InteractiveFly: GeneBrief

brain-specific homeobox: Biological Overview | References

The Drosophila optic lobe comprises a wide variety of neurons forming laminar and columnar structures similar to the mammalian brain. The Drosophila optic lobe may provide an excellent model to investigate various processes of brain development. However, it is poorly understood how neuronal specification is regulated in the optic lobe to form a complicated structure. This study shows that the Brain-specific-homeobox (Bsh) protein, which is expressed in the lamina and medulla ganglia, is involved in specifying neuronal identity. Bsh is expressed in L4 and L5 lamina neurons and in Mi1 medulla neurons. Analyses of loss-of-function and gain-of-function clones suggest that Bsh is required and largely sufficient for Mi1 specification in the medulla and L4 specification in the lamina. Additionally, Bsh is at least required for L5 specification. In the absence of Bsh, L5 is transformed into glial cells (Hasegawa, 2013).

The establishment of precise neuronal circuits is essential for correct brain function. Complex neuronal circuits contain various types of neurons that are connected intricately with one another. Processes that result in the formation of the correct circuits include the specification of neuronal types, the extension of axons to the appropriate places, and the formation of synapses with their correct partners. The specification of neuronal types is an important process in the making of complete neuronal circuits (Hasegawa, 2013).

The Drosophila visual system may serve as a powerful model for neuronal circuit formation because it has only a limited number of neurons but forms sufficiently complex neuronal circuits that can be analyzed comprehensively. In addition, neurogenetic tools that are available in Drosophila allow artificial manipulation of neuronal activity in temporal ly and spatially restricted manner. However, the full picture of development of the Drosophila optic lobe awaits further investigation (Hasegawa, 2013).

The Drosophila retina is composed of 750-800 ommatidia that contain eight types of photoreceptor neurons, denoted as R1-R8. The visual information received in the retina is transmitted to the optic lobe, which is composed of four ganglia; the lamina, medulla, lobula and lobula plate. The complex neuronal circuits in the visual center process various types of visual information, such as motion, color and shape. This paper focuses on the development of the lamina and medulla (Hasegawa, 2013).

The development of the lamina has been studied in some detail. During the third instar larva, photoreceptor neurons extend retinal axons (R axons) to the optic lobe and deliver the inductive signal Hedgehog (Hh) to the lamina precursor cells (LPCs). LPCs divide and become lamina neurons and activate the expression of Dachshund (Dac) and EGF receptor. Spitz, an EGF ligand, is delivered by R axons, received by EGFR and promotes further differentiation of lamina neurons, including the expression of Elav and the formation of lamina columns. Finally, the five types of lamina neurons, L1-L5, become closely associated with R1-R8 axons, forming a lamina cartridge. Although the differentiation of lamina neurons is understood to some extent, how the distinction among L1-L5 neurons is regulated remains unclear (Hasegawa, 2013).

The second visual ganglion, the medulla, contains 40,000 neurons forming tangentially oriented stratifications, which are defined as 10 layers. Medulla neurons are classified by their pattern of arborizations. Some neurons arborize only in the medulla (medulla intrinsic neurons, Mi-neurons), and some send projections back to the lamina (lamina wide-field neurons, Lawf-neurons). Other neurons arborize in both the medulla and the lobula (transmedullary neurons, Tm-neurons) and in the lobula complex (transmedullary Y neurons, TmY neurons). The medulla is the largest part of the optic lobe and is thought to process both color and motion. Although the medulla is considered to play an important role in visual processing, the developmental mechanisms of the medulla remain elusive. During the third instar larva, neuroblasts (NBs) located in the outer proliferation center divide to make ganglion mother cells (GMCs), which divide to produce differentiated neurons. Expression of specific transcription factors in a subset of medulla neurons was examined (Hasegawa, 2011; Morante, 2008). It has been reported previously that neurons produced from NBs express different types of transcription factors according to their birth order to form a concentric expression pattern (Hasegawa, 2011). However, how the differentiation of the medulla neurons is controlled is still unclear (Hasegawa, 2013).

The Bsx family transcription factors are widely conserved homeodomain proteins that are involved in various neuronal processes. For example, mouse Bsx regulates hyperphagia, locomotory behavior (Sakkou, 2007), growth, and nursing (McArthur, 2007). Xenopus Bsx links daily cell cycle rhythms to pineal photoreceptors (D'Autilia, 2010). The Drosophila Bsx protein, Brain-specific homeobox (Bsh), is expressed in the embryonic brain (Jones, 1993), and in the lamina and medulla neurons of larvae and adults (Choe, 2006; Chu, 2006; Hasegawa, 2011; Huang, 1998a; Huang, 1998b; Poeck, 2001; Zhu, 2009). However, the molecular function of Drosophila bsh has not been studied (Hasegawa, 2013).

A previous paper showed that Bsh is expressed in the medulla and that Bsh-positive neurons differentiate into a single type of medulla intrinsic neuron, Mi1. Moreover, Hth, which is expressed in Mi1 together with Bsh, is essential for Mi1 neuron identity (Hasegawa, 2011). This study shows that Bsh is also required for Mi1 neuron specification in the medulla. bsh mutant neurons were transformed to Tm-type neurons, and overexpression of Bsh induced Mi1-like neurons. Moreover, Bsh expression was required for L4 neuronal specification in the L4 neurons of the lamina, and overexpression of Bsh in the lamina induced L4-like neurons. Therefore, Bsh may have roles in neuronal type specification in both the lamina and the medulla. Relatively weak Bsh expression found in L5 lamina neurons may be required for neuronal differentiation of L5. In the absence of bsh, L5 cells were transformed into glial cells (Hasegawa, 2013).

The previous study showed that Hth and Bsh are expressed in a concentric manner in the larval medulla. Bsh is expressed in the outer half of the Hth domain, and these Bsh/Hth double-positive neurons differentiate into a single neuronal type, Mi1 (Hasegawa, 2011). bsh is predicted to encode two isoforms of homeodomain proteins, long Bsh-PB and short Bsh-PA lacking the N-terminal domain of Bsh-PB The homeodomain is located in the C-terminal region that is shared by both isoforms. No conserved motifs are found in the N-terminal region, which is deleted in Bsh-PA (Hasegawa, 2013).

This study has shown that Bsh is expressed in Mi1 neurons, which differentiates into Tm-type neurons in bsh mutant clones. A similar neuronal transformation from local interneuron to projection neuron is observed in hth mutant clones (Hasegawa, 2011). The number of Hth positive cells was decreased in bsh homozygous animals. However, Hth expression was not affected in bsh mutant clones at larval and adult stages, suggesting that the neuronal type change observed in bsh mutants may not have been caused by reduced expression of Hth. The previous paper showed that residual Bsh is still observed in transformed hth mutant neurons. Moreover, expressing UAS-bshPB could not rescue the defect of the hth mutation, suggesting that Bsh alone cannot induce Mi1 neuron identity and both Hth and Bsh are required for Mi1 neuron identity. By contrast, Bsh expression under the control of drf-Gal4 induced Mi1-like neurons. These results seem inconsistent, however, Mi1-like neurons induced by ectopic Bsh expression were still abnormal compared to endogenous Mi1. Expressing Bsh together with Hth induced Mi1-like neurons that were more similar to endogenous Mi1, implying that both Hth and Bsh are required to generate a complete Mi1 neuron (Hasegawa, 2013).

Because Bsh is a homeodomain protein, the neuronal transformation observed in bsh mutants may be a homeotic transformation. Similar neuronal transformation is observed in mouse Hox gene mutants (Jung, 2010). However, the transformations observed in bsh mutant clones are striking compared to those observed in mouse Hox mutants. It is not yet known what happens downstream of Bsh and what kind of genes are expressed in transformed Tm-type neurons. It may be that Bsh represses the expression of unknown transcription factors that specify Tm-type neurons. Identification of such transcription factors will lead to insights into the mechanism of neuronal transformation found in bsh mutant (Hasegawa, 2013).

Bsh is expressed in L4 and L5 neurons. The results suggest that bsh mutation transforms L4 neurons into L3-like neurons, and that ectopic Bsh expression induces L4-like neurons, suggesting that Bsh is necessary and sufficient for L4 neuron specification. Although induction and development of lamina neurons are understood to some extent, almost nothing is known about neuronal type specification in the lamina. It is possible to speculate that Ap acts downstream of Bsh. However, Ap alone could not induce L4-like neurons (data not shown), suggesting that Ap is not sufficient for L4 neuron formation. Ap may cooperate with Bsh and/or other factors to specify L4 neuron identity (Hasegawa, 2013).

In bsh mutant lamina neurons, transformation into L3-like neurons was observed most often. It is not known whether this transformation is specifically oriented to L3. Other mutations that transform, for example, L1 neuron into other neurons may reveal whether L3 is the ground state of lamina neurons. At third instar, L1-L5 all express Dac, but during metamorphosis Dac expression disappears in L2 and is reduced in L5. It will be interesting to determine the mechanism that regulates the changes in Dac expression. The combination of Dac, Bsh, and Ap expression can specify L2, L4 and L5 but cannot distinguish between L1 and L3. Therefore, there must be other transcription factors that distinguish between L1 and L3 (Hasegawa, 2013).

Using L5-Gal4, it was unexpectedly found that L5 neurons are transformed to glial cells in bsh mutant clones. The transformed glial cells are found along the endogenous glial cell layers that are situated adjacent to the L5 cells in adult and pupal lamina. They are located in the lamina cortex and ensheath lamina neuron cell bodies, and are closely positioned to L5, suggesting that they are most likely proximal satellite glial cells (Edwards, 2012). Transformation of L5 to glia may suggest that their developmental mechanisms are coupled to each other. It will be interesting to see if there is developmental relationship between L5 lamina neurons and proximal satellite glial cells in the developing optic lobe (Hasegawa, 2013).

Bsx regulates hyperphagia and locomotory behavior by regulating the expression of Npy and Agrp in the arcuate nucleus of the mouse hypothalamus (Sakkou, 2007). Bsx expression is repressed by repressor element silencing transcription factor (REST) in non-neuronal cells (Park, 2007). Xenopus Bsx links daily cell cycle rhythms with pineal photoreceptors (D'Autilia, 2010), but its downstream targets are not known. Xbsx expression peaks during the night and represses proliferation. Xbsx knockdown prevents the cell cycle exit of photoreceptor precursors that eventually undergo apoptosis. Xbsx overexpression increases the cell cycle exit of photoreceptor precursors and promotes their differentiation. Therefore, Bsx family proteins may have a general role in cell fate determination. If Bsh target genes are studied in Drosophila, insights into the molecular function of Bsh family proteins may be obtained in the future (Hasegawa, 2013).

These results show that Bsh is expressed in the Mi1 medulla neuron and essential for Mi1 neuron identity. Overexpression of Bsh induces Mi1-like neurons that show some of the features of the Mi1 neuron. In the lamina, Bsh is expressed in the L4 and L5 neurons and is required for L4 and L5 neuron specification. Overexpression of Bsh can induce L4-like neurons. Bsh may have roles in specifying neuronal identity in both the lamina and the medulla (Hasegawa, 2013).

A temporal mechanism that produces neuronal diversity in the Drosophila visual center

The brain consists of various types of neurons that are generated from neural stem cells; however, the mechanisms underlying neuronal diversity remain uncertain. A recent study demonstrated that the medulla, the largest component of the Drosophila optic lobe, is a suitable model system for brain development because it shares structural features with the mammalian brain and consists of a moderate number and various types of neurons. The concentric zones in the medulla primordium that are characterized by the expression of four transcription factors, including Homothorax (Hth), Brain-specific homeobox (Bsh), Runt (Run) and Drifter (Drf/Vvl), correspond to types of medulla neurons. This study examined the mechanisms that temporally determine the neuronal types in the medulla primordium. For this purpose, transcription factors were sought that are transiently expressed in a subset of medulla neuroblasts (NBs, neuronal stem cell-like neural precursor cells) and identified five candidates [Hth, Klumpfuss (Klu), Eyeless (Ey), Sloppy paired (Slp) and Dichaete (D)]. The results of genetic experiments at least explain the temporal transition of the transcription factor expression in NBs in the order of Ey, Slp and D. The results also suggest that expression of Hth, Klu and Ey in NBs trigger the production of Hth/Bsh-, Run- and Drf-positive neurons, respectively. These results suggest that medulla neuron types are specified in a birth order-dependent manner by the action of temporal transcription factors that are sequential ly expressed in NBs (Suzuki, 2013).

In the embryonic central nervous system, the heterochronic transcription factors suchas Hb, Kr, Pdm, Cas and Grh are expressed in NBs to regulate the temporal specification of neuronal identity. They regulate each other to achieve sequential changes in their expression in NBs without cell-extrinsic factors. However, expression of the embryonic heterochronic genes was not detected in the medulla NBs.Instead this study found that Hth, Klu, Ey, Slp and D are transiently and sequentially expressed in medulla NBs. The expression of Hth and Klu was observed in lateral NBs, while that of Ey/Slp and D was observed in intermediate and medial NBs, respectively. These observations suggest that the expression of heterochronic transcription factors changes sequentially as each NB ages, as observed in the development of the embryonic central nervous system (Suzuki, 2013).

This study demonstrates that at least three of the temporal factors Ey, Slp and D regulate each other to form a genetic cascade that ensures the transition from Ey expression to D expression in the medulla NBs. Ey expression in NBs activates Slp, while Slp inactivates Ey expression. Similarly, Slp expression in NBs activates D expression, while D inactivates Slp expression. In fact, the expression of Slp is not strong in newer NBs in which Ey is strongly expressed, but is up regulated in older NBs in which Ey is weakly expressed in the wildtype medulla. A similar relationship is found between Slp and D, supporting the idea that Ey, Slp and D regulate each other's expression to control the transition from Ey-expression to D-expression. In the embryonic central nervous system, similar interaction is mainly observed between adjacent genes of the cascade hb-Kr-pdm-cas-grh, and this concept may also be applied to the medulla primordium. The expression pattern and function of Ey, Slp and D suggest that they are adjacent to each other in the cascade of transcription factor expression in medulla NBs (Suzuki, 2013).

However, no such relationship was found between Hth, Klu and the other temporal factors.The sequential expression of Hth and Klu could be regulated by an unidentified mechanism that is totally different from the genetic cascade that controls the transition through Ey-Slp-D. Or, there might be unidentified temporal factors that are expressed in lateral NBs which act upstream of Hth and Klu to regulate their expression. It is necessary to identify additional transcription factors that are transiently expressed in medulla NBs (Suzuki, 2013).

The expression of concentric transcription factors in the medulla neurons correlates with the temporal sequence of neuron production from the medulla NBs (Hasegawa, 2011). In the larval medulla primordium, the neurons are located in the order of Hth/Bsh-, Run- and Drf-positive cells from inside to outside, and these domains are adjacent to each other (Hasegawa, 2011). Given that NBs generate neurons toward the center of the developing medulla, Hth/Bsh-positive neurons are produced at first, and then Run-positive and Drf-positive neurons. Thus Hth/Bsh, Run and Drf were used as markers to examine roles of Hth, Klu, Ey, Slp and D expressed in NBs in specifying types of medulla neurons. The continuous expression of Hth and Ey from NBs to neurons and the results of clonal analyses that visualize the progeny of NBs expressing each one of the temporal transcription factors suggest that the temporal windows of NBs expressing Hth, Klu and Ey approximately correspond to the production of Hth/Bsh-, Run- and Drf- positive neurons, respectively. Indeed, the results of the genetic study suggest that Hth and Ey are necessary and sufficient to induce the production of Hth/Bsh- and Drf-positive neurons,respectively (Hasegawa, 2011, 2013). Ectopic Klu expression at least induces the produc tion of Run-positive neurons (Suzuki, 2013).

Slp and D expression in NBs may correspond to the temporal windows that produce medulla neurons in the outer domains of the concentric zones, which are most likely produced after the production of Drf-positive neurons. The results at least suggest that Slp is necessary and sufficient and D is sufficient to repress the production of Drf-positive neurons. Identification of additional markers that are expressed in the outer concentric zones compared to the Drf-positive domain would be needed to elucidate the roles of Slp and D in specification of medulla neuron types (Suzuki, 2013).

D mutant clones did not produce any significant phenotype except for derepression of Slp expression in NBs. Drf expression in neurons was not affected either. Since D is a Sox family transcription factor, SoxN, another Sox family transcription factor, is a potential candidate molecule that acts together with D in the medulla NBs. However, its expression was found in neuroepithelia cells and lateral NBs that overlap with Hth-positive cells but not with D-positive cells. All the potential heterochronic transcription factors examined in this study are expressed in three to five cell rows of NBs. Nevertheless, one NB has been observed to produce one Bsh- positive and one Run-positive neuron (Hasegawa, 2011). Therefore, the expression pattern of the heterochronic transcription factors is not sufficient to explain the stable production of one Bsh-positive and one Run-positive neuron from a single NB.The combinatorial action of multiple temporal factors expressed in NBs may play important roles in the specification of Bsh- and Run- positive neurons (Suzuki, 2013).

Another possible mechanism that guarantees the production of a limited number of the same neuronal type from multiple rows of NBs expressing a temporal transcription factor could be a mutual repression between concentric transcription factors expressed in medulla neurons. For example, Hth/Bsh, Run and Drf may repress each other to restrict the number of neurons that express either of these transcription factors. However, expression of Run and Drf was not essentially affected in hth mutant clones and in clones expressing Hth (Hasegawa, 2011). Similarly, expression of Hth and Drf was not essentially affected in clones expressing run RNAi under the control of AyGal4, in which Run expression is eliminated. Hth and Run expression was not affected in drf mutant clones (Hasegawa, 2011). These results suggest that Hth/Bsh, Run and Drf do not essentially regulate each other during the formation of concentric zones in the medulla (Suzuki, 2013).

During embryonic development, the heterochronic genes that are expressed in NBs (hb-Kr-pdm-cas-grh) are maintained and act in GMCs to specify neuronal type. Similarly, Hth and Ey are continuously expressed from NBs to neurons, suggesting that their expression may also be inherited through GMCs (Hasegawa, 2011). However, this type of regulatory mechanism may be somewhat modified in the case of Klu, Slp and D (Suzuki, 2013).

Klu is expressed in NBs and GMCs, but not in neurons. Slp and D are predominantly detected in NBs and neurons visualized by Dpn and Elav, respectively. Occasionally, however, expression of D was found in putative GMCs, which are situated between NBs and neurons. Additionally, both D-positive and D-negative cells were found among Miranda-positive GMCs. Slp expression was not found in Miranda-positive GMCs. Finally, D is expressed in medulla neurons forming a concentric zone in addition to its expression in medial NBs. However, D expression was abolished in slp mutant NBs but remained in the mutant neurons, suggesting that D expression in medulla neurons is not inherited from the NBs. These results suggest that Slp and D expression are not maintained from NBs to neurons and that not all the temporal transcription factors expressed in NBs are inherited through GMCs. However, it is possible to speculate that Klu, Slp and D regulate expression of unidentified transcription factors in NBs that are inherited from NBs to neurons through GMCs (Suzuki, 2013).

Integration of temporal and spatial patterning generates neural diversity

In the Drosophila optic lobes, 800 retinotopically organized columns in the medulla act as functional units for processing visual information. The medulla contains over 80 types of neuron, which belong to two classes: uni-columnar neurons have a stoichiometry of one per column, while multi-columnar neurons contact multiple columns. This study shows that combinatorial inputs from temporal and spatial axes generate this neuronal diversity: all neuroblasts switch fates over time to produce different neurons; the neuroepithelium that generates neuroblasts is also subdivided into six compartments by the expression of specific factors (see The OPC neuroepithelium is patterned along its dorsal-ventral axis). Uni-columnar neurons are produced in all spatial compartments independently of spatial input; they innervate the neuropil where they are generated. Multi-columnar neurons are generated in smaller numbers in restricted compartments and require spatial input; the majority of their cell bodies subsequently move to cover the entire medulla. The selective integration of spatial inputs by a fixed temporal neuroblast cascade thus acts as a powerful mechanism for generating neural diversity, regulating stoichiometry and the formation of retinotopy (Erclik, 2017).

The optic lobes, composed of the lamina, medulla and the lobula complex, are the visual processing centres of the Drosophila brain. The lamina and medulla receive input from photoreceptors in the compound eye, process information and relay it to the lobula complex and central brain. The medulla, composed of ~40,000 cells, is the largest compartment in the optic lobe and is responsible for processing both motion and colour information. It receives direct synaptic input from the two colour-detecting photoreceptors, R7 and R8. It also receives input from five types of lamina neuron that are contacted directly or indirectly by the outer photoreceptors involved in motion detection (Erclik, 2017).

Associated with each of the ~800 sets of R7/R8 and lamina neuron projections are 800 medulla columns defined as fixed cassettes of cells that process information from one point in space. Columns represent the functional units in the medulla and propagate the retinotopic map established in the compound eye. Each column is contributed to by more than 80 neuronal types, which can be categorized into two broad classes. Uni-columnar neurons have arborizations principally limited to one medulla column and there are thus 800 cells of each uni-columnar type. Multi-columnar neurons possess wider arborizations, spreading over multiple columns. They compare information covering larger receptor fields. Although they are fewer in number, their arborizations cover the entire visual field (Erclik, 2017).

The medulla develops from a crescent-shaped neuroepithelium, the outer proliferation centre (OPC). During the third larval instar, the OPC neuroepithelium is converted into lamina on its lateral side and into medulla neuroblasts on its medial side. A wave of neurogenesis moves through the neuroepithelial cells, transforming them into neuroblasts; the youngest neuroblasts are closest to the neuroepithelium while the oldest are adjacent to the central brain. Neuroblasts divide asymmetrically multiple times to regenerate themselves and produce a ganglion mother cell that divides once more to generate medulla neurons. Recent studies have shown that six transcription factors are expressed sequentially in neuroblasts as they age: neuroblasts first express Homothorax (Hth), then Klumpfuss (Klu), Eyeless (Ey), Sloppy-paired 1 (Slp1), Dichaete (D) and Tailless (Tll). This temporal series is reminiscent of the Hb --> Kr --> Pdm --> Cas --> Grh series observed in Drosophila ventral nerve cord neuroblasts that generates neuronal diversity in the embryo. Indeed, distinct neurons are generated by medulla neuroblasts in each temporal window. Further neuronal diversification occurs through Notch-based asymmetric division of ganglion mother cells. In total, over 20 neuronal types can theoretically be generated using combinations of temporal factors and Notch patterning mechanisms. However, little is known about how the OPC specifies the additional ~60 neuronal cell types that constitute the medulla (Erclik, 2017).

To understand the logic underlying medulla development, late larval brains were stained with 215 antibodies generated against transcription factors and 35 genes were identifiied that are expressed in subsets of medulla progenitors and neurons. The OPC neuroepithelial crescent can be subdivided along the dorsal-ventral axis by the mutually exclusive expression of three homeodomain-containing transcription factors: Vsx1 is expressed in the central OPC (cOPC), Rx in the dorsal and ventral posterior arms of the crescent (pOPC), and Optix in the two intervening 'main arms' (mOPC). These three proteins are regionally expressed as early as the embryonic optic anlage and together mark the entire OPC neuroepithelium with sharp, non-overlapping boundaries. Indeed, these three regions grow as classic compartments: lineage trace experiments show that cells permanently marked in the early larva in one OPC region do not intermingle at later stages with cells from adjacent compartments. Of note, Vsx1 is expressed in cOPC progenitor cells and is maintained in a subset of their neuronal progeny whereas Optix and Rx are not expressed in post-mitotic medulla neurons. The OPC can be further subdivided into dorsal (D) and ventral (V) halves: a lineage trace with hedgehog-Gal4 (hh-Gal4) marks only the ventral half of the OPC, bisecting the cOPC compartment. As hedgehog is not expressed in the larval OPC, this dorsal-ventral boundary is set up in the embryo. Thus, six compartments (ventral cOPC, mOPC and pOPC and their dorsal counterparts) exist in the OPC. The pOPC compartment can be further subdivided by the expression of the wingless and dpp signalling genes. Cells in the wingless domain behave in a very distinct manner from the rest of the OPC, and have been described elsewhere (Erclik, 2017).

The Hth --> Klu --> Ey --> Slp1 --> D --> Tll temporal progression is not affected by the compartmentalization of the OPC epithelium; the same neuroblast progression throughout the entire OPC. Thus, in the developing medulla, neuroblasts expressing the same temporal factors are generated by developmentally distinct epithelial compartments (Erclik, 2017).

To test whether the intersection of the dorsal-ventral and temporal neuroblast axes leads to the production of distinct neural cell types, focus was placed on the progeny of Hth neuroblasts, which maintain Hth expression. In the late third instar, Hth neurons are found in a crescent that mirrors the OPC (see Distinct neuronal cell types are generated along the dorsal-ventral axis of the OPC). The Notch^ON (N^ON) progeny of Hth⁺ ganglion mother cells express Bsh and Ap, and they are distributed throughout the entire medulla crescent. In contrast, the Notch^OFF (N^OFF) progeny, which are Bsh⁻Hth⁺ neurons, express different combinations of transcription factors, and can be subdivided into three domains along the dorsal-ventral axis: (1) in the cOPC, N^OFFHth⁺ neurons express Vsx1, Seven-Up (Svp) and Lim3; (2) in the pOPC, these neurons also express Svp and Lim3, but not Vsx1; (3) in the ventral pOPC exclusively, these neurons additionally express Teashirt (Tsh). N^OFFHth⁺ cells are not observed in the mOPC. Rather, Cleaved-Caspase-3⁺ cells are intermingled with Bsh⁺ neurons. When cell death is prevented, Bsh⁺Hth⁺ cells become intermingled with neurons that express the N^OFF marker Lim3, confirming that the N^OFFHth⁺ progeny undergo apoptosis in the mOPC (Erclik, 2017).

It was therefore possible to distinguish three regional populations of Hth neurons (plus one that is eliminated by apoptosis) and a fourth population that is generated throughout the OPC. The neuronal identity of each of these populations was identified, as follows. (1) Bsh is a specific marker of Mi1 uni-columnar interneurons that are generated in all regions of the OPC. (2) To determine the identity of Hth⁺N^OFF cOPC-derived neurons, Hth⁺ single cell flip-out clones were generated (using hth-Gal4) in the adult medulla. The only Hth⁺ neurons that are also Vsx1⁺Svp⁺ are Pm3 multi-columnar local neurons. (3) For Hth⁺N^OFF pOPC-derived neurons, 27b-Gal4 was used; it drives expression in larval pOPC Hth⁺N^OFF neurons and is maintained to adulthood. Flip-out clones with 27b-Gal4 mark Pm1 and Pm2 neurons, as well as Hth^- Tm1 uni-columnar neurons that come from a different temporal window. Both Pm1 and Pm2 neurons (but not Tm1) express Hth and Svp. Pm1 neurons also express Tsh, which only labels larval ventral pOPC neurons (Erclik, 2017).

Thus, in addition to uni-columnar Mi1 neurons generated throughout the OPC, Hth neuroblasts generate three region-specific neuronal types: multi-columnar Pm3 neurons in the cOPC; multi-columnar Pm1 neurons in the ventral pOPC; and multi-columnar Pm2 neurons in the dorsal pOPC (Erclik, 2017).

To determine the contribution of the temporal and spatial factors to the generation of the different neuronal fates, the factors were mutated them and whether neuronal identity was lost was examined. To test the temporal axis, hth was mutated. As previously reported, Bsh expression is lost in hth mutant clones. Loss of hth in clones also leads to the loss of the Pm3 marker Svp without affecting expression of Vsx1, indicating that Vsx1 is not sufficient to activate Svp and can only do so in the context of an Hth⁺ neuroblast. Hth is also required for the specification of Pm1 and Pm2 in the pOPC as Svp and Tsh expression is lost in hth mutant larval clones. Ectopic expression of Hth in older neuroblasts is not able to expand Pm1, 2 or 3 fates (on the basis of the expression of Svp) into later born neurons, although it is able to expand Bsh expression. Thus, temporal input is necessary for the specification of all Hth⁺ neuronal fates but only sufficient for the generation of Mi1 neurons (see Temporal and spatial inputs are required for neuronal specification in the medulla. ) (Erclik, 2017).

Next, whether regional inputs are necessary and/or sufficient to specify neuronal fates in the progeny of Hth⁺ neuroblasts was determined. In Vsx1 RNA interference (RNAi) clones, Svp expression is lost in the cOPC but Bsh is unaffected. Additionally, Hth⁺Lim3⁺ cells are absent, suggesting that N^OFF cells undergo apoptosis in these clones. Conversely, ectopic expression of Vsx1 leads to the expression of Svp in mOPC Hth⁺ neurons but does not affect Bsh expression. Therefore, Vsx1 is both necessary and sufficient for the specification of Pm3 fates in the larva. However, unlike the temporal factor Hth, Vsx1 does not affect the generation of Mi1 neurons (Erclik, 2017).

In Rx whole mutant larvae and in mutant clones, Svp⁺Lim3⁺Hth⁺ larval neurons (that is, Pm1 and Pm2 neurons) in the pOPC are lost. Additionally, the Pm1 marker Tsh is lost in ventral pOPC Hth⁺ cells. Consistent with the Vsx1 mutant data, larval Bsh expression is not affected by the loss of Rx. In adults, the Pm1/Pm2 markers (Svp, Tsh and 27b-Gal4) are lost in the medulla (Erclik, 2017).

Ectopic expression of Rx leads to the activation of Svp in mOPC Hth⁺ neurons, but does not affect the expression of Bsh. It also leads to the activation of Tsh, but only in the ventral half of mOPC Hth⁺ neurons, suggesting that a ventral factor acts together with Rx to specify ventral fates. Taken together, the above data show that Rx is both necessary and sufficient for the specification of Pm1/2 neurons but (like Vsx1) does not affect the generation of Mi1 neurons (Erclik, 2017).

Finally, the role of the mOPC marker Optix in neuronal specification was examined. In Optix mutant clones, Svp is ectopically expressed in the mOPC, but Bsh expression is not affected. Of note, these ectopic Svp⁺ neurons fail to express the region-specific Pm markers Vsx1 or Tsh (in ventral clones), which suggests that they assume a generic Pm fate. Conversely, ectopic expression of Optix leads to the loss of Svp expressing neurons in both the cOPC and pOPC but does not affect Bsh. These N^OFF neurons die by apoptosis as no Lim3⁺ neurons are found intermingled with Bsh⁺Hth⁺N^ON neurons. When apoptosis is prevented in mOPC-derived neurons, Svp is not derepressed in the persisting Hth⁺N^OFF neurons, which suggests that Optix both represses Svp expression and promotes cell death in Hth⁺N^OFF neurons (Erclik, 2017).

The above data demonstrate that input from both the temporal and regional axes is required to specify neuronal fates. The temporal factor Hth is required for both Mi1 and Pm1/2/3 specification. The spatial genes are not required for the specification of N^ON Mi1 neurons, consistent with the observation that Mi1 is generated in all OPC compartments. The spatial genes, however, are both necessary and sufficient for the activation (Vsx1 and Rx) or repression (Optix) of the N^OFF Pm1/2/3 neurons. Thus, Hth⁺ neuroblasts generate two types of progeny: N^OFF neurons that are sensitive to spatial input (Pm1/2/3) and N^ON neurons that are refractory to spatial input (Mi1). Vsx1 expression in the cOPC is only maintained in Hth⁺N^OFF neurons, suggesting that spatial information may be 'erased' in Mi1, thus allowing the same neural type to be produced throughout the OPC (Erclik, 2017).

Do spatial genes regulate each other in the neuroepithelium? In Vsx1 mutant clones, Optix (but not Rx) is derepressed in the cOPC epithelium. Conversely, ectopic Vsx1 is sufficient to repress Optix in the mOPC and Rx in the pOPC. Similarly, Optix, but not Vsx1, is derepressed in Rx mutant clones in the pOPC epithelium and ectopic Rx is sufficient to repress Optix in the mOPC (but not Vsx1 in the cOPC). In Optix mutant clones, neither Vsx1 nor Rx are derepressed in the mOPC epithelium, but ectopic Optix is sufficient to repress both Vsx1 in the cOPC and Rx in the pOPC. The observation that Optix is not necessary to suppress Vsx1 or Rx in the mOPC neuroepithelium is surprising because Svp is activated in a subset of Hth⁺ neurons in the mOPC in Optix mutant clones. Nevertheless, when cell death in the mOPC is abolished, the ectopic undead N^OFF neurons express Lim3 but not Svp, which confirms that Optix represses Svp expression in mOPC neurons. Taken together, these results support a model in which Optix is sufficient to repress Vsx1 and Rx, to promote the death of Hth⁺N^OFF neurons and to repress Pm1/2/3 fates (see Spatial genes cross-regulate each other in the OPC neuroepithelium). Vsx1 and Rx act to promote Pm3 (Vsx1) or Pm1/2 (Rx) fates but can only do so in the absence of Optix (Erclik, 2017).

These results suggest that multi-columnar neurons are generated at specific locations in the medulla crescent. However, since these neurons are required to process visual information from the entire retina in the adult medulla, how does the doral-ventral position of neuronal birth in the larval crescent correlate with their final position in the adult? Lineage-tracing experiments were performed with Vsx1-Gal4 to permanently mark neurons generated in the cOPC and with Optix-Gal4 for mOPC neurons, and the position of the cell bodies of these neurons was analyzed. In larvae, neurons from the cOPC or from the mOPC remain located in the same dorsal-ventral position where they were born. However, in adults, both populations have moved to populate the entire medulla cortex along the dorsal-ventral axis (see Neuronal movement during medulla development is restricted to multi-columnar cell types). The kinetics of cell movement during development was analyzed by following cOPC neurons. Neurons born in the cOPC remain tightly clustered until 20 h after puparium formation (P20), after which point the cell bodies spread throughout the medulla cortex. By P30 the neurons are distributed over the entire dorsal-ventral axis of the medulla cortex. In the adult, most neurons derived from the cOPC neuroepithelium are located throughout the cortex although there is an enrichment of neurons in the central region of the cortex (Erclik, 2017).

To determine whether these observed movements involve the entire neuron or just the cell body, the initial targeting of cOPC or mOPC-derived neurons in larvae was examined before the onset of cell movement. In larvae, both populations send processes that target the entire dorsal-ventral axis of the medulla neuropi. Therefore, medulla neurons first send projections to reach their target columns throughout the entire medulla. Later, remodelling of the medulla results in extensive movement of cell bodies along the dorsal-ventral axis, leading to their even distribution in the cortex (Erclik, 2017).

What is the underlying logic behind why some neurons move while others do not? Markers were studied for the Mi1 (Bsh), Pm2 (Hth⁺Svp⁺), Pm1 (Hth⁺Svp⁺Tsh⁺), and Pm3 (Vsx1⁺Svp⁺Hth⁺) populations of neurons through pupal stages and up to the adult. Mi1 neurons are generated evenly throughout the larval OPC and remain regularly distributed across the dorsal-ventral axis at all stages. The lineage-tracing experiment was repeated with Vsx1-Gal4 to follow Mi1 neurons produced by the cOPC. These Mi1 neurons remain exclusively in the centre of the adult medulla cortex, demonstrating that they do not move. In contrast, Pm3 neurons remain tightly clustered in the central region until P20, at which point they move to occupy the entire cortex (Erclik, 2017).

However, not all multi-columnar neurons have cell bodies that move to occupy the entire medulla cortex. Unlike Mi1 and Pm3, adult Pm1 and Pm2 cell bodies are not located in the adult medulla cortex but instead in the medulla rim, at the edges of the cortex. Pm1 and Pm2 markers remain clustered at the ventral (Pm1) or dorsal (Pm2) posterior edges of the medulla cortex throughout all pupal stages. In the adult, both populations occupy the medulla rim from where they send long horizontal projections that reach the entire dorsal-ventral axis of the medulla neuropil. The pOPC may be a specialized region where many of the medulla rim cell types are generated. Even though most of cOPC-derived neurons move during development, a cOPC-derived multi-columnar neuron (TmY₁₄) was identified that sends processes targeting the entire dorsal-ventral length of the medulla neuropil but whose cell bodies remain in the central medulla cortex in the adult (Erclik, 2017).

Thus, the four populations of Hth neurons follow different kinetics: Mi1 neurons are born throughout the OPC and do not move; Pm3 neurons are born centrally and then move to distribute throughout the entire cortex; and Pm1/Pm2 neurons are born at the ventral or dorsal posterior edges of the OPC and occupy the medulla rim in adults (Erclik, 2017).

It is noted that uni-columnar Mi1 neurons, whose cell bodies do not move, reside in the distal cortex whereas multi-columnar Pm3 neurons, which move, reside in the proximal cortex. The hypothesis was thus tested that neurons whose cell bodies are located distally in the medulla cortex represent uni-columnar neurons generated homogeneously throughout the OPC that do not move. In contrast, proximal neurons, which are fewer in number and are generated in specific subregions of the medulla OPC, would be multi-columnar and move to their final position (Erclik, 2017).

It was first confirmed that neurons that move have their cell bodies predominantly in the proximal medulla cortex. The cell body position of neurons born ventrally that have moved dorsally was analyzed using the hh-Gal4 lineage trace: in the adult, the cell bodies found dorsally are mostly in the proximal medulla cortex, whereas the cell bodies in the ventral region are evenly distributed throughout the distal-proximal axis of the ventral cortex. They probably represent both distal uni-columnar neurons that did not move as well as proximal multi-columnar neurons that remained in the ventral region (Erclik, 2017).

Next the pattern of movement of Tm2 uni-columnar neurons from the ventral and dorsal halves of the OPC was analyzed using the hh-based lineage-trace. The cell bodies of Tm2 neurons are located throughout the dorsal-ventral axis in the adult medulla cortex but are co-labelled with the hh lineage marker only in the ventral half. Thus, like Mi1, Tm2 uni-columnar neurons do not move. Furthermore, uni-columnar Tm1 neurons, labelled by 27b-Gal4, are born throughout the dorsal-ventral axis of the OPC crescent with distal cell bodies in the adult, suggesting that they also remain where they were born (Erclik, 2017).

Conversely, it was asked whether neurons that are specified in only one region, such as the Vsx⁺ neurons of the cOPC, are multi-columnar in morphology. By sparsely labelling cOPC-derived neurons using the Vsx1-Gal4 driver, 13 distinct cell types were characterized that retain Vsx1 expression in the adult medulla. Strikingly, all are multi-columnar in morphology, further supporting the model that it is the multi-columnar neurons that move during pupal development (Erclik, 2017).

Finally, MARCM clones were generated in the OPC neuroepithelium and visualized using cell-type-specific Gal4 drivers in the adult medulla. Two classes of adult clone distribution were observed: clones in which neurons are tightly clustered, and clones in which neurons are dispersed. Consistent with the model, the clustered clones are those labelled with uni-columnar neuronal drivers, whereas the dispersed clones are those labelled with a multi-columnar driver (Erclik, 2017).

Taken together, these data demonstrate that neurons that do not move are uni-columnar (with cell bodies in the distal cortex), whereas most multi-columnar neurons (with cell bodies in the proximal cortex) move (Erclik, 2017).

This study shows that combinatorial inputs from the temporal and spatial axes act together to promote neural diversity in the medulla. Previous work has shown that a temporal series of transcription factors expressed in medulla neuroblasts allows for a diversification of the cell types generated by the neuroblasts as they age. This study now shows that input from the dorsal-ventral axis leads to further diversification of the neurons made by neuroblasts; at a given temporal stage, neuroblasts produce the same uni-columnar neuronal type globally as well as smaller numbers of multi-columnar cell types regionally. This situation is reminiscent of the mode of neurogenesis in the Drosophila ventral nerve cord, in which each neuroblast also expresses a (different) temporal series of transcription factors that specifies multiple neuronal types in the lineage. Spatial cues from segment polarity, dorsal-ventral and Hox genes then intersect to impart unique identities to each of the lineages. However, neuroblasts from the different segments give rise to distinct lineages to accommodate the specific function of each segment. In contrast, in the medulla, the entire OPC contributes to framing the repeating units that form the retinotopic map. It is therefore likely that each neuroblast produces a common set of neurons that connect to each pair of incoming R7 and R8 cells, or L1-L5 lamina neurons. This serves to produce 800 medulla columns with a 1:1 stoichiometry of medulla neurons to photoreceptors. The medulla neurons that are produced by neuroblasts throughout the dorsal-ventral axis of the OPC are thus uni-columnar The production of the same neuronal type along the entire OPC could be achieved by selectively 'erasing' spatial information in uni-columnar neurons, as observed in Mi1 neurons (Erclik, 2017).

Regional differences in the OPC confer further spatial identities to neuroblasts with the same temporal identity, and lead to specific differences in the lineages produced in the compartments along the dorsal-ventral axis of the medulla. These differences produce smaller numbers of multi-columnar neurons whose stoichiometry is much lower than 1:1. The majority of these neurons move during development to be uniformly distributed in the adult medulla cortex. This combination of regional and global neuronal specification in the medulla presents a powerful mechanism to produce the proper diversity and stoichiometry of neuronal types and generate the retinotopic map (Erclik, 2017).

Homeodomain proteins hierarchically specify neuronal diversity and synaptic connectivity

How the human brain generates diverse neuron types that assemble into precise neural circuits remains unclear. Using Drosophila lamina neuron types (L1-L5), this study showed that the primary homeodomain transcription factor (HDTF) brain-specific homeobox (Bsh) is initiated in progenitors and maintained in L4/L5 neurons to adulthood. Bsh activates secondary HDTFs Ap (L4) and Pdm3 (L5) and specifies L4/L5 neuronal fates while repressing the HDTF Zfh1 to prevent ectopic L1/L3 fates (control: L1-L5; Bsh-knockdown: L1-L3), thereby generating lamina neuronal diversity for normal visual sensitivity. Subsequently, in L4 neurons, Bsh and Ap function in a feed-forward loop to activate the synapse recognition molecule DIP-β, thereby bridging neuronal fate decision to synaptic connectivity. Expression of a Bsh:Dam, specifically in L4, reveals Bsh binding to the DIP-β locus and additional candidate L4 functional identity genes. It is proposed that HDTFs function hierarchically to coordinate neuronal molecular identity, circuit formation, and function. Hierarchical HDTFs may represent a conserved mechanism for linking neuronal diversity to circuit assembly and function (Xu, 2024a).

HDTFs are evolutionarily conserved factors in specifying neuron-type specific structure and function. In C. elegans, some HDTFs function as terminal selectors, controlling the expression of all neuronal identity genes and diversifying neuronal subtypes, while other HDTFs act downstream of terminal selectors to activate a subset of identity genes. This study shows that the Bsh primary HDTF functions for L4/L5 fate specification by promoting expression of the Ap and Pdm3 secondary HDTFs and suppressing the HDTF Zfh1 to inhibit ectopic L1/L3 fate, thereby generating lamina neuronal diversity. In L4, Bsh and Ap act in a feed-forward loop to drive the expression of synapse recognition molecule DIP-β, thereby bridging neuronal fate decision to synaptic connectivity. DamID data provides support for several hundred Bsh direct binding targets that also show enriched expression in L4 neurons; these Bsh targets include predicted and known L4 identity genes as well as pan-neuronal genes, similar to the regulatory logic first observed in C. elegans. HDTFs are widely expressed in the nervous system in flies, worms, and mammals. By characterizing primary and secondary HDTFs according to their initiation order, it may be possible decode conserved mechanisms for generating diverse neuron types with precise circuits assembly (Xu, 2024a).

How can a single primary HDTF Bsh activate two different secondary HDTFs and specify two distinct neuron fates: L4 and L5? In an accompanying work (Xu, 2024b), Notch signaling was shown to be activated in newborn L4 but not in L5. This is not due to an asymmetric partition of a Notch pathway component between sister neurons, as is common in most regions of the brain, but rather due to L4 being exposed to Delta ligand in the adjacent L1 neurons; L5 is not in contact with the Delta+ L1 neurons and thus does not have active Notch signaling. While Notch signaling and Bsh expression are mutually independent, Notch is necessary and sufficient for Bsh to specify L4 fate over L5. The NotchON L4, compared to NotchOFF L5, has a distinct open chromatin landscape which allows Bsh to bind distinct genomic loci, leading to L4-specific identity gene transcription. It is proposed that Notch signaling and HDTF function are integrated to diversify neuronal types (Xu, 2024a).

DamID (this work) and a scRNAseq dataset were used to identify genomic loci containing both Bsh direct binding sites and L4-enriched expression. Genes that have Bsh:Dam binding peaks but are not detected in L4 scRNA sequencing data at 48h or 60h APF might be due to the following reasons: they are transcribed later, at 60h - 76h APF; the algorithm that was used to detect Bsh:Dam peaks and call the corresponding genes is not 100% accurate; some regulatory regions are outside the stringent +/− 1 kb association with genes; Bsh may act as transcription repressor; TFs generally act combinatorially as opposed to alone and that many required specific cooperative partner TFs to also be bound at an enhancer for gene activation; and scRNAseq data is not 100% accurate for representing gene transcription (Xu, 2024a).

Does the primary HDTF Bsh control all L4 neuronal identity genes? It seems likely, as Bsh:Dam shows binding to L4-transcribed genes that could regulate L4 neuronal structure and function, including the functionally validated synapse recognition molecule DIP-β. Furthermore, Bsh and Ap were found to form a feed-forward loop to control DIP-β expression in L4 neurons. Similarly, in C. elegans, terminal selectors UNC-86 and PAG-3 form a feed-forward loop with HDTF CEH-14 to control the expression of neuropeptide FLP-10, NLP-1 and NLP-15 in BDU neurons, suggesting an evolutionarily conserved approach, using feed-forward loops, for terminal selectors to activate neuronal identity genes. An important future direction would be testing whether Bsh controls the expression of all L4 identity genes via acting with Ap in a feed-forward loop. One intriguing approach would be profiling the Ap genome-binding targets in L4 during the synapse formation window and characterizing the unique and sharing genome-binding targets of Bsh and Ap in L4 neurons. Further, it would be interesting to test whether the primary HDTF Bsh functions with Ap to maintain neuron type-specific morphology, connectivity, and function properties in adults (Xu, 2024a).

Newborn neurons are molecularly distinct prior to establishing their characteristic morphological or functional attributes. The primary HDTF Bsh was discovered to be specifically expressed in newborn L4 and L5 neurons and is required to specify L4 and L5 fates, suggesting that identifying differentially expressed factors in newborn neurons is essential to decoding neuron type specification. It is noted that primary and secondary TFs may be HDTFs as well as non-HDTFs. For example, the primary HDTF Zfh1 is required to activate Svp in L1 and Erm in L3, neither of which are HDTF, though Erm has a significant function in L3 axon targeting. This suggests that the primary HDTF can activate non-HDTFs to initiate neuron identity features. Recent work in Drosophila medulla found that a unique combination of TFs (a mix of HDTFs and non-HDTFs) is required to control neuron identity features. It would be important to dissect whether there is hierarchical expression and function within these TF combinations and to test whether HDTFs activate non-HDTFs (Xu, 2024a).

Evolution can drive a coordinated increase in neuronal diversity and functional complexity. It is hypothesized that there was an evolutionary path promoting increased neuronal diversity by the addition of primary HDTF Bsh expression. This is based on the finding that the loss of a single HDTF (Bsh) results in reduced lamina neuron diversity (only L1-3), which may represent a simpler ancestral brain. A similar observation was described in C. elegans where the loss of a single terminal selector caused two different neuron types to become identical, which was speculated to be the ancestral ground state, suggesting phylogenetically conserved principles observed in highly distinct species. An interesting possibility is that evolutionarily primitive insects, such as silverfish, lack Bsh expression and L4/L5 neurons, retaining only the core motion detection L1-L3 neurons. These findings provide a testable model whereby neural circuits evolve more complexity by adding the expression of a primary HDTF (Xu, 2024a).

Concentric zones, cell migration and neuronal circuits in the Drosophila visual center

The Drosophila optic lobe comprises a wide variety of neurons, which form laminar neuropiles with columnar units and topographic projections from the retina. The Drosophila optic lobe shares many structural characteristics with mammalian visual systems. However, little is known about the developmental mechanisms that produce neuronal diversity and organize the circuits in the primary region of the optic lobe, the medulla. This study describes the key features of the developing medulla and reports novel phenomena that could accelerate understanding of the Drosophila visual system. The identities of medulla neurons are pre-determined in the larval medulla primordium, which is subdivided into concentric zones characterized by the expression of four transcription factors: Drifter, Runt, Homothorax and Brain-specific homeobox (Bsh). The expression pattern of these factors correlates with the order of neuron production. Once the concentric zones are specified, the distribution of medulla neurons changes rapidly. Each type of medulla neuron exhibits an extensive but defined pattern of migration during pupal development. The results of clonal analysis suggest homothorax is required to specify the neuronal type by regulating various targets including Bsh and cell-adhesion molecules such as N-cadherin, while drifter regulates a subset of morphological features of Drifter-positive neurons. Thus, genes that show the concentric zones may form a genetic hierarchy to establish neuronal circuits in the medulla (Hasegawa, 2011).

Concentric genes are expressed in a defined subset of medulla neurons throughout development, suggesting that a part of neuronal identities are pre-determined in the larval medulla primordium. The data suggest that Drf-positive neurons produce nine types of medulla neurons, including lobula projection and medulla intrinsic neurons, while Hth-positive neurons produce at least four types of neurons, including lamina projection and medulla intrinsic neurons. In Hth-positive neurons, Bsh is exclusively expressed in medulla intrinsic Mi1 neurons. A hth mutation caused the neuron to switch type, while a drf mutation affected subsets of morphological features of Drf-positive neurons. Thus, roles of concentric genes may be functionally segregated to form a genetic hierarchy. Apparently, other concentric genes must exist in addition to the four genes reported in this study. Because there are many neurons outside of the Drf domain in the larval medulla, some concentric genes may be expressed in the outer zones. Some transcription factors may have expression patterns that differ from those of concentric genes, and their combined expression may specify restricted subtypes of medulla neurons. For example, apterous (ap) and Cut are widely expressed in medulla neurons. Cut was co- expressed in subsets of Drf-positive neurons, while ap was expressed in all Drf- and Bsh/Hth-positive neurons (Hasegawa, 2011).

Early-born medulla neurons express the inner concentric genes, while late born neurons express the outer ones. Thus, concentric gene expression correlates with neuronal birth order. However, it is still unknown how concentric gene expression is specified. It would be possible to speculate that genes controlling temporal specification of neurons are expressed in NBs to control the concentric gene expression. However, the genes that are known to control neuronal birth order in the embryonic CNS were not expressed in larval medulla NBs. In addition to local temporal mechanisms, such as birth order, global and spatial mechanisms governed by morphogen gradient may also play a role in determining medulla cell type. In addition to birth order or a morphogen gradient, mutual repression among concentric genes may be essential in establishing defined concentric zones. Except for rare occasions, de-repression of other concentric genes was not induced in clones mutant for hth or drf. Additionally, ectopic hth expression did not compromise Drf and Run expression. These results may suggest that unidentified genes act redundantly with these genes to repress expression of other concentric genes and that weak Hth expression in NBs does not play roles in temporal specification of medulla neurons (Hasegawa, 2011).

Various types of cell migration play important roles during vertebrate neurogenesis. Although Drosophila has been a powerful model of neural development, extensive neuronal migrations coupled with layer formation found in this study have not been previously reported. The current findings may establish a model to understand molecular mechanisms that govern brain development via neuronal migrations (Hasegawa, 2011).

It is important to know whether the migration of medulla neurons occurs actively or passively. The distribution of cell bodies in the adult medulla cortex was not random, but organized according to cell type. In particular, the Mi1 neurons identified by Bsh expression migrated outwards and were eventually located in the outermost area of the adult medulla cortex, which was affected in hth mutant clones. The observation that defined localization of cell bodies is under the control of genetic program may not be explained by passive migration. Repression of apoptosis by expressing p35 under the control of elav-Gal4 did not compromise migration of Bsh- and Drf-positive neurons, suggesting that apoptosis is not a driving force of the migration. If neurons migrate actively in an organized manner, what regulates the pattern of migration? In many cases, glial cells play important roles in neuronal migration. Indeed, glial cells and their processes were identified in the medulla cortex. Glial cells or other cell types could provide cues for neuronal migration (Hasegawa, 2011).

The medulla neurons project axons near their targets forming subsets of dendrites in the larval brain; the cell bodies migrated in the presence of preformed neurites during pupal development. During or following cell body migration, additional dendrites were formed along the axonal shafts. Therefore, cell body migration may somehow contribute to circuit formation in the medulla. Indeed, similar strategies have been reported in sensory neurons of C. elegans and cerebellar granule cells in mammals. Thus, cell body migration in the presence of neurites may be a general conserved mechanism of circuit formation. Cell body migration may also allow developing cells to receive inductive cues provided by cells in the vicinity of the medulla cortex. For example, glial cells placed on the surface of the brain may trigger the expression of specific genes (e.g., ChAT) in Mi1 cells that are located in the outermost area of the adult medulla cortex (Hasegawa, 2011).

In adults, Mi1 neurons have arborization sites at M1 and M5, which coincide with the L1 lamina neuron terminals. In Golgi studies, Mi1 neurons were found in all parts of the retinotopic field. Indeed, the number of Bsh expressing medulla neurons was about 800, a figure similar to the number of ommatidial units. Therefore, the Mi1 neurons identified by Bsh expression are most probably columnar neurons with direct inputs from L1 neurons. Because L1 is known to have inputs from R1-6, which processes motion detection, Mi1 may participate in the motion detection circuit (Hasegawa, 2011).

If the genetic codes that specify each type of neuron are found, it may encourage the functional study of defined neurons. In the medulla, bsh-Gal4 is solely expressed by Mi1 neurons. Although the expression of Bsh is also observed in L4/5 lamina neurons, intersectional strategies such as split Gal4 may enable the activity of Mi1 to be specifically manipulated by inducing expression of neurogenetic tools like shibire^ts. This could provide insight into high-resolution functional neurobiology in the Drosophila visual system (Hasegawa, 2011).

Development of the mammalian central nervous system reiteratively establishes cell identity, directs cell migration and assembles neuronal layers, processes similar to the patterns observed during medulla development. In the cerebral cortex, neurons are generated within the ventricular or subventricular zones and migrate outwards, leaving their birthplace along the radial glial fibers. Later-born neurons migrate radially into the cortical plate, past the deep layer neurons and become the upper layers. The layers of the cortex are thus created inside-out. In the developing spinal cord, neuronal types are specified according to morphogen gradients. Within each domain along the dorsoventral axis, neuronal and glial types are specified according to their birth order. The spinal cord neurons then migrate extensively along the radial, tangential and rostrocaudal axes. Therefore, the initial organization of spinal cord neurons is disrupted in the mature system (Hasegawa, 2011).

The medulla shares intriguing similarities with the mammalian central nervous system. For example, the concentric zones established in the larval medulla resemble the dorsoventral subdivisions of the spinal cord. Extensive migrations of medulla neurons disrupt concentric zones, as observed in the spinal cord. However, this study found that the locations of cell bodies were organized according to neuronal type, a distribution that may be similar to the cortical organization of the cerebral cortex. Thus, the development of the medulla may share characteristics with various forms of neurogenesis found in the mammalian central nervous system. A comprehensive study of important features of neurogenesis will now be possible using the Drosophila visual center and powerful tools of Drosophila genetics. Unveiling all aspects of development in the medulla will not only shed light into the functional neurobiology of the visual system, but also elucidate the developmental neurobiology of vertebrates and invertebrates (Hasegawa, 2011).

Photoreceptor neurons find new synaptic targets when misdirected by overexpressing runt in Drosophila

As a neuron differentiates, it adopts a suite of features specific to its particular type. Fly photoreceptors are of two types: R1-R6, which innervate the first optic neuropile, the lamina; and R7-R8, which innervate the second, the medulla. Photoreceptors R1-R6 normally have large light-absorbing rhabdomeres, express Rhodopsin1, and have synaptic terminals that innervate the lamina. In Drosophila melanogaster, the yeast GAL4/UAS system was used to drive exogenous expression of the transcription factor Runt in subsets of photoreceptors, resulting in aberrant axonal pathfinding and, ultimately, incorrect targeting of R1-R6 synaptic terminals to the medulla, normally occupied by terminals from R7 and R8. Even when subsets of their normal R1-R6 photoreceptor inputs penetrate the lamina, to terminate in the medulla, normal target cells within the lamina persist and maintain expression of cell-specific markers. Some R1-R6 photoreceptors form reciprocal synaptic inputs with their normal lamina targets, whereas supernumerary terminals targeted to the medulla also form synapses. At both sites, tetrad synapses form, with four postsynaptic elements at each release site, the usual number in the lamina. In addition, the terminals at both sites are invaginated by profiles of glia, at organelles called capitate projections, which in the lamina are photoreceptor sites of vesicle endocytosis. The size and shape of the capitate projection heads are identical at both lamina and medulla sites, although those in the medulla are ectopic and receive invaginations from foreign glia. This uniformity indicates the cell-autonomous determination of the architecture of its synaptic organelles by the presynaptic photoreceptor terminal (Edwards, 2009).

Expression of lamina monopolar cell-specific proteins, such as BSH in L5 (Poeck, 2001) and FasII in L1 and L3, reveals that these protein markers continue to be expressed despite the mistargeting of photoreceptors. Their expression suggests that cell fate is properly established and maintained in at least three subtypes of monopolar cells and possibly others. In mutant flies, the arrangement of proximally located BSH-labeled L5 neurons is disordered and there appear to be fewer cells overall, presumably because of a reduction in lamina size. FasII expression is also maintained in L1 and L3 at least up to P+60% pupal development, after which time protein expression is downregulated. Although expression of neuron-specific markers Elav, Dachshund, and BSH (Choe, 2006) has been reported in the lamina cells of pathfinding mutants, such expression has previously been examined shortly after lamina cell differentiation, in the third-instar larva. This study reports evidence that the fates of these neurons are maintained through development and that the cells persist in the adult lamina (Edwards, 2009).

The wingless gene is required for embryonic brain development in Drosophila.

The role of the wingless gene has been studied in the embryonic brain development of Drosophila. wingless is expressed in a large domain in the anlage of the protocerebrum and also transiently in smaller domains in the anlagen of the deutocerebrum and tritocerebrum. In the protocerebral and deutocerebral anlagen, wg-expressing neuroblasts are first observed at late stage 10. At both stages 11 and 12, three groups of wg-immunoreactive cells are observed in the embryonic brain. These are referred to as wg-b1, wg-b2 and wg-b3 for cells in the protocerebrum, deutocerebrum and tritocerebrum respectively. At stage 12, the wg-b1 domain represents a significant part of the protocerebral anlage. The wg-b1 domain remains prominent throughout the rest of embryogenesis. At embryonic stage 15, the anterior end of the embryonic brain is Wg positive, with the wg-b1 domain forming a 'cap' around the most anterior part of each hemisphere. The wg-b2 domain consists of markedly fewer cells than the wg-b1 domain. This small domain is formed of two groups of cells that are separated by a small distance. Wg immunoreactivity in the wg-b2 domain disappears at late stage 12. The wg-b3 domain comprises only a few cells and Wg immunoreactivity in the wg-b3 domain also ceases at late stage 12 (Richter, 1998).

Elimination of the wingless gene in null mutants has dramatic effects on the developing protocerebrum; although initially generated, approximately one half of the protocerebrum is deleted in wingless null mutants by apoptotic cell death at late embryonic stages. To characterize this deleted part of the brain in more detail, immunocytochemical labelling studies were carried out for the Brain specific homeobox (Bsh) protein and the Engrailed protein in wg mutant embryos. Bsh is expressed exclusively in the the developing brain; at embryonic stage 15, Bsh immunoreactivity is found in three small groups of cells in the developing protocerebrum. In the mutants, only two groups of Bsh-positive cells remain in the protocerebrum; the Bsh-immunoreactive cells, which are the most anterior cells in the wild-type protocerebrum, are missing in the mutant. En immunostaining in the wild-type embryonic brain labels three small cell clusters at borders of the protocerebral, deutocerebral and tritocerebral anlage, and also a small group of cells in the embryonic protocerebrum, which form the En secondary head spot. In stage 15 mutants, the most anterior group of En positive cells (En secondary head spot) is missing; however, the En cluster in the posterior protocerebrum is still present. Before stage 13, no obvious signs of structural defects in the protocerebrum are observed in wg mutants (Richter, 1998).

Using temperature sensitive mutants, a rescue of the mutant phenotype can be achieved by stage-specific expression of functional Wingless protein during embryonic stages 9-10. This time period correlates with that of neuroblast specification but preceeds the generation and subsequent loss of protocerebral neurons. Ectopic wingless over-expression in gain-of-function mutants results in a dramatically oversized CNS. The oversized brains are patterned to a certain degree, in that the neuromeres can be identified and major axon tracts, like the longitudinal tract, are visible. To investigate the oversized brains further, the expression pattern of the brain specific homeobox (bsh) gene was studied. In the wild type, bsh is expressed in distinct domains of the protocerebrum. In the oversized brains, bsh expressing cells are scattered throughout the anterior brain and the region that expresses bsh is increased more than twofold. This indicates that the embryonic protocerebrum has increased that much in size. It is concluded that wingless is required for the development of the anterior protocerebral brain region in Drosophila. It is proposed that an important role for wingless in this part of the developing brain is the determination of neural cell fate. Interestingly, ectopic expression of Wnt-1 in the mouse spinal cord also causes massive overgrowth of the spinal cord tissue. This overexpression is caused by increased mitosis (Richter, 1998).

A retinal axon fascicle uses spitz, an EGF receptor ligand, to construct a synaptic cartridge in the brain of Drosophila

Photoreceptor axons arriving in the Drosophila brain organize their postsynaptic target field into a precise array of five neuron 'cartridge' ensembles. Hedgehog, an initial inductive signal transported along retinal axons from the developing eye, induces postsynaptic precursor cells to express the Drosophila homolog of the epidermal growth factor receptor (Egfr). HH alone is not sufficient for this cartridge assembly process, which depends on the presence of retinal axons. The Egfr ligand Spitz, a signal for ommatidial assembly in the compound eye, is transported to retinal axon termini in the brain where it acts as a local cue for the recruitment of five cells into a cartridge ensemble. Hedgehog and Spitz thus bring about the concerted assembly of ommatidial and synaptic cartridge units, imposing the 'neurocrystalline' order of the compound eye on the postsynaptic target field (Huang, 1998b).

In the mutant sine oculis, where only a few ommatidia may form in the eye disc, lamina development is restricted to the immediate vicinity of the small number of axons that grow into the lamina target field. The reduced number of arrays of retinal axons in these animals induces a proportionately reduced field of Dachshund-positive cells. A subset of these cells expresses the neuronal marker Elav. The onset of Dac expression is under the control of Hedgehog and neuronal differentiation (as indicated by Elav expression) involves a distinct signal. Is the putative signal for neuronal differentiation restricted to the immediate vicinity of a retinal axon fascicle? When axon fascicles enter a large field of postmitotic lamina precursor cells (LPCs) induced by hh+ somatic clones, Elav-positive cells are found only in the immediate vicinity of retinal axon fascicles. This local inductive effect is also observed for the expression of the gene argos (aos). aos is a direct transcriptional target of Egf receptor activation and encodes a secreted EGF-like product that can act as a negative regulator of Egfr signaling. In the lamina, Aos displays a punctate distribution surrounding the Elav-positive cartridge cells. As in the case of Elav, Hh is not sufficient to induce aos expression in the absence of retinal axons. Retinal axons thus appear to harbor a locally acting neuronal differentiation signal that is distinct from Hh. The local induction of Aos suggests that this signal may act via the Egf receptor (Huang, 1998b).

The Drosophila homolog of the Egf receptor is strongly and specifically expressed by LPCs within the lamina target field. The onset of Egf receptor expression coincides with the terminal division of LPCs at the lamina furrow and the appearance of early markers such as Dac. Egf receptor immunoreactivity is found at higher levels among the older LPCs at the posterior of the lamina. The expression of the EGF receptor in the lamina depends on retinal innervation and is not detected in mutant animals, such as eyes absent (eya), that lack photoreceptor cells. Hh is sufficient for the onset of Egfr expression, as determined by ectopically expressing hh⁺ in the brain of an 'eyeless' animal. R cell differentiation is blocked by the eya¹ mutation or by maintaining hh^ts2 animals at the nonpermissive temperature from a time point in early larval development. Ectopic hh⁺ expression induces the expression of the EGF receptor in its normal anterior-to-posterior gradient. This and additional experiments show that Hh is both necessary and sufficient for the onset of Egf receptor expression in the lamina (Huang, 1998b).

The notion that Egfr activity might play a role in cartridge neuron differentiation is suggested by the observation that elav expression in prospective L1-L5 neurons coincides with the expression of aos, a transcriptional target of Egfr activation in many tissues. To determine whether Egf receptor activity is required for cartridge neuron differentiation, a dominant-negative form of the Egf receptor (DN-Egfr) was used to block Egfr signal reception. In the developing eye, strong ectopic expression of DN-Egfr prevents the formation of ommatidial cell clusters. This effect of DN-Egfr is suppressed by a wild-type Egfr transgene, consistent with the notion that the truncated receptor acts by interfering with Egfr signal reception. Widespred induction of DN-Egfr results in a normal array of photoreceptor axons innervating the lamina target field and induces an apparently normal field of DAC-positive LPCs. However, Elav-positive cells are absent from the lamina in these animals. Large DN-Egfr-expressing somatic clones also lack Elav-positive cells when they include the lamina target field. These observations indicate that Egfr signal reception is required for cartridge neuron differentiation but not for the early steps of lamina development that are under Hh control (Huang, 1998b).

In the developing eye, the Egfr ligand Spitz is required for the differentiation of all ommatidial cell types, with the exception of the founding R8 cell. Spi is expressed initially by the R8 cell and later by additional cells as they join the ommatidial unit. Spi antigen is found on retinal axons as they project into the lamina. Within the developing lamina, Spi is found on retinal axon fascicles immediately adjacent to Elav-positive cartridge neurons. Spi is thus present at the right time and place to be an Egfr-activating ligand required for cartridge neuron differentiation. Additional experiments show that spi is required for cartridge formation (Huang, 1998b).

Spi is synthesized as a transmembrane molecule. An artificially truncated form of Spi (secreted Spi, or sSpi), containing most of the extracellular portion of the molecule, has been shown to activate the Egf receptor both in vivo and in cell culture. In the developing eye, ubiquitous expression of sSpi induces the differentiation of ommatidial precursors without their assembly into ommatidial cell clusters. sSpi expression likewise can trigger ectopic neuronal differentiation within the lamina. To determine whether these ectopic neurons included a bona fide L-neuron cell type, the specimens were stained with an antibody against the Brain specific homeobox (Bsh) protein. Bsh expression is an early marker of L5 differentiation and coincides with the onset of elav expression in a single medial layer in the posterior two-thirds of the lamina. With the expression of sSpi, ectopic Bsh-positive neurons are found throughout the three medial cell layers of the lamina. These include cells at the anterior of the lamina, where Bsh-positive cells are not seen in the wild type. Thus, LPCs that are normally destined for elimination by apoptosis or that undergo neuronal differentiation precociously can assume a proper L-neuron identity. In sum, these data indicate that spi⁺ activity is sufficient for the onset of cartridge neuron differentiation in the lamina (Huang, 1998b).

The role of an individual ommatidial fascicle as the "founder" of a cartridge ensemble, together with the precision of axon pathfinding in this system, serve to impose the "neurocrystalline" order of the compound eye on the developing postsynaptic field. This mechanism yields a precise numerical match of ommatidial and cartridge units. The component axons of an ommatidial fascicle might additionally make important individual contributions to the specification of the number and type of postsynaptic cells in a cartridge. For example, individual R axons may make important individual contributions to the spatial and temporal pattern of Spi expression. A dynamic interplay between the extracellular levels of SPI and its negative regulator, Aos, might provide the tight localization of spi⁺ activity necessary for this axon-cell signaling. Following cartridge neuron differentiation, a remarkable feat of "axon-shuffling" occurs as the six R1-R6 axons of an ommatidial fascicle separate and migrate laterally to form their synaptic connections in a set of six neighboring cartridges. In the adult lamina, a synaptic cartridge thus receives its complement of R1-R6 axons from six ommatidial units whose axons did not contribute to its induction. The assembly of this precise circuitry nonetheless relies on the order imposed on the lamina during its initial inductive phase. A test of this notion may provide a significant insight into the establishment of precise synaptic circuitry in this and other systems (Huang, 1998b).

A new Drosophila homeobox gene, bsh, is expressed in a subset of brain cells during embryogenesis

Homeobox genes have been shown to control the determination of positional, tissue and cellular identity during the development of the fruitfly. Because genes involved in the determination of internal structures derived from neural, mesodermal and endodermal tissues may have been overlooked in conventional genetic screens, this study undertook the identification of new homeobox genes expressed in these internal tissues. The characterization is discribed of one of these new Drosophila homeobox genes, called brain-specific-homeobox (bsh). In embryos, bsh is expressed exclusively in the brain. bsh protein accumulates in approximately 30 cells in each brain hemisphere. One of these bsh expressing cells is closely associated with the terminus of the larval visual nerve (Bolwig's nerve). While deletions of chromosomal interval containing the bsh gene show no dramatic changes in embryonic brain morphology, the expression pattern of the bsh gene suggests that it may play a highly specialized role in the determination and function of cell type in the Drosophila brain (Jones, 1993).

Search PubMed for articles about Bsh

Choe, K. M., Prakash, S., Bright, A. and Clandinin, T. R. (2006). Liprin-α is required for photoreceptor target selection in Drosophila. Proc. Natl. Acad. Sci. 103(31): 11601-6. PubMed ID: 16864799

Chu, T., Chiu, M., Zhang, E. and Kunes, S. (2006). A C-terminal motif targets Hedgehog to axons, coordinating assembly of the Drosophila eye and brain. Dev Cell 10: 635-646. PubMed ID: 16678778

D'Autilia, S., Broccoli, V., Barsacchi, G. and Andreazzoli, M. (2010). Xenopus Bsx links daily cell cycle rhythms and pineal photoreceptor fate. Proc Natl Acad Sci U S A 107: 6352-6357. PubMed ID: 20308548

Edwards, T. N. and Meinertzhagen, I. A. (2009). Photoreceptor neurons find new synaptic targets when misdirected by overexpressing runt in Drosophila. J Neurosci 29: 828-841. PubMed ID: 19158307

Edwards, T. N., Nuschke, A. C., Nern, A. and Meinertzhagen, I. A. (2012). Organization and metamorphosis of glia in the Drosophila visual system. J Comp Neurol 520: 2067-2085. PubMed ID: 22351615

Erclik, T., Li, X., Courgeon, M., Bertet, C., Chen, Z., Baumert, R., Ng, J., Koo, C., Arain, U., Behnia, R., Rodriguez, A. D., Senderowicz, L., Negre, N., White, K. P. and Desplan, C. (2017). Integration of temporal and spatial patterning generates neural diversity. Nature [Epub ahead of print]. PubMed ID: 28077877

Hasegawa, E., Kitada, Y., Kaido, M., Takayama, R., Awasaki, T., Tabata, T. and Sato, M. (2011). Concentric zones, cell migration and neuronal circuits in the Drosophila visual center. Development 138: 983-993. PubMed ID: 21303851

Hasegawa, E., Kaido, M., Takayama, R. and Sato, M. (2013). Brain-specific-homeobox is required for the specification of neuronal types in the Drosophila optic lobe. Dev Biol 377: 90-99. PubMed ID: 23454478

Huang, Z. and Kunes, S. (1998a). Signals transmitted along retinal axons in Drosophila: Hedgehog signal reception and the cell circuitry of lamina cartridge assembly. Development 125: 3753-3764. PubMed ID: 9729484

Huang, Z., Shilo, B. Z. and Kunes, S. (1998b). A retinal axon fascicle uses spitz, an EGF receptor ligand, to construct a synaptic cartridge in the brain of Drosophila. Cell 95: 693-703. PubMed ID: 9845371

Jones, B. and McGinnis, W. (1993). A new Drosophila homeobox gene, bsh, is expressed in a subset of brain cells during embryogenesis. Development 117: 793-806. PubMed ID: 8101170

Jung, H., Lacombe, J., Mazzoni, E. O., Liem, K. F., Jr., Grinstein, J., Mahony, S., Mukhopadhyay, D., Gifford, D. K., Young, R. A., Anderson, K. V., Wichterle, H. and Dasen, J. S. (2010). Global control of motor neuron topography mediated by the repressive actions of a single hox gene. Neuron 67: 781-796. PubMed ID: 20826310

McArthur, T. and Ohtoshi, A. (2007). A brain-specific homeobox gene, Bsx, is essential for proper postnatal growth and nursing. Mol Cell Biol 27: 5120-5127. PubMed ID: 17485440

Morante, J. and Desplan, C. (2008). The color-vision circuit in the medulla of Drosophila. Curr Biol 18: 553-565. PubMed ID: 18403201

Park, S. Y., Kim, J. B. and Han, Y. M. (2007). REST is a key regulator in brain-specific homeobox gene expression during neuronal differentiation. J Neurochem 103: 2565-2574. PubMed ID: 17944879

Poeck, B., Fischer, S., Gunning, D., Zipursky, S. L. and Salecker, I. (2001). Glial cells mediate target layer selection of retinal axons in the developing visual system of Drosophila. Neuron 29: 99-113. PubMed ID: 11182084

Richter, S., Hartmann, B. and Reichert, H. (1998). The wingless gene is required for embryonic brain development in Drosophila. Dev. Genes Evol. 208(1): 37-45. PubMed ID: 9518523

Sakkou, M., Wiedmer, P., Anlag, K., Hamm, A., Seuntjens, E., Ettwiller, L., Tschop, M. H. and Treier, M. (2007). A role for brain-specific homeobox factor Bsx in the control of hyperphagia and locomotory behavior. Cell Metab 5: 450-463. PubMed ID: 17550780

Suzuki, T., Kaido, M., Takayama, R. and Sato, M. (2013). A temporal mechanism that produces neuronal diversity in the Drosophila visual center. Dev Biol 380: 12-24. PubMed ID: 23665475

Xu, C., Ramos, T. B., Marshall, O. J., Doe, C. Q. (2024b). Notch signaling and Bsh homeodomain activity are integrated to diversify Drosophila lamina neuron types. Elife, 12 PubMed ID: 38193901

Xu, C., Ramos, T. B., Rogers, E. M., Reiser, M. B., Doe, C. Q. (2024a). Homeodomain proteins hierarchically specify neuronal diversity and synaptic connectivity. Elife, 12 PubMed ID: 38180023

Zhu, Y., Nern, A., Zipursky, S. L. and Frye, M. A. (2009). Peripheral visual circuits functionally segregate motion and phototaxis behaviors in the fly. Curr Biol 19: 613-619. PubMed ID: 19303299

date revised: 20 December 2024

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