Gene name - sloppy paired 1 and sloppy paired 2
Cytological map position - 24C1
Function - Transcription factor
Symbol - Slp1 and Slp2
Genetic map position - 2-8
Classification - Fork head domain
Cellular location - nuclear
|Recent literature||Hang, S. and Gergen, J.P. (2017). Different modes of enhancer-specific regulation by Runt and Even-skipped during Drosophila segmentation. Mol Biol Cell [Epub ahead of print]. PubMed ID: 28077616
The initial metameric expression of the Drosophila sloppy paired 1 (slp1) gene is controlled by two distinct cis-regulatory DNA elements that interact in a non-additive manner to integrate inputs from transcription factors encoded by the pair-rule segmentation genes. This study performed Chromatin Immuno-Precipitation (ChIP) on reporter genes containing these elements in different embryonic genotypes to investigate the mechanism of their regulation. The Distal Early Stripe Element (DESE) mediates both activation and repression by Runt. The differential response of DESE to Runt was found to be due to an inhibitory effect of Fushi tarazu (Ftz) on P-TEFb recruitment and the regulation of RNA Polymerase II (Pol II) pausing. The Proximal Early Stripe Element (PESE) is also repressed by Runt, but in this case Runt prevents PESE-dependent Pol II recruitment and pre-initiation complex (PIC) assembly. PESE is also repressed by Even-skipped (Eve) but interestingly this repression involves regulation of P-TEFb recruitment and promoter-proximal Pol II pausing. These results demonstrate that the mode of slp1 repression by Runt is enhancer-specific whereas the mode of repression of the slp1 PESE enhancer is transcription factor-specific. The study proposes a model based on these differential regulatory interactions that accounts for the non-additive interactions between the PESE and DESE enhancers during Drosophila segmentation.
|Santiago, I. J., Zhang, D., Saras, A., Pontillo, N., Xu, C., Chen, X., Weirauch, M. T., Mistry, M., Ginty, D. D., Pecot, M. Y. and Peng, J. (2021). Drosophila Fezf functions as a transcriptional repressor to direct layer-specific synaptic connectivity in the fly visual system. Proc Natl Acad Sci U S A 118(13). PubMed ID: 33766917
The layered compartmentalization of synaptic connections, a common feature of nervous systems, underlies proper connectivity between neurons and enables parallel processing of neural information. However, the stepwise development of layered neuronal connections is not well understood. The medulla neuropil of the Drosophila visual system, which comprises 10 discrete layers (M1 to M10), where neural computations underlying distinct visual features are processed, serves as a model system for understanding layered synaptic connectivity. The first step in establishing layer-specific connectivity in the outer medulla (M1 to M6) is the innervation by lamina (L) neurons of one of two broad, primordial domains that will subsequently expand and transform into discrete layers. Previous work found that the transcription factor dFezf (Earmuff) cell-autonomously directs L3 lamina neurons to their proper primordial broad domain before they form synapses within the developing M3 layer. This study shows that dFezf controls L3 broad domain selection through temporally precise transcriptional repression of the transcription factor slp1 (sloppy paired 1). In wild-type L3 neurons, slp1 is transiently expressed at a low level during broad domain selection. When dFezf is deleted, slp1 expression is up-regulated, and ablation of slp1 fully rescues the defect of broad domain selection in dFezf-null L3 neurons. Although the early, transient expression of slp1 is expendable for broad domain selection, it is surprisingly necessary for the subsequent L3 innervation of the M3 layer. DFezf thus functions as a transcriptional repressor to coordinate the temporal dynamics of a transcriptional cascade that orchestrates sequential steps of layer-specific synapse formation.
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).
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 eyJ5.71 null background. eyJ5.71 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, slpS37A, 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 NON 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 NOFF daughter maintains the neuroblast transcription factor expression, whereas the NON 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+ NON daughter of D+ GMCs. By contrast, the D+ Ap− neurons in the deeper layers (corresponding to the NOFF 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 NOFF 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 NON 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 NOFF 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 NON 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 NOFF progeny of the last-born Ey+ GMCs, Toy is also expressed in Ap+ (NON) 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).
Bases in 5' UTR -107 for slp1; 301 for slp2
Bases in 3' UTR - 399 for slp1; 267 for slp2
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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