homothorax: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

Gene name - homothorax

Synonyms -

Cytological map position - 85F16--86B6

Function - transcription factor

Keywords - segmentation, Hox cofactor

Symbol - htx

FlyBase ID: FBgn0001235

Genetic map position - 3-[49]

Classification - homeodomain and HM domain

Cellular location - cytoplasmic and nuclear

NCBI link: Entrez Gene

homothorax orthologs: Biolitmine

Recent literature
Sharma, P. P., Tarazona, O. A., Lopez, D. H., Schwager, E. E., Cohn, M. J., Wheeler, W. C. and Extavour, C. G. (2015). A conserved genetic mechanism specifies deutocerebral appendage identity in insects and arachnids. Proc Biol Sci 282. PubMed ID: 25948691
The segmental architecture of the arthropod head is one of the most controversial topics in the evolutionary developmental biology of arthropods. The deutocerebral (second) segment of the head is putatively homologous across Arthropoda, as inferred from the segmental distribution of the tripartite brain and the absence of Hox gene expression of this anterior-most, appendage-bearing segment. While this homology statement implies a putative common mechanism for differentiation of deutocerebral appendages across arthropods, experimental data for deutocerebral appendage fate specification are limited to winged insects. Mandibulates (hexapods, crustaceans and myriapods) bear a characteristic pair of antennae on the deutocerebral segment, whereas chelicerates (e.g. spiders, scorpions, harvestmen) bear the eponymous chelicerae. In such hexapods as the fruit fly, Drosophila melanogaster, and the cricket, Gryllus bimaculatus, cephalic appendages are differentiated from the thoracic appendages (legs) by the activity of the appendage patterning gene homothorax (hth). This study shows that embryonic RNA interference against hth in the harvestman Phalangium opilio results in homeonotic chelicera-to-leg transformations, and also in some cases pedipalp-to-leg transformations. In more strongly affected embryos, adjacent appendages undergo fusion and/or truncation, and legs display proximal defects, suggesting conservation of additional functions of hth in patterning the antero-posterior and proximo-distal appendage axes. Expression signal of anterior Hox genes labial, proboscipedia and Deformed is diminished, but not absent, in hth RNAi embryos, consistent with results previously obtained with the insect G. bimaculatus. These results substantiate a deep homology across arthropods of the mechanism whereby cephalic appendages are differentiated from locomotory appendages.

Kumar, R., Chotaliya, M., Vuppala, S., Auradkar, A., Palasamudrum, K. and Joshi, R. (2015). Role of Homothorax in region specific regulation of Deformed in embryonic neuroblasts. Mech Dev [Epub ahead of print]. PubMed ID: 26409112
The expression and regulation of Hox genes in developing central nervous system (CNS) lack important details like specific cell types where Hox genes are expressed and the transcriptional regulatory players involved in these cells. This study has investigated the expression and regulation of Drosophila Hox gene Deformed (Dfd) in specific cell types of embryonic CNS. Using Dfd neural autoregulatory enhancer, it was found that Dfd autoregulates itself in cells of mandibular neuromere. The role was investigated of a Hox cofactor Homothorax (Hth) for its role in regulating Dfd expression in CNS. Hth was found to exhibit a region specific role in controlling the expression of Dfd, but has no direct role in mandibular Dfd neural autoregulatory circuit. These results also suggest that homeodomain of Hth is not required for regulating Dfd expression in embryonic CNS.

Beh, C. Y., El-Sharnouby, S., Chatzipli, A., Russell, S., Choo, S. W. and White, R. (2016). Roles of cofactors and chromatin accessibility in Hox protein target specificity. Epigenetics Chromatin 9: 1. PubMed ID: 26753000
The regulation of specific target genes by transcription factors is central to understanding of gene network control in developmental and physiological processes, yet how target specificity is achieved is still poorly understood. This is well illustrated by the Hox family of transcription factors as their limited in vitro DNA-binding specificity contrasts with their clear in vivo functional specificity. This study generated genome-wide binding profiles for three Hox proteins, Ubx, Abd-A and Abd-B, following transient expression in Drosophila Kc167 cells, revealing clear target specificity and a striking influence of chromatin accessibility. In the absence of the TALE class homeodomain cofactors Exd and Hth, Ubx and Abd-A bind at a very similar set of target sites in accessible chromatin, whereas Abd-B binds at an additional specific set of targets. Provision of Hox cofactors Exd and Hth considerably modifies the Ubx genome-wide binding profile enabling Ubx to bind at an additional novel set of targets. Both the Abd-B specific targets and the cofactor-dependent Ubx targets are in chromatin that is relatively DNase1 inaccessible prior to the expression of Hox proteins/Hox cofactors. It is concluded that there is a strong role for chromatin accessibility in Hox protein binding, and the results suggest that Hox protein competition with nucleosomes has a major role in Hox protein target specificity in vivo.
Neto, M., Naval-Sanchez, M., Potier, D., Pereira, P. S., Geerts, D., Aerts, S. and Casares, F. (2017). Nuclear receptors connect progenitor transcription factors to cell cycle control. Sci Rep 7(1): 4845. PubMed ID: 28687780
The specification and growth of organs is controlled simultaneously by networks of transcription factors. While the connection between these transcription factors with fate determinants is increasingly clear, how they establish the link with the cell cycle is far less understood. This study investigated this link in the developing Drosophila eye, where two transcription factors, the MEIS1 homologue hth and the Zn-finger tsh, synergize to stimulate the proliferation of naive eye progenitors. Experiments combining transcriptomics, open-chromatin profiling, motif analysis and functional assays indicate that these progenitor transcription factors exert a global regulation of the proliferation program. Rather than directly regulating cell cycle genes, they control proliferation through an intermediary layer of nuclear receptors of the ecdysone/estrogen-signaling pathway including EcR, Ftz-f1 and Hr46/DHR3. This regulatory subnetwork between hth, tsh and nuclear receptors might be conserved from Drosophila to mammals, as a significant co-overexpression of their human homologues was found in specific cancer types.
Chang, L. W., Tseng, I. C., Wang, L. H. and Sun, Y. H. (2020). Isoform-specific functions of an evolutionarily conserved 3 bp micro-exon alternatively spliced from another exon in Drosophila homothorax gene. Sci Rep 10(1): 12783. PubMed ID: 32732884
Micro-exons are exons of very small size (usually 3-30 nts). Some micro-exons are alternatively spliced. Their functions, regulation and evolution are largely unknown. This study presents an example of an alternatively spliced 3 bp micro-exon (micro-Ex8) in the homothorax (hth) gene in Drosophila. Hth is involved in many developmental processes. It contains a MH domain and a TALE-class homeodomain (HD). It binds to another homeodomain Exd via its MH domain to promote the nuclear import of the Hth-Exd complex and serve as a cofactor for Hox proteins. The MH and HD domains in Hth as well as the HTh-Exd interaction are highly conserved in evolution. The alternatively spliced micro-exon lies between the exons encoding the MH and HD domains. This study provides clear proof that the micro-Ex8 is produced by alternative splicing from a 48 bp full-length exon 8 (FL-Ex8) and the micro-Ex8 is the first three nt is FL-Ex8. The micro-Ex8 is the ancient form and the 3 + 48 organization of alternatively spliced overlapping exons only emerged in the Schizophora group of Diptera and is absolutely conserved in this group. Several strategies were used to test the in vivo function of the two types of isoforms, and the micro-Ex8 and FL-Ex8 isoforms were found to have largely overlapping functions but also have non-redundant functions that are tissue-specific, which supports their strong evolutionary conservation. Since the different combinations of protein interaction of Hth with Exd and/or Hox can have different DNA target specificity, this finding of alternatively spliced isoforms adds to the spectrum of structural and functional diversity under developmental regulation.
Garaulet, D. L., Moro, A. and Lai, E. C. (2021). A double-negative gene regulatory circuit underlies the virgin behavioral state. Cell Rep 36(1): 109335. PubMed ID: 34233178
Virgin females of many species conduct distinctive behaviors, compared with post-mated and/or pregnant individuals. In Drosophila, this post-mating switch is initiated by seminal factors, implying that the default female state is virgin. However, it was recently shown that loss of miR-iab-4/8-mediated repression of the transcription factor Homothorax (Hth) within the abdominal ventral nerve cord (VNC) causes virgins to execute mated behaviors. This study used genomic analysis of mir-iab-4/8 deletion and hth-microRNA (miRNA) binding site mutants (hth[BSmut]) to elucidate doublesex (dsx) as a critical downstream factor. Dsx and Hth proteins are highly complementary in CNS, and Dsx is downregulated in miRNA/hth[BSmut] mutants. Moreover, virgin behavior is highly dose sensitive to developmental dsx function. Strikingly, depletion of Dsx from very restricted abdominal neurons (SAG-1 cells) abrogates female virgin conducts, in favor of mated behaviors. Thus, a double-negative regulatory pathway in the VNC (miR-iab-4/8 -| Hth -| Dsx) specifies the virgin behavioral state.
Mishra, A. K., Fritsch, C., Voutev, R., Mann, R. S. and Sprecher, S. G. (2021). Homothorax controls a binary Rhodopsin switch in Drosophila ocelli. PLoS Genet 17(7): e1009460. PubMed ID: 34314427
Visual perception of the environment is mediated by specialized photoreceptor (PR) neurons of the eye. Each PR expresses photosensitive opsins, which are activated by a particular wavelength of light. In most insects, the visual system comprises a pair of compound eyes that are mainly associated with motion, color or polarized light detection, and a triplet of ocelli that are thought to be critical during flight to detect horizon and movements. It is widely believed that the evolutionary diversification of compound eye and ocelli in insects occurred from an ancestral visual organ around 500 million years ago. Concurrently, opsin genes were also duplicated to provide distinct spectral sensitivities to different PRs of compound eye and ocelli. In the fruit fly Drosophila melanogaster, Rhodopsin1 (Rh1) and Rh2 are closely related opsins that originated from the duplication of a single ancestral gene. However, in the visual organs, Rh2 is uniquely expressed in ocelli whereas Rh1 is uniquely expressed in outer PRs of the compound eye. It is currently unknown how this differential expression of Rh1 and Rh2 in the two visual organs is controlled to provide unique spectral sensitivities to ocelli and compound eyes. This study shows that Homothorax (Hth) is expressed in ocelli and confers proper rhodopsin expression. Hth was shown to control a binary Rhodopsin switch in ocelli to promote Rh2 expression and repress Rh1 expression. Genetic and molecular analysis of rh1 and rh2 supports that Hth acts through their promoters to regulate Rhodopsin expression in the ocelli. Finally, this study also showed that when ectopically expressed in the retina, hth is sufficient to induce Rh2 expression only at the outer PRs in a cell autonomous manner. It is therefore proposed that the diversification of rhodpsins in the ocelli and retinal outer PRs occurred by duplication of an ancestral gene, which is under the control of Homothorax.
Winant, M., Buhler, K., Clements, J., De Groef, S., Hens, K., Vulsteke, V. and Callaerts, P. (2022). Genome-wide analysis identifies Homothorax and Extradenticle as regulators of insulin in Drosophila Insulin-Producing cells. PLoS Genet 18(9): e1010380. PubMed ID: 36095003
Drosophila Insulin-Producing Cells (IPCs) are the main production site of the Drosophila Insulin-like peptides or dilps which have key roles in regulating growth, development, reproduction, lifespan and metabolism. To better understand the signalling pathways and transcriptional networks that are active in the IPCs publicly available transcriptome data of over 180 highly inbred fly lines were queried for dilp expression, and dilp expression was used as the input for a Genome-wide association study (GWAS). This resulted in the identification of variants in 125 genes that were associated with variation in dilp expression. The function of 57 of these genes in the IPCs was tested using an RNAi-based approach. IPC-specific depletion of most genes was found to result in differences in expression of one or more of the dilps. Then on one of the candidate genes with the strongest effect on dilp expression, Homothorax, a transcription factor known for its role in eye development, was examined further. Homothorax and its binding partner Extradenticle were found to be involved in regulating dilp2, -3 and -5 expression; genetic depletion of both TFs shows phenotypes associated with reduced insulin signalling. Furthermore, evidence is provided that other transcription factors involved in eye development are also functional in the IPCs. In conclusion, this study showed that this expression level-based GWAS approach identified genetic regulators implicated in IPC function and dilp expression.
Buffry, A. D., Kittelmann, S. and McGregor, A. P. (2023). Characterisation of the role and regulation of Ultrabithorax in sculpting fine-scale leg morphology. Front Cell Dev Biol 11: 1119221. PubMed ID: 36861038
Hox genes are expressed during embryogenesis and determine the regional identity of animal bodies along the antero-posterior axis. However, they also function post-embryonically to sculpt fine-scale morphology. To better understand how Hox genes are integrated into post-embryonic gene regulatory networks, this study further analysed the role and regulation of Ultrabithorax (Ubx) during leg development in Drosophila melanogaster. Ubx regulates several aspects of bristle and trichome patterning on the femurs of the second (T2) and third (T3) leg pairs. Repression of trichomes in the proximal posterior region of the T2 femur by Ubx is likely mediated by activation of the expression of microRNA-92a and microRNA-92b by this Hox protein. Furthermore, this study identified a novel enhancer of Ubx that recapitulates the temporal and regional activity of this gene in T2 and T3 legs. Transcription factor (TF) binding motif analysis was used in regions of accessible chromatin in T2 leg cells to predict and functionally test TFs that may regulate the Ubx leg enhancer.The role of the Ubx co-factors Homothorax (Hth) and Extradenticle (Exd) in T2 and T3 femurs was also tested. Several TFs were found that may act upstream or in concert with Ubx to modulate trichome patterning along the proximo-distal axis of developing femurs and that the repression of trichomes also requires Hth and Exd. Taken together our results provide insights into how Ubx is integrated into a post-embryonic gene regulatory network to determine fine-scale leg morphology.
Rader, A. E., Bayarmagnai, B., Frolov, M. V. (2023). Combined inactivation of RB and Hippo converts differentiating Drosophila photoreceptors into eye progenitor cells through derepression of homothorax. Dev Cell, 58(21):2261-2274 PubMed ID: 37848027
The retinoblastoma (RB) and Hippo pathways interact to regulate cell proliferation and differentiation. However, the mechanism of interaction is not fully understood. Drosophila photoreceptors with inactivated RB and Hippo pathways specify normally but fail to maintain their neuronal identity and dedifferentiate. Single-cell RNA sequencing was performed to elucidate the cause of dedifferentiation and to determine the fate of these cells. Dedifferentiated cells were found to adopt a progenitor-like fate due to inappropriate activation of the retinal differentiation suppressor homothorax (hth) by Yki/Sd. This results in the activation of a distinct Yki/Hth transcriptional program, driving photoreceptor dedifferentiation. Rbf physically interacts with Yki and, together with the GAGA factor, inhibits the hth expression. Thus, RB and Hippo pathways cooperate to maintain photoreceptor differentiation by preventing inappropriate expression of hth in differentiating photoreceptors. This work highlights the importance of both RB and Hippo pathway activities for maintaining the state of terminal differentiation.

Before discussing homothorax, a general statement on the related topic of Hox genes: a comprehensive understanding of the regulation of Hox gene function remains a persistent problem for developmental biologists because of the apparent promiscuity with which Hox genes bind to promoters. Transcription of Hox genes are regulated by Polycomb and Trithorax groups of proteins. The phenomenon termed phenotypic suppression suggests that the activities of some HOX proteins are achieved through a mechanism by which some HOX proteins suppress others. Phosphorylation appears to modulate Hox activity, providing still another mechanism to explain Hox cellular specificity. Hox protein DNA binding specificity can be regulated by interaction with other homeodomain proteins, specifically Extradenticle and Exd vertebrate homologs known as Pbx proteins physically interact with Hox genes in targeted promoters.

Yet another layer of regulation is suggested by the observation that Extradenticle subcellular localization is regulated. In the embryonic midgut, both Decapentaplegic (Dpp) and Wingless (Wg) signaling pathways control the subcellular localization of Extradenticle protein. Exd protein is predominantly nuclear in endoderm cells close to the Dpp- and Wg-secreting cells of the visceral mesoderm, but Exd protein is found in the cytoplasm in more distant endoderm cells. Both dpp and wg are required for the nuclear localization of Exd in the endoderm; ectopic expression of dpp and wg expands the domain of nuclear Exd (Mann, 1996). Nuclear accumulation of Exd occurs in a highly regulated pattern. For example, Exd is present at high levels within the nucleus of visceral mesoderm cells at the positions where the gastric caeca and all the midgut constrictions will form. Exd is also present throughout the underlying endoderm, but it is cytoplasmic at both ends of the midgut and only accumulates in nuclei in the central zone. Although it is broader than the region of labial expression, the zone for nuclear accumulation of Exd in the endoderm is similarly centered around parasegment 7 (Aspland, 1997).

In order to identifiy additional factors contributing to Hox specificity, Drosophila mutations that affect embryonic patterning have been examined. Mutations were sought that alter antero-posterior pattern without affecting the expression of the trunk Hox genes. Because of the long history of Drosophila developmental genetics such genes can be found simply through examinations of previously characterized genetic mutants. homothorax is one such mutant; further analysis showed it to be a gene required for nuclear transport of Exd (Rieckhof, 1997).

Mutation in hth appears to phenocopy extradenticle mutants for which the maternal and zygotic exd functions are eliminated. In addition to similar transformations observed in the embryonic cuticle, both hth and exd mutant embryos show a loss of engrailed expression in the ectoderm of stage 12 and older embryos. If the two genes act in the same pathway, then one gene might be epistatic to the other. In fact, when exd function is eliminated, the hth genotype appears to be irrelevant. These data suggest that many of the functions of hth require exd (Rieckhof, 1997).

To test whether hth function is required for Exd's nuclear localization, hth mutant embryos were immunostained with anti-Exd antibody. Throughout embryogenesis, Exd is observed primarily in the cytoplasm in hth mutants. The requirement of Hth for Exd's nuclear localization is apparent in many embryonic tissues, including the ectoderm, visceral mesoderm, and endoderm. This requirement is observed in cells where the signaling molecules Wg and Dpp contribute to Exd's nuclear translocation (e.g., the endoderm). It has been suggested that Wg and Dpp both regulate expression of hth in the domains in which Exd nuclear function is required (Rieckhof, 1997).

How does Exd interact with both Hox and Hth proteins and what is the role of the Hth homeodomain? It has been suggested that for some binding sites, Hth is displaced from the Hth-Exd complex upon the formation of an Exd-Hox-DNA complex, while for other binding sites, the Hth-Exd interaction is maintained, resulting in a Hth-Exd-Hox-DNA complex. The Hth homeodomain and the sequence of the binding site may determine if Hth is displaced or not. Consistent with this idea, it has been observed that in the presence of Exd and the appropriate Hox protein, Hth can bind some Exd-Hox binding sites, but not all. Thus, some, but not all, Exd-Hox binding sites also contain a Hth binding site. In the future, it will be important to determine the function of Hth binding sites in vivo, especially those located close to Exd-Hox binding sites (Rieckhof, 1997).

The Distal-less gene is known for its role in proximodistal patterning of Drosophila limbs. However, Distal-less has a second critical function during Drosophila limb development, that of distinguishing the antenna from the leg. The antenna-specifying activity of Distal-less is genetically separable from the proximodistal (PD) patterning function because certain Distal-less allelic combinations exhibit antenna-to-leg transformations without proximodistal truncations. Distal-less has been shown to act in parallel with homothorax (a previously identified antennal selector gene) to induce antennal differentiation. While mutations in either Distal-less or homothorax cause antenna-to-leg transformations, neither gene is required for the others expression, and both genes are required for antennal expression of spalt. Coexpression of Distal-less and homothorax activates ectopic spalt expression and can induce the formation of ectopic antennae at novel locations in the body, including the head, the legs, the wings and the genital disc derivatives. Ectopic expression of homothorax alone is insufficient to induce antennal differentiation from most limb fields, including those of the wing. Distal-less therefore is required for more than induction of a proximodistal axis upon which homothorax superimposes antennal identity. hth encodes a TALE-class homeodomain protein required for the nuclear localization of a PBC-class homeodomain protein encoded by extradenticle. Based on their genetic and biochemical properties, it is proposed that Homothorax and Extradenticle may serve as antenna-specific cofactors for Distal-less (Dong, 2000).

Animals heterozygous for Dll null alleles exhibit partial antenna-to-leg transformations, indicating that Dll levels may be important for antennal determination. Weak hypomorphic combinations of Dll alleles also lead to partial transformation of the third antennal segment (a3) and the arista into leg-like structures. Intermediate hypomorphic combinations of Dll alleles transform the medial antenna toward leg and exhibit distal truncations. Strong combinations of Dll alleles exhibit more severe antennal truncations. These same allelic combinations result in progressively more severe truncations of the distal leg. Notably, the antenna-to-leg transformations are not a property of a specific subset of Dll alleles, but are observed with all Dll alleles when assayed in appropriate combinations. For the transformation phenotype to be apparent, Dll PD function must be largely intact. This is likely due to the fact that the PD axis must be manifest in order for either antennal or leg identity to occur. The fact that transformation is observed without limb truncation indicates that the antennal selector function is more sensitive to Dll dosage than its PD function. Together, the results of Dll phenotypic analysis indicate that Dll is required for antennal identity, as well as for limb outgrowth (Dong, 2000).

Antp represses hth, thereby restricting hth expression to the proximal region of the leg. Antp also represses sal in the leg. Because antennal sal expression is dependent upon both Dll and hth, it was hypothesized that Antp repression of sal might be mediated via Antp repression of hth, which in turn prevents the overlap of Dll and Hth in the distal leg. Consistent with this possibility, Sal is expressed in Antp null clones in the Dll domain where hth is derepressed. It was therefore thought likely that Antp may be repressing sal expression indirectly by preventing hth from being expressed in the Dll domain of the leg. Since both Dll and Hth are required for antennal differentiation, by preventing the coexpression of Dll and Hth, Antp can preclude antennal development. The explanation for this favored by the authors is that when Hth is ectopically expressed using the GAL4/UAS system, Dll expression is downregulated in the cells producing Hth. These cells would then have Hth, but insufficient Dll. If both are required for antennal differentiation, antennal differentiation would not be possible. Consistent with this idea, a decrease in Dll expression in leg cells ectopically expressing Hth is seen (Dong, 2000).

Could Dll form a functional complex with Hth and Exd in the antenna? Given that Dll and Hth cooperate to regulate antennal differentiation, it is of interest to elucidate the molecular basis of this synergy. Exd and its vertebrate counterpart, Pbx, are known cofactors for a variety of homeodomain proteins, including Labial, Engrailed and Ultrabithorax. Hth is required for retention of Exd in the nucleus and may form part of the functional Exd/Hox complex. Vertebrate homologs of Hth, the Meis and Prep proteins, have been shown to form trimeric complexes with Hox and Pbx proteins. Several lines of evidence now support the idea that Exd and Hth are cofactors for the Dll homeodomain protein in the developing Drosophila antenna. These include: (1) the similar antenna-to-leg transformation phenotypes of Dll, hth and exd mutants; (2) the known physical interactions of Exd and Hth with other homeodomain proteins; (3) the fact that Dll and hth function in parallel to regulate antennal development, and (4) the fact that ectopically expressing Hth can mimic loss of Dll function in the antenna. Testing whether Dll, Hth and Exd interact physically and whether such a complex activates antennal enhancers will be important steps toward understanding limb development and tissue-specific gene regulation (Dong, 2000).

Homothorax, Eyeless and Teashirt act in combination in eye development

In Drosophila, the development of the compound eye depends on the movement of a morphogenetic furrow (MF) from the posterior (P) to the anterior (A) of the eye imaginal disc. Several subdomains along the A-P axis of the eye disc have been described that express distinct combinations of transcription factors. One subdomain, anterior to the MF, expresses two homeobox genes, eyeless (ey) and homothorax, and the zinc-finger gene teashirt (tsh). Evidence suggests that this combination of transcription factors may function as a complex and that their combination plays at least two roles in eye development: it blocks the expression of later-acting transcription factors in the eye development cascade, and it promotes cell proliferation. A key step in the transition from an immature proliferative state to a committed state in eye development is the repression of hth by the BMP-4 homolog Dpp (Bessa, 2002).

Anterior to the MF, at least three cell types can be distinguished by the patterns of Hth, Ey, and Tsh expression. The most anterior domain in the eye field, which is next to the antennal portion of the eye-antennal imaginal disc, expresses Hth, but not Tsh or Ey. In a slightly more posterior domain, all three of these factors are coexpressed (region II). In a more posterior domain, Tsh and Ey, but not Hth, are coexpressed. This domain, which also expresses hairy, is equivalent to the pre-proneural (PPN) domain. The MF, marked by the expression of Dpp, is immediately posterior to the PPN domain, and therefore abuts Tsh + Ey-expressing cells (Bessa, 2002).

Domain II is the only region of the eye-antennal imaginal disc that strongly expresses all three of these transcription factors. Posterior to the MF, Hth, but not Tsh or Ey, is expressed in cells committed to become pigment cells. Hth and Ey, but not Tsh, are coexpressed in a narrow row of margin cells that frame the eye field and separate the main epithelium of the eye disc from the peripodial membrane. Finally, Hth is also strongly expressed in peripodial cells, whereas Ey and Tsh are weakly expressed in a subset of these cells (Bessa, 2002).

The expression patterns of So, Dac, and Eya were also examined in wild-type eye discs. All three of these transcription factors are expressed in the PPN domain but not in domain II. Their expression domains have the same anterior limit but different posterior limits. Furthermore, the anterior limits of their expression domains are not sharp, but instead decrease gradually as Hth levels increase. Thus, cells in the PPN domain express So, Dac, and Eya as well as Tsh, Ey, and Hairy. Anterior to the PPN domain there is a gradual transition into domain II, where cells express Hth, Ey, and Tsh, but not So, Eya, Dac, or Hairy (Bessa, 2002).

In late second/early-third-instar eye discs, before or just as the MF is initiated, most eye disc cells express tsh, hth, and ey, although the levels of Hth are lower close to the posterior margin. Therefore, at this stage of development most eye disc cells express the same combination of transcription factors as domain II of third-instar discs. In both cases, these cells are uncommitted and dividing asynchronously (Bessa, 2002).

The overlapping expression patterns of ey, hth, and tsh in domain II raised the possibility that their gene products could be functioning together. As a first test of this idea, it was determined whether their protein products could interact with each other in vitro and in vivo. Histidine (his)-tagged Hth, alone or together with its partner protein Extradenticle (Exd), specifically binds to 35S-labeled Ey and Tsh in vitro. In vivo, it was found that both Exd and Tsh could be coimmunoprecipitated (IP) from wild-type embryos with Hth. Ey and Hth could not be IPed from wild-type embryos, perhaps because the number of cells coexpressing these transcription factors is too few. These results suggest that Hth, Exd, Tsh, and Ey have the potential to interact with each other in vivo. However, additional experiments are required to definitively test this idea (Bessa, 2002).

Tests were made of the ability of these factors to regulate each other's expression in the eye disc. Clones of cells were generated that express the yeast transcription factor Gal4 in flies containing UAS-Ey, UAS-Hth, or UAS-Tsh transgenes. These clones were generated during the second instar, when all three of these genes are coexpressed throughout the eye disc, and they were analyzed during the third instar, when the Hth expression pattern is distinct from the Tsh and Ey expression patterns. Tsh or Ey overexpressing clones in the PPN domain up-regulate Hth. The ability to maintain Hth expression was limited to the PPN domain; Ey- or Tsh-expressing clones within or posterior to the MF did not alter Hth expression. Hth can maintain Ey and Tsh expression posterior to its normal expression domain. Although this effect was not limited to the PPN domain, it was only observed in ~50% of the clones generated during the second instar, suggesting that other factors or the timing of clone induction limit this response. In addition, ectopic Tsh can also induce Ey expression in a subset of the eye imaginal disc. Together with the protein interaction experiments, the ability of these transcription factors to maintain or induce each other's expression suggests that these proteins may function together in eye development (Bessa, 2002).

Expression of Tsh or Ey maintains Hth expression in the PPN domain, where Tsh and Ey are already expressed. This result is interpreted as suggesting that hth is under two competing controls: maintenance by Tsh and Ey and repression by other factors, in particular the Dpp pathway, and that expressing higher levels of Tsh or Ey can shift this balance in favor of maintenance (Bessa, 2002).

The patterns of Tsh, Ey, and Hth expression in the anterior of the eye disc suggest that hth is repressed by a signal coming from the MF. A good candidate for this signal is Dpp because it can act at long range ahead of the furrow. This idea was tested by eliminating the activity of the Dpp pathway by generating clones of cells mutant for Dpp's downstream transcription factor, mothers against Dpp (mad). mad- clones de-repress hth, consistent with the idea that Dpp represses hth. De-repression of hth was observed in mad- clones that touched the posterior margin of the eye disc as well as in clones within the PPN domain. However, mad- clones close to the MF only partially de-repressed hth, suggesting that other signals present in the MF and acting at short range can also repress hth. One such signal may be Hh, which is sufficient for furrow propagation in the absence of Dpp signaling (Bessa, 2002).

The de-repression of hth in mad- clones suggests that Dpp, expressed from the MF, acts at long range to repress hth. In contrast, tsh and ey are expressed in cells adjacent to the MF, suggesting that these genes are not as sensitive to repression by Dpp. To test this directly, the Dpp pathway was activated in clones of cells by expressing an activated form of the Dpp receptor, Thick veins (Tkv*). Expression of Tkv* completely represses hth, but fails to repress ey. tsh was also not repressed in most Tkv* clones. However, tsh expression was reduced in some clones, suggesting that high levels of Dpp activity may be able to repress tsh. The complete repression of hth, but not ey or tsh, by Tkv* is consistent with the idea that Dpp represses hth, but not ey or tsh, as the MF moves anteriorly. Because ey and tsh are also repressed as the MF moves, there must be another signal coming from the furrow that acts at short range to repress these genes. This signal could be Dl, Hh, or a third, as-yet unidentified, signal (Bessa, 2002).

The complementary patterns of Hth versus So, Eya, and Dac at the transition between domain II and the PPN domain suggested that these factors may also be playing a role in hth repression. To test this idea, clones of cells mutant for eya were examined. eya- clones de-repress hth. Part of this de-repression is probably due to the fact that dpp expression requires eya. However, the de-repression of hth is observed in all eya- cells, even in cells that are next to wild-type, dpp-expressing cells. Thus, Dpp expressed in wild-type neighboring cells is not able to repress hth in adjacent eya- cells. These data suggest that eya is required for Dpp to repress hth in the PPN domain. hth was also de-repressed in dac- clones, suggesting that dac also plays a role in hth repression (Bessa, 2002).

In wild-type eye discs, the anterior edge of the PPN domain, as defined by hairy expression, abuts the posterior edge of Hth expression. This observation suggests that hairy, which is an activated target of Dpp ahead of the MF, might be repressed by Hth. In support of this idea, ectopic Hth expression represses hairy. In addition, in some, but not all, anterior hth- clones hairy was de-repressed. These data suggest that the anterior limit of the PPN domain, defined by hairy expression, is controlled by hth (Bessa, 2002).

In contrast to the clear repression of hairy by Hth, in most cases ectopic expression of Hth is unable to repress dac or eya. Similarly, ectopic expression of Tsh is also generally unable to repress these genes. The few cases in which repression of eya by Tsh or Hth were observed were in the PPN domain, which, significantly, is where these transcription factors are able to maintain each other's expression. In contrast, repression of eya or dac was not observed in clones expressing Tsh or Hth posterior to the MF (Bessa, 2002).

Because Hth is coexpressed and can interact in vitro with Tsh and Ey, the possibility was considered that combinations of these transcription factors might be required to repress eya and dac. Consistent with this idea, it was found that the simultaneous expression of Tsh and Hth efficiently represses eya and dac expression. Importantly, the dual expression of Tsh and Hth is maintained by Ey expression; consequently, these clones expressed all three of these transcription factors. Other pairs of these transcription factors (Hth + Ey and Tsh + Ey) were also tested, and it was found that they can also partially repress eya (Bessa, 2002).

The above results suggest that the combination of Hth + Ey + Tsh, which is normally present in domain II, is able to repress the expression of eya. To test if hth normally plays a role in the repression of these genes, hth- clones were examined. Although hth- clones anterior to the MF are rare, it was found that both dac and eya are de-repressed in anterior hth- clones (Bessa, 2002).

In summary, these data suggest that the combination of the factors expressed in domain II is necessary and sufficient to repress eya and dac. In contrast, Hth is sufficient to repress the pre-proneural gene hairy. Conversely, eya and dac, together with Dpp, repress hth as the MF advances. It is suggested that one function for this reciprocal antagonism may be to prevent premature and uncoordinated differentiation anterior to the MF. However, as the MF advances, hth must be repressed to allow differentiation to occur (Bessa, 2002).

These experiments have shed new light on the nature and function of the cells anterior to the MF. Two domains anterior to the hairy-expressing PPN domain have been defined. One of these domains (II) expresses three transcription factors: Ey, which was already known to play a central role in eye development; Hth, which also plays a role in suppressing eye development in the ventral head, and Tsh, which, because of its ability to induce ectopic eyes elsewhere in the head, has also been implicated in eye development. The results suggest that, although these cells have not committed to become a particular cell type, they are predisposed to become eye tissue. Furthermore, it is suggested that the combination of Hth, Ey, and Tsh performs at least two functions during eye development: it represses the expression of later-acting transcription factors in the eye development cascade, and it promotes cell proliferation. Each of these points is discussed and these findings are integrated with the current view of eye development (Bessa, 2002).

The definition of the PPN domain stems from the observation that the induction of neural cell fates in the eye disc requires at least two signals downstream of Hh. The first signal is Dpp, which creates a zone of cells ahead of the MF, termed the PPN domain, which is competent to receive a second, proneural-inducing signal. Cells in the PPN domain express high levels of hairy. Only cells that receive the Dpp signal are able to respond to the second, shorter-acting signal. This second signal is Dl, which is expressed by cells in and behind the furrow and is required for the down-regulation of hairy. In addition to Dl, neural induction, in particular the initiation of ato expression, may also require another signal that is transduced by the ser/thr kinase raf (Bessa, 2002).

hth has been linked to the PPN domain in three ways: (1) in wild-type eye discs, hth expression abuts hairy expression; (2) Hth represses hairy -- these data suggest that hth defines the anterior limit of hairy expression (3) Dpp is a repressor of hth. Together, these results suggest that the anterior limit of the PPN domain is defined by hth expression, and that, as the MF moves anteriorly, hth is repressed by Dpp, allowing the PPN domain and hairy expression to shift anteriorly. In these experiments, only some anterior hth- clones de-repressed hairy. This result is interpreted as suggesting that hairy expression is both activated by Dpp and repressed by hth. Consequently, hth- cells that do not receive enough Dpp would still be unable to express hairy (Bessa, 2002).

In the absence of Dpp signaling, the MF is still able to progress across the eye disc because other signals, such as Hh, are sufficient for furrow progression. This is consistent with the inference that other short-range signals present in the MF can also repress hth. However, the furrow moves more slowly when it confronts cells that cannot respond to Dpp. The slower progression of the furrow could, in part, be because these cells express Hth. Interestingly, Dpp is not expressed in the MF during retinal morphogenesis in the beetle Tribolium or the grasshopper Schistocerca. The use of dpp in eye development may have been necessary in faster-growing insects like Drosophila to increase the speed of eye morphogenesis. Cells at the lateral and posterior edges of the Drosophila eye disc and in the far anterior of the disc continue to express hth and contribute to non-eye portions of the adult head. Moreover, eya- eye disc cells continue to express hth and contribute to non-eye regions of the head. Taken together, these observations suggest that changing the potency of Dpp's ability to repress hth could be used as a way to both modulate the pace of eye development and to control the ratio of eye-to-head tissue (Bessa, 2002).

These experiments suggest that one of the functions mediated by Ey-Hth-Tsh is to repress eya and dac. This proposal stems from both ectopic expression experiments, showing that the coexpression of Ey, Hth, and Tsh represses these genes, and from loss-of-function experiments, showing that hth- clones anterior to the MF de-repress these genes. Similarly, hth is de-repressed in both eya- and dac- clones, suggesting that this antagonism exists in both directions. Interestingly, the antagonism between these two sets of genes is analogous to that observed in other appendages. In the leg, hth and tsh are required for the development of proximal fates, and have been shown to be mutually antagonistic with dac and Distal-less (Dll), two genes required for intermediate and distal leg fates, respectively. Similarly, in the wing, hth and tsh are required for proximal wing fates, and oppose the activity of vestigial (vg), which is required for more distal wing fates (Bessa, 2002).

It is proposed that the putative Ey-Hth-Tsh complex promotes cell proliferation in early eye discs and in cells anterior to the PPN domain in third-instar discs. This suggestion is based on three observations. (1) In young discs, when most of the growth of the eye disc occurs and before the MF initiates, all eye disc cells express all three of these transcription factors. (2) hth- clones are only rarely observed anterior to the MF. The lack of hth- clones observed in this region of the eye disc suggests that hth is playing an important role in either the survival or proliferation of these cells (Bessa, 2002).

(3) Linking this combination of transcription factors with the growth of the eye disc stems from the observation that, when coexpressed, these factors can induce cell proliferation. This was most readily observed in clones that include cells at the edge of the eye disc. These cells may be unique in the eye disc because they express wg. Interestingly, activation of the wg pathway by generating axin- clones in the eye disc also induces proliferation and the maintenance of ey, hth, and tsh expression. Thus, proliferating eye disc cells express hth, ey, and tsh and are in a state in which the wg pathway is activated. It is speculated that this state, which can be induced by the expression of Tsh, Ey, and Hth at the edge of the eye disc, mimics the normal state of eye disc cells during the second instar, when the disc is growing most rapidly. Consistent with this idea, anterior hth expression in the eye disc is autonomously lost in dishevelled- (dsh-) clones, showing that these cells require wg signaling to maintain their anterior identity (Bessa, 2002).

The proliferation-inducing ability of Ey, Tsh, and Hth is interesting in light of the fact that the mammalian homologs of hth, the meis genes, are proto-oncogenes. The proliferation observed in Drosophila may require wg signaling and the coexpression of tsh, which has been implicated in modulating wg signaling during Drosophila development. Given these findings, it will be of interest to determine if the oncogenic potential of the meis genes also depends on the activities of wg and/or tsh homologs (Bessa, 2002).

Transcription factors often act in unique combinations to elicit distinct biological outputs. The combination examined here is Ey-Hth-Tsh. Because Hth and Tsh are also required for leg and wing development, Ey must make this combination specific for eye development. It is suggested that this combination of factors is used transiently during eye development to promote the proliferation of eye disc cells and to prevent the premature expression of later-acting transcription factors that are required for eye development. Consistent with this second role, ectopic expression of Hth blocks eye development. Similarly, forcing the expression of Ey can also interfere with eye development. The ability of these factors to repress eye development may in part be due to the ability of the Ey-Hth-Tsh combination to repress eya and dac (Bessa, 2002).

In addition to the functions suggested here, Ey is also important for promoting eye morphogenesis and has been called the master regulator of eye development. In fact, Ey is likely to be a direct activator of so. It is speculated that the eye-activating functions of Ey may be carried out in cells that express a different combination of transcription factors from those present in domain II. Cells in the PPN domain, for example, express Ey and Tsh, but not Hth. These cells also express so. It is therefore possible that Ey activates so in the PPN domain. In contrast, Ey-Hth-Tsh appears to repress eya, dac, and, by inference, so. Since all three of these factors are DNA-binding proteins, one possibility is that they are part of a specific DNA-binding complex that directly regulates these, as well as other, target genes in domain II. A different set of target genes may be regulated by Ey (+/-Tsh) in the absence of Hth. A second possibility is that the regulation observed in this study is indirect. Finally, the results are also consistent with a model in which Hth binds to Ey and blocks its ability to bind DNA. Such a mechanism has been proposed to account for repression of eye development by the Hox protein Antennapedia (Antp). Toy, a second Pax6 family member in flies, may also be part of the combinatorial control of eye development described here. An assessment of Toy's role is not possible at present, but will be important to characterize in the future (Bessa, 2002).

The progression of the MF across the eye is an elegant mechanism for gradually changing the combination of transcription factors as development proceeds. So, Eya, and Dac also have the ability to positively activate each other's expression, as is the case with Hth, Ey, and Tsh. Thus, both ahead of and behind the MF, eye disc cells are in different, but relatively stable states, in part because the factors expressed within these regions -- Hth, Tsh, and Ey in domain II and Eya, So, and Dac posterior to the MF -- can reinforce each other's expression. These two states are important for promoting proliferation and differentiation, respectively. Signals coming from the MF convert one state into another, and a key to flipping this switch is the repression of hth. Remarkably, in the vertebrate retina, Sonic hedgehog, a homolog of Drosophila Hh, is expressed in a wave-like fashion as retina cells differentiate. Furthermore, Pax6, the vertebrate ey homolog, is required to keep retinal cells multipotent: this is reminiscent of the uncommitted state of anterior cells in the fly eye disc. Given these intriguing parallels, it will be very interesting to determine if homologs of hth and tsh play analogous roles in the vertebrate retina before the initiation of differentiation (Bessa, 2002).

A double-negative gene regulatory circuit underlies the virgin behavioral state

Virgin females of many species conduct distinctive behaviors, compared with post-mated and/or pregnant individuals. In Drosophila, this post-mating switch is initiated by seminal factors, implying that the default female state is virgin. However, it was recently shown that loss of miR-iab-4/miR-iab-8-mediated repression of the transcription factor Homothorax (Hth) within the abdominal ventral nerve cord (VNC) causes virgins to execute mated behaviors. This study used genomic analysis of mir-iab-4/8 deletion and hth-microRNA (miRNA) binding site mutants (hth[BSmut]) to elucidate doublesex (dsx) as a critical downstream factor. Dsx and Hth proteins are highly complementary in CNS, and Dsx is downregulated in miRNA/hth[BSmut] mutants. Moreover, virgin behavior is highly dose sensitive to developmental dsx function. Strikingly, depletion of Dsx from very restricted abdominal neurons (SAG-1 cells) abrogates female virgin conducts, in favor of mated behaviors. Thus, a double-negative regulatory pathway in the VNC (miR-iab-4/8 -| Hth -| Dsx) specifies the virgin behavioral state (Garaulet, 2021).

Females of diverse invertebrate and vertebrate species coordinate multiple behavioral programs with their reproductive state. Mature female virgins are receptive to male courtship and copulation, but following mating and/or pregnancy, they decrease sexual activity and modulate behaviors to generate and foster their children. Behavioral remodeling associated with the female reproductive state includes increased aggression and nest building in avians and mammals and decreased male acceptance, increased egg-laying, and appetitive/metabolic changes in insects. The genetic and neurological control of this process has been intensively studied in fruit flies, where sexual activity induces the post-mating switch, a host of behavioral changes collectively known as post-mating responses (PMRs) (Garaulet, 2021).

In Drosophila, as in other species, 'virgin' is typically considered the default behavioral state, because factors that induce PMRs are transferred in seminal fluids during copulation. Among these, Sex Peptide (SP) is necessary and sufficient to drive most female post-mated behaviors. SP signals via uterine SP sensory neurons (SPSNs). Some SPSN+ neurons contact abdominal interneurons in the ventral nerve cord (VNC) that express myoinhibitory peptide, which input into a restricted population of ascending neurons (SP abdominal ganglion [SAG] neurons) that project to the posterior brain, including pC1 neurons. This outlines an ascending flow of information for how a seminal fluid peptide can alter female brain activity. The brain integrates this with auditory and visual cues to coordinate diverse behaviors mediated by distinct lineages of descending neurons and VNC populations that modulate specific behaviors according to internal state and external stimuli (Garaulet, 2021).

Recently, it was found that post-transcriptional suppression of the homeobox gene homothorax (hth) within the VNC is critical to implement the virgin behavioral state (Garaulet, 2020). Of note, deletion of the Bithorax Complex (BX-C) locus mir-iab-4/8, point mutations of their binding sites in hth, or deletion of the hth neural-specific 3' UTR extension bearing many of these microRNA (miRNA) sites all cause mutant female virgins to perform mated behaviors. Thus, the failure to integrate two post-transcriptional regulatory inputs at a single target gene prevents females from appropriately integrating their sexual internal state with external behaviors (Garaulet, 2021).

Recognition of the transcription factor Hth as a target of regulatory circuits for virgin behavior implies that downstream loci may serve as a functional output for this process. This study used molecular genetic profiling to identify a critical requirement for Doublesex (Dsx) to implement the female virgin behavioral state. Dsx has been well studied with respect to differentiation of sexually dimorphic traits, but its roles in post-mitotic neurons are little known. This study found that expression of Dsx in the VNC mediates virgin behavior, and that modulation of Dsx in only a few abdominal VNC neurons is sufficient to convert the suite of female virgin behaviors into mated conducts (Garaulet, 2021).

Recent work established how miRNA mediated suppression of the transcription factor Hth to safeguard the virgin female behavioral state (Garaulet, 2020). Using engineered alleles and spatio-temporal hth manipulations, this study demonstrated a developmental requirement for post-transcriptional regulation of Hth within the abdominal ganglion of the CNS for female behavior. However, Hth was not required in otherwise wild-type VT-switch neurons for execution of virgin behaviors, implying that expression of Hth in the abdominal VNC must normally be prevented. This involves integration of two mechanisms: a high density of BX-C miRNA binding sites (miR-iab-4/8) within the hth-HD 3' UTR, as well as a neural-specific 3' UTR elongation, which unveils many of these sites only on neural hth isoforms (Garaulet, 2021).

This study has extended this regulatory axis by showing that loss of BX-C miRNAs, acting through derepressed Hth, leads to downregulation of the Dsx in the abdominal VNC. Dsx is well-known as a master sex determination transcription factor, and it shows localized expression in specific CNS domains. However, although the activity of Dsx-expressing neurons per se has been implicated in the switch in females, the functions of Dsx in post-mitotic neurons are less well defined. This work reveals that Dsx itself is a central component in specifying virgin behavior, because its restricted suppression in as few as four (SAG-1+) neurons is sufficient to induce post-mated behaviors. It remains to be better defined how SAG-1 neurons are affected by depletion of Dsx. No overt differentiation defects were observed, but an effect of masculinization cannot be ruled out. Otherwise, the recent work suggests an activity defect in a general population of switch neurons in the miRNA mutant (Garaulet, 2020), but more direct analysis of dsx-depleted SAG-1 neurons awaits (Garaulet, 2021).

Altogether, in contrast with highly branched regulatory networks that are bioinformatically inferred to lie downstream of individual miRNAs, this study revealed a linear, double-negative regulatory cascade comprising miRNAs and two transcription factors (see SAG-1 neurons specifically require Dsx for a suite of female virgin behaviors). These findings provide impetus to assess possible direct regulation of Dsx by Hth, as well as to elucidate Dsx targets that are relevant to female behavioral control. Overall, this study expands a genetic hierarchy that is essential for females to couple the virgin internal state with appropriate behaviors (Garaulet, 2021).

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).


Recently, a large number of ultraconserved (uc) sequences have been identified in noncoding regions of human, mouse, and rat genomes that appear to be essential for vertebrate and amniote ontogeny. Similar methods were used to identify ultraconserved genomic regions between the insect species Drosophila melanogaster and Drosophila pseudoobscura, as well as the more distantly related Anopheles gambiae. As with vertebrates, ultraconserved sequences in insects appear to occur primarily in intergenic and intronic sequences, and at intron-exon junctions. The sequences are significantly associated with genes encoding developmental regulators and transcription factors, but are less frequent and are smaller in size than in vertebrates. The longest identical, nongapped orthologous match between the three genomes was found within the homothorax (hth) gene. This sequence spans an internal exon-intron junction, with the majority located within the intron, and is predicted to form a highly stable stem-loop RNA structure. Real-time quantitative PCR analysis of different hth splice isoforms and Northern blotting showed that the conserved element is associated with a high incidence of intron retention in hth pre-mRNA, suggesting that the conserved intronic element is critically important in the post-transcriptional regulation of hth expression in Diptera (Glazov, 2005: full text of article).

In a recent study that identified highly evolutionary conserved sequences in three genomes of Diptera species an ultraconserved element found at an internal exon-intron junction of the Drosophila melanogaster homothorax (hth) gene was described that appeared to be involved in the control of hth pre-mRNA splicing. A possible role was discussed of RNA secondary structure at this site in the regulation of hth pre-mRNA splicing. This study identified a shorter evolutionary conserved intronic element within the hth gene that is located downstream of the first element and has sequence complementarity to it. Intramolecular interactions between these two elements would give rise to alternative RNA secondary structures, which in turn may result in differential control of homothorax pre-mRNA splicing. Additional comparative genomic data from several newly available insect genomes is provided supporting the original conclusion that these conserved elements are important in the post-transcriptional regulation of homothorax gene expression in Diptera (Glazov, 2006).


Amino Acids - 458

Structural Domains

Two regions of Drosophila Hth are very similar to the myeloid ecotropic insertion site 1 (Meis1), a murine homeobox protein. One region in the N-terminal third of the protein, termed the Homothorax-MEIS (HM) domain, is identical to MEIS1 in 105/119 amino acids. A second region includes the homeodomain and is identical to MEIS1 in 69/73 amino acids. The Hth homeodomain belongs to an atypical class that is characterized by an extra three amino acids between helices 1 and 2. Exd's homeodomain is also in this class, and the Hth and Exd homeodomains are either identical (or similar) in 27 (37)/63 amino acids (Rieckhof, 1997).

A new Caenorhabditis elegans homeobox gene, ceh-25, is described that belongs to the TALE superclass of atypical homeodomains, which are characterized by three extra residues between helix 1 and helix 2. ORF and PCR analysis reveals a novel type of alternative splicing within the homeobox. The alternative splicing occurs such that two different homeodomains can be generated, which differ in their first 25 amino acids. ceh-25 is an ortholog of the vertebrate Meis genes and it shares a new conserved domain of 130 amino acids with them. A thorough analysis of all TALE homeobox genes was performed and a new classification is presented. Four TALE classes are identified in animals: PBC, MEIS, TGIF and IRO (Iroquois); two types in fungi: the mating type genes (M-ATYP) and the CUP genes; and two types in plants: KNOX and BEL. The IRO class has a new conserved motif downstream of the homeodomain. For the KNOX class, a conserved domain, the KNOX domain, was defined upstream of the homeodomain. Comparison of the KNOX domain and the MEIS domain shows significant sequence similarity revealing the existence of an archetypal group of homeobox genes that encode two associated conserved domains. Thus TALE homeobox genes were already present in the common ancestor of plants, fungi and animals and represent a branch distinct from the typical homeobox genes (Burglin, 1999).

homothorax: Evolutionary Homologs | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

date revised:  25 April 2024

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