InteractiveFly: GeneBrief

scarecrow: Biological Overview | References

Gene name - scarecrow

Synonyms -

Cytological map position - 80F-80F

Function - Transcription factor

Keywords - optic lobe, pharyngeal primordia, central nervous system, brain, regulates Pdf neuropeptide expression controlling circadian rhythms

Symbol - scro

FlyBase ID: FBgn0287186

Genetic map position - chr3L:24,724,162-24,758,036

Classification - Homeodomain

Cellular location - nuclear

NCBI links: EntrezGene, Nucleotide, Protein
scro orthologs: Biolitmine
Recent literature
Contreras, E. G., Glavic, A., Brand, A. H. and Sierralta, J. (2021). The serine protease homologue, Scarface, is sensitive to nutrient availability and modulates the development of the Drosophila blood brain barrier. J Neurosci. PubMed ID: 34210781
The adaptable transcriptional response to changes in food availability not only ensures animal survival, but also lets progressing with embryonic development. Interestingly, the central nervous system is preferentially protected to periods of malnutrition, a phenomenon known as 'brain sparing'. However, the mechanisms that mediates this response remains poorly understood. To get a better understanding of this, Drosophila melanogaster was used as a model, analysing the transcriptional response of neural stem cells (neuroblasts) and glia of the blood-brain barrier (BBB), from larvae of both sexes, during nutrient restriction using targeted DamID. Differentially expressed genes were found in both neuroblasts and glia of the BBB, although the effect of nutrient deficiency was primarily observed in the BBB. The function of a nutritional sensitive gene expressed in the BBB, the serine protease homologue, scarface (scaf), was characterized. Scaf is expressed in subperineurial glia in the BBB in response to nutrition. Tissue-specific knockdown of scaf increases subperineurial glia endoreplication and proliferation of perineurial glia in the blood-brain barrier. Furthermore, neuroblast proliferation is diminished upon scaf knockdown in subperineurial glia. Interestingly, re-expression of Scaf in subperineurial glia is able to enhance neuroblast proliferation and brain growth of animals in starvation. Finally, this study shows that loss of scaf in the blood-brain barrier increases the sensitivity to drugs in adulthood suggesting a physiological impairment. It is proposed that Scaf integrates the nutrient status to modulate the balance between neurogenesis and growth of the BBB, preserving the proper equilibrium between the size of the barrier and the brain.

scarecrow (scro) gene encodes a Drosophila homolog of mammalian Nkx2.1 that belongs to an evolutionally conserved NK2 family. Nkx2.1 has been well known for its role in the development of hypothalamus, lung, thyroid gland, and brain. However, little is known about biological roles of scro. To understand scro functions, this study generated two types of knock-in mutant alleles, substituting part of either exon-2 or exon-3 for EGFP (or Gal4) by employing the CRISPR/Cas9 genome editing tool. Using these mutations, spatio-temporal expression patterns of the scro gene and its mutant phenotypes were characterized. Homozygous knock-in mutants are lethal during embryonic and early larval development. In developing embryos, scro is exclusively expressed in the pharyngeal primordia and numerous neural clusters in the central nervous system (CNS). In postembryonic stages, the most prominent scro expression is detected in the larval and adult optic lobes, suggesting that scro plays a role for the development and/or function of this tissue type. Notch signaling is the earliest factor known to act for the development of the optic lobe. scro mutants lacked mitotic cells and Delta expression in the optic anlagen, and showed altered expression of several proneural and neurogenic genes including Delta and Notch. Furthermore, scro mutants showed grossly deformed neuroepithelial (NE) cells in the developing optic lobe and severely malformed adult optic lobes, the phenotypes of which are shown in Notch or Delta mutants, suggesting scro acting epistatic to the Notch signaling. From these data it is proposed that scro plays an essential role for the development of the optic lobe, possibly acting as a regional specification factor (Yoo, 2020).

Scro was first identified 20 years ago, and it was shown to be a fly homolog of mammalian Nkx2.1, which was discovered originally for its transcriptional activity on the thyroid specific genes (Guazzi, 1990; Zaffean, 2000). Scro displays approximately 44% identity to Nkx2.1 (ID 21869, mouse) and 47% to Nkx2.4 (ID 644524, mouse), a paralog of Nkx2.1, suggesting that scro is homologous to both genes. Studies in zebrafish showed that Nkx2.1 and Nkx2.4 have partially overlapping functions in the development of the hypothalamus (Manoli, 2014). In Drosophila, scro expression was found in the pharyngeal primordia and in many distinct clusters of neural precursor cells in the embryonic CNS, leading to a speculation that scro function might be involved in the development of these tissues (Zaffran, 2000). However, no further studies followed to explore in vivo roles of scro. One of the main reasons is the lack of useful mutations for this gene. Because the scro locus is situated in a heterochromatized region of the third chromosome that is known for a genetically inactive 'cold-spot', genetic manipulations of this gene have been a challenge (Yoo, 2020).

There are four annotated transcript types (RA/RB/RC/RF) from the scro locus, and two start codons are predicted to be used for translation; one in E2 for RA/RB/RC and the other in E3 for RF. To further delve into scro's function, scroΔE2 and scroΔE3 knock-in alleles in which a reporter gene (EGFP or Gal4) replaces either part of E2 (scroΔE2) or E3 (scroΔE3). The scroΔE2 was designed not only to disrupt but also reflect the expression of RA/RB/RC transcripts (collectively refered to as RA-type). In this mutant, RF expression was unaffected. In contrast, the scroΔE3 was intended to disrupt the expression of all four transcript types but to report expression patterns of RF isoform only. However, contrary to prediction, mutant phenotypes and complementation tests suggest that scroΔE3 is not a null but rather a weaker allele than scroΔE2, and currently little is known about a cause of this unexpected result (Yoo, 2020).

Both types of scro knock-in mutants are homozygous lethal, suggesting that scro plays a vital function. Undisturbed RF expression in scroΔE2 suggests that the vital function is mainly provided by the RA-type isoforms. Based on prominent scro expression in the pharyngeal primordia and significantly less food consumption by scro mutant larvae, it is speculated that subnormal feeding activity due to aberrant development and/or function of pharyngeal and foregut tissue might contribute at least partly to the lethality. It was also noticed that numerous clusters of scro-expressing neurons are present in the larval CNS including the pars intercerebralis (PI) region which is considered to be analogous to vertebrate hypothalamus-pituitary axis. PI is a major neuroendocrine center that regulates metabolism and growth in flies. Therefore, defective neuroendocrine system and/or subnormal activities of the CNS might impinge on a lethal cause of the scro mutants (Yoo, 2020).

It is noted that the knock-in reporters may not completely recapitulate the endogenous scro expression for the following reason. Both scroΔE2 and scroΔE3 knock-ins produce a transcript containing a reporter-coding ORF that starts shortly after the endogenous start codon (AUG). It is known that when two AUGs are closely situated in a single mRNA, 43S pre-initiation complex (PIC) starts translation at the first AUG of upstream ORF (uORF). If uORF is short due to early termination codon, PIC often re-initiates the translation at the downstream AUG, the event of which is referred to as 'leaky scanning'. In the case of E1-E2/Reporter from scroΔE2 and E1-E3/Reporter from scroΔE3, translation from the first native AUG terminates shortly, raising the probability of the re-initiation by PIC at the reporter AUG. On the other hand, another E1-E2-E3/Reporter transcript type produced also in scroΔE3 mutants contains three start codons; two native ones in E2 and E3 and the third one in the reporter. In this case, the most upstream AUG in E2 is expected to be used first; however, the resulting lengthy uORF (173 codons) would likely suppress the re-initiation of translation at the downstream reporter-AUG. Hence, the E1-E3/Reporter transcript should be considered the main source for the reporter expression that was observed with the scroΔE3. Despite such complicated molecular events concerning the translation efficiency of the knock-in transcripts, comparable expression patterns between the knock-in reporters and in situ hybridization (Zaffran, 2000) indicate that at least scroΔE2 recapitulates most, if not all, endogenous scro expression patterns. Moreover, scro knockdown by scroΔE2-Gal4 phenocopied the lethal phenotype of scro knock-in mutants, further supporting the fidelity of this reporter. Therefore it is argued that scro knock-in alleles are certainly a valuable tool to further understand scro's expression and functions (Yoo, 2020).

Reporter expression patterns by scroΔE2 generally agree with those by scroΔE3 particularly in the postembryonic CNS, implying that both RA-type and RF transcripts are simultaneously produced in the same neural cells. However, dissimilar expression patterns was also observed between the two reporters. For instance, embryonic expression is detected only in scroΔE2, implying that the RA-type transcript is a key player during embryogenesis. In addition, some neural clusters in the adult central brain and larval VNC are unique to either scroΔE2 or scroΔE3. These results suggest both common and distinct roles played by scro isoforms. Differential expression patterns and distinct functions of individual isoforms are not unprecedented in other NK genes. For instance, only one of two isoforms of Nkx2.1 is known to play a major role in pulmonary differentiation and development. Similarly, two isoforms of vnd gene perform different roles for the embryonic nervous system development and the differentiation of photoreceptor cells during metamorphosis (Yoo, 2020).

This study has shown that scro's function is critical for the OL development. Although the underlying molecular mechanisms remain to be investigated, it is hypothesized that scro might act as a specification factor of the OL precursor cells during embryogenesis. Members of the NK family have been shown to act as specification factors or in upstream event essential for development of a certain tissue (or cell) type. For instance, Drosophila VNC develops from three columns of neuroectodermal cells along the dorsoventral (DV) axis: ventral, intermediate, and dorsal. vnd induces ventral fates and represses intermediate fates through the regulation of an array of proneural genes including achaete, scute, and l'sc. Loss of vnd transforms ventral into intermediate column identity. In mammalian telencephalon development, Nkx2.1 is critical for the specification of the ventral pallidum, as Nkx2.1-deficiency causes the transformation of pallidum to dorsal striatum (Sussel, 1999; van den Akker, 2008). Furthermore, Nkx2.1 is important for the specification of post-mitotic GABAergic neurons generated in the medial ganglionic eminence (Butt, 2008; Du, 2008; Yoo, 2020 and references therein).

These data provide some clues about what roles scro plays for the OL development. It is known that the Notch signaling is required for fate determination of the embryonic NE cells of the optic placode during and after its invagination and for later differentiation of NE cells into the OL neuropils. According to the current data, scro seems to act epistatic to the Notch signaling. For instance, Delta expression in the optic anlagen is significantly reduced in scro mutants, suggesting that Delta expression is under control of scro. Besides, the Notch-expressing NE cells in scro mutants do not show a typical columnar shape and layer arrangement, and their progeny cells fail to form proper neural projections. These results suggest that scro function is required for the NE cells to establish characteristic morphology and arrangement of NE cells and proper differentiation of progeny cells. In addition, hypomorphic scro mutants fail to form the lamina structure and show a substantially smaller medulla region partly due to defective neural projections of medulla-like cells. Interestingly, similar phenotypes were described for the loss-of-function of Notch or Delta (Yoo, 2020).

scro mutants also show significantly reduced svp and cas expression but elevated expression of Notch, achaete, lethal of scute, and dpn, suggesting that an array of proneural and neural genes involved in cell fate determination of NE and/or NBs is directly or indirectly influenced by scro both positively and negatively. Lastly, following larval hatching, the NE cells actively proliferate through symmetric division and form the outer and inner optic anlage (OOA and IOA), which further develop to outer (OPC) and inner (IPC) proliferation centers, respectively. This study found that scro expression is restricted to the IOA in L2 stage, but Delta expression and mitotic activities of proliferating NE cells at this stage are absent from both optic anlagen in scroΔE2 mutants. A plausible explanation for such contradiction is that the fates of embryonic NE cells are altered in scro mutants, thus lacking their cellular properties to proliferate and form both IOA and OOA. Testing this hypothesis will be a future subject of study. In summary, this study generated scro knock-in alleles and characterized developmental and spatial scro expression patterns and mutant phenotypes, leading to the uncovering of a novel function of Drosophila scro for the development of adult visual processing nervous system, possibly as a regional specification factor (Yoo, 2020).

A homeobox transcription factor Scarecrow (SCRO) negatively regulates Pdf neuropeptide expression through binding an identified cis-acting element in Drosophila melanogaster

In Drosophila, transcriptional feedback loops contribute to intracellular timekeeping mechanisms responsible for daily rhythms. Pigment-dispersing factor (PDF) is the major neuropeptide produced by latero-ventral neurons (LNvs) that function as a central pacemaker for circadian locomotor activity rhythms. PDF synchronizes other clock neurons thereby playing an essential role in the maintenance and coordination of circadian locomotor rhythms. However, the underlying molecular mechanism of the LNvs-specific Pdf expression is not well understood. Using Pdf promoter-bashing experiment, a cis-acting Pdf regulatory element (PRE) was identified that is sufficient for driving Pdf expression in the LNvs. A homeobox transcription factor, scarecrow (SCRO) was identified as a direct binding factor to PRE. Furthermore, transgenic expression of scro in the clock neurons abolished Pdf expression and circadian locomotor activity rhythms, and such repressive function requires DNA-binding homeodomain, but none of the other conserved domains. scro is predominantly expressed in the optic lobe and various clusters of cells in other areas of the central nervous system. A homozygous scro-null mutant generated by CRIPSR is lethal during embryonic and early larval development, suggesting that scro plays a vital role during early development (Nair, 2020).

Cell autonomous clock comprising of transcriptional and translational feedback loops drives biological rhythms in almost all living organisms. Transcriptional feedback loops in Drosophila melanogaster consist of two interlocking molecular loops involving core-clock regulators, Period (Per), Timeless (Tim), Clock (Clk), and Cycle (Cyc) proteins. Clk and Cyc bind to E boxes of per and tim in the per/tim feedback loop and vrille (vri) and Par domain1ε (Pdp1ε) in the Clk feedback loop. Several post-translational modifiers involving kinases and phosphatases in turn regulate stability and activity of Per and Tim. Such intracellular network of the molecular feedback loops generate output rhythms that manifest in physiological and behavioral rhythms of animals by regulating clock-controlled genes and through extracellular signaling between cells using secreted neuropeptides (Nair, 2020).

Pigment-dispersing factor (PDF), a structural homolog of crustacean pigment-dispersing hormones (PDH), is a well-characterized clock-associated neuropeptide in Drosophila. Expression of the Pdf gene is restricted to sixteen neurons out of the approximately 150 clock neurons in the adult brain, and these PDF neurons consist of two distinct groups; the small and large lateral neurons ventral (s- and l-LNvs). PDF peptide and PDF neurons are critical for the maintenance of circadian locomotor activity rhythms. Pdf-null mutants (Pdf01) are largely arrhythmic under constant darkness (DD), and a minor portion of rhythmic Pdf01 flies have shorter free-running periods than wild-type flies. Moreover, PDF receptor-null mutants exhibit behavioral defects similar to those of Pdf01, further providing evidence for the crucial role of PDF signaling in regulating circadian rhythms. PDF is also required for the coordination of molecular rhythms in other pacemaker cells (Nair, 2020).

Given the importance of PDF in the regulation of circadian locomotor activity rhythms, it is essential to maintain PDF levels in the s-LNvs. Notably, Pdf transcription, as well as post-translational regulation, is affected by core-clock factors. Previous work has shown that Pdf mRNA expression in the s-LNvs is largely absent in the ClkJrk and cyc0 mutants, implying that Clk and Cyc proteins act as transcriptional activators of the Pdf gene. However, the lack of E-box sequence, a known regulatory element for Clk and Cyc, in the Pdf upstream sequence suggests that Clk and Cyc are indirect regulators of Pdf. Such indirect activation could involve Per and Tim, as the expression of these clock regulators is directly regulated by Clk and Cyc. However, transcript levels of Pdf are not affected in per01 and tim01 mutants, indicating that there are other factors involved in the direct transcriptional regulation of Pdf. In the larval lateral neurons, a bZIP transcriptional repressor Vrille was shown to suppress PDF peptide but not Pdf mRNA level (Nair, 2020).

In addition to the positive regulation, a previous study suggested that Pdf expression can be negatively regulated. Transgenic introduction of a genomic fragment containing Drosophila virilis Pdf coding region (for short, DvPdf) and 1.9-kb upstream region into the D. melanogaster genome resulted in the DvPdf expression in non-PDF clock neurons, LNds and 5th s-LNv, and the endogenous PDF neurons in adult CNS. Reporter gene expression driven by DvPdf-Gal4, which was made with the 1.9-kb upstream regulatory region, showed the same results as did the DvPdf transgene. The ectopic DvPdf expression in non-PDF neurons is perhaps because negative regulators of Pdf do not act on the DvPdf regulatory region owing to the sequence divergence. This result raised the possibility that Pdf transcription is normally inactive in the LNds, and 5th s-LNv neurons. Thus, spatial regulation of Pdf seems to require both positive and negative gene regulation (Nair, 2020).

To gain insight into the transcriptional regulatory mechanisms of Pdf, this study first identified the minimal regulatory element required for Pdf expression. Furthermore, it was found that a transcription factor, Scarecrow (Scro), binds to this element through its homeodomain. Transgenic expression of scro downregulates Pdf, suggesting that Scro is potentially involved in the transcriptional regulation of Pdf (Nair, 2020).

Spatio-temporal patterns of gene expression are primarily governed by the interaction between cis-regulatory elements and their cognate binding factors. Expression of neuropeptide genes are stereotypically restricted to defined sets of neurons in the CNS, which often requires multiple cis-elements that work individually or in combination to regulate the expression in a cell type–specific manner (Nair, 2020).

Cell type-specific regulation of a neuropeptide gene has been well studied for FMRFamide. Subsets of OL neurons begin to produce FMRFamide during metamorphosis and such an expression requires a regulatory sequence consisting of 10-bp tandem repeats. The anatomical position and developmental acquisition of the neuropeptide phenotype are similar between FMRFamide-OL neurons and PDF l-LNvs. Another similarity is the presence of tandem repeats (13-bp in the PRE), and hence, the PRE was anecdotally proposed to play a role in Pdf expression in l-LNvs during metamorphosis. The current experiments, however, revealed that PRE is crucial for the Pdf expression not only in l-LNvs but also in s-LNvs. It has not been able to separate the regulatory elements for these two types of neurons. Since ClkJrk and cyc0 mutations abolish Pdf expression mostly in s-LNvs, not l-LNvs, transcriptional regulatory mechanisms are likely to be different between the two neuronal groups (Nair, 2020).

The data show that Pdf expression in a group of Ab neurons is controlled by cis-acting elements that are distinct from PRE. Moreover, Pdf expression in Ab neurons is controlled differentially between larva and adult stages, as separate regulatory regions have been found acting in these two developmental stages. According to developmental studies, PDF immunoreactivity in Ab neurons changes noticeably during metamorphosis. The larval immunoreactivity is weakened or even absent during the first half of pupal development, and then, adult-like immunoreactivity appears at later pupal stage. This developmental gap might indicate transcriptional reorganization during metamorphosis to control timely production of PDF for adult-specific functions (Nair, 2020).

Scro was found to be a binding factor to PRE, and transgenically expressed Scro downregulates Pdf transcription. Multiple lines of evidence presented in this study support that the HD of Scro is crucial for binding to PRE. Since PRE is essential for driving endogenous Pdf expression, it is predicted that there are yet unknown positive transcriptional regulators interacting with PRE. Perhaps overexpressed SCRO competes for the PRE with these positive factors, thereby inhibiting Pdf transcription. The other two domains conserved in the NK2 family of proteins, NK2 and eh1/TN, are not required for Pdf repression. In the case of VND, the interaction of TN domain with Groucho, a global co-repressor, is essential for the VND's transcriptional repression of several target genes in developing embryos . Moreover, the NK2 domain seems to stabilize Groucho-VND interaction. However, the data indicate that Scro-mediated Pdf repression does not involve Gro, suggesting that the modes of Scro action are dissimilar to those of VND (Nair, 2020).

Scro belongs to the NK2 homeobox family, members of which are widespread in vertebrates and invertebrates. The closest vertebrate homolog of Scro is Nkx2.1, which is also referred to as TTF1 (thyroid transcription factor 1). Nkx2.1/TTF1 is important for the development of lung and thyroid glands. It is also important for the CNS development, as it is required for the migration of interneurons from medial ganglionic eminence to the striatum and cerebral cortex in the developing telencephalon. Consistent with such diverse developmental roles, Nkx2.1-null mutant mice die at birth due to abnormalities in forebrain development and respiratory failure. A recent study showed that a number of genes are either upregulated or downregulated in the developing forebrain of the Nkx2.1 mutant mouse, suggesting that Nkx2.1 can act as an activator and a repressor. In this respect, it would be anticipated that SCRO acts similarly depending on the tissue/cell types or genes to be regulated (Nair, 2020).

scro is an essential gene in Drosophila. Based on the early lethality of scro-null mutant, it is proposed that scro is important for embryonic and early larval development. Striking expression of scro in the developing optic lobe suggests that scro is particularly important for the post-embryonic development of the visual system and perhaps maintenance of it in adults. Expression in various cell clusters in the central brain also indicates other roles of SCRO in these brain cells. Work on single cell transcriptomics has indicated that scro is expressed in a subgroup of dopaminergic neurons in the protocerebral anterior medial cluster (PAM DANs), suggesting that scro is potentially involved in the development and/or functions of these neurons. Endogenous scro function in association with the biological clock system remains to be elucidated. RNA-seq data from isolated clock neurons did not detect scro, implying no direct role for scro in the central clock network. Nevertheless, the current data raise the possibility that mis-regulated transcription factors can cause aberrant biological rhythms via altered regulation of clock-associated genes. Additionally, it would be interesting to assess if there is any role for scro in circadian entrainment to light-dark cycles through the visual system, given its prominent expression in the optic lobe (Nair, 2020).

NanoDam identifies novel temporal transcription factors conserved between the Drosophila central brain and visual system

Temporal patterning of neural progenitors is an evolutionarily conserved strategy for generating neuronal diversity. Type II neural stem cells in the Drosophila central brain produce transit-amplifying intermediate neural progenitors (INPs) that exhibit temporal patterning. However, the known temporal factors cannot account for the neuronal diversity in the adult brain. To search for new temporal factors, NanoDam, which enables rapid genome-wide profiling of endogenously-tagged proteins in vivo with a single genetic cross, was developed. Mapping the targets of known temporal transcription factors with NanoDam identified Homeobrain and Scarecrow (ARX and NKX2.1 orthologues) as novel temporal factors. Homeobrain and Scarecrow define middle-aged and late INP temporal windows and play a role in cellular longevity. Strikingly, Homeobrain and Scarecrow have conserved functions as temporal factors in the developing visual system. NanoDam enables rapid cell type-specific genome-wide profiling with temporal resolution and can be easily adapted for use in higher organisms (Tang, 2021).

The nervous system is generated by a relatively small number of neural stem cells (NSCs) and progenitors that are patterned both spatially and temporally. Spatial patterning confers differences between populations of NSCs, while changes in gene expression over time direct the birth order and subtype identity of neuronal progeny. Temporal transcription factor cascades determine neuronal birth order in the Drosophila embryonic central nervous system (CNS) and the larval central brain and optic lobe. In the central brain, Type II NSCs generate transit-amplifying intermediate neural progenitors (INPs), which divide asymmetrically to self-renew and generate daughter cells (ganglion mother cells or GMCs) in a manner analogous to human outer radial glial cells. GMCs in turn undergo a terminal cell division, generating neurons or glial cells that contribute to the adult central complex. The sequential divisions of INPs increase the quantity of neurons, which in turn creates a platform for generating wider neuronal diversity: eight Type II NSCs in each brain lobe give rise to at least 60 different neuronal subtypes. The tight control of progenitor temporal identity is crucial for the production of neuronal subtypes at the appropriate time and in the correct numbers. The INPs produced by the six dorsal-medial Type II lineages (DM1-6) sequentially express the temporal transcription factors Dichaete (D, a member of the Sox family), Grainy head (Grh, a Grh/CP2 family transcription factor) and Eyeless (Ey, a homologue of Pax6). These temporal factors were discovered initially by screening Type II lineages for restricted expression of neural transcription factors, using 60 different antisera. This non-exhaustive approach was able to find a fraction of the theoretically necessary temporal factors, leaving the true extent of temporal regulation and the identity of missing temporal factors open. Furthermore, the cross-regulatory interactions predicted in a temporal cascade, in which each temporal transcription factor activates expression of the next temporal factor and represses expression of the temporal factor preceding it, are not fulfilled solely by D, Grh and Ey (Tang, 2021).

Three further factors contribute to INP temporal progression, but they are expressed broadly rather than in discrete temporal windows: Osa, a SWI/SNF chromatin remodelling complex subunit, and two further transcription factors, Odd-paired (Opa) and Hamlet (Ham). Therefore, there must exist other transcription factors that are expressed in defined temporal windows and exhibit the regulatory interactions expected in a temporal cascade. It was postulated that other temporal factors, that contribute to generating the diversity of neuronal subtypes arising within each INP lineage, remain to be identified (Tang, 2021).

Given the feed-forward and feed-back transcriptional regulation previously observed in temporal transcription cascades, it was surmised that novel temporal factors would be amongst the transcriptional targets of D, Grh or Ey. Therefore, a novel approach, NanoDam, was devised to identify the genome-wide targets of transcription factors within their normal expression windows in vivo without cell isolation, cross linking or immunoprecipitation. Temporal factors are expressed transiently in a small pool of rapidly dividing progenitor cells. NanoDam provides a simple, streamlined approach to obtain genome-wide binding profiles in a cell-type-specific and temporally restricted manner (Tang, 2021).

Using NanoDam, the transcriptional targets of D, Grh and Ey were determined in INPs and, by performing single cell RNA sequencing, determined which of the directly bound loci were activated or repressed. Next, which of the target loci encoded transcription factors was assessed and whether these were expressed in restricted temporal windows within INPs. Where in the INP transcriptional cascade these factors acted was surveyed and whether they cross regulate the expression of other temporal transcription factor genes was ascertained, as expected for temporal factors. Finally, it was shown that the newly discovered temporal factors play the same roles, and exhibit the same cross regulatory interactions, in the temporal cascade in the developing visual system. This is particularly striking as theINPs and the NSCs of the developing optic lobe have different cells of origin and yet the mechanism they use to generate neural diversity is conserved (Tang, 2021).

Temporal patterning leads to the generation of neuronal diversity from a relatively small pool of neural stem or progenitor cells. Temporal regulation is achieved by the restricted expression of temporal transcription factors within precise developmental windows. The onset and duration of each temporal window in neural stem or progenitor cells must be regulated tightly in order for the appropriate subtypes of neurons to be generated at the correct time to establish functional neuronal circuits (Tang, 2021).

This study focused on the INPs of the Type II NSC lineages that generate the central complex of the Drosophila brain. Previously, INPs were shown to express sequentially the temporal factors D, Grh and Ey. Given the expectation that other temporal factors remained to be discovered, and that these were likely to be the transcriptional targets of the known temporal factors, a new technique called was used NanoDam to profile the binding targets of D, Grh and Ey with cell-type specificity and within their individual temporal windows (Tang, 2021).

NanoDam enables genome-wide profiling of any endogenously tagged chromatin-binding protein with a simple genetic cross, bypassing the need to generate Dam-fusion proteins, or the need for specific antisera or cell isolation. Furthermore, NanoDam profiles binding only in cells where the tagged protein is normally expressed. Binding within a subset of the protein's expression pattern can be achieved by controlling NanoDam with specific GAL4 drivers. To date, collaborative efforts have produced more than 3900 Drosophila lines expressing GFP-tagged proteins in their endogenous patterns. Approximately 93% of all transcription factors have been GFP-tagged in lines that are publicly available at stock centres. Lines that are not yet available can be rapidly generated by CRISPR/Cas9-mediated tagging (Tang, 2021).

NanoDam is thus a versatile tool that can be used as a higher throughput method to profile genome-wide binding sites of any chromatin associated protein. NanoDam can be readily adapted for use in other organisms to facilitate simpler and easier in vivoprofiling experiments, as hads been demonstrated previously for TaDa (Tang, 2021).

By combining the power of NanoDam with scRNA-seq, it was possible to identify scro and hbn as novel temporal factors in the INPs of Type II NSC lineages. It was shown that hbn and scro regulate the maintenance and transition of the middle-aged and late temporal windows. The mammalian homologues of ey (Pax6), hbn (Arx) and scro (Nkx2.1) are restricted to distinct progenitor populations in the developing mouse forebrain. This study found that scro regulates the late INP identity by repression of Ey. Interestingly, the loss of Nkx2.1 in the mouse forebrain leads to aberrant expression in ventral regions of the dorsal factor Pax6, suggesting that the repressive relationship between scro and ey may be conserved between Nkx2.1 and Pax6. Not all relationships appear to be conserved, however. It esd found that Hbn promotes progression through the middle-aged temporal stage and that maintenance of the middle-aged temporal window is regulated in part by interactions between Hbn and Grh. Arx mutant mice exhibit loss of upper layer (later-born) neurons but no change in the number of lower layer (early-born) neurons (Tang, 2021).

Intriguingly, the novel temporal factors identified in the INPs were also temporally expressed in optic lobe NSCs and the regulatory relationships between scro and Ey appeared to be conserved. This suggests that similar regulatory strategies may be shared between neural stem cells or progenitor cells inorder to regulate longevity and neuronal subtype production. The remarkable conservation of the regulatory interactions of scroin two different progenitor cell types with different origins in the Drosophila brain may also be translated to the context of mammalian neurogenesis, highlighting the possibility of a more generalised regulatory network used by stem and progenitor cells to regulate cell fate, progeny fate and proliferation (Tang, 2021).

The Type II lineages in Drosophila divide in a very similar manner to the outer radial glia (oRGs) that have been attributed to the rapid evolutionary expansion of the neocortex seen in humans and other mammals. Interestingly, oRGs show a shortened cell cycle length in primates in comparison to rodent progenitors, which increase cell cycle duration as development progresses . Investigating whether oRGs use temporally expressed factors to control longevity and cell cycle dynamics at different developmental stages in order to regulate neuronal subtype generation would be important for understanding neocortex development (Tang, 2021).

There is significant heterogeneity between the Type II lineages and this study has identified differences in the regulatory relationships of hbn and scro. For example, misexpression of Ey leads to an increase in scro in all lineages except DM 2 and 3, where scro expression is reduced. To date, Hbn is the only factor identified that activates Grh in DM1, the lineage that does not normally express Grh. The heterogeneity between lineages may be a consequence of variations in combinatorial binding of temporal factors, as the NanoDam data indicate. Although INPs share temporal factors, different DM lineages display subtle to striking differences when the temporal cascade is manipulated, demonstrating the likelihood that each DM employs unique temporal cascades. Combinatorial binding would enable more complex regulatory interactions that could refine or sub-divide temporal windows in the INPs (Tang, 2021).

The NK-2 homeobox gene scarecrow (scro) is expressed in pharynx, ventral nerve cord and brain of Drosophila embryos

Members of the NK homeobox family have been widely conserved during evolution. This study describes the sequence and expression of a novel Drosophila NK-2 homeobox gene, named scarecrow (scro), which shows considerable homology to vertebrate Nkx-2.1. During embryogenesis, scro expression is initially observed in the pharyngeal primordia and later maintained in the pharynx. During band germ retraction, scro expression appears in two bilateral clusters of procephalic neuroblasts that give rise to distinct neuronal clusters in the brain. In addition, scro expression is observed in segmental clusters of neuronal precursors in the ventral nerve cord. In larval stages, scro expression occurs in portions of the optic lobe regions. These observations indicate that scro and vertebrate Nkx2.1 share similarities both in terms of their sequence and their expression patterns (Zaffran, 2000).

Functions of Scarecrow orthologs in other species

Nkx2.1 regulates the generation of telencephalic astrocytes during embryonic development

The homeodomain transcription factor Nkx2.1 (NK2 homeobox 1) controls cell differentiation of telencephalic GABAergic interneurons and oligodendrocytes. This study shows that Nkx2.1 also regulates astrogliogenesis of the telencephalon from embryonic day (E) 14.5 to E16.5. Moreover this study identified the different mechanisms by which Nkx2.1 controls the telencephalic astrogliogenesis. In Nkx2.1 knockout (Nkx2.1(-/-)) mice a drastic loss of astrocytes is observed that is not related to cell death. Further, in vivo analysis using BrdU incorporation reveals that Nkx2.1 affects the proliferation of the ventral neural stem cells that generate early astrocytes. Also, in vitro neurosphere assays showed reduced generation of astroglia upon loss of Nkx2.1, which could be due to decreased precursor proliferation and possibly defects in glial specification/differentiation. Chromatin immunoprecipitation analysis and in vitro co-transfection studies with an Nkx2.1-expressing plasmid indicate that Nkx2.1 binds to the promoter of glial fibrillary acidic protein (GFAP), primarily expressed in astrocytes, to regulate its expression. Hence, Nkx2.1 controls astroglial production spatiotemporally in embryos by regulating proliferation of the contributing Nkx2.1-positive precursors (Minocha, 2017).

nkx2.1 and nkx2.4 genes function partially redundant during development of the zebrafish hypothalamus, preoptic region, and pallidum

During ventral forebrain development, orthologs of the homeodomain transcription factor Nkx2.1 control patterning of hypothalamus, preoptic region, and ventral telencephalon. However, the relative contributions of Nkx2.1 and Nkx2.4 to prosencephalon development are poorly understood. This study analyzed functions of the previously uncharacterized nkx2.4-like zgc:171531 as well as of the presumed nkx2.1 orthologs nkx2.1a and nkx2.1b in zebrafish forebrain development. The results show that zgc:171531 and nkx2.1a display overlapping expression patterns and a high sequence similarity. Together with a high degree of synteny conservation, these findings indicate that both these genes indeed are paralogs of nkx2.4. As a result, zgc:171531 was named nkx2.4a, and the name of nkx2.1a to nkx2.4b, and of nkx2.1b was changed to nkx2.1. In nkx2.1, nkx2.4a, and nkx2.4b triple morpholino knockdown (nkx2TKD) embryos a loss of the rostral part of prosomere 3 and its derivative posterior tubercular and hypothalamic structures were observed. Furthermore, there was a loss of rostral and intermediate hypothalamus, while a residual preoptic region still develops. The reduction of the ventral diencephalon was accompanied by a ventral expansion of the dorsally expressed pax6, revealing a dorsalization of the basal hypothalamus. Within the telencephalon a loss of pallidal markers was observed, while striatum and pallium are forming. At the neuronal level, nkx2TKD morphants lacked several neurosecretory neuron types, including avp, crh, and pomc expressing cells in the hypothalamus, but still form oxt neurons in the preoptic region. These data reveals that, while nkx2.1, nkx2.4a, and nkx2.4b genes act partially redundant in hypothalamic development, nkx2.1 is specifically involved in the development of rostral ventral forebrain including the pallidum and preoptic regions, whereas nkx2.4a and nkx2.4b control the intermediate and caudal hypothalamus (Manoli, 2014).

NKX2.1 specifies cortical interneuron fate by activating Lhx6

In the ventral telencephalon, the medial ganglionic eminence (MGE) is a major source of cortical interneurons. Expression of the transcription factor NKX2.1 in the MGE is required for the specification of two major subgroups of cortical interneurons - those that express parvalbumin (PV) or somatostatin (SST) - but direct targets of NKX2.1 remain to be established. This study found that electroporation of Nkx2.1 cDNA into the ventral telencephalon of slice cultures from Nkx2.1-/- mouse embryos, followed by transplantation into neonatal cortex to permit postnatal analysis of their fate, rescues the loss of PV- and SST-expressing cells. The LIM-homeobox gene Lhx6 is induced by this rescue experiment, and gain- and loss-of-function studies suggest that Lhx6 is necessary and sufficient to rescue these and other interneuron phenotypes in cells transplanted from Nkx2.1-/- slices. Finally, NKX2.1 protein binds a highly conserved sequence in the Lhx6 promoter, and this sequence appears to mediate the direct activation of Lhx6 by NKX2.1. The slice transfection and transplantation methods employed in this study are beginning to uncover embryonic mechanisms for specifying neuronal fates that only become definable postnatally (Du, 2008).

Comparative functional analysis provides evidence for a crucial role for the homeobox gene Nkx2.1/Titf-1 in forebrain evolution

Knockout of the Nkx2.1 (Titf-1) homeobox gene in the mouse leads to severe malformation and size reduction of the basal telencephalon/preoptic area and basal hypothalamus, indicating an important role of this gene in forebrain patterning. This study shows that abrogation of the orthologous gene in the frog Xenopus laevis by way of morpholino knockdown also affects the relative size of major regions in both the telencephalon (subpallium versus pallium) and diencephalon (hypothalamus versus thalamus). Remarkably, while a similar effect on the telencephalon was noted previously in Nkx2.1-knockout mice, the effect on the diencephalon seems to be specific for Xenopus. This difference may be explained by the partially dissimilar expression of the orthologous genes in the forebrain of Xenopus and mouse. In both species Nkx2.1 is expressed in the basal telencephalon/preoptic area and basal hypothalamus, but in Xenopus this gene is additionally expressed in the alar hypothalamus. Phylogenetic comparison of Nkx2.1 expression in the forebrain suggests that the expression in the basal telencephalon-preoptic region and alar hypothalamus appeared in the transition from jawless to jawed vertebrates, but the alar hypothalamic expression was later dramatically reduced during evolution to birds and mammals. This study suggests that changes in the regulation of Nkx2.1 expression have played an important role on the evolution of forebrain development, and emphasizes the potential of the combined analysis of expression and function of master control genes in different vertebrates for unraveling the origin of brain complexity and diversity (van den Akker, 2008).

The requirement of Nkx2-1 in the temporal specification of cortical interneuron subtypes

Previous work has demonstrated that the character of mouse cortical interneuron subtypes can be directly related to their embryonic temporal and spatial origins. The relationship between embryonic origin and the character of mature interneurons is likely reflected by the developmental expression of genes that direct cell fate. However, a thorough understanding of the early genetic events that specify subtype identity has been hampered by the perinatal lethality resulting from the loss of genes implicated in the determination of cortical interneurons. This study employed a conditional loss-of-function approach to demonstrate that the transcription factor Nkx2-1 is required for the proper specification of specific interneuron subtypes. Removal of this gene at distinct neurogenic time points results in a switch in the subtypes of neurons observed at more mature ages. This strategy reveals a causal link between the embryonic genetic specification by Nkx2-1 in progenitors and the functional attributes of their neuronal progeny in the mature nervous system (Butt, 2008).

Loss of Nkx2.1 homeobox gene function results in a ventral to dorsal molecular respecification within the basal telencephalon: evidence for a transformation of the pallidum into the striatum

The telencephalon is organized into distinct longitudinal domains: the cerebral cortex and the basal ganglia. The basal ganglia primarily consists of a dorsal region (striatum) and a ventral region (pallidum). Within the telencephalon, the anlage of the pallidum expresses the Nkx2.1 homeobox gene. A mouse deficient in Nkx2.1 function does not form pallidal structures, lacks basal forebrain TrkA-positive neurons (probable cholinergic neurons) and has reduced numbers of cortical cells expressing GABA, DLX2 and calbindin that migrate from the pallidum through the striatum and into the cortex. Evidence is presented that these phenotypes result from a ventral-to-dorsal transformation of the pallidal primordium into a striatal-like anlage (Sussel, 1999).


Search PubMed for articles about Drosophila Scarecrow

Butt, S. J., Sousa, V. H., Fuccillo, M. V., Hjerling-Leffler, J., Miyoshi, G., Kimura, S. and Fishell, G. (2008). The requirement of Nkx2-1 in the temporal specification of cortical interneuron subtypes. Neuron 59(5): 722-732. PubMed ID: 18786356

Du, T., Xu, Q., Ocbina, P. J. and Anderson, S. A. (2008). NKX2.1 specifies cortical interneuron fate by activating Lhx6. Development 135(8): 1559-1567. PubMed ID: 18339674

Guazzi, S., Price, M., De Felice, M., Damante, G., Mattei, M. G. and Di Lauro, R. (1990). Thyroid nuclear factor 1 (TTF-1) contains a homeodomain and displays a novel DNA binding specificity. EMBO J 9(11): 3631-3639. PubMed ID: 1976511

Manoli, M. and Driever, W. (2014). nkx2.1 and nkx2.4 genes function partially redundant during development of the zebrafish hypothalamus, preoptic region, and pallidum. Front Neuroanat 8: 145. PubMed ID: 25520628

Minocha, S., Valloton, D., Arsenijevic, Y., Cardinaux, J. R., Guidi, R., Hornung, J. P. and Lebrand, C. (2017). Nkx2.1 regulates the generation of telencephalic astrocytes during embryonic development. Sci Rep 7: 43093. PubMed ID: 28266561

Nair, S., Bahn, J. H., Lee, G., Yoo, S. and Park, J. H. (2020). A homeobox transcription factor Scarecrow (SCRO) negatively regulates Pdf neuropeptide expression through binding an identified cis-acting element in Drosophila melanogaster. Mol Neurobiol. PubMed ID: 31950355

Sussel, L., Marin, O., Kimura, S. and Rubenstein, J. L. (1999). Loss of Nkx2.1 homeobox gene function results in a ventral to dorsal molecular respecification within the basal telencephalon: evidence for a transformation of the pallidum into the striatum. Development 126(15): 3359-3370. PubMed ID: 10393115

Tang, J. L. Y., Hakes, A. E., Krautz, R., Suzuki, T. Contreras, E. G., Fox, P. M. and Brand, A. H. (2021). NanoDam identifies novel temporal transcription factors conserved between the Drosophila central brain and visual system Biorxiv

van den Akker, W. M., Brox, A., Puelles, L., Durston, A. J. and Medina, L. (2008). Comparative functional analysis provides evidence for a crucial role for the homeobox gene Nkx2.1/Titf-1 in forebrain evolution. J Comp Neurol 506(2): 211-223. PubMed ID: 18022953

Yoo, S., Nair, S., Kim, H. J., Kim, Y., Lee, C., Lee, G. and Park, J. H. (2020). Knock-in mutations of scarecrow, a Drosophila homolog of mammalian Nkx2.1, reveal a novel function required for development of the optic lobe in Drosophila melanogaster. Dev Biol. PubMed ID: 32061586

Zaffran, S., Das, G. and Frasch, M. (2000). The NK-2 homeobox gene scarecrow (scro) is expressed in pharynx, ventral nerve cord and brain of Drosophila embryos. Mech Dev 94(1-2): 237-241. PubMed ID: 10842079

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date revised: 5 August 2021

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