BIOLOGICAL OVERVIEW

Two closely linked genes, sloppy paired 1 and 2, share the combined characteristics of gap, pair rule and segment polarity genes. Because slp2 is dispensible, particularly in the head, slp1 will be the major concern in this discussion, and will be referred to as sloppy paired (slp). The two genes are first expressed in the head where they act like gap genes, but their action in the trunk is more like pair rule and segment polarity genes.

Expression of slp1 in the head is independent of the pair rule genes, which regulate segment polarity genes in the trunk. Combinatorial inputs from gap genes establish the domains of segment polarity genes in the head. The gap genes, in combination with sloppy paired , define seven regions. Thus gap genes subdivide the head into the various segments which define the adult head stucture (Grossniklaus, 1994).

In the trunk, sloppy paired is expressed in the posterior cells of each parasegment, acting like a segment polarity gene. This expression abuts cells expressing engrailed. Sloppy paired activates wingless and represses engrailed. This function is critical for the sharp boundary of expression between en and wg (Cadigan, 1994a).

Sloppy paired and Even-skipped are involved in cell fate determination and segmentation in the Drosophila mesoderm. In wild-type embryos, slp1 first appears during gastrulation (stage 6) in a pattern of 7 stripes. slp is expressed in both ectoderm and mesoderm. A second set of stripes appears between the first 7 such that a regular 14-stipe pattern is generated by stage 7. The primordia for heart, fat body, and visceral and somatic muscles arise in specific areas of each segment in the Drosophila mesoderm. The primordium of the somatic muscles, which expresses high levels of twist, a crucial factor of somatic muscle determination, is lost in sloppy-paired mutants. The effect of slp on Twist levels is probably partly, but not completely mediated by wingless. wg mutant embryos show a premature and ectopic decay of Twist, but not to the same degree as seen in slp embryos. Whereas patches of cells expressing high levels of Twist are initially established in wg mutant embryos, no Twist is seen in the trunk region of slp mutant embryos after stage 11. At the same time that twist expression is lost in slp mutants, the primordium of the visceral muscles is expanded (Riechmann, 1997).

bagpipe and serpent expressing mesodermal domains corresponding to the ectodermal even-skipped domains, alternate with the sloppy-paired expressing high-twist mesodermal domains. Ectodermal even-skipped is thought to act through engrailed and subsequently hedgehog to promote bagpipe expression in cardiac and dorsal muscle and serpent in the fat body (Azpiazu, 1996). Ectodermal Dpp is required for the maintenance of mesodermal tinman, which in turn activates bap expression in the eve domain. The visceral muscle and fat body primordia require even-skipped for their development and the mesoderm is thought to be unsegmented in even-skipped mutants. However, it has been found that even-skipped mutants retain the segmental modulation of the expression of twist. Both the domain of even-skipped function and the level of twist expression are regulated by sloppy-paired; eve serves reciprocally to regulate the slp domain. sloppy-paired thus controls segmental allocation of mesodermal cells to different fates (Riechmann, 1997).

Wingless (Wg) and other Wnt proteins play a crucial role in a number of developmental decisions in a variety of organisms. In the ventral nerve cord of the Drosophila embryo, Wg, signaling from row 5 is non-autonomously required for the formation and specification of a neuronal precursor cell, NB4-2. NB4-2 gives rise to a well-studied neuronal lineage, the RP2/sib lineage. While the various components of the Wg-signaling pathway are also required for generating NB4-2, the target gene(s) of this pathway in the signal-receiving cell is not known. In this paper, it is shown that sloppy paired 1 and sloppy paired 2 function as the downstream targets of the Wg signaling to generate the NB4-2 cell. Thus, while the loss-of-function mutations in wg and slp have the same NB4-2 formation and specification defects, these defects in wg mutants can be rescued by expressing slp genes from a heterologous promoter. The fact that slp genes function downstream of the Wg signaling is also indicated by the result that expression of slp genes is lost from the neuroectoderm in wg mutants and that ectopic expression of wg induces ectopic expression of slp. Finally, Gooseberry (Gsb) prevents Wg from specifying NB4-2 identity to the wg-expressing NB5-3. In this paper, it is shown that gsb interacts with slp and prevents Slp from specifying NB4-2 identity in NB5.3. Overexpression of slp overcomes this antagonistic interaction and respecifies NB5-3 as NB4-2. This respecification, however, can be suppressed by a simultaneous overexpression of gsb at high levels. This mechanism appears to be responsible for specifying NB5-3 identity to a row 5 neuroblast and preventing Wg from specifying NB4-2 identity to that neuroblast (Bhat, 2000).

NB4-2 is delaminated from an equivalence group of 4-6 neuroectodermal cells during the second wave of neuroblast delamination in mid stage 9 (approximately 4.5 hours old) of embryogenesis. It is located in the 4th column along the anterior-posterior axis and 2nd row along the medio-lateral axis within each hemisegment. The NB4-2 undergoes its first asymmetric division approximately 1.5 hours after formation to self renew and to generate its first GMC, GMC-1 (this GMC-1 is also called GMC4-2a: the first GMC generated from NB4-2). The GMC-1 divides about 1.5 hours later to generate two cells, the larger RP2 and the smaller sib. The RP2 cell migrates to its specific position within the anterior commissure and projects its axon antero-ipsilaterally to the intersegmental nerve bundle (ISN) and innervates muscle #2 on the dorsal musculature. The sib cell migrates to a position posterior and more dorsal to RP2. NB4-2, GMC-1, RP2 and RP2-sib cells can be reliably identified by their gene expression pattern, physical sizes and position within the half-segment (Bhat, 2000).

In the epidermis, mutation in slp genes result in a fusion of abdominal segments A1-A2, A3-A4, A5-A6 and A7-A8 (characteristic of pair-rule mutants) and replacement of naked cuticle by denticle belts, a wg-type of segment polarity phenotype. During the patterning of the epidermis, slp genes function upstream of wg to maintain wg expression (Cadigan, 1994a and b). Since wg is also required for the formation and specification of NB4-2 identity, it is possible that the effect of loss of slp genes on NB4-2 is mediated via its effect on wg expression. Therefore, to determine the precise temporal requirement of slp for maintaining wg expression during neurogenesis, the expression of wg in slp mutant embryos was first examined. In slp mutants the wg expression begins to fade from the neuroectoderm initially in even-numbered parasegments approximately 3.75 hours of development (stage 7, early germ band extension). This fading is particularly prominent in abdominal segments. By approximately 4.5 hours of development (early stage 9), wg expression in these parasegments is completely lost. By contrast, in odd numbered parasegments, wg expression is nearly as high as in wild type during early stage 9 (approximately 4.5 hours of development), and is only lost by approximately 6-6.5 hours of development (stage 10). These results are consistent with the previous findings (Cadigan, 1994a and b) and show that slp genes function upstream of wg and positively regulate wg expression (Bhat, 2000).

Studies using a temperature-sensitive allele of wg have revealed that Wg activity is required for the specification of NB4-2 identity at approximately 4 hours of development (between early to mid-stage 8, at 22°C). However, in slp mutants the expression of wg is still high in the odd-numbered parasegments around the time of NB4-2 specification and the expression of wg is lost in these parasegments only by approximately 6.5 hours of development (stage 10), nearly 2.5 hours after the specification of NB4-2 identity. Thus the loss of Wg expression from odd-numbered parasegments is well past the temporal requirement of wg for NB4-2 specification. Therefore, at least in the odd-numbered parasegments, the specification of NB4-2 identity in slp mutants must occur earlier than the decay of wg expression. Therefore, it is concluded that the loss of NB4-2 identity in slp mutants is unlikely due to the loss of wg expression, at the least in the odd-numbered parasegments, and possibly in the even-numbered parasegments as well (Bhat, 2000).

While the evidence to support the conclusion that slp genes regulate expression of wg in the epidermis is quite strong (Cadigan, 1994a and b), the evidence that the wg-signaling controls the expression of slp in the CNS is also equally strong. (1) The slp genes are expressed not only in the wg-expressing row 5 cells but also in the Wg-negative, but Wg-receiving row 4 cells. (2) The expression of slp is affected in wg mutant embryos. That is, staining of wg mutant embryos show that the expression of slp is lost from the Wg-receiving row 4 neuroectodermal cells. This result is also supported by the western analysis of embryo extract from wg mutants in which the level of Slp protein is found to be greatly reduced. (3) Consistent with the above result, the ectopic expression of wg induces ectopic expression of slp in the neuroectoderm. (4) In slp mutants, just as in wg mutants, the formation and identity specification of a well-studied neuronal precursor cell, NB4-2, is affected; this defect in wg mutants can be rescued by the expression of slp genes from a heterologous promoter. Moreover, a similar relationship also appears to exist between slp and wg during mesoderm specification. For instance, in both wg and slp mutants, the specification of heart cells (derived from mesoderm) is affected and this defect in wg mutants can be rescued by expressing slp genes from a heterologous promoter. These results therefore indicate that wg is a positive regulator of slp expression not only during neurogenesis but also in other processes such as mesoderm specification (Bhat, 2000).

An intriguing aspect of regulation of slp genes by wg is the finding that this regulation is restricted primarily to the neuroectoderm but not extended to the neuroblasts that are derived from these neuroectodermal cells, with one exception: the NB4-2. Thus, while row 4 neuroectodermal cells in wg mutant are missing slp1 expression, row 4 neuroblasts other than NB4-2 have slp1 expression. The induction/maintenance of slp1 expression in these neuroblasts must, therefore, necessarily be under the control of some other pathway. Alternatively, the Wg-signaling pathway is redundant in these neuroblasts. These results are consistent with the finding that the induction of an ectopic gsb-stripe by gain-of-function wg occurs only in the neuroectodermal cells but not in the underneath neuroblasts. In summary, these results reveal a hitherto unsuspected relationship between wg and slp in the CNS that is the opposite of their relationship in the epidermis (Bhat, 2000).

Using a temperature-sensitive allele of wg, it has been shown that the requirement of Wg in the CNS for NB4-2 formation and specification is between late stage 7 and early stage 8 and precedes Wg requirement for epidermal patterning. Moreover, it is the neuroectodermal expression of wg that regulates NB4-2 formation and identity specification. Thus, while the timing of decay of wg expression in slp mutants in even-numbered parasegments coincides with the requirements of wg for NB4-2 formation and specification, it is not so in the odd-numbered parasegments. Thus, the odd-numbered parasegments in slp mutants have wg expression during the time Wg is required for NB4-2 formation and specification. Since the loss of NB4-2 lineage in slp mutants is not parasegment-specific and the expression of slp in NB4-2 and its precursor neuroectodermal cells is lost in wg mutants, it must be that slp genes are downstream of wg in these CNS cells (Bhat, 2000).

The Wg signal regulates the specification of NB4-2 identity via Armadillo and Pangolin. The Arm-Pan signaling complex must activate certain downstream target gene(s), presumably transcription factors, and these transcription factors then initiate a program that mediates the formation and specification of NB4-2. slp genes function as downstream targets of the wg signaling, regulating both the NB4-2 formation as well as the identity specification during neurogenesis. This conclusion is based on the following facts: (1) the loss of function effect for slp genes has the same effect as the loss of function for wg on NB4-2 lineage; (2) the loss of wg activity in row 5 cells in the CNS leads to a loss of slp expression from the Wg-receiving NB4-2 and its precursor cell; (3) the loss of NB4-2 in wg mutants can be rescued by the expression of slp genes from a heterologous promoter during the time when wg is known to be required for the process. It is acknowledged that the slp genes might be either the direct targets of the Wg-signaling pathway (i.e. Arm-Pan complex directly activating slp genes), or instead there may be additional genes in between pan and the slp genes. While this issue has not been resolved here, the rescue of the NB4-2 lineage defect in wg mutants by expressing slp genes from a heterologous promoter reveals that the Wg-signaling pathway must ultimately activate slp genes, and the slp genes then regulate the formation and specification of NB4-2 (Bhat, 2000).

Temporal patterning of Drosophila medulla neuroblasts controls neural fates

In the Drosophila optic lobes, the medulla processes visual information coming from inner photoreceptors R7 and R8 and from lamina neurons. It contains approximately 40,000 neurons belonging to more than 70 different types. This study describes how precise temporal patterning of neural progenitors generates these different neural types. Five transcription factors - Homothorax, Eyeless, Sloppy paired, Dichaete and Tailless - are sequentially expressed in a temporal cascade in each of the medulla neuroblasts as they age. Loss of Eyeless, Sloppy paired or Dichaete blocks further progression of the temporal sequence. Evidence is provided that this temporal sequence in neuroblasts, together with Notch-dependent binary fate choice, controls the diversification of the neuronal progeny. Although a temporal sequence of transcription factors had been identified in Drosophila embryonic neuroblasts, this work illustrates the generality of this strategy, with different sequences of transcription factors being used in different contexts (Li, 2013).

In the developing medulla, the wave of conversion of neuroepithelium into neuroblasts makes it possible to visualize neuroblasts at different temporal stages in one snapshot, with newly generated neuroblasts on the lateral edge and the oldest neuroblasts on the medial edge of the expanding crescent shaped neuroblast region. An antibody screen was conducted for transcription factors expressed in the developing medulla and five transcription factors, Hth, Ey, Slp1, D and Tll, were identified that are expressed in five consecutive stripes in neuroblasts of increasing ages, with Hth expressed in newly differentiated neuroblasts, and Tll in the oldest neuroblasts. This suggests that these transcription factors are sequentially expressed in medulla neuroblasts as they age. Neighbouring transcription factor stripes show partial overlap in neuroblasts with the exception of the D and Tll stripes, which abut each other. Previous studies have reported that Hth and Ey< were expressed in medulla neuroblasts, but they had not been implicated in controlling neuroblast temporal identities. Hth and Tll also show expression in the neuroepithelium (Li, 2013).

To address whether each neuroblast sequentially expresses the five transcription factors, their expression was examined in the neuroblast progeny. Hth, Ey and Slp1 are expressed in three different layers of neurons that correlate with birth order, that is, Hth in the first-born neurons of each lineage in the deepest layers; Ey or Slp1 in correspondingly more superficial layers, closer to the neuroblasts. This suggests that they are born sequentially in each lineage. D is expressed in two distinct populations of neurons. The more superficial population inherit D from D⁺ neuroblasts. D⁺ neurons in deeper layers (corresponding to the Hth and Ey layers) turn on D expression independently and will be discussed later. Single neuroblast clones were generated, and the expression of the transcription factors was examined in the neuroblast and its progeny. Single neuroblast clones in which the neuroblast is at the Ey⁺ stage include Ey⁺ GMCs/neurons as well as Hth⁺ neurons. This indicates that Ey⁺ neuroblasts have transited through the Hth⁺ stage and generated Hth⁺ neurons. Clones in which the neuroblast is at the D⁺ stage contain Slp1⁺ GMCs and Ey⁺ neurons, suggesting that D⁺ neuroblasts have already transited through the Slp⁺ and Ey⁺ stages. This supports the model that each medulla neuroblast sequentially expresses Hth, Ey, Slp1 and D as it ages, and sequentially produces neurons that inherit and maintain expression of the transcription factor (Li, 2013).

slp1 and slp2 are two homologous genes arranged in tandem and function redundantly in embryonic and eye development. Slp2 is expressed in the same set of medulla neuroblasts as Slp1. Slp1 and Slp2 are referred to collectively as Slp (Li, 2013).

Tll is expressed in the oldest medulla neuroblasts. The oldest Tll⁺ neuroblasts show nuclear localization of Prospero (Pros), suggesting that they undergo Pros-dependent cell-cycle exit at the end of their life, as in larval nerve cord and central brain neuroblasts. Tll⁺ neuroblasts and their progeny express glial cells missing (gcm), and the progeny gradually turn off Tll and turn on Repo, a glial-specific marker. These cells migrate towards deeper neuronal layers and take their final position as glial cells around the medulla neuropil. Thus, Tll⁺ neuroblasts correspond to previously identified glioblasts between the optic lobe and central brain that express gcm and generate medulla neuropil glia. Clones in which the neuroblast is at the Tll⁺ stage contain Hth⁺ neurons and Ey⁺ neurons, among others, confirming that Tll⁺ neuroblasts represent the final temporal stage of medulla neuroblasts rather than a separate population of glioblasts. Therefore, these data clearly show that medulla neuroblasts sequentially express five transcription factors as they age. The four earlier temporal stages generate neurons that inherit and maintain the temporal transcription factor present at their birth, although a subset of neurons born during the Ey, Slp or D neuroblast stages lose expression of the neuroblast transcription factor. At the final temporal stage, neuroblasts switch to glioblasts and then exit the cell cycle (Li, 2013).

Whether cross-regulation among transcription factors of the neuroblast temporal sequence contributes to the transition from one transcription factor to the next was examined. Loss of hth or its cofactor, extradenticle (exd), does not affect the expression of Ey and subsequent progression of the neuroblast temporal sequence (Li, 2013).

ey-null mutant clones were generated using a bacterial artificial chromosome (BAC) rescue construct recombined on a chromosome containing a Flip recombinase target (FRT) site in an ey^J5.71 null background. ey^J5.71 homozygous mutant larvae were also tested. In both cases, Slp expression is lost in neuroblasts, along with neuronal progeny produced by Slp⁺ neuroblasts, marked by the transcription factor Twin of eyeless (Toy, see below). However, neuroblast division is not affected, and Hth remains expressed in only the youngest neuroblasts and first-born neurons. Targeted ey RNA interference (RNAi) using a Vsx-Gal4 driver that is expressed in the central region of the neuroepithelium and neuroblasts gives the same phenotype. This suggests that Ey is required to turn on the next transcription factor, Slp, but is not required to repress Hth (Li, 2013).

In clones of a deficiency mutation, slp^S37A, that deletes both slp1 and slp2, neuroblasts normally transit from Hth⁺ to Ey⁺, but older neuroblasts maintain the expression of Ey and do not progress to express D or Tll, suggesting that Slp is required to repress ey and activate D (Li, 2013).

Similarly, in D mutant clones, neuroblasts are also blocked at the Slp⁺ stage, and do not turn on Tll, indicating that D is required to repress slp and activate tll. Finally, in tll mutant clones, D expression is not expanded into oldest neuroblasts, suggesting that tll is not required for neuroblasts to turn off D. Thus, in the medulla neuroblast temporal sequence, ey, slp and D are each required for turning on the next transcription factor. slp and D are also required for turning off the preceding transcription factor (Li, 2013).

Gain-of-function phenotypes of each gene were studied. However, misexpression of Hth, Ey, Slp1 or Slp2, or D in all neuroblasts or in large neuroblast clones is not sufficient to activate the next transcription factor or repress the previous transcription factor in neuroblasts. Only misexpressing tll in all neuroblasts is sufficient to repress D expression (Li, 2013).

In summary, cross-regulation among transcription factors is required for at least some of the transitions. No cross-regulation was observed between hth and ey. Because ey is already expressed at low levels in the neuroepithelium and in Hth⁺ neuroblasts, an as yet unidentified factor might gradually upregulate ey and repress hth to achieve the first transition. As tll is sufficient but not required to repress D expression, additional factors must act redundantly with Tll to repress D (Li, 2013).

The temporal sequence of neuroblasts described above could specify at least four neuron types plus glia (in fact more than ten neuron types plus glia considering that neuroblasts divide several times at each stage with overlaps between neighbouring temporal transcription factors). As this is not sufficient to generate the 70 medulla neuron types, it was asked whether another process increases diversity in the progeny neurons born from a neuroblast at a specific temporal stage. Apterous (Ap) is known to mark about half of the 70 medulla neuron types. In the larval medulla, Ap is expressed in a salt-and-pepper manner in subsets of neurons born from all temporal stages. In the progeny from Hth⁺ neuroblasts, all neurons seem to maintain Hth, with a subset also expressing Ap. However, only half of the neurons born from neuroblasts at other transcription factor stages maintain expression of the neuroblast transcription factor. For instance, in the progeny of Ey⁺ neuroblasts, Ey⁺ neurons are intermingled with about an equal number of Ey⁻ neurons that instead express Ap. Neuroblast clones contain intermingled Ey⁺ and Ap⁺ neurons. This is also true for the progeny of Slp⁺ neuroblasts: Slp1⁺ neurons are intermingled with Slp1⁻ Ap⁺ neurons. In the progeny of D⁺ neuroblasts, D and Ap are co-expressed in the same neurons, and they are intermingled with neurons that express neither D nor Ap. Neurons in deeper neuronal layers (corresponding to the Ey⁺ and Hth⁺ neuron layers) also express D independently, and these neurons are Ap⁻. The expression of Ap is stable from larval to adult stages (Li, 2013).

The intermingling of Ap⁺ and Ap⁻ neurons raised the possibility that asymmetric division of GMCs gives rise to one Ap⁺ and one Ap⁻ neuron. Two-cell clones were generated to visualize the two daughters of a GMC. In every case, one neuron is Ap⁺ and the other is Ap^-, suggesting that asymmetric division of GMCs diversifies medulla neuron fates by controlling Ap expression (Li, 2013).

Asymmetric division of GMCs in Drosophila involves Notch (N)-dependent binary fate choice. In the developing medulla, the N pathway is involved in the transition from neuroepithelium to neuroblast, and loss of Su(H), the transcriptional effector of N signalling, leads to faster progression of neurogenesis and neuroblast formation. However, Su(H) mutant neuroblasts still follow the same transcription factor sequence and generate GMCs and neuronal progeny, allowing analysis of the effect of loss of N function on GMC progeny diversification. Notably, neurons completely lose Ap expression in Su(H) mutant clones. All mutant neurons born during the Hth⁺ stage still express Hth, but not Ap, suggesting that the N^ON daughters of Hth⁺ GMCs are the neurons expressing both Ap and Hth. In contrast to wild-type clones, all Su(H) mutant neurons born during the Ey⁺ neuroblast stage express Ey and none express Ap. Similarly, all mutant neurons born during the Slp⁺ neuroblast stage express Slp1 but lose Ap. These data suggest that, for Ey⁺ or Slp⁺ GMCs, the N^OFF daughter maintains the neuroblast transcription factor expression, whereas the N^ON daughter loses this expression but expresses Ap. In the wild-type progeny born during the D⁺ neuroblast stage, Ap⁺ neurons co-express D. Both D and Ap are lost in Su(H) mutant clones in the D⁺ neuroblast progeny, confirming that D is transmitted to the Ap⁺ N^ON daughter of D⁺ GMCs. By contrast, the D⁺ Ap⁻ neurons in the deeper layers (corresponding to the N^OFF progeny born during the Ey⁺ and Hth⁺ neuroblast stages, see above) are expanded in Su(H) mutant clones at the expense of Ap⁺ neurons. Therefore, the deeper layer of D expression is turned on independently in the N^OFF daughters of Hth⁺ and Ey⁺ GMCs (Li, 2013).

Finally, in wild type, a considerable amount of apoptotic cells were observed dispersed among neurons, suggesting that one daughter of certain GMCs undergoes apoptosis in some of the lineages. Together these data suggest that Notch-dependent asymmetric division of GMCs further diversifies neuronal identities generated by the temporal sequence of transcription factors (Li, 2013).

How does the neuroblast transcription factor temporal sequence, together with the Notch-dependent binary fate choice, control neuronal identities in the medulla? Transcription factor markers specifically expressed in subsets of medulla neurons, but not in neuroblasts, were examined including Brain-specific homeobox (Bsh) and Drifter (Dfr), as well as other transcription factors identified in the antibody screen, for example, Lim3 and Toy. Bsh is required and sufficient for the Mi1 cell fate, and Dfr is required for the morphogenesis of nine types of medulla neurons, including Mi10, Tm3, TmY3, Tm27 and Tm27Y (Hasegawa, 2011). Investigation were carried out to identify at which neuroblast temporal stage these neurons were born by examining co-expression with the inherited neuroblast transcription factors. Then whether the neuroblast transcription factors regulate expression of these markers and neuron fates was investigated. The results for each neuroblast stage are described below (Li, 2013).

Bsh is expressed in a subset of Hth⁺ neurons, suggesting that Bsh is in the N^ON daughter of Hth⁺ GMCs. Indeed, Bsh expression is lost in both Su(H) and hth mutant clones. Thus, both Notch activity and Hth are required for specifying the Mi1 fate, consistent with the previous report that Hth is required for the Mi1 fate. Ectopic expression of Hth in older neuroblasts is also sufficient to generate ectopic Bsh⁺ neurons, although the phenotype becomes less pronounced in later parts of the lineage. These data suggest that Hth is necessary and sufficient to specify early born neurons, but the competence to do so in response to sustained expression of Hth decreases over time. This is similar to embryonic CNS neuroblasts, where ectopic Hb is only able to specify early born neurons during a specific time window (Li, 2013).

Lim3 is expressed in all Ap⁻ progeny of both Hth⁺ and Ey⁺ neuroblasts. Toy and Dfr are expressed in subsets of neurons born from Ey⁺ neuroblasts, as indicated by their expression in the Ey⁺ neuron progeny layer. The most superficial row of Ey⁺ Ap⁻ neurons express Toy (and Lim3), suggesting that they are the N^OFF progeny of the last-born Ey⁺ GMCs. Dfr is co-expressed with Ap in two or three rows of neurons that are intermingled with Ey⁺ neurons, suggesting that they are the N^ON progeny from Ey⁺ GMCs. In addition to these Ap⁺ Dfr⁺ neurons, Dfr is also expressed in some later-born neurons that are Ap⁻ but express another transcription factor: Dachshund (Dac), in specific sub-regions of the medulla crescent (Li, 2013).

Whether Ey in neuroblasts regulates Dfr expression in neurons was tested. As expected, Dfr-expressing neurons are lost in ey-null mutant clones, suggesting that they require Ey activity in neuroblasts, even though Ey is not maintained in Ap⁺ Dfr⁺ neurons. Furthermore, in slp mutant clones in which neuroblasts remain blocked in the Ey⁺ state, the Ap⁺ Dfr⁺ neuron population is expanded into later-born neurons, suggesting that the transition from Ey⁺ to Slp⁺ in neuroblasts is required for shutting off the production of Ap⁺ Dfr⁺ neurons. In addition, Ap⁺ Dfr⁺ neurons are lost in Su(H) mutant clones. Thus, Ey expression in neuroblasts and the Notch pathway together control the generation of Ap⁺ Dfr⁺ neurons (Li, 2013).

In addition to its expression with Ey in the N^OFF progeny of the last-born Ey⁺ GMCs, Toy is also expressed in Ap⁺ (N^ON) neurons in more superficial layers generated by Slp⁺ and D⁺ neuroblasts. Consistently, in Su(H) mutant clones, an expansion of Toy⁺ Ey⁺ neurons is seen in the Ey progeny layer, followed by loss of Toy in the Slp and D progeny layer (Li, 2013).

Tests were performed to see whether Slp is required for the neuroblasts to switch from generating Toy⁺ Ap⁻ neurons, progeny of Ey⁺ neuroblasts, to generating Toy⁺ Ap⁺ neurons. Indeed, in slp mutant clones, the Toy⁺ Ap⁺ neurons largely disappear, whereas Toy⁺ Ap⁻ neurons expand (Li, 2013).

WAp and Toy expression was examined in specific adult neurons. OrtC1-gal4 primarily labels Tm20 and Tm5 plus a few TmY10 neurons, and these neurons express both Ap and Toy. To examine whether Slp is required for the specification of these neuron types, wild-type or slp mutant clones were generated using the mosaic analysis with a repressible cell marker (MARCM) technique by heat-shocking for 1 h at early larval stage, and the number of OrtC1-gal4-marked neurons in the adult medulla was examined. In wild-type clones, OrtC1-gal4 marks ~100 neurons per medulla. By contrast, very few neurons are marked by OrtC1-gal4 in slp mutant clones. Slp is unlikely to directly regulate the Ort promoter because Slp expression is not maintained in Ap⁺ Toy⁺ neurons. Furthermore, the expression level of OrtC1-gal4 in lamina L3 neurons is not affected by slp mutation. These data suggest that loss of Slp expression in neuroblasts strongly affects the generation of Tm20 and Tm5 neurons (Li, 2013).

In summary, these data show that the sequential expression of transcription factors in medulla neuroblasts controls the birth-order-dependent expression of different neuronal transcription factor markers, and thus the sequential generation of different neuron types (Li, 2013).

Although a temporal transcription factor sequence that patterns Drosophila nerve cord neuroblasts was reported more than a decade ago, it was not clear whether the same or a similar transcription factor sequence patterns neural progenitors in other contexts. The current identification of a novel temporal transcription factor sequence patterning the Drosophila medulla suggests that temporal patterning of neural progenitors is a common theme for generating neuronal diversity, and that different transcription factor sequences might be recruited in different contexts (Li, 2013).

There are both similarities and differences between the two neuroblast temporal sequences. In the Hb-Kr-Pdm-Cas-Grh sequence, ectopically expressing one gene is sufficient to activate the next gene, and repress the previous gene, but these cross-regulations are not necessary for the transitions, with the exception of Castor. In the Hth-Ey-Slp-D-Tll sequence, removal of Ey, Slp or D does disrupt cross-regulations necessary for temporal transitions (except the Hth-Ey transition). However, in most cases these cross-regulations are not sufficient to ensure temporal transitions, suggesting that additional timing mechanisms or factors are required (Li, 2013).

For simplicity, the medulla neuroblasts are represented as transiting through five transcription factor stages, whereas in fact the number of stages is clearly larger than five. First, neuroblasts divide more than once while expressing a given temporal transcription factor, and each GMC can have different sub-temporal identities. Furthermore, there is considerable overlap between subsequent temporal neuroblast transcription factors: neuroblasts expressing two transcription factors are likely to generate different neuron types from neuroblasts expressing either one alone (Li, 2013).

Although the complete lineage of medulla neuroblasts is still being investigated, this study shows how a novel temporal sequence of transcription factors is required to generate sequentially the diverse neurons that compose the medulla. The requirement for transcription factor sequences in the medulla and in embryonic neuroblasts suggests that this is a general mechanism for the generation of neuronal diversity. Interestingly, the mammalian orthologue of Slp1, FOXG1, acts in cortical progenitors to suppress early born cortical cell fates. Thus, transcription-factor-dependent temporal patterning of neural progenitors might be a common theme in both vertebrate and invertebrate systems (Li, 2013).

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).

A Notch-dependent transcriptional mechanism controls expression of temporal patterning factors in Drosophila medulla

Temporal patterning is an important mechanism for generating a great diversity of neuron subtypes from a seemingly homogenous progenitor pool in both vertebrates and invertebrates. Drosophila neuroblasts are temporally patterned by sequentially expressed Temporal Transcription Factors (TTFs). These TTFs are proposed to form a transcriptional cascade based on mutant phenotypes, although direct transcriptional regulation between TTFs has not been verified in most cases. Furthermore, it is not known how the temporal transitions are coupled with the generation of the appropriate number of neurons at each stage. This study used neuroblasts of the Drosophila optic lobe medulla to address these questions and show that the expression of TTFs Sloppy-paired 1/2 (Slp1/2) is directly regulated at the transcriptional level by two other TTFs and the cell-cycle dependent Notch signaling through two cis-regulatory elements. It was also shown that supplying constitutively active Notch can rescue the delayed transition into the Slp stage in cell cycle arrested neuroblasts. These findings reveal a novel Notch-pathway dependent mechanism through which the cell cycle progression regulates the timing of a temporal transition within a TTF transcriptional cascade (Ray, 2022).

Drosophila neuroblasts are temporally patterned by sequentially expressed TTFs. Although the expression pattern and mutant phenotypes suggest that TTFs form a transcriptional cascade, direct transcriptional regulation between TTFs has not been demonstrated in most cases. This work has characterized two enhancers of the slp genes that enable the expression of Slp1 and Slp2 in medulla neuroblasts. The u8772 220 bp enhancer is activated at an earlier stage relative to the d5778 850 bp enhancer. In these two enhancers, sites were identified for the previous TTF -Ey and Scro-a TTF expressed at around the same time as Slp1. Deleting either enhancer alone did not eliminate the expression of endogenous Slp1 and Slp2, suggesting that they act partially redundantly with one another. Deletion of both enhancers completely eliminates Slp1 and Slp2 expression in medulla neuroblasts but does not affect their expression in lamina neurons or glia, confirming the specificity and necessity of these two enhancers. Using GFP reporter assays, this study has shown that mutation of Ey binding sites in these enhancers abolishes reporter expression similar to genetic experiments where a loss of GFP reporter was reported within ey RNAi clones. The results are also consistent with previous studies that showed a complete loss of endogenous Slp1/2 expression in UAS-ey-RNAi expressing neuroblasts. This study also confirmed the in vivo binding of Ey to the identified enhancers of Slp by Dam-ID sequencing. The expression of the TTF Scro is initiated simultaneously as Slp1/2, and it has been shown that loss of Scro significantly reduces Slp expression level. Mutation of most probable Scro binding sites on the u8772 220 bp or d5778 850 bp enhancers led to a dramatic reduction of GFP reporter expression. Thus, the combined effect of mutating Scro binding sites on both enhancers recapitulates the observed impact of Scro knock-down on endogenous Slp1/2 expression, which is reduced expression of Slp1/2 in neuroblasts expressing UAS scro-RNAi and a consequent loss of neural fates specified by Slp1/2 in their progeny. It is interesting to note that the observation of multiple enhancers regulating Slp1/2 expression is consistent with regulation of Slp1/2 in other developmental contexts. Previous studies have noted the presence of multiple enhancers of Slp1/2 expression in the vicinity of the slp1 and slp2 coding loci. Many of these regulatory DNA segments function as stripe enhancers enabling Slp1/2 to function as pair-rule genes during embryonic segmentation. Although these enhancers share some overlapping functions and domains of activation, a full complement of stripe enhancers is required for maintaining parasegment boundaries and wingless expression (Ray, 2022).

It was previously demonstrated that although Ey is necessary for activating Slp1/2 expression it is not sufficient. There is always a time delay after the start of Ey expression to the start of Slp expression to ensure the sufficient duration of the Ey window. How is the timing controlled? From analyses of the slp1/2 enhancer sequences, several binding sites were found for the CSL transcription factor Su(H), most prominently known as a central component of the ternary Notch transcription complex and the primary DNA binding component. To confirm the involvement of the Notch pathway in regulating Slp1/2, we observed the effects of knocking down key Notch pathway components on endogenous Slp1/2 expression. In all cases a delay was observed in the expression of Ey and a further delay in the transition to the Slp1/2 stage in neuroblasts expressing the RNAi knockdowns. Mutating Su(H) binding sites in the u8772 220bp enhancer led to a loss or reduction of GFP reporter expression in neuroblasts. However, mutating Su(H) binding sites in the d5778 850 bp enhancer did not decrease the reporter expression. These results suggest that Notch signaling directly regulates Slp expression through the u8772 220 bp enhancer, but not the d5778 850 bp enhancer, and this is consistent with the delayed expression driven by the d5778 850 bp enhancer. However, Ey still plays a more critical role in activating Slp1/2 expression than the Notch pathway, since Slp1/2 are still expressed albeit later in the absence of Su(H) and other Notch components, and Notch signaling requires Ey to speed up the Ey to Slp transition. As with Ey, this study confirmed Su(H) binding to the u872 220 bp enhancer using DamID-seq. Thus, this work provided strong evidence that N signaling, a general signaling pathway involved in neuroblast development, regulates the timing of activation of a TTF gene directly. In addition, the results also raised the interesting hypothesis that Notch signaling might be involved in facilitating all temporal transitions, because the turning on of Opa and Ey is also delayed, and a further and further delay was observed in turning on of later TTFs. Whether Notch signaling regulates other TTF expression directly or indirectly still awaits further investigation. (Ray, 2022).

What might explain the delay in Slp1/2 expression in the absence of Notch signaling? Recent developments in single-molecule Fluorescence In Situ Hybridization (smFISH) technology and live imaging techniques using the MS2-MCP system have enabled studying the transcription process in molecular detail. Imaging transcription driven by Notch responsive enhancers in native contexts has shown this process to be inherently 'bursty', i.e., episodes of transcription (enhancer 'On' state) are punctuated with gaps in activity (enhancer in 'Off' state) (Falo-Sanjuan et al., 2019; Lee et al., 2019). The dosage of NICD modulates the duration of the 'On' phase in one context studied by live imaging (Falo-Sanjuan et al., 2019; Lee et al., 2019). Additionally, binding of tissue-specific regional factors to these Notch responsive enhancers may prime these enhancers and help synchronize transcription and sustain a steady transcriptional output upon Notch binding to enhancers; this helps integrate important positional cues and the perception of context (Falo-Sanjuan et al., 2019). Applying these insights to our system, we suggest that Ey may act by priming the Notch-responsive enhancer of slp providing crucial contextual information, and this is required for Notch to further activate Slp1/2 transcription, and speed up the transition. (Ray, 2022).

Notch target genes and Dpn are transcriptional repressors that act partially redundantly to maintain neuroblast identity. In type II NBs, Dpn depends on Notch signaling, and loss of Dpn causes premature differentiation. However, in type I NBs, Dpn is not lost when Notch signaling is lost, and Notch signaling seems dispensable for the self-renewing abilities of NBs. In the medulla neuroblasts, this study also observed that in Su(H) mutant clones, the clone size and neuroblast proliferation are not significantly affected. On the other hand, N signaling was observed to be dependent on cell-cycle progression, and the Notch target gene is lost when cell cycle progression is blocked. (Ray, 2022).

In the medulla, blocking cell cycle progression in neuroepithelial cells prematurely transforms them into neuroblasts, and these neuroblasts seem to be arrested or severely delayed in the TTF cascade. When the cell cycle was arrested or slowed down later in neuroblasts to preserve Ey expression, Slp expression was is still delayed. Therefore, cell cycle progression also has a role in the Ey to Slp transition. Further, this study showed that supplying Notch signaling is sufficient to rescue the delay in the Ey to Slp transition caused by cell cycle defect. Thus at the Ey to Slp transition, the cell cycle effect is mediated through the direct regulation of Slp transcription by Notch signaling. Taken together, these results suggest that in Ey stage neuroblasts, Ey is required to initiate Slp expression but not sufficient to activate it to a strong level right away, and after each asymmetric division, activation of Notch signaling in the neuroblast enhances Slp expression, until Slp expression reaches a certain level to repress Ey expression and make the transition. This can be part of a mechanism to coordinate the TTF temporal transition with the cell cycle progression to generate the appropriate number of neural progenies at a given temporal stage (Ray, 2022).

GENE STRUCTURE

Bases in 5' UTR -107 for slp1; 301 for slp2

Bases in 3' UTR - 399 for slp1; 267 for slp2

PROTEIN STRUCTURE

Amino Acids - 322 for slp1; 445 for slp2

Structural Domains and Evolutionary Homologs

Slp1 and Slp2 are structurally and functionally related. They belong to a novel class of putative transcription factors containing a fork head domain, also found in mammalian hepatocyte transcription factors (Grossniklaus, 1992). The FKH domains of SLP-1 and SLP-2 are more closely related to each other than they are to Forkhead or the mammalian homolog.

Drosophila sloppy-paired 1 and 2 both have Forkhead domains (Hacker, 1992). Sloppy paired belongs to a different class of forkhead domain proteins than does Forkhead itself and HNF-3beta. Sloppy paired's closest homolog is BF-1. Another forkhead homolog in Drosophila has been discovered, the crocodile gene, required for the establishment of head structures. Crocodile's closest mammalian homolog is FD1 belonging to a different class of forkhead domain proteins than does Forkhead. Forkhead belongs to the same class as HNF-3alpha, HNF-3beta, HNF-3gamma, XFKH1/XFD1, and XFD1/pintallavis (Sasaki, 1993 and Hacker, 1995).

date revised: 5 August 2021

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