, wing, or haltere discs. In the eye disc, toy expression is restrict to the undifferentiated part that lies anterior to the morphogenetic furrow (Czerny, 1999).

In contrast to toy, ey is first expressed during late germband extension. ey transcripts are then detected in every segment of the developing ventral nerve cord, whereas toy expression is still absent in this structure. Later in embryogenesis, both genes are expressed in the ventral nervous system, although in different sets of cells. Moreover, ey is initially expressed only in a few cells of the developing brain at germband extension; when compared to toy, its expression remains more regionalized in both brain hemispheres during further development. However, the expression of toy and ey is very similar, if not identical, in the developing visual system, with the exception that during embryogenesis, toy is already expressed in the posterior procephalic region from where the optic primordia originate. Thus, ey and toy are coexpressed in the optic lobe and eye primordia of the late embryo as well as in the undifferentiated part of the eye imaginal discs of third instar larvae (Czerny, 1999)

In the absence of a characterized toy mutation, the epistatic relationship between toy and ey was determined by several different criteria demonstrating that Toy functions directly upstream of ey in the eye developmental pathway and that both proteins fulfill nonredundant functions in compound eye development: (1) toy is normally expressed in the developing eye discs of eyeless mutants. Consequently, toy cannot compensate for the loss of Ey activity in ey mutant flies, indicating that Toy and Ey fulfill nonredundant functions in eye development. (2) Targeted expression of Toy activates ey transcription at the ectopic expression site, whereas misexpression of Ey does not induce toy transcription in heterologous imaginal discs. Hence, toy acts upstream of ey but not vice versa. (3) toy requires ey function for activating the eye developmental pathway, since targeted misexpression of Toy is unable to induce the formation of ectopic eyes in eyeless mutant flies. However, Ey can induce ectopic eye development in the absence of toy, since toy expression is not activated in Ey-induced ectopic eyes. (4) Toy directly regulates ey gene by binding to Pax-6 sites, which are present in the eye-specific enhancer of ey and are essential for enhancer activation at the onset of eye development in the embryo. In conclusion, Toy and Ey have different functions and presumably regulate different sets of target genes during compound eye development. It is proposed that ey is a key target gene of Toy in the developing eye disc. Following activation by Toy, Ey regulates, in turn, downstream genes like sine oculis and eyes absent to further activate the eye development pathway (Czerny, 1999).

A role for eyeless in adult Drosophila brain development and function has been described. eyeless expression is detected in neurons, but not glial cells, of the mushroom bodies, the medullar cortex, the lateral horn, and the pars intercerebralis. Furthermore, severe defects in adult brain structures essential for vision, olfaction, and for the coordination of locomotion are provoked by two newly isolated mutations of eyeless that result in truncated proteins. Consistent with the morphological lesions, defective walking behavior has been observed for these eyeless mutants (Callaerts, 2001).

In the adult, strong immunopositive nuclear staining for Eyeless is observed in the Kenyon cells (mushroom body somata), in the medulla cortex, in neurons in the pars intercerebralis, and in neurons in the lateral horn. In situ hybridization with eyeless probes gives the same expression pattern. No eyeless expression is seen in glial cells. In the larval brain, strong expression is observed in the outer proliferation center of the optic lobes, in the mushroom body neuroblasts, and in Kenyon cell (mushroom body neuron) somata, as well as in other cells in the central brain (Callaerts, 2001).

Several candidate eyeless alleles were identified in a screen for dominant enhancers of an eye loss phenotype induced by ectopic expression of the homeotic Proboscipedia (HoxA2/B2) protein. The ey^JD and ey^DIDa mutations were identified as ey alleles through complementation analysis of previously characterized eyeless alleles. The majority of ey^JD homozygous mutant animals display severe eye defects, with eye sizes reduced to less than 25% the size of wild-type. ey^DIDa homozygotes have slightly weaker eye phenotypes. Homozygosity for ey^JD and ey^DIDa is associated with 85% and 13% lethality, respectively. Hemizygosity for ey^JD and ey^DIDa leads to an increase in lethality to 94% and 29%. Transheterozygous combinations of ey^JD and ey^DIDa with another eyeless allele display a reduced lethality, variable eye phenotypes, and severe mushroom body and central complex defects. Very severe mushroom body defects are also observed in homozygous ey^JD and ey^DIDa flies, and in transheterozygotes of ey^JD and ey^DIDa with Df(4)J2 (Callaerts, 2001).

The molecular nature of the defects underlying the new eyeless alleles is characterized by PCR amplification and sequencing. ey^JD and ey^DIDa have exonic mutations. ey^DIDa is caused by a microdeletion and frameshift in exon 9 encoding the C-terminal domain of the Ey protein. The predicted mutant protein is 682 amino acids long (compared to 838 in wild-type), with the last 29 amino acids of the mutant protein unrelated to the wild-type sequence. ey^JD is due to a point mutation in the homeobox (position 1382 in the cDNA), resulting in a stop codon, predicted to encode a protein truncated after the first helix of the homeodomain with a total length of 432 amino acids. The ey^JD allele leads to the production of a protein of the predicted, reduced size. Surprisingly, no Ey protein could be detected in the ey^DIDa samples. This lack of protein is not due to destabilization of the mRNA. These two new alleles, ey^DIDa and ey^JD, are the first identified in Drosophila that affect the Ey protein product. They may thus reveal previously undetected aspects of normal eyeless function (Callaerts, 2001).

The brains of homozygous ey^DIDa and ey^JD mutant flies are consistently smaller in size when compared to the heterozygous controls, which appeared normal. All homozygous individuals have clearly recognizable defects in the optic lobes, the central complex, and the mushroom bodies (Callaerts, 2001).

The optic lobe consists of four distinct neuropils; lamina, medulla (proximal and distal), lobula, and lobula plate. The lamina and medulla are connected via the first optic chiasm, and the medulla and lobula/lobula plate via the second optic chiasm. Several defects in the eyeless mutant optic lobes were detected. The lamina often appears smaller and flatter in mutants. The medulla is reduced in size and mispositioned, presumably due to incomplete rotation relative to the lamina. The medullar cortex is severely under-developed. The serpentine layer, which separates the distal and proximal part of the medulla, appears disorganized. The remaining two neuropils of the optic lobe, the lobula and lobula plate, are also reduced in size, the internal chiasm appears abnormal, and ectopic fiber bundles that appear to originate directly from the lamina can be observed in the lobula/lobula plate complex. Occasionally, a fusion of the optic lobe with the central brain was observed (Callaerts, 2001).

At the gross-morphological level, each of the two mirror-symmetrical mushroom bodies is a three-armed structure. Each mushroom body consists of about 2,500 Kenyon cells. Their cell bodies lie dorsocaudally in the brain cortex. Their dendrites constitute the calyx, and the axons form the peduncle and the different mushroom body lobes. In a frontal plane just anterior to the ellipsoid body of the central complex, the alpha and alpha' lobes point dorsally and the beta, beta', and gamma lobes point medially toward each other. The third arm of each mushroom body is formed by the peduncle extending obliquely from the dorsally and most posteriorly residing calyx to the comparatively anterior and ventral branching point of the lobes. The calyces are embedded in the dorsal cortex layer of the brain. The mushroom body lobes are mostly not recognizable in flies homozygous for either ey allele, though immunohistochemistry with anti-Fas II antibody reveals residual traces of the lobes. Peduncles are discernible in most brains, but are greatly reduced in diameter. The calyces of the mutant mushroom bodies are clearly diminished in size. Both ey alleles have similar qualitative effects on the mushroom bodies (Callaerts, 2001).

The central complex resides between the brain hemispheres just dorsal to the esophagus. It comprises four strongly connected neuropil regions. The anterior-most ellipsoid body is of torus shape, and it resides in the anterior concavity of the fan-shaped body. The paired noduli are located ventral to the ellipsoid body and dorsal to the esophagus. Finally, the protocerebral bridge is found posterior to the fan-shaped body at the border between cortex and protocerebral neuropil, and is flanked laterally by the calyces of the mushroom bodies. The central complex region was strongly disordered in all homozygous ey mutant flies examined, with the defects more pronounced in ey^JD flies. For both alleles, the ellipsoid body appears to be fused with the fan-shaped body. Both neuropil regions are not well separated from the surrounding protocerebral neuropil in ey^DIDa brains, and are almost fused with it in ey^JD brains. The protocerebral bridge is disintegrated into several chunks of neuropil. Despite this marked disorganization, the total volume of the central complex was nearly unchanged in the ey mutants. In addition to the described phenotypes, strongly perturbed Fas II-positive neuronal projections are observed in ellipsoid and fan-shaped bodies of all mutant brains analyzed (Callaerts, 2001).

The central complex has been identified as a higher center for the control of walking behavior. Walking behavior of the homozygous ey alleles was studied as one measure of brain function at two different levels of resolution: (1) In the object fixation task 'Buridan's paradigm', the test fly is seen as a point-like object while walking between two inaccessible landmarks, and information is gathered about its walking activity, speed, and orientation behavior; (2) on a stepping analyzer, the actions of the single legs are resolved and their coordination studied. Regardless of the experimental situation, homozygous flies of both alleles are extremely reluctant to walk. This was particularly surprising in Buridan's paradigm, a situation that prompts normal flies to walk spontaneously, sometimes for hours. For ey mutant flies, spontaneous walking activity is already very low at the beginning, then fades further during the 15 min tests, as is typical of flies with a defective central complex. The homozygous test flies show almost no variation in their mean step length, which stays about as low as during slow walking (i.e., long stepping periods). Their range of stepping frequencies is limited at the fast end to 13 steps per second, whereas control heterozygotes, like wild-type flies, carried out up to 16 steps/s with every leg. The coordination of swing phases is normal and resembles the usual alternating tripod gait. A plot of the relative differences in step length and swing phase duration between mutants and controls reveals a 15% smaller step length in ey mutants, a reduction which is not explained by a mean shortening of swing phases. The smaller step size therefore is explained by a lower swing speed of legs during swing phases of almost normal duration. This specific walking defect has been previously found in other central complex structural mutants with a disrupted protocerebral bridge (Callaerts, 2001).

Drosophila Pax6 promotes development of the entire eye-antennal disc, thereby ensuring proper adult head formation

Paired box 6 (Pax6) is considered to be the master control gene for eye development in all seeing animals studied so far. In vertebrates, it is required not only for lens/retina formation but also for the development of the CNS, olfactory system, and pancreas. Although Pax6 plays important roles in cell differentiation, proliferation, and patterning during the development of these systems, the underlying mechanism remains poorly understood. In the fruit fly, Drosophila melanogaster, Pax6 also functions in a range of tissues, including the eye and brain. This report describes the function of Pax6 in Drosophila eye-antennal disc development. Previous studies have suggested that the two fly Pax6 genes, eyeless (ey) and twin of eyeless (toy), initiate eye specification, whereas eyegone (eyg) and the Notch (N) pathway independently regulate cell proliferation. This study shows that Pax6 controls eye progenitor cell survival and proliferation through the activation of teashirt (tsh) and eyg, thereby indicating that Pax6 initiates both eye specification and proliferation. Although simultaneous loss of ey and toy during early eye-antennal disc development disrupts the development of all head structures derived from the eye-antennal disc, overexpression of N or tsh in the absence of Pax6 rescues only antennal and head epidermis development. Furthermore, overexpression of tsh induces a homeotic transformation of the fly head into thoracic structures. Taking these data together, this study demonstrates that Pax6 promotes development of the entire eye-antennal disc and that the retinal determination network works to repress alternative tissue fates, which ensures proper development of adult head structures (Zhu, 2017).

In contrast to vertebrates that have a single Pax6 gene, the Drosophila genome contains two Pax6 homologs, ey and toy. Both genes are expressed broadly throughout the entire eye-antennal disc but are later limited to a far more restricted domain within the undifferentiated cells of the eye field. Whereas most studies on Pax6 in the eye-antennal disc have focused on the developing compound eye, several reports have hinted at a role for both genes outside of the eye. However, the underlying mechanism of how Ey/Toy promote eye-antennal disc development has been elusive. This is, in part, because of the use of single Pax6 mutants to study development. The phenotypes associated with individual mutants are variable and often restricted to the eye. Several studies have suggested that Ey and Toy function redundantly to each other. This finding most likely explains the variability of phenotype severity and penetrance. Thus, the combined loss of both Ey/Toy may be a more accurate reflection of the effect that Pax6 loss has on Drosophila development. Indeed, this appears to be the case as it is reported that the combined loss of both ey and toy leads to the complete loss of all head structures that are derived from the eye antennal disc. This study attempted to determine the mechanism by which Ey/Toy support eye-antennal disc development (Zhu, 2017).

Previous studies in the fly eye proposed that Pax6 is concerned solely with eye specification, whereas Notch signaling and other retinal determination proteins, such as Eyg, Tsh, and Hth, control cell proliferation and tissue growth. This study proposes an alternate model in which Ey/Toy are in fact required for cell survival and proliferation in addition to eye specification. The data indicate that Ey/Toy regulate growth of the eye-antennal disc through Tsh, N/Eyg, and additional N-dependent proliferation promoting genes. It is proposed that on simultaneous removal of Ey and Toy the eye-antennal disc fails to develop, in part, because the expression of eyg and tsh is lost in complete absence of Pax6. Expression of tsh and activation of the N pathway are sufficient to restore tissue growth to the eye-antennal disc. Support for this model linking Ey/Toy to cell proliferation via Eyg and Tsh comes from studies showing that eyg loss-of-function mutants display a headless phenotype identical to that seen in the ey/toy double knockdowns, that cells lacking eyg do not survive in the eye disc, and overexpression of Tsh causes overproliferation (Zhu, 2017).

The results also show that the combined loss of Ey and Toy affects the number of cells that are in S and M phases of the cell cycle. This observation directly supports the model that Ey/Toy control growth of the eye-antennal disc and is consistent with studies in vertebrates that demonstrate roles for Pax6 in the proliferation of neural progenitors within the brain. Earlier studies observed cells undergoing apoptosis in Pax6 single-mutant eye-antennal discs and showed that blocking cell death alone can partially rescue the head defects of the ey^D and toy^hdl mutants. Although this study shows that retinal progenitor cells lacking both Pax6 proteins undergo even greater levels of apoptosis, blocking cell death does not restore the eye-antennal disc. What accounts for the differences in the two experiments? In the ey^D and toy^hdl rescue experiments, each genotype contained wild-type copies of the other Pax6 paralog, but this study has knocked down both Pax6 genes simultaneously. Another possible difference is that Pax6 levels are being reduced while the ey^D and toy^hdl mutants are likely functioning as dominant negatives. It is concluded from these results that a reduction in cell proliferation but not elevated apoptosis levels is the proximate cause for the complete loss of the eye-antennal disc (Zhu, 2017).

Although the activation of Tsh and the Notch pathway can restore antennal and head epidermal development, neither factor is capable of restoring eye development to the ey/toy double-knockdown discs. This is most likely because both Pax6 genes are also required for the specification of the eye. In particular, Ey/Toy are required for the activation of several other retinal determination genes, including so, eya, and dac. Thus, the results suggest that Notch signaling, Eyg, and Tsh can restore nonocular tissue growth to the eye field but cannot compensate for the Pax6 requirement in eye specification (Zhu, 2017).

Finally, the results using the double knockdown of ey/toy are consistent with the dosage effects that are seen in mammalian Pax6 mutants. Although mutations in ey have just eye defects, the combined loss of ey/toy lacks all head structures. Mice that are heterozygous for Pax6 mutations have small eyes, whereas those that are homozygous completely lack eyes, have severe CNS defects, and die prematurely. Similarly, human patients carrying a single mutant copy of Pax6 suffer from aniridia, whereas newborns that are homozygous for the mutant Pax6 allele have anophthalmia, microcephaly, and die very early as well. As a master control gene of eye development, Pax6 appears to initiate both retinal specification and proliferation. These data demonstrate that the functions of Ey and Toy in the eye-antennal disc are redundant and dependent upon gene dosage, thereby making the roles of Pax6 in the Drosophila similar to what is observed in vertebrates where Pax6 controls both specification and proliferation of the brain and retina in a dosage-sensitive manner (Zhu, 2017).

Konstantinides, N., Holguera, I., ...., Walldorf, U., Roussos, P. and Desplan, C. (2022). A complete temporal transcription factor series in the fly visual system. Nature 604(7905): 316-322. PubMed ID: 35388222

A complete temporal transcription factor series in the fly visual system

The brain consists of thousands of neuronal types that are generated by stem cells producing different neuronal types as they age. In Drosophila, this temporal patterning is driven by the successive expression of temporal transcription factors (tTFs). This study used single-cell mRNA sequencing to identify the complete series of tTFs that specify most Drosophila optic lobe neurons. It was verified that tTFs regulate the progression of the series by activating the next tTF(s) and repressing the previous one(s), and also identify more complex mechanisms of regulation. Moreover, the temporal window of origin and birth order of each neuronal type in the medulla was established Finally, this study describes the first steps of neuronal differentiation and shows that these steps are conserved in humans. That terminal differentiation genes, such as neurotransmitter-related genes, are present as transcripts, but not as proteins, in immature larval neurons (Konstantinides, 2022).

The brain is the most complex organ of the animal body. The human brain consists of over 80 billion neurons that belong to probably thousands of neuronal types. As neural stem cells age, temporal patterning allows them to generate different neuronal types in the correct order and stoichiometry. Temporal patterning in neuronal systems was first described in the Drosophila ventral nerve cord (VNC), in which a cascade of tTFs is expressed in embryonic neural stem cells (neuroblasts) as they divide and age. This concept was later expanded to the Drosophila optic lobe, with a different tTF series. It was later suggested that tTFs also contribute to the generation of neuronal diversity in different mammalian neuronal tissues, such as the retina and the cortex. However, series of tTFs are incomplete, as they were discovered by relying on existing antibodies. To generate a comprehensive description of the tTFs patterning a neural structure, a single-cell mRNA-sequencing (scRNA-seq) analysis was performed of the larval fly optic lobe (Konstantinides, 2022).

The Drosophila optic lobe is an ideal system to address how neuronal diversity is generated and how neurons proceed to differentiate. It is an experimentally manageable, albeit complex structure, for which there exists a very comprehensive catalogue of neuronal cell types. Meticulous research from the past decades has identified multiple cell types in the optic lobes based solely on morphological characters. Recent research made use of elaborate molecular genetic tools, as well as scRNA-seq, to expand the number of neuronal cell types to around 200, based on both morphology and molecular identity. Importantly, the neuroblasts that generate the medulla, which is the largest optic lobe neuropil containing around 100 neuronal types, are formed by a wave of neurogenesis over a period of days and progress through the same tTF temporal series. This means that, at any given developmental stage from mid third larval stage (L3) to early pupal stages (P15), the neurogenic region contains neuroblasts at all developmental stages (Konstantinides, 2022).

To study neuroblast and neuronal trajectories, a scRNA-seq analysis was performed of the optic lobes. 49,893 single-cell transcriptomes were obtained from 40 L3 optic lobes. The outer proliferation centre (OPC) neuroepithelium generates two optic lobe neuropils: the medulla from the medial side and the lamina from the lateral side. Medulla neuroepithelium, neuroblasts, intermediate precursors (known as ganglion mother cells (GMCs)) and neurons were arranged in a uniform manifold approximation and projection (UMAP) plot following a progression that resembled their differentiation in vivo. Similarly, lamina neuroepithelium, precursor cells and neurons were also arranged following a similar differentiation trajectory but in the opposite orientation of that of the medulla. The neuroblasts and the neurons that are generated from the inner proliferation centre followed a different trajectory in the UMAP plot (Konstantinides, 2022).

The larval single-cell dataset was merged with the annotated early P15 stage single-cell dataset. The P15 neurons mapped at the tip of each of the neuronal trajectories, which enabled identification of the corresponding neuronal types. Neurons were identified from all the neuropils of the optic lobe (lamina, medulla, lobula and lobula plate), as well as a small number of neuroblasts and neurons from the central brain that were probably retained when microdissecting the optic lobe (Konstantinides, 2022).

Next, expression was looked at of the known spatial TFs in the OPC neuroepithelium and tTFs in the neuroblasts: the spatial TFs Vsx1, Optix and Rx25 were expressed in largely non-overlapping subsets of neuroepithelial cells, and the tTFs Homothorax (Hth), Eyeless (Ey), Sloppy-paired (Slp), D and Tll were expressed in neuroblast subsets that were temporally organized in the UMAP plot (Konstantinides, 2022).

Thus, the UMAP plot recapitulated both proliferation and differentiation axes in the developing tissue: the UMAP horizontal axis represents differentiation status, whereas the vertical axis represents neuroblasts progressing through their tTF series (Konstantinides, 2022).

The larval scRNA-seq dataset provided the opportunity to look for all potential tTFs in an unbiased manner. The medulla neuroblast cluster was isolated from the scRNA-seq data and Monocle was used to reconstruct its developmental trajectory. Hth, Ey, Slp1/2, D and Tll were expressed in the previously described temporal order along the trajectory. The expression dynamics of all Drosophila TFs was examined and 14 candidate tTFs were identified, the expression of which was restricted to a specific pseudotime window, including the 6 previously known tTFs. Using antibodies or in situ hybridization for the eight newly discovered candidate tTFs and those already known in medulla neuroblasts, it was shown that their expression is indeed limited to restricted temporal windows, therefore defining new temporal windows as the neuroblasts progress through divisions (Konstantinides, 2022).

The previously known tTFs (except for Hth) contribute to the progression of the series by activating the next tTF in the cascade and repressing the previous one. To test which of the newly identified tTFs were involved in the progression of the temporal series, tTF mutant neuroblast MARCM (mosaic analysis with a repressible cell marker) clones or tTF RNA interference (RNAi) knockdowns were generated using the MZVUM-Gal4 line that is expressed in the Vsx1 domain of the OPC. Hth is expressed in the neuroepithelium and young neuroblasts, and is not required for Ey activation. Two factors were identified that regulate the expression of Ey in different ways: Erm is required to activate Ey and to inhibit Hth, whereas Opa is required for the correct timing of Ey activation. Opa also activates the expression of Oaz, which does not regulate the expression of any of the tTFs. Opa expression is repressed by Erm. Once Ey expression is initiated at the correct time by the combined action of Erm and Opa, Ey represses the expression of its activators. Thus, Erm is essential for the progression of the cascade, whereas Opa contributes to the correct timing of the expression of the next tTFs (Konstantinides, 2022).

Previous work has shown that Ey activates Slp, which in turn inhibits Ey. However, the developmental trajectory of neuroblasts uncovered a more complex situation. First, Ey activates Hbn. Hbn then represses Ey and activates Slp. Hbn also activates Scro and a second wave of Opa expression. Hbn then inhibits the expression of Erm and Scro inhibits the expression of Ey. Finally, Slp inhibits Hbn, Opa and Oaz (Konstantinides, 2022).

D expression requires both Slp and Scro. Previous work showed that in slp-mutant clones D is not expressed. Similarly, when scro was knocked down by RNAi, D was not activated. Scro is therefore important for the progression of the series, as it inhibits Ey and activates the expression of D. It remains expressed until the end of the neuroblast life. Once D is activated, it inhibits Slp and activates BarH1, which in turn activates Tll. Finally, similar to the inhibitory interaction between Tll and D previously described, Tll is sufficient but not necessary to inhibit BarH1 (Konstantinides, 2022).

This study has therefore identified most, if not all, tTFs in a developing neuronal system and show that these tTFs participate in the progression of the temporal series. Many of these interactions were confirmed by analysing the effect of tTF mis-expression on the temporal cascade (Konstantinides, 2022).

Besides their participation in the progression of the temporal series, tTFs regulate neuronal identity. Some tTFs are maintained in the neuronal subsets that are generated during their temporal window, whereas others are expressed only in newly born neurons. tTFs activate the expression of downstream neuronal transcription factors that regulate effector genes in the absence of the tTF. To test how tTFs regulate neuronal identity, whether knocking down the expression of the tTFs in neuroblasts affects the expression of neuronal transcription factors was tested. The loss of hth, ey and slp in neuroblasts leads to the loss of Bsh-, Vvl- and Toy-positive neurons, respectively. Hbn was shown to be required for the specification of Toy-, Traffic-jam (Tj)- and Orthodenticle (Otd)-positive neurons and Opa is required for the generation of TfAP-2-positive neurons. Thus, Hbn and Opa, as well as Hth, Ey and Slp, regulate neuronal diversity not only by allowing the temporal series to progress, but also by regulating the expression of neuronal transcription factors (Konstantinides, 2022).

The identified tTFs define at least 11 temporal windows in which different neurons (and glia) are generated. As they are generated, newly born neurons displace earlier born neurons away from the parent neuroblast, creating a columnar arrangement of neuronal cell bodies in the medulla cortex that represent birth order: early born neurons are located close to the emerging medulla neuropil, whereas late born neurons are closer to the surface of the brain. Neurons born in each temporal window express downstream effectors of tTFs (such as Bsh, Runt (Run) and Vvl) that were termed concentric genes due to their pattern of expression). The expression of tTFs in GMCs, and concentric genes that were previously described as well as those described in this work, in scRNA-seq neuronal clusters, together with cluster relative proximity in the UMAP plot, were used to assign the 105 neuronal clusters that comprise the medulla dataset to their predicted temporal window of origin. Proximal medulla neurons are generated in the Hth and Hth/Opa temporal windows, whereas distal medulla neurons are generated starting from the Ey temporal window. By contrast, transmedullary neurons are generated throughout most of the neuroblast life (Opa, Ey/Hbn and Slp temporal windows). Importantly, co-expression of some concentric genes is restricted to subregions of the medulla cortex, which enabled assigning the spatial origin to several medulla neuron clusters (Konstantinides, 2022).

To assess the status of all neuronal types, the expression of Apterous (Ap), which is expressed in the NotchON progeny of each GMC, was examined. Among the 105 neuronal types, 64 were NotchOFF and 41 were NotchON. As a given GMC division generates one NotchON and one NotchOFF neuron, Ap+ and Ap- neurons are intermingled in the medulla cortex. Thus, the position in the medulla cortex of concentric TFs expressed in NotchON and NotchOFF neurons enables inferrence of sister neurons, for example, Run neurons are probably sisters of TfAP-2 neurons, whereas early-born Vvl neurons are probably sisters of Knot (Kn) neurons (Konstantinides, 2022).

Finally, neurotransmitter identity was assigned to all of the medulla clusters at L3 and P15 stages. Ap expression is highly correlated with cholinergic identity, as nearly all Ap+-that is, NotchON-clusters in the dataset express ChAT and therefore have cholinergic identity, whereas most of the NotchOFF clusters are either GABAergic (most of them express Lim3)18 or glutamatergic (most of them express Tj or Fd59A). Interestingly, all the NotchOFF neurons from the same temporal window express the same neurotransmitter, independently of their spatial origin. This suggests that the temporal origin of medulla neurons and their Notch status instructs shared TF expression and neurotransmitter identity, and therefore function. In summary, this study has defined the temporal (and spatial) origin, birth order and Notch identity of all medulla cell types and highlighted the role of tTFs in regulating the generation of neural diversity (Konstantinides, 2022).

To study the first steps of neuronal differentiation after specification, the clusters from pupal stages (P15, P30, P40, P50 and P70) corresponding to the Mi1 cells were merged with the L3 scRNA-seq cluster and the GMCs most closely linked to them in the UMAP plot. Their differentiation trajectory was reconstructed, groups of genes (modules) were identified that co-vary along the entire trajectory from L3 to P70 and the Gene Ontology (GO) terms enriched in each gene module were sought. The timing of differentiation appears to follow a specific path. At L3, cell cycle genes and DNA replication genes are first expressed, as expected, from the division of GMCs. This is closely followed by genes involved in translation. Then, genes related to dendrite development and axon guidance are upregulated from late L3 until P30, stages during which the neurons direct their neurites to the appropriate neuropils. Genes that are important for neuronal function, such as neurotransmitter-related genes, synaptic transmission proteins, as well as ion channels start to be expressed as early as L3, reaching a plateau that is maintained until P15. Their expression then increases again until adulthood, when their products support neuronal function. This timing of differentiation was observed not only for Mi1 but could be generalized to all optic lobe neurons. These results indicate that not only is neuronal identity specified during the first hours of neuronal development, but their neuronal function (as indicated by the upregulation of chemical synaptic transmission terms) is also implemented very early, although the function is not required until much later. As this was unexpected, whether neurotransmitter mRNA expression observed as early as L3 was also translated was examined. Neurotransmitter-related genes, ChAT, VGlut and Gad1 mRNA are all expressed in the scRNA-seq data in non-overlapping neuronal sets and are maintained in the adult. However, protein expression at L3 was not observed. This suggests that their transcription represents a commitment to a specific neurotransmitter identity early in their development, but that other factors prevent premature translation of these mRNAs until they are needed at later stages of development (Konstantinides, 2022).

Next, whether the Drosophila optic lobe neuronal differentiation trajectory was similar to human neuronal differentiation was examined. This study generated single-nucleus RNA-seq data from the human fetal cortical plate at gestational week 19. Monocle was used to reconstruct their developmental trajectory from apical progenitors to intermediate progenitors and postmitotic neurons and identified gene modules that were co-regulated along the trajectory. GO analysis uncovered a notable similarity to Drosophila. Then the expression of the GO terms that were expressed at different stages of the differentiation trajectory in Drosophila was plotred on the human cortical differentiation trajectory. Very similar dynamics were observed; the main difference was the absence of enrichment for ribosome assembly and translation-related GO terms at early stages. This could potentially be explained by the slower development of human neurons compared with those of Drosophila, leading to a slower increase in size and the fact that the divisions of the radial glia are more symmetric31 compared with those of optic lobe neuroblasts. Despite this difference, these results show that neurons follow a similar differentiation trajectory in Drosophila and humans (Konstantinides, 2022).

Although temporal patterning is a universal neuronal specification mechanism, it is unclear how it has evolved. This study investigated whether the medulla tTFs were conserved in mouse cortical radial glia using a published scRNA-seq dataset. None of the medulla neuroblast tTFs were expressed in strict temporal windows in ageing radial glia, with the exception of PAX6 (orthologue of Ey), which was enriched in older progenitors. Reciprocally, the Drosophila orthologues of the mouse temporally expressed TFs were not expressed temporally in the developing optic lobe (Konstantinides, 2022).

The mouse orthologues of the Drosophila VNC tTFs Ikzf1, Pou2f1/Pou2f2 and Casz1 are expressed temporally in mouse retinal progenitors. The expression was looked at of the optic lobe tTFs in the mouse retina in a published scRNA-seq dataset. PAX6 was constitutively expressed, MEIS2 (orthologue of Hth), ZIC5 (orthologue of Opa) and SOX12 (orthologue of D) were expressed at embryonic stage 12, while NR2E1, the orthologue of Tll (which is expressed when neuroblasts become gliogenic), was expressed late, when retinal progenitors become gliogenic and start generating Muller glia. The lack of a strict conservation of tTFs between flies and mice indicates that the acquisition of the specific temporal series occurred independently in each phylum (Konstantinides, 2022).

The comprehensive series of transcription factors described in this work and their regulatory interactions temporally pattern a developing neural structure. Most tTFs are expressed in overlapping windows, creating combinatorial codes that differentiate neural stem cells of different ages and therefore provide them with the ability to generate diverse neurons after every division. They were conservatively assigned into 11 distinct temporal windows (ten of which generate neurons) that-when integrated with spatial patterning (six spatial domains) and the Notch binary cell fate decision-can explain the generation of approximately 120 cell types, which is close to the entire neuronal type diversity of the Drosophila medulla. Moreover, this study determined the downstream TFs that were expressed in neurons produced temporally, which enabled establishment of the birth order of all medulla neurons. Moreover, a detailed transcriptomic description is provided of the first steps in the differentiation trajectory of a neuron. Terminal differentiation genes are expressed within the first 20 h of neuronal life, approximately 2-4 days before their protein products can fulfil their function. Why these genes are expressed so early remains unclear, but it is hypothesized that this reflects the commitment of neurons to a specific function. This study also shows that all neurons follow the same route for differentiation and that this is similar to the differentiation process in developing human cortical neurons. Thus, understanding the mechanisms of neuronal differentiation in flies can generate insight for the equivalent process in humans (Konstantinides, 2022).

GENE STRUCTURE

The transcription unit spans approximately 16 kb. The gene encodes two transcripts which differ with respect to their first exons. These are spliced to exon three, which is shared. The N-terminal paired domain is coded for by three exons, and is present in both proteins (Quiring, 1994).

cDNA clone length - 2832

Bases in 5' UTR - 98+

Exons - Eight exons encode each splice variant.

Bases in 3' UTR - 229

PROTEIN STRUCTURE

Amino Acids - Two coding variants are noted: cDNA E10 codes for a protein of 838 amino acids, and cDNA D1 codes for a protein of 857 amino acids.

Structural Domains

eyeless has an N-terminal paired domain and a central homeodomain. eyeless is homologous to the mouse Small eye (Pax-6) gene, and to the Aniridia gene in humans. These genes share extensive sequence identity, with 94% sequence identity in the paired domain and 90% identity in the homeodomain. The vertebrate genes are expressed similarly in the developing nervous system and in the eye during morphogenesis (Quering, 1994). In the human paired box, there is a 14 amino acid alternatively spliced extra exon, not present in the fly. One splice site in the homeodomain is shared with humans, but a second, present in the fly, is absent in humans (Quiring, 1994).

Pax6, a transcription factor containing the bipartite paired DNA-binding domain, has critical roles in development of the eye, nose, pancreas, and central nervous system. The 2.5 A structure of the human Pax6 paired domain with its optimal 26-bp site reveals extensive DNA contacts from the amino-terminal subdomain, the linker region, and the carboxy-terminal subdomain. The Pax6 structure not only confirms the docking arrangement of the amino-terminal subdomain as seen in cocrystals of the Drosophila Paired Pax protein, but also reveals some interesting differences in this region and helps explain the sequence specificity of paired domain-DNA recognition. In addition, this structure gives the first detailed information about how the paired linker region and carboxy-terminal subdomain contact DNA. The extended linker makes minor groove contacts over an 8-bp region, and the carboxy-terminal helix-turn-helix unit makes base contacts in the major groove. The structure and docking arrangement of the carboxy-terminal subdomain of Pax6 is remarkably similar to that of the amino-terminal subdomain, and there is an approximate twofold symmetry axis relating the polypeptide backbones of these two helix-turn-helix units. The structure of the Pax6 paired domain-DNA complex provides a framework for understanding paired domain-DNA interactions, for analyzing mutations that map in the linker and carboxy-terminal regions of the paired domain, and for modeling protein-protein interactions of the Pax family proteins (Xu, 1999).

Pax6 genes encode transcription factors with two DNA-binding domains that are highly conserved during evolution. In Drosophila, two Pax6 genes function in a pathway in which twin of eyeless directly regulates eyeless, which is necessary for initiating the eye developmental pathway. To investigate the gene duplication of Pax6 that occurred in holometabolous insects like Drosophila and silkworm, different truncated forms of toy and small eyes (sey, the mouse Pax6 gene) were used, and their capacity to induce ectopic eye development was tested in an ey-independent manner. Even though the Paired domains of TOY and SEY have DNA-binding properties that differ from those of the Paired domain of EY, they all are capable of inducing ectopic eye development in an ey mutant background. One of the main functional differences between toy and ey lies in the C-terminal region of their protein products, implying differences in their transactivation potential. Furthermore, only the homeodomain (HD) of EY is able to downregulate the expression of Distal-less (Dll), a feature that is required during endogenous eye development. These results suggest distinct functions of the two DNA-binding domains of TOY and EY, and significant evolutionary divergence between the two Drosophila Pax6 genes (Punzo, 2004; full text of article).

The results strongly suggest that the functional differences between ey and toy are not only due to their different DNA-binding specificities and changes in the cis-regulatory sequences of their PDs, but also to interactions with different co-factors through their C termini. Recent studies showed that the transcriptional activator Pax5 is converted into a repressor by interaction with the groucho protein through its C terminus and its octapeptide. Similarly, the EY-CT, which differs strongly from that of TOY, is likely to interact with a different set of co-factors to confer specific activation or repression of target genes. This hypothesis is supported by the analysis of the CT. Only the EY-CT, and not that of TOY, is capable of inducing ectopic eyes on the antenna, and only the EY-HD with an EY-CT is able to confer DLL repression, which is required for normal eye development. Thus, these experiments provide new insights into the evolutionary divergence of the two Pax6 genes in Drosophila, and their role in eye and head development (Punzo, 2004).

date revised: 2 December 2018 

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.