nubbin/POU domain protein 1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - nubbin

Synonyms - dOct1, dPOU-19, twain, POU domain protein 1

Cytological map position - 33-F1

Function - transcription factor

Keyword(s) - CNS, PNS and endoderm

Symbol - nub

FlyBase ID:FBgn0085424

Genetic map position - 2 -[46]

Classification - homeodomain and POU domain

Cellular location - nuclear



NCBI link: Entrez Gene

nub orthologs: Biolitmine

Recent literature
Corty, M.M., Tam, J. and Grueber, W.B. (2016). Dendritic diversification through transcription factor-mediated suppression of alternative morphologies. Development 143: 1351-1362. PubMed ID: 27095495
Summary:
Neurons display a striking degree of functional and morphological diversity, and the developmental mechanisms that underlie diversification are of significant interest for understanding neural circuit assembly and function. This study finds that the morphology of Drosophila sensory neurons is diversified through a series of suppressive transcriptional interactions involving the POU domain transcription factors Pdm1 (Nubbin) and Pdm2, the homeodomain transcription factor Cut, and the transcriptional regulators Scalloped and Vestigial. Pdm1 and Pdm2 are expressed in a subset of proprioceptive sensory neurons and function to inhibit dendrite growth and branching. A subset of touch receptors show a capacity to express Pdm1/2, but Cut represses this expression and promotes more complex dendritic arbors. Levels of Cut expression are diversified in distinct sensory neurons by selective expression of Scalloped and Vestigial. Different levels of Cut impact dendritic complexity and, consistent with this, it was found that Scalloped and Vestigial suppress terminal dendritic branching. This transcriptional hierarchy therefore acts to suppress alternative morphologies to diversify three distinct types of somatosensory neurons. 

Dantoft, W., Lundin, D., Esfahani, S.S. and Engström, Y. (2016). The POU/Oct transcription factor Pdm1/nub is necessary for a beneficial gut microbiota and normal lifespan of Drosophila. J Innate Immun 8: 412-426. PubMed ID: 27231014
Summary:
Maintenance of a stable gut microbial community relies on a delicate balance between immune defense and immune tolerance. This study used Drosophila to study how the microbial gut flora is affected by changes in host genetic factors and immunity. Flies with a constitutively active gut immune system, due to a mutation in the POU transcriptional regulator Pdm1/nubbin (nub) gene, have higher loads of bacteria and a more diverse taxonomic composition than controls. In addition, the microbial composition shifts considerably during the short lifespan of the nub1 mutants. This shift is characterized by a loss of relatively few OTUs (operational taxonomic units) and a remarkable increase in a large number of Acetobacter spp. and Leuconostoc spp. Treating nub1 mutant flies with antibiotics prolongs their lifetime survival by more than 100%. Immune gene expression is also persistently high in the presence of antibiotics, indicating that the early death is not a direct consequence of an overactive immune defense but rather an indirect consequence of the microbial load and composition. Thus, changes in host genotype and an inability to regulate the normal growth and composition of the gut microbiota lead to a shift in the microbial community, dysbiosis and early death. 

Lindberg, B. G., Tang, X., Dantoft, W., Gohel, P., Seyedoleslami Esfahani, S., Lindvall, J. M. and Engstrom, Y. (2018). Nubbin isoform antagonism governs Drosophila intestinal immune homeostasis. PLoS Pathog 14(3): e1006936. PubMed ID: 29499056
Summary:
Gut immunity is regulated by intricate and dynamic mechanisms to ensure homeostasis despite a constantly changing microbial environment. This study shows that the POU/Oct gene nubbin (nub) encodes two transcription factor isoforms, Nub-PB and Nub-PD, which antagonistically regulate immune gene expression in Drosophila. Global transcriptional profiling of adult flies overexpressing Nub-PB in immunocompetent tissues revealed that this form is a strong transcriptional activator of a large set of immune genes. Further genetic analyses showed that Nub-PB is sufficient to drive expression both independently and in conjunction with nuclear factor kappa B (NF-kappaB), JNK and JAK/STAT pathways. Similar overexpression of Nub-PD did, conversely, repress expression of the same targets. Strikingly, isoform co-overexpression normalized immune gene transcription, suggesting antagonistic activities. RNAi-mediated knockdown of individual nub transcripts in enterocytes confirmed antagonistic regulation by the two isoforms and that both are necessary for normal immune gene transcription in the midgut. Furthermore, enterocyte-specific Nub-PB expression levels had a strong impact on gut bacterial load as well as host lifespan. Overexpression of Nub-PB enhanced bacterial clearance of ingested Erwinia carotovora carotovora 15. Nevertheless, flies quickly succumbed to the infection, suggesting a deleterious immune response. In line with this, prolonged overexpression promoted a proinflammatory signature in the gut with induction of JNK and JAK/STAT pathways, increased apoptosis and stem cell proliferation. These findings highlight a novel regulatory mechanism of host-microbe interactions mediated by antagonistic transcription factor isoforms.
Tang, X., Zhao, Y., Buchon, N. and Engstrom, Y. (2018). The POU/Oct transcription factor Nubbin controls the balance of intestinal stem cell maintenance and differentiation by isoform-specific regulation. Stem Cell Reports 10(5):1565-1578. PubMed ID: 29681543
Summary:
Drosophila POU/Oct transcription factors are required for many developmental processes, but their putative regulation of adult stem cell activity has not been investigated. This stuy shows that Nubbin (Nub)/Pdm1, homologous to mammalian OCT1/POU2F1 and related to OCT4/POU5F1, is expressed in gut epithelium progenitor cells. The nub-encoded protein isoforms, Nub-PB and Nub-PD, play opposite roles in the regulation of intestinal stem cell (ISC) maintenance and differentiation. Depletion of Nub-PB in progenitor cells increased ISC proliferation by derepression of escargot expression. Conversely, loss of Nub-PD reduced ISC proliferation, suggesting that this isoform is necessary for ISC maintenance, analogous to mammalian OCT4/POU5F1 functions. Furthermore, Nub-PB is required in enteroblasts to promote differentiation, and it acts as a tumor suppressor of Notch RNAi-driven hyperplasia. It is suggested that a dynamic and well-tuned expression of Nub isoforms in progenitor cells is required for maintaining gut epithelium homeostasis.
Meng, J. L., Wang, Y., Carrillo, R. A. and Heckscher, E. S. (2020). Temporal transcription factors determine circuit membership by permanently altering motor neuron-to-muscle synaptic partnerships. Elife 9. PubMed ID: 32391795
Summary:
How circuit wiring is specified is a key question in developmental neurobiology. Previously, using the Drosophila motor system as a model, the classic temporal transcription factor Hunchback was found to act in NB7-1 neuronal stem cells to control the number of NB7-1 neuronal progeny form functional synapses on dorsal muscles (Meng, 2019). However, it is unknown to what extent control of motor neuron-to-muscle synaptic partnerships is a general feature of temporal transcription factors. Additional temporal transcription factor manipulations-prolonging expression of Hunchback in NB3-1-were performed, as well as precociously expressing Pdm and Castor in NB7-1. Confocal microscopy, calcium imaging, and electrophysiology were used to show that in every manipulation there are permanent alterations in neuromuscular synaptic partnerships. These data show temporal transcription factors, as a group of molecules, are potent determinants of synaptic partner choice and therefore ultimately control circuit membership.
BIOLOGICAL OVERVIEW

One of the major challenges in developmental biology is to understand the regulatory events that generate neuronal diversity. During Drosophila embryonic neural lineage development, cellular temporal identity is established in part by a transcription factor (TF) regulatory network that mediates a cascade of cellular identity decisions. Two of the regulators essential to this network are the POU-domain TFs Nubbin and Pdm-2, encoded by adjacent genes collectively known as pdm. The focus of this study is the discovery and characterization of cis-regulatory DNA that governs their expression. Phylogenetic footprinting analysis of a 125 kb genomic region that spans the pdm locus identified 116 conserved sequence clusters. To determine which of these regions function as cis-regulatory enhancers that regulate the dynamics of pdm gene expression, this study tested each for in vivo enhancer activity during embryonic development and postembryonic neurogenesis. The screen revealed 77 unique enhancers positioned throughout the noncoding region of the pdm locus. Many of these activated neural-specific gene expression during different developmental stages and many drove expression in overlapping patterns. Sequence comparisons of functionally related enhancers that activate overlapping expression patterns revealed that they share conserved elements that can be predictive of enhancer behavior. To facilitate data accessibility, the results of this analysis are catalogued in cisPatterns, an online database of the structure and function of these and other Drosophila enhancers. These studies reveal a diversity of modular enhancers that most likely regulate pdm gene expression during embryonic and adult development, highlighting a high level of temporal and spatial expression specificity. In addition, clusters of functionally related enhancers were discovered throughout the pdm locus. A subset of these enhancers share conserved elements including sequences that correspond to known TF DNA binding sites. Although comparative analysis of the nubbin and pdm-2 encoding sequences indicate that these two genes most likely arose from a duplication event, only partial evidence of sequence duplication between their enhancers was found, suggesting that after the putative duplication their cis-regulatory DNA diverged at a higher rate than their coding sequences (Ross, 2015).

This study found 41 enhancers that directed embryonic expression, an overlapping set of 46 activated larval expression, and another overlapping set of 46 activated expression in the adult CNS. While many of these enhancers were activated only in the nervous system, a subset activated reporter gene expression outside of the nervous system, including in larval appendages and in the trachea. Roughly a third of the tested CSCs did not exhibit any detectable cis-regulatory activity in the nervous system. Since this study focused on identifying neural enhancers, the possibility exists that some or all of these CSCs that lack neural system activity may regulated gene expression in the larval and adult tissues that were not examined (Ross, 2015).

There are other online resources of documented enhancers in the Drosophila genome, namely, FlyLight and Vienna Tiles. While these cis-regulatory libraries provide useful information, the coverage of the pdm locus in these databases is not complete. For example, FlyLight analysis did not detect 14 enhancers that flank the nub transcribed sequence. These include those located upstream to the nub long transcript (nub-12 and nub-13), its first intron (nub-28), second exon (nub-32a), second intron (nub-32b, nub-32c, nub-33, nub-36, nub-40b, nub-41, nub-42, nub-44, and nub-45a), and third intron (nub-49b). The FlyLight library also does not include seven pdm-2 enhancers: located in the upstream region (pdm2-21); within the second intron (pdm2-27 and pdm2-28) and lacks information regarding its downstream region (pdm2-45, pdm2-46, pdm2-47 and pdm2-48). Vienna Tiles also provides only partial coverage of the pdm locus, omitting the following 11 pdm locus enhancers: nub-58a, nub-58b, pdm2-13, pdm2-17, pdm2-21, pdm2-22, pdm2-23a, pdm2-31b, pdm2-32, pdm-33, and pdm2-48 . While the Vienna Tiles database provides information on embryonic and adult enhancers, it does not supply information on cis-regulatory activity during larval development. In addition, based on the current analysis, most of the reporter transgenes in these two libraries contain multiple enhancers. For example, he Vienna Tiles enhancer denoted as VT6436 enhancer is made up of two embryonic enhancers (nub-28 and nub-29) (Ross, 2015).

Analysis of the pdm locus enhancers identified four functionally related enhancers (nub-46, nub-49b, pdm2-34, and pdm2-37a) that activated expression during NB lineage development. The nub-46 and pdm2-34 enhancers are both located in the third intron of the nub and pdm-2 long transcript, respectively, whereas nub-49b and pdm2-37a are positioned immediately 5' to the transcriptional start site of their respective short isoform. While the nub-46 and pdm2-34 enhancers drove overlapping but nonidentical expression during embryonic and larval NB lineage development, nub-49b and pdm2-37a regulated similar expression patterns during postembryonic NB lineage development. Analysis of nub-46 and pdm2-34 revealed that these enhancers share multiple conserved DNA elements, albeit in largely unique configurations. Although these observations suggest these enhancers are related, additional studies are needed to further resolve subtle differences between their regulatory activities (Ross, 2015).

Comparative analysis of the nub and pdm-2 coding sequences revealed that their sequence relationship was mostly limited to the exons that encode their POU domains and homeodomains. In contrast, no evidence of collinearity was detected within their noncoding regions, suggesting that they have diverged at a faster rate than the coding sequences. Only one pdm ortholog was found in the mosquito, whereas the medfly and housefly carry both genes. Given this observation and accounting for the divergence of Drosophila from these distant Diptera, the pdm duplication event may have occurred in the Dipteran line between 100 and 260 million (Ross, 2015).

Given the presence of the pdm genes in the medfly and housefly genomes, it was asked whether some or all of the Drosophila CSCs could also be identified in these distant species. Submitting the D. melanogaster genomic sequences surrounding nub and pdm-2 to BLAST searches using the medfly and housefly genomes revealed sequences conserved in the three Dipteran species within several pdm locus CSCs (see Three-way alignment of ultraconserved sequences in conserved sequence clusters identified in Drosophila, housefly, and medfly) that were typically found within their longest conserved sequence blocks (CSBs). For example, a 48 bp sequence within the pdm2-26 CSC that is conserved in all drosophilids, in addition to the medfly and housefly (see The pdm2-26 enhancer contains ultraconserved sequences detected in multiple Diptera)(Ross, 2015).

These studies revealed that two-thirds of the CSCs function as cis-regulatory enhancers that regulate gene expression in a diverse array of spatiotemporal aspects, which taken together reflect pdm expression domains. These observations suggest that the pdm genes are dynamically regulated by multiple cis-regulatory modules, and that these enhancers are more amenable to evolutionary restructuring than their protein encoding exons. This is in agreement with recent reviews on the evolution of Dipteran enhancers highlighting the flexibility of enhancers to maintain their function after loss and/or gain of TF DNA binding sites. Also consistent with these observations, functionally related enhancers were found within the pdm locus that share conserved sequences, albeit in different arrangements and orientations (Ross, 2015).

From a mechanistic perspective, these observations suggest that enhancer behavior can be predicted based on the combination of the conserved elements shared among functionally related enhancers. Similar observations have been made by others. Hierarchical clustering analysis of shared conserved sequences revealed that pdm SOG enhancers may be grouped based on shared elements that are for the most part not present within other pdm locus CSCs. A similar analysis of adult median neurosecretory cell (mNSC) enhancers revealed that they grouped together, as evidenced by sharing of conserved sequence elements, which were largely absent in non-mNSC CSCs with the pdm locus. While further work is required to determine whether these shared elements are important for enhancer activity, these findings suggest a level of structural complexity in the presence and clustering of enhancers that requires further analysis. To construct a better representation of enhancer structure and thus cis-regulatory prediction, one would ideally prefer to use a larger training set of enhancers to improve the accuracy of prediction. These approaches will be addressed in future studies (Ross, 2015).

One of the principal findings of this study is the discovery of 77 enhancers that exhibit a remarkably diverse range of cis-regulatory activities during embryonic and postembryonic development. The biological significance of this enhancer diversity most likely reflects the diversity of the developmental programs in which these transcription factors participate. Functionally related enhancers that share multiple conserved DNA sequences were also identified, and these enhancers could be classified using hierarchical clustering techniques. In addition, this analysis has revealed that the collinearity between the pdm genes is predominantly confined to their POU domain and homeodomain exons, suggesting that their noncoding sequences are diverging at a faster rate than their coding sequences. These results should provide further insight into the regulatory logic that controls cis-regulatory function and thus gene regulation (Ross, 2015).

Mutational analysis of a Drosophila neuroblast enhancer governing nubbin expression during CNS development

While developmental studies of Drosophila neural stem cell lineages have identified transcription factors (TFs) important to cell identity decisions, currently only an incomplete understanding exists of the cis-regulatory elements that control the dynamic expression of these TFs. Previous studies have identified multiple enhancers that regulate the POU-domain TF paralogs nubbin and pdm-2 genes. Evolutionary comparative analysis of these enhancers reveals that they each contain multiple conserved sequence blocks (CSBs) that span TF DNA-binding sites for known regulators of neuroblast (NB) gene expression in addition to novel sequences. This study functionally analyzes the conserved DNA sequence elements within a NB enhancer located within the nubbin gene and highlights a high level of complexity underlying enhancer structure. Mutational analysis has revealed CSBs that are important for enhancer activation and silencing in the developing CNS. Adjusting the number and relative positions of the TF binding sites within these CSBs alters enhancer function (Ross, 2018).

A previous enhancer-reporter transgene survey identified an enhancer (denoted as nub-46) that recapitulated nub expression during embryonic cephalic lobe and VNC NB lineage development (Ross, 2015). As an initial step to functionally characterize the nub-46 enhancer, its conserved sequence blocks were identified by comparative evolutionary analysis using 12 Drosophila species, including D. melanogaster, D. simulans, D. sechellia, D. yakuba, D. erecta, D. ananassae, D. persimilis, D. pseudoobscura, D. willistoni, D. virilis, D. mojavensis, and D. grimshawi. This analysis revealed that nub-46 is made up of 11 CSBs. While many of its conserved elements are novel, a CSB, denoted as 'C', containing two adjacent 9-mer sequences (TAAAAATTG and CATAAAAAA) corresponds to the DNA-binding site motifs for Cas (Ross, 2018).

The nub-46 enhancer-reporter transgene expression is dynamic during embryonic CNS development. Transient nub-46 activation is observed at the cellular blastoderm stage, followed by progressive NB reactivation during embryonic neurogenesis. At stage 9, nub-46 regulates transgene reporter expression in several NBs per ventral cord hemisegment, and enhancer activity is detected in a subset of cephalic lobe NBs. Later in CNS development enhancer/reporter expression is detected in additional cephalic lobe and ventral cord NB lineages. After embryonic stage 13, nub-46 cis-regulatory activity is downregulated in both the brain and ventral cord (Ross, 2018).

To delimit the boundaries of the nub-46 enhancer, both 5' and 3' deletions were generated of the full nub-46 enhancer CSB cluster and the in vivo cis-regulatory activity of these truncated fragments was examined via enhancer-reporter transgenes. This analysis revealed that the centrally located CSBs were sufficient for embryonic CNS expression. However, compared to the full-length enhancer, a reduced enhancer/reporter activity was observed for the core that contains elements 'C' through 'I'. These findings demonstrate that the core fragment consists of activator and repressor sequences required for its wild-type spatial and temporal regulatory dynamics (Ross, 2018).

Given that Cas is a negative regulator of pdm gene expression in embryonic NBs, it was predicted that the putative Cas DNA-binding motifs within nub-46 are required to deactivate enhancer activity. Expression of nub-46 enhancer activity partially overlaps endogenous Cas protein expression in stage 13 embryos. To determine whether the putative Cas binding-motifs function as Cas binding sites, the regulatory activity was examined of a nub-46 deletion that lacks a 40 bp conserved region containing the two Cas motifs. Deletion of the Cas DNA-binding sites triggers ectopic enhancer activity in the cephalic lobes during stage 13, suggesting that the 'C' CSB functions as a repressor element during cephalic lobe development. Interestingly, no significant ectopic enhancer activity was observed in the developing VNC. Therefore, removal of the nub-46 'C' CSB does not completely account for the repressive action of Cas on the nub-46 enhancer, especially in the VNC, and other direct or indirect effects of Cas action on the nub should be considered (Ross, 2018).

While the 'C' element may contain repressor DNA-binding sites, it remained unknown how the nub-46 enhancer is activated in the embryonic CNS. To address this question, the effects of internal deletions within the nub-46 enhancer were further examined. Each of the 10 remaining CSBs were individually removed. Enhancers with these individual deletions were tested in two independent transgenic lines. The wild type control enhancer activity was tested under the same conditions and at the same time as the deletion mutants. It was observed that nub-46 variants lacking either the 'B' (AGAACGCAAT) element or 'E' (CTACCTGAG) element displayed only a modest reduction in enhancer activity compared to the wild-type. Surprisingly, it wasfound that singular removal of other CSBs had only subtle effects on enhancer activity during embryonic NB lineage development, suggesting that these CSBs may be either required at later time points or are functionally redundant (Ross, 2018).

Given that other cis-regulatory enhancers contain a combination of repeat and unique sequence elements, it was hypothesized that nub-46 activation may result from a complex set of multiple inputs. Indeed, self-alignment of conserved sequences within nub-46 revealed that the enhancer is made up of 11 distinct repeat and palindromic elements. Upon closer inspection, seven of the 10 repeat elements were found to be located within the 'C' element, and that many of these repeats were also found in CSBs 'D,' 'H,' and 'I' of the enhancer core (Ross, 2018).

Next, whether the repeat elements within the core are required for enhancer activation was assessed. Loss of the 'C' element does not significantly affect onset of enhancer-reporter expression during embryonic VNC development. Among the six repeats identified within the 'C' element, nearly all are present in the 'D,' 'H,' and 'I' elements, and it was speculated that these may compensate for the loss of repeats in the nub-46 [C]- mutant. To test this hypothesis, the core was truncated to exclude the 'C' element (denoted as the [C -] and then further removed all elements containing repeats ('D,' 'H,' and 'I' elements) from the core enhancer (referred to as [CDHI]-. Surprisingly, removal of these CSBs had little or no effect on enhancer activity. One possible explanation for the lack of any significant effect of element 'C' (and other elements containing repeat sequences) on enhancer activation is that activator sequences are located within elements lacking repeats (elements 'E,' 'F,' and 'G'). To investigate whether the 'E' (CTACCTGAG), 'F' (GGGGTGTCAAATACCAGC), and 'G' (TACCGTA) elements are required for enhancer activation, all three elements were removed from the enhancer [CEFG]- and it was observed that deletion of these resulted in complete loss of reporter activity, suggesting that 'E,' 'F,' and 'G', containing only unique sequences, are required to activate reporter expression. To determine whether a subset of these elements is necessary for enhancer function, the effect was tested of different combinations of internal deletions on cis-regulatory activity during embryonic neurogenesis. While removal of either the 'E,' 'F,' or 'G' elements had little or no effect on enhancer function, only the combined loss of 'E' and 'F' compromised core activity. Notably, however, increased enhancer activity was identified with loss of 'F' and 'G', whereas loss of all three non-repeat elements disrupted enhancer function. Individual deletion of non-repeat CSBs exhibited minor reduction in enhancer activity within brain lineages (Ross, 2018).

Given that all three elements lacking repeat sequences are essential for enhancer function, it was next asked whether enhancer function is modified by the multiplicity of these sequences. To explore this, core enhancers were synthesized that contain three copies of either element, substituting each into the positions of the other two non-repeat elements. Construct expression was also examined during multiple stages of CNS development. Whenthe 'F' and 'G' elements were replaced with 'E' elements, increasing the number of 'E' elements to three, higher enhancer activity was observed within subsets of NBs compared to the wild-type during stage 11. However, by stage 13, higher levels of enhancer activity were also observed throughout the CNS. Notably, ectopic expression was also observed within putative PNS lineages during stage 14. It should be noted that additional co-localization experiments using cell lineage markers would be needed to substantiate the ectopic expression. Increasing the number of 'F' elements also altered core enhancer activity, but the effect was limited to a subset of lateral VNC NBs and dorso-anterior cephalic lobe cells during early stage 12. These differences were not apparent at stage 13 and stage 14. Increasing the number of 'G' elements resulted in diminished expression at all three stages examined (Ross, 2018).

The principal findings of this study are the identification of a core sequence within the nub-46 NB enhancer that is sufficient to recapitulate the embryonic expression pattern of nubbin and that novel non-repeated conserved sequences are required for enhancer activity. This study has delimited the target of Cas repression to a CSB containing two adjacent 9-mer sequences corresponding to the TF DNA-binding motif for Cas in CSB 'C.' Nevertheless, the possibility still exists that Cas is not the only repressor of nub-46 during embryonic CNS development (Ross, 2018).

Also activator CSBs were identified that contain uniquely represented sequences within the enhancer, suggesting that the enhancer may be regulated by as yet uncharacterized TF activators that play a role in the temporal regulation of nubbin. These data suggests that multiple copies of either 'E' or 'F' can function as an activator within the enhancer core. While previous studies have suggested that clusters of repeat regulatory sequences are an important aspect of enhancer regulation, this study points to unique non-repeated motifs as targets of transcriptional activators. While these initial observations revealed altered expression outside the spatial/temporal boundaries of nub-46 activity, further experiments using cell-type specific markers are needed to confirm this ectopic expression (Ross, 2018).

The Drosophila ortholog of mammalian transcription factor Sox9 regulates intestinal homeostasis and regeneration at an appropriate level

Balanced stem cell self-renewal and differentiation is essential for maintaining tissue homeostasis, but the underlying mechanisms are poorly understood. This study identified the transcription factor SRY-related HMG-box (Sox) 100B, which is orthologous to mammalian Sox8/9/10, as a common target and central mediator of the EGFR/Ras and JAK/STAT signaling pathways that coordinates intestinal stem cell (ISC) proliferation and differentiation during both normal epithelial homeostasis and stress-induced intestinal repair in Drosophila. The two stress-responsive pathways directly regulate Sox100B transcription via two separate enhancers. Interestingly, an appropriate level of Sox100B is critical for its function, as its depletion inhibits ISC proliferation via cell cycle arrest, while its overexpression also inhibits ISC proliferation by directly suppressing EGFR expression and additionally promotes ISC differentiation by activating a differentiation-promoting regulatory circuitry composed of Sox100B, Sox21a, and Pdm1. Thus, this study reveals a Sox family transcription factor that functions as a stress-responsive signaling nexus that ultimately controls tissue homeostasis and regeneration (Jin, 2020).

Homeostatic renewal of many adult tissues requires balanced stem cell proliferation and differentiation, a process that is commonly compromised in cancer and in tissue degenerative diseases. The intestinal epithelium in adult Drosophila midgut provides a genetically tractable system for understanding the underlying mechanisms of tissue homeostasis and regeneration driven by resident stem cells. The intestinal stem cells (ISCs) of the Drosophila midgut normally divide to renew themselves and give rise to two different types of progenitor cells that respectively differentiate into enterocyte cells (ECs) and enteroendocrine cells (EEs). Normally, ISCs divide occasionally and thereby maintain the ongoing renewal of the epithelium, a slow process that takes approximately 2-4 weeks. However, upon damage or infection, ISCs are able to rapidly divide to facilitate accelerated epithelial repair in as fast as two days (Jin, 2020).

Extensive studies have implicated the JAK/STAT and the EGFR/Ras/mitogen-activated protein kinase (MAPK) as the two major signaling pathways that regulate ISC proliferation and differentiation during both normal epithelial homeostasis and stress-induced intestinal repair. The EGFR signaling is considered to play a predominant role in the regulation of ISC proliferation because it is required for the JAK/STAT signaling activation-induced ISC proliferation, whereas the JAK/STAT signaling is not essential for EGFR/Ras signaling activation-induced ISC proliferation. The EGFR signaling is also important for remodeling of the differentiated cells, including the exclusion of damaged/aged ECs and incorporation of new cells. The JAK/STAT pathway is also essential for ISC differentiation. ISCs with compromised JAK/STAT activity generate progenitor cells that are incapable of further differentiation. Despite the importance of the two signaling pathways in controlling intestinal homeostasis, their downstream targets-which integrate pathway activities to coordinate ISC proliferation and differentiation-remain elusive (Jin, 2020).

Sox (SRY-related HMG-box) family transcription factors (TFs) are known to have diverse roles in cell-fate specification and differentiation in multicellular organisms. In mouse-small intestine, Sox9, a SoxE subfamily member, is expressed in ISCs to regulate ISC proliferation and differentiation, but whether it acts as an oncogene or a tumor suppressor is still in debate. In Drosophila midgut, Sox21a, a SoxB2 subfamily member, is specifically expressed in ISCs and transient progenitor cells, and is essential for progenitor cell differentiation into mature cells. This study identified Sox100B, the Drosophila ortholog of Sox9, as a common downstream gene target for both the JAK/STAT and the EGFR signaling in regulating ISC proliferation and differentiation. This study also revealed that an appropriate level of Sox100B is critical for its function in regulating ISC proliferation, in that it may allow it to serve as an important mediator for a balanced process of ISC proliferation and differentiation, thereby maintaining intestinal homeostasis (Jin, 2020).

Although it has been well established that in the Drosophila midgut, the stress-responsive JAK/STAT signaling and EGFR/Ras/MAPK signaling are the two major signaling pathways that regulate ISC proliferation and differentiation, the downstream signaling targets that coordinate ISC proliferation and differentiation for intestinal regeneration are still yet to be identified. Sox100B identified in this study may represent such a key target. First, the expression of Sox100B is regulated by both JAK/STAT- and EGFR-signaling pathways. Normally Sox100B is expressed specifically in ISCs and EBs, where JAK/STAT- and EGFR/Ras/MAPK-pathway activities are high, and its expression is highly dependent on the activity of JAK/STAT- and EGFR/Ras/MAPK-signaling activities. Second, similar to the functions of JAK/STAT and EGFR signaling, Sox100B is critically required for both ISC proliferation and differentiation. The sustained EGFR/Ras/MAPK activity in EBs is important for the initiation of DNA endoreplication during the process of EC differentiation, and the sustained JAK/STAT signaling activity in EBs is essential for terminal differentiation toward both EC and EE lineage. Depletion of Sox100B causes ISC quiescence, similar to that caused by the disruption of EGFR signaling, as well as arrest of EB differentiation, similar to that caused by the disruption of JAK/STAT signaling. Third, an appropriate level of Sox100B expression appears to be critical for intestinal homeostasis. This effect by the expression level, as well as its responsiveness to JAK/STAT, EGFR, and potentially other stress-induced signaling activities (not shown), such as Wnt and Hippo signaling, may position Sox100B as a central mediator that coordinates ISC proliferation and differentiation during intestinal homeostasis and regeneration in Drosophila (Jin, 2020).

Sox100B is a Sox family group-E transcription factor, homolog of mammalian Sox8/9/10. In mouse small intestine, Sox9 is expressed in stem cells and progenitor cells at the base of crypts, and loss of Sox9 in the intestinal epithelium causes ISC hyperplasia and failure of Paneth cell differentiation. Interestingly, in the stem cell zone, Sox9 is expressed at low levels in ISCs and high levels in the quiescent or reserved stem cells that are also considered as the secretory progenitors. A possible explanation for these observations is that a low level of Sox9 sustains actively dividing ISCs, while an increase of SOX9 converts these proliferating ISCs into quiescent ISCs that will eventually differentiate into Paneth cells. Similarly, Sox9 is also implicated in regulating colorectal cancer cells, but there are conflicting data regarding whether Sox9 functions as an oncogene or a tumor suppressor. These seemingly contradictory results can be reconciled with a proposed model that Sox9 functions at an appropriate level, with a critical dose of Sox9 that exhibits proliferation-promoting activity, while increasing or decreasing this dose both result in proliferation-inhibitory activity. It is worthy to note that the differentiation-promoting function of Sox9 could potentially further complicate the interpretation of the mutant phenotype. It has been shown in Drosophila gut that defects in differentiation can induce a stressed microenvironment that promotes cell proliferation and propels tumor development (Jin, 2020).

The results of this study suggest many aspects of functional conservation of this Sox E subfamily gene in ISCs from Drosophila to mammals. Sox100B regulates both ISC proliferation and differentiation in the Drosophila intestine, and in terms of regulating ISC proliferation, Sox100B also requires an appropriate expression level. This study has demonstrated that this modulation of Sox100B expression is largely due to a negative feedback mechanism, in which increased Sox100B caused by elevated EGFR/Ras/MAPK signaling in turn suppresses the expression of EGFR, thereby leading to damped EGFR-signaling activity. Of note, contradictory data were recently reported on the roles of Sox100B and Sox21a in regulating ISC proliferation: both a proliferation-promoting role and a tumor-suppressive role for Sox21a in ISCs have been reported; as for the role of Sox100B, a previous study showed in an RNAi genetic screen that Sox100B is required for P.e.-induced ISC proliferation, whereas another study showed that depletion of Sox100B by RNAi causes increased ISC proliferation. Consideration of the effects caused by different levels of Sox100B expression that was observed in the present study may help resolve understanding of apparently disparate functions for these genes as central coordinators of both ISC proliferation and differentiation. It is proposed that, normally, a low level of Sox protein expression sustains ISC proliferation. A transient increase of Sox protein may not only promote cell cycle exit but also activate programs for terminal differentiation, thereby leading to a coordinated ISC proliferation and differentiation and, consequently, a coherent process of epithelial renewal (Jin, 2020).

This study demonstrates that Sox100B directly regulates Sox21a to promote differentiation. One important downstream target of Sox100B and Sox21a appears to be Pdm1, a known EC-fate-promoting factor. Interestingly, overexpression of Pdm1 in progenitor cells rapidly shuts down both Sox100B and Sox21a expression, indicating a negative feedback mechanism. Therefore, the induced Sox100B-Sox21a-Pdm1 axis in the differentiating ECs not only promotes cell differentiation, but also acts in a feedback mechanism to turn down EGFR and JAK/STAT signaling activities, thereby allowing ECs to terminally differentiate. This differentiation-promoting axis might also have a role in turning down ISC-specific programs, which are independently regulated by EGFR or JAK/STAT signaling pathways. For example, downregulation of the stem-cell-factor Esg is required for EB differentiation, and ectopic expression of Pdm1 is able to antagonize Esg expression in progenitor cells. These kinds of feedback regulation could be a common strategy used for initiation and finalization of a cell-differentiation program (Jin, 2020).

In summary, this study identified the transcription factor Sox100B as a major effector downstream of JAK/STAT and EGFR pathways that acts at an appropriate level to coordinate ISC proliferation and differentiation during both normal intestinal homeostasis and during damage- and infection-induced intestinal regeneration in Drosophila. With the 'just-right' effect endowed by a feedback mechanism, Sox100B behaves as a homeostatic sensor in the intestinal epithelium that coordinates stem cell proliferation with stem cell differentiation under various environmental conditions. It is proposed that this expressional and functional modulation associated with Sox family transcription factors may be a general mechanism for maintaining tissue homeostasis and regeneration in many organs, including those in mammals, and that deregulation of this mechanism may lead to tissue degeneration or cancer development (Jin, 2020).

A novel temporal identity window generates alternating Eve(+)/Nkx6(+) motor neuron subtypes in a single progenitor lineage

Spatial patterning specifies neural progenitor identity, with further diversity generated by temporal patterning within individual progenitor lineages. In vertebrates, these mechanisms generate 'cardinal classes' of neurons that share a transcription factor identity and common morphology. In Drosophila, two cardinal classes are Even-skipped (Eve)(+) motor neurons projecting to dorsal longitudinal muscles, and Nkx6(+) motor neurons projecting to ventral oblique muscles. Cross-repressive interactions prevent stable double-positive motor neurons. The Drosophila neuroblast 7-1 (NB7-1) lineage uses a temporal transcription factor cascade to generate five distinct Eve(+) motor neurons; the origin and development of Nkx6(+) motor neurons remains unclear. This study used a neuroblast specific Gal4 line, sparse labelling and molecular markers to identify an Nkx6(+) VO motor neuron produced by the NB7-1 lineage. Lineage analysis to birth-date the VO motor neuron to the Kr(+) Pdm(+) neuroblast temporal identity window. Gain- and loss-of-function strategies to test the role of Kr(+) Pdm(+) temporal identity and the Nkx6 transcription factor in specifying VO neuron identity. Lineage analysis identifies an Nkx6(+) neuron born from the Kr(+) Pdm(+) temporal identity window in the NB7-1 lineage, resulting in alternation of cardinal motor neuron subtypes within this lineage (Eve>Nkx6>Eve). Co-overexpression of Kr/Pdm generates ectopic VO motor neurons within the NB7-1 lineage - the first evidence that this TTF combination specifies neuronal identity. Moreover, the Kr/Pdm combination promotes Nkx6 expression, which itself is necessary and sufficient for motor neuron targeting to ventral oblique muscles, thereby revealing a molecular specification pathway from temporal patterning to cardinal transcription factor expression to motor neuron target selection. Thus this study shows that one neuroblast lineage generates interleaved cardinal motor neurons fates; that the Kr/Pdm TTFs form a novel temporal identity window that promotes expression of Nkx6; and that the Kr/Pdm > Nkx6 pathway is necessary and sufficient to promote VO motor neuron targeting to the correct ventral muscle group (Seroka, 2020).

Neural diversity from flies to mice arises from two major developmental mechanisms. First, neural progenitors acquire a unique and heritable spatial identity based on their position along the rostrocaudal or dorsoventral body axes. Second, temporal patterning based on neuronal birth-order results in individual progenitors producing a diverse array of neurons and glia. Temporal patterning is best characterized in Drosophila; neural progenitors (neuroblasts) located in the ventral nerve cord, central brain, and optic lobes all undergo temporal patterning, in which the neuroblast sequentially expresses a cascade of TTFs that specify distinct neuronal identities. Although all neuroblasts undergo temporal patterning, the TTFs are different in each region of the brain. Similar mechanisms are used in the mammalian cortex, retina, and spinal cord, although many TTFs remain to be identified (Seroka, 2020).

A major open question is how transient expression of TTFs like Kr and Pdm lead to long-lasting specification of molecular and morphological neuronal diversity. Good candidates for integrating spatial and temporal cues to consolidate motor neuron identity are homeodomain transcription factors expressed in post-mitotic motor neurons. In vertebrates, dorsoventral domains of the spinal cord are partitioned into 12 distinct cardinal classes of neurons - each characterized by development from a common progenitor domain, expression of unique homeodomain transcription factors with cross-repressive interactions to stabilize boundaries, and generating neurons with common morphology. This nomenclature is adapted to define Eve+ and Nkx6+ (Flybase: HGTX) motor neurons as two 'cardinal classes' of motor neurons: each class expresses a homeodomain transcription factor (Eve or Nkx6) with cross-repressive interactions, and each class consists of motor neurons with related neuronal morphology (Eve+ motor neurons project to dorsal and lateral longitudinal muscles; Nkx6+ motor neurons project to ventral muscle groups) (Seroka, 2020).

The Drosophila neuroblast 7-1 (NB7-1) is arguably the best characterized system for understanding TTF expression and function. Similar to most other ventral nerve cord neuroblasts, NB7-1 expresses the canonical TTF cascade Hb-Kr-Pdm-Cas with each TTF inherited by the GMCs born during an expression window, and transiently maintained in the two post-mitotic neurons produced by each GMC. The TTF cascade generates diversity among the five Eve+ U1-U5 motor neuron progeny of NB7-1: Hb specifies U1 and U2, Kr specifies U3, Pdm specifies U4, and Pdm/Cas together specify U5. Identifying TTF target genes, including transcription factors and cell surface molecules, will provide a comprehensive view of how developmental determinants direct neuronal morphology and synaptic partner choices (Seroka, 2020).

It has long been thought that the cardinal classes of motor neurons derive from distinct progenitors; Eve+ motor neurons derive from NB7-1, NB1-1, and NB4-2 whereas Hb9+ or Nkx6+ motor neurons derive from NB3-1 and others. However, DiI labeling of NB7-1 identified a potentially unknown motor neuron innervating ventral muscles, which is distinct from dorsal and lateral longitudinal muscles targeted by the Eve+ motor neurons. The observed ventral projection in this lineage could reflect transient exuberant outgrowths that are lost during larval life, or they could be due to an uncharacterized motor neuron that forms stable synapses with ventral muscles (Seroka, 2020).

This study shows that a newly discovered Kr/Pdm TTF window generates an Nkx6+ Eve-motor neuron, born between U3 and U4 in the NB7-1 lineage, that projects to ventral oblique (VO) muscles. It was also shown that overexpression of Kr/Pdm together, or Nkx6 alone, generates ectopic VO motor neurons based on molecular marker expression. Finally, this study demonstrates that Nkx6 is required for proper motor neuron axon targeting to ventral oblique muscles. These results establish a genetic pathway from TTFs (Kr/Pdm), to a cardinal motor neuron transcription factor (Nkx6) to motor axonal targeting. Also the unexpected discovery was made that a single progenitor can alternate production of different cardinal motor neuron classes (Seroka, 2020).

Kr/Pdm co-expression has been detected in several neuroblast lineages, but until now there has not been evidence that this TTF combination could specify neuronal identity. Previous work showed that the Kr/Pdm window generates a Kr/Pdm GMC, and this study shows that this GMC generates an Nkx6+ ventral-projecting motor neuron. It is unknown whether Kr/Pdm directly or indirectly activate nkx6 expression. The nkx6 gene lies in a 45kb region devoid of genes, and there are only a few, sparse predicted Kr or Pdm binding sites in this genomic expanse. How do Kr and Pdm together specify one fate (Nkx6+ VO motor neuron) whereas Kr or Pdm alone specify completely different fates (Eve+ dorsal motor neurons)? It is likely that Kr/Pdm together activate a different suite of target genes than either alone. For example, Kr/Pdm together may directly activate nkx6 expression, whereas neither alone has that potential. The emergence of single cell transcriptome and ChIP studies will help to reveal how the combination of Kr/Pdm TTFs generates different cell fate output compared to Kr or Pdm alone (Seroka, 2020).

The production of an Nkx6+ VO motor neuron in Kr/Pdm window interrupts the sequential production of Eve+ dorsal motor neurons in the NB7-1 lineage, resulting in an Eve > Nkx6 > Eve alternation of cardinal motor neuron production within the lineage. This is unusual, as in most cases neurons with similar morphology or function are produced together in a lineage. In mammals, progenitors generate neurons first, followed by glia; no examples are known of neuron>glia>neuron production from a single lineage. Similarly, Drosophila central brain neuroblast lineages produce the mushroom body γ neurons, then α'/ β' neurons, and lastly α/β neurons, with no evidence for alternating or interspersed fates. In the abdominal NB3-3 lineage, the early-born cells are in a mechanosensitive circuit, whereas the late-born cells are in a proprioceptive circuit. The only possible example of interleaved production of two morphological classes of neurons is in the Drosophila lateral antennal lobe neuroblast lineage, which alternate between uniglomerular and multiglomerular (AMMC) projection neurons. The use of clonal and temporal labeling tools will be needed to examine additional lineages to determine the prevalence of lineages producing temporally interleaved neuronal subtypes as in the NB7-1 lineage (Seroka, 2020).

Overexpression of Kr/Pdm or Nkx6 can induce only 2-3 ectopic VO motor neurons within the NB7-1 lineage. Clearly not all neurons in the lineage are competent to respond to these transcription factors. Early-born temporal identities specified by Hb and Kr (U1-U3) are unaffected by Kr/Pdm or Nkx6 overexpression, which is similar to previous data showing that early temporal fates are not affected by overexpression of later TTFs in multiple lineages. It remains a puzzle why the Kr+ U3 neuron does not switch to a VO fate upon overexpression of Kr/Pdm. There may need to be an equal level of Kr and Pdm to specify VO fate, although this would not explain why Kr/Pdm overexpression converts the Pdm+ U4 motor neuron to a VO fate. Alternatively, there may be an early chromatin landscape that blocks access to relevant Pdm target loci (Seroka, 2020).

It is noted that the assay of VO neuronal identity was done in newly-hatched larvae. Although motor circuits are functional at this time, larvae grow for five more days. These is no data on whether the ectopic VO motor neurons are functional or are maintained through the life of the larvae. This would be an important question for the future (Seroka, 2020).

Nkx6 and Eve have cross-repressive interactions (Broihier, 2004), but with some limitations: early-born Eve+ motor neurons are not affected by Nkx6 overexpression. Wild type animals even show sporadic expression of Nkx6 in the Eve+ U2 motor neuron, but in these neurons it has no effect on Eve expression, nor does it promote targeting to ventral oblique muscles. There appears to be a mechanism to block endogenous or overexpressed Nkx6 function in the early lineage of neuroblasts producing Eve+ motor neurons. The mechanism 'protecting' early-born Eve+ neurons from Nkx6 repression of Eve is unknown. Early lineages may lack an Nkx6 cofactor; Nkx6 could act indirectly via an intermediate transcription factor missing in early lineages; the early TTFs Hb or Kr may block Nkx6 function; or the eve locus could be in a subnuclear domain inaccessible to Nkx6 (Seroka, 2020).

Nkx6 promotes motor neuron specification in both Drosophila and vertebrates. In Drosophila, loss of Nkx6 reduces ventral projecting motor neuron numbers and increases the number of Eve+ neurons, while overexpression increases ventral projecting motor neuron numbers at the expense of Eve+ neurons. In vertebrates the Nkx6 family members Nkx6.1/Nkx6.2 appear to play a broader role in motor neuron specification. Nkx6.1/Nkx6.2 show early expression throughout the pMN domain; mice mutant for both Nkx6 family members lack most somatic motor neurons; and Nkx6.1 overexpression in chick or zebrafish can induce ectopic motor neurons. It would be interesting to investigate whether vertebrate Nkx6.1/Nkx6.2 are required to suppress a specific motor neuron identity, similar to the antagonistic relationship between Nkx6 and Eve in Drosophila (Seroka, 2020).

Neuroblasts in all regions of the Drosophila CNS (brain, ventral nerve cord, optic lobe) use TTF cascades to generate neuronal diversity, yet less is known about TTF target genes. It is likely that TTFs induce expression of suites of transcription factors that persist in neurons and confer their identity. Examples may include the 'morphology transcription factors' that specify adult leg motor neuron dendrite projections, but in this case it remains unknown whether these transcription factors control all other aspects of adult motor neuron identity. It is possible that 'morphology transcription factors' are one module downstream of a broader regulatory tier similar to the terminal selector genes in C. elegans (Seroka, 2020).

This study has identified a linear pathway from Kr/Pdm to Nkx6 which specifies VO motor neuron identity. TTFs could act by two non-mutually exclusive mechanisms: inducing a stable combinatorial codes of transcription factors that consolidate neuronal identity, or by altering the chromatin landscape to have a heritable, long lasting effect on motor neuron gene expression. The observation that Nkx6 is maintained in the VO neuron after fading of Kr/Pdm expression supports the former mechanism. Identification of Kr/Pdm or Nkx6 target genes would give a more comprehensive understanding of TTF specification of neuronal identity (Seroka, 2020).

The results presented in this work lead to several interesting directions. Other embryonic VNC lineages exhibit a Kr/Pdm window; does this window generate neurons in these lineages? Are there common features to neurons born in the Kr/Pdm window? Furthermore, do ectopic VO neurons make functional presynapses with the ventral oblique muscles, and do they have the normal inputs to their dendritic postsynapses? In only a few cases has it been shown the TTF-induced neurons are functionally integrated into the appropriate circuits [30]. Kr and Pdm orthologs have been identified in vertebrates. Looking for dual expression of Kr and Pdm orthologs in vertebrates may reveal a role in specifying temporal identity, similar to evidence for Hb and Cas TTFs having vertebrate orthologs that specify temporal identity (Seroka, 2020).

Earlier treatment of Nubbin/Pdm-1 in The Interactive Fly

pdm-1 and pdm-2 are closely linked genes. They are coordinately regulated, with overlapping functions. Their expression is mostly ectodermal and both are essential for proper neuron development. The effects of mutation show that pdm-2 is quantitatively more important in neuroblast differentiation than pdm-1 (Yang, 1995). There is significant independently regulated endodermal expression for pdm-1 as well as expression of pdm-1in the wing.

pdm-1, more properly termed nubbin is expressed in both the anterior and posterior midgut primordia and in the developing endoderm. However, pdm-1 is not expressed in the central domain of the gut. This lack of central domain expression is thought to be due to both Ubx and dpp expression in the visceral mesoderm. Two gaps in pdm-1 expression are found: one in the anterior domain, underlying a region of dpp expression, and the second in parasegment 7, underlying a region of Ubx induced dpp expression. No developmental defects are known to result from pdm-1 expression in endoderm (Affolter, 1995).

Expression of pdm-1 in the wing disc is ubiquitous. In spite of this, it appears to be required locally in the wing hinge. Removal of pdm-1 activity from the hinge region results in a severe wing phenotype, while removal from more distal regions results in a less severe disruption. It is hypothesized that the effects in the hinge region are due to a disruption in the proximal-distal axis (Ng, 1995). pdm-1 expression in the wing disc is regulated by wingless which has a primary role in specifying the proximal-distal axis of the wing (Ng, 1996).

Cell interactions mediated by Notch-family receptors have been implicated in the specification of tissue boundaries in vertebrate and insect development. Although Notch ligands are often widely expressed, tightly localized activation of Notch is critical for the formation of sharp boundaries. Evidence is presented that the POU domain protein Nubbin contributes to the formation of a sharp dorsoventral (DV) boundary in the Drosophila wing. Nubbin represses Notch-dependent target genes and sets a threshold for Notch activity that defines the spatial domain of boundary-specific gene expression (Neumann, 1998).

Certain features of the abnormal wings in flies mutant for nubbin suggest a possible role for Nubbin protein in spatially limiting Notch activity at the DV boundary of the wing. The row of sensory bristles that makes up the wing margin is disorganized in nubbin wing mutants, suggesting a defect in Wingless or Notch activity. In preparations where the wing margin is viewed edge on, this disorganization reflects a broadening of the region where bristles form. Margin bristles are normally specified in cells very close to the DV boundary, reflecting a requirement for high levels of Wingless signaling activity. The broadening of the margin suggests that Wingless might be ectopically expressed in nubbin mutant wing discs. Wingless is normally expressed in a stripe of two to three cells straddling the DV boundary. In nubbin mutant discs, this stripe is widened considerably. Expression of the Notch targets vestigial and cut are similarly expanded at the DV boundary in nubbin mutants (Neumann, 1998).

To determine whether the effect on bristle specification is a direct consequence of removing nubbin activity, clones of nubbin mutant cells were generated in a wild-type background. Ectopic wing margin bristles are found in nubbin mutant clones located near the endogenous wing margins. The nubbin mutant clones show ectopic expression of neuralized, a molecular marker for precursors of the sensory neurons that innervate the bristles. The nubbin mutant clones misexpress wingless and vestigial. The largely autonomous effect of nubbin mutant clones on bristle specification may be due to the relatively low levels of Wg protein expressed in nubbin mutant clones. Together with the results on cut expressioon, these observations suggest that Notch target genes are transcriptionally up-regulated in nubbin mutant cells near the DV boundary (Neumann, 1998).

To test whether ectopic activation of these genes in nubbin mutant clones directly depends on Notch signaling activity, clones of cells were generated that were simultaneously mutant for nubbin and Suppressor of Hairless [Su(H)]. Su(H) encodes a DNA binding protein that mediates transcriptional activity of Notch target genes. Su(H) is autonomously required for the expression of wingless, vestigial, and cut at the DV boundary and binds directly to the vestigial DV boundary enhancer. Clones of cells mutant for both nubbin and Su(H) do not ectopically activate wingless, demonstrating that ectopic expression of wingless in nubbin mutant cells depends on activity of the Notch pathway. To confirm that Nubbin acts downstream of Notch, a test was performed to see whether overexpression of Nubbin could suppress the effects of a ligand-independent form of Notch. When Nubbin is coexpressed with such a constitutively active Notch, ectopic Wingless expression is strongly reduced. Together, these observations suggest that Nubbin may act as a direct repressor of Notch-dependent target gene expression. These findings argue that the effects of Nubbin are unlikely to be mediated by indirect effects on the expression of Notch ligands (Neumann, 1998).


GENE STRUCTURE

pdm-1 and pdm-2 are both transcribed in the same direction with respect to the centromere: distal to proximal. They are separated by 60 kb (Yeo, 1995).

Bases in 5' UTR - 192

Exons - four

Bases in 3' UTR - 485


PROTEIN STRUCTURE

Characterization of cDNA clones reveal two alternative forms of PDM-1 transcripts, which differ in a translated first exon, but have identical second and third exons (Ng, 1995).

Amino Acids - 498

Structural Domains

PDM-1 has a homeodomain (the POU homeodomain) and a POU domain (Lloyd, 1991).

The 75 amino acid POU-specific (POUs) domain and a 60 amino acid carboxy-terminal homeo (POUh) domain are joined by a hypervariable linker segment that can vary from 15 to 56 amino acids in length in different POU domain proteins. Thus the POU domain is not a single structural domain; indeed, the POUs and POUh segments form separate structurally independent domains. The POUs and POUh domains are, however, always found together and have therefore coevolved. Both POUs and POUh domains contain helix-turn-helix motifs. The POUs-domain structure is very similar to that of lambda and 434 bacteriophage proteins, but there are significant differences in the length of the first alpha helix, and the "turn" connecting the two HTH alpha helices is also longer. Both POUs and POUh bind DNA, and the length of the linker regulates the efficacy of binding various DNA sequence motifs, especially because POUs and POUh DNA binding sites have different spacings in different promoter elements (Herr, 1995).


nubbin/POU domain protein 1: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 22 December 2018 

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

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