EvoprintHD of Scratch

BIOLOGICAL OVERVIEW

Differentiation can be thought of as a progressive unfolding of developmental fates and elaboration of complexity. Developmental biologists are becoming increasingly aware of a second, equally essential process; the repression of alternative cell fates. Ultimately a breakdown in this process leads to an excess of unwanted products of differentiation.

scratch is a prime example of a gene required for this second developmental process, in this example the suppression of genes promoting non-neuronal cell fates. scratch is expressed in the CNS, PNS, eye and brain.

Analysis of the promoters of deadpan and scratch reveal separate regulatory regions confering expression in the central and peripheral nervous systems. This separate regulation represents an enigma, since regulation by proneural genes of the achaete-scute complex could jointly regulate pan-neural expression. The number of scratch-expressing cells is reduced but not eliminated in proneural mutants. The most parsimonius explanation is that there are unknown regulators of scratch restricted to the CNS or PNS. Such putative regulators are likely to be a repressor of expression in postmitotic CNS neurons, which acts on the PNS element to make these elements completely PNS specific. So in order to maintain orderly development in the fly, even repressors are regulated by repressors (Emery, 1995).

cDNA clone length - 6.5 kb

Exons - two

PROTEIN STRUCTURE

Amino Acids - 644

Structural Domains

There are 5 zinc-fingers near the carboxyl domain (Roark, 1995)

Evolutionary Homologs

Scratch is most closely related to Snail, a gene essential for mesodermal determination, and Escargot, a gene that controls polyploidy during imaginal disc growth. Xenopus protein Snail and chick protein Slug are also part of this family (Roark, 1995).

The ces-1 and ces-2 genes of C. elegans control the programmed deaths of specific neurons. Genetic evidence suggests that ces-2 functions to kill these neurons by negatively regulating the protective activity of ces-1. ces-2 encodes a protein closely related to the vertebrate PAR family of bZIP transcription factors, and a ces-2/ces-1-like pathway may play a role in regulating programmed cell death in mammalian lymphocytes. ces-1 encodes a Snail family zinc finger protein, most similar in sequence to the Drosophila neuronal differentiation protein Scratch. An element important for ces-1 regulation is defined and evidence is providd that CES-2 can bind to a site within this element and thus may directly repress ces-1 transcription. These results suggest that a transcriptional cascade controls the deaths of specific cells in C. elegans (Metzstein, 1999).

Members of the Snail family of zinc finger transcription factors are known to play critical roles in neurogenesis in invertebrates, but none of these factors has been linked to vertebrate neuronal differentiation. Expression of a mammalian Snail family member is restricted to the nervous system. Human and murine Scratch (Scrt) share 81% and 69% identity to Drosophila Scrt and the Caenorhabditis elegans neuronal antiapoptotic protein, CES-1, respectively, across the five zinc finger domain. Expression of mammalian Scrt is predominantly confined to the brain and spinal cord, appearing in newly differentiating, postmitotic neurons and persisting into postnatal life. Additional expression is seen in the retina and, significantly, in neuroendocrine (NE) cells of the lung. In a parallel fashion, hScrt expression is detected in lung cancers with NE features, especially small cell lung cancer. hScrt shares the capacity of other Snail family members to bind to E-box enhancer motifs, which are targets of basic helix-loop-helix (bHLH) transcription factors. hScrt directly antagonizes the function of heterodimers of the proneural bHLH protein achaete-scute homolog-1 and E12, leading to active transcriptional repression at E-box motifs. Thus, Scrt has the potential to function in newly differentiating, postmitotic neurons and in cancers with NE features by modulating the action of bHLH transcription factors critical for neuronal differentiation (Nakakura, 2001).

Like other Snail family members, hScrt is a nuclear protein that functions as a transcriptional repressor. Repressor activity resides within the N-terminal non-zinc finger region. However, the conserved N-terminal eight amino acids that hScrt shares with other SNAG domain containing proteins are not required for repressor function. This observation is in contrast to other reports ascribing important repressor function to the N-terminal twenty amino acids of the SNAG domain of vertebrate Snail, Slug, Smuc, and Gfi1 proteins. Though modest nuclear targeting activity has been described for this N-terminal region of Gfi1, effective nuclear localization depends on the full-length protein, including the zinc finger domain. The N-terminal non-zinc finger region of hScrt is not sufficient for proper expression in the nucleus; conversely, information within the zinc finger domain is necessary and sufficient to effect nuclear localization (Nakakura, 2001).

During vertebrate development, Mash1 and Neurogenin1 and -2 are transiently expressed in proliferating neurons of the nervous system and exhibit determination and differentiation functions. mScrt is expressed in an adjacent layer characteristic of newly differentiating neurons and hScrt can repress hASH1-E12-mediated reporter transactivation. Taken together, Scrt may modulate the effects of ASH1-E12 on common target genes, thereby potentially affecting neuronal determination and differentiation. Because Scrt expression is more widespread than Mash1, it is possible that Scrt may interact functionally with other bHLH transcription factors, such as the Neurogenins. Functional interactions occur between Escargot and Scute-Daughterless, as well as Smuc and MyoD-E12. Thus, interactions between Snail family and bHLH factors may be a common theme in development. Further studies of Scrt should provide insight into programs of neural differentiation that appear conserved in normal and neoplastic tissues (Nakakura, 2001).

Members of the Snail family of zinc finger transcription factors are known to play critical roles in neurogenesis in invertebrates, but none of these factors has been linked to vertebrate neuronal differentiation. Reported here is the isolation of a gene encoding a mammalian Snail family member that is restricted to the nervous system. Human and murine Scratch (Scrt) share 81% and 69% identity to Drosophila Scrt and the Caenorhabditis elegans neuronal antiapoptotic protein, CES-1, respectively, across the five zinc finger domain. Expression of mammalian Scrt is predominantly confined to the brain and spinal cord, appearing in newly differentiating, postmitotic neurons and persisting into postnatal life. Additional expression is seen in the retina and, significantly, in neuroendocrine (NE) cells of the lung. In a parallel fashion, hScrt expression is detected in lung cancers with NE features, especially small cell lung cancer. hScrt shares the capacity of other Snail family members to bind to E-box enhancer motifs, which are targets of basic helix-loop-helix (bHLH) transcription factors. hScrt directly antagonizes the function of heterodimers of the proneural bHLH protein achaete-scute homolog-1 and E12, leading to active transcriptional repression at E-box motifs. Thus, Scrt has the potential to function in newly differentiating, postmitotic neurons and in cancers with NE features by modulating the action of bHLH transcription factors critical for neuronal differentiation (Nakakura, 2002).

Mammalian Scrt is the first vertebrate Snail family member known to be expressed highly and specifically in neural tissues. Outside the nervous system and lung NE cells, significant levels of Scrt expression are detected. In flies, scrt is expressed in dividing neuronal precursors and persists in postmitotic neurons. In contrast, mScrt transcripts were not detected in regions known to contain proliferating neurons -- the VZ of the telencephalon, spinal cord, and retina. Rather, prominent domains of mScrt expression were seen adjacent to the VZ in these tissues. Specifically, mScrt expression is detected in a distribution similar to BrdUrd-/Tuj1+ neurons in the telencephalon. Thus, mScrt expression in the CNS appears to be confined to newly differentiating, postmitotic neurons, suggesting a potential role in neuronal differentiation (Nakakura, 2002).

Because Snail family proteins share DNA binding specificity to the hepatanucleotide sequence ACAGGTG, whether hScrt exhibits similar binding properties was investigated. hScrt protein was produced by IVT. In an electrophoretic mobility gel shift assay, hScrt bound to an oligonucleotide containing the wild-type Snail family consensus binding site. Specificity of binding was supported by absence of a shift when a mutant probe containing three base changes was used. Unlabeled wild-type oligonucleotide competes with labeled probe for binding in a dose-dependent manner. This competition is specific; unlabeled mutant oligonucleotide does not compete for binding, even at 2,000-fold molar excess to labeled probe (Nakakura, 2002).

Because the Snail consensus binding sequence contains the E-box motif CAGGTG recognized by bHLH transcription factors, the ability of hASH1 to bind to the same sequence was tested. When either hASH1 or E12 (a heterodimerizing partner for bHLH proteins) was used alone, no or only a modest shift of the wild-type Snail probe was seen, respectively. However, a mixture containing hASH1 and E12 produced a distinct shifted band of the wild-type probe. No shift was detected with the mutant probe. Competition for binding of the wild-type probe was observed with an excess of unlabeled wild-type, but not mutant, oligonucleotide in a dose-dependent manner. Therefore, hASH1-E12 heterodimers can bind specifically to similar sequences as hScrt (Nakakura, 2002).

Because hScrt shares DNA binding specificity with hASH1-E12 and both hScrt and hASH1 are expressed in similar normal tissues and lung cancer cell lines, whether they could interact functionally was tested. Before testing the actions of various hScrt constructs in this context, their subcellular expression was detected by confocal microscopy. Wild-type hScrt is expressed in the nucleus. An hScrt mutant (Delta Zinc Fingers, amino acids 1-190) containing the N-terminal half but lacking all five zinc fingers was expressed diffusely throughout the nucleus and cytoplasm. However, a mutant containing only the zinc finger domain of hScrt (Delta N terminus, amino acids 173-348) localizes to the nucleus indistinguishably from the wild-type protein. Therefore, a critical nuclear localization signal is present within the zinc finger region of hScrt, and the N-terminal SNAG-like domain is dispensable for nuclear localization (Nakakura, 2002).

To determine whether hScrt exhibits active repressor activity, various hScrt domains were fused in-frame with the GAL4 DNA binding domain and targeted to a reporter containing five GAL4 binding sites upstream of the TK promoter. Expression of all constructs was verified by Western blot. GAL4-fusion constructs containing full-length hScrt (GAL4-hScratch), hScrt lacking all five zinc fingers (GAL4-Delta Zinc Fingers), and hScrt minus the first eight amino acids of the SNAG domain (GAL4-Delta 8) all repressed reporter activity as effectively as a construct containing the known repressor MeCP2. In contrast, when the first 40 amino acids of hScrt were expressed as a GAL4-fusion protein (GAL4-N terminus), no repression was seen. Therefore, hScrt repressor activity resides in the non-zinc finger region and is not dependent on the conserved N-terminal eight amino acids of the SNAG domain, which has been reported to be important for repressor activity in Snail family and other zinc finger transcription factors (Nakakura, 2002).

During vertebrate development, Mash1 and Neurogenin1 and -2 are transiently expressed in proliferating neurons of the nervous system and exhibit determination and differentiation functions. mScrt is expressed in an adjacent layer characteristic of newly differentiating neurons and hScrt can repress hASH1-E12-mediated reporter transactivation. Taken together, Scrt may modulate the effects of ASH1-E12 on common target genes, thereby potentially affecting neuronal determination and differentiation. Because Scrt expression is more widespread than Mash1, it is possible that Scrt may interact functionally with other bHLH transcription factors, such as the Neurogenins. Others have reported functional interactions between Escargot and Scute-Daughterless, as well as Smuc and MyoD-E12 in vitro. Thus, interactions between Snail family and bHLH factors may be a common theme in development. Further studies of Scrt should provide insight into programs of neural differentiation that appear conserved in normal and neoplastic tissues (Nakakura, 2002).

Promoter Structure

The scrt promoter has separable elements responsible for PNS and CNS expression. The proximal region is responsible for the full range of expression in the PNS, while an upstream region gives CNS expression during late waves of neuroblast delamination. Another fragment, between between proximal and distal regions drives expression in both PNS and CNS. The proximal region also drives expression later in subsets of cells in the CNA and along the ventral midline (Emery, 1995).

Targets of Activity

Scratch functions to repress expression of various genes promoting non-neuronal cell fates. These targets include torpedo (the EGF receptor), orthodenticle, scabrous, hedgehog, and hairy (Roark, 1995).

DEVELOPMENTAL BIOLOGY

Embryonic

scratch transcripts are first detected in a single row of ectodermal cells flanking the ventral midline. During early germ band extention [Images], scratch-expressing cells delaminate and contribute to the first of three rows of S1 neuroblasts in the ventral CNS. It is subsequently expressed in all S1 neuroblasts, as well as neuroblasts formed during later rounds of segregation. scratch continues to be expressed in ganglion mother cells (GMCs), the immediate progeny of neuroblasts, and later in postmitotic neurons.

Unlike scratch, deadpan is not expressed in GMCs. In the peripheral nervous system scratch is first expressed in primary sensory mother cells, then in secondary precursor cells, and finally in postmitotic neurons. scratch is expressed prior to deadpan in the first row of neuroblasts, but subsequently deadpan expression precedes scratch in the CNS and sensory mother cells. Likewise snail and scratch expression partially overlap. Ectopic expression of scratch generates extra neurons (Roark, 1995).

Wild-type zygotic scrt expression first appears in the S1 neuroblasts as they delaminate. As in the case of dpn, scrt expression extends to each subsequent wave of neuroblast delamination. Unlike dpn, however, scrt continues to be expressed in GMCs. It is likely that expression in GMCs derived from divisions of the S1 neuroblasts obscures the rosette pattern of neuroblast expression observed with dpn. By stage 12, most or all cells in the CNS express scrt, with strongest expression in a group of 5-7 cells per abdominal hemisegment arranged in a crescent. A small subset of cells along the ventral midline also expresses scrt at this time. Expression in the PNS begins with delamination of the earliest precursors and continues as subsequent precursors segregate from the epithelium. Postmitotic cells in both the CNS and PNS express scrt prior to their morphological differentiation. In the PNS, where all postmitotic neurons can be identified, it is possible to determine that scrt is expressed in every neuron. In the CNS, there are still some GMCs present after germband retraction, and it is possible that scrt is expressed in these cells and/or in glia as well as in postmitotic neurons (Emery, 1995).

Larval

scratch is expressed in neuronal cells of the wing disc, in developing neurons in pupal wings, in cells posterior to the morphogenic furrow, in the eye-antennal disc, in leg neuronal precursors, and in many cells of the third-instar larval brain and ventral nerve cord (Roark, 1995).

Effects of mutation or deletion

scratch null mutants have scarred facets in the eye, a phenotype that gives the gene its name. It is also a male sterile mutation, producing defective spermatids (Roark, 1995).

scratch interacts genetically with deadpan. These two genes have similar pan-neural expression patterns but encode unrelated proteins. Loss of function of either of these genes alone does not lead to obvious morphological disruption of the embryonic nervous system. Under optimal conditions, animals homozygous null for each of these genes occasionally complete development and eclose. In contrast, animals null for both genes never hatch and frequently exhibit a dramatic reduction of the nervous system. In addition, axon projections are frequently disorganized. Double mutants have missing and disorganized longitudinal and commissural axon tracts. CNS defects are evident early during neurogenesis as there are fewer hunchback expressing cells contributing to the S1 wave of neuroblasts than in wild type embryos (Roark, 1995).

REFERENCES

Emery, J.F. and Bier, E. (1995). Specificity of CNS and PNS regulatory subelements comprising pan-neural enhancers of the deadpan and scratch genes is achieved by repression. Development 121: 3549-3560

Roark, M., et al. (1995). scratch, a pan-neural gene encoding a zinc finger protein related to snail, promotes neuronal development. Genes & Dev. 9: 2384-98

Metzstein, M. M. and Horvitz, H. R. (1999). The C. elegans cell death specification gene ces-1 encodes a snail family zinc finger protein. Mol. Cell 4(3): 309-19.

Nakakura, E. K., et al. (2001). Mammalian Scratch: A neural-specific Snail family transcriptional repressor. Proc. Natl. Acad. Sci. 98: 4010-4015. 11274425

Nakakura, E. K., et al. (2002). Mammalian Scratch participates in neuronal differentiation in P19 embryonal carcinoma cells. Brain Res. Mol. Brain Res. 95(1-2): 162-6. 11687288

date revised:  15 February 2003

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