InteractiveFly: GeneBrief

Sp1: Biological Overview | References

BIOLOGICAL OVERVIEW

The appendages of arthropods and vertebrates are not homologous structures, although the underlying genetic mechanisms that pattern them are highly conserved. Members of the Sp family of transcription factors are expressed in the developing limbs and their function is required for limb growth in both insects and chordates. Despite the fundamental and conserved role that these transcription factors play during appendage development, their target genes and the mechanisms in which they participate to control limb growth are mostly unknown. This study analyzed the individual contributions of two Drosophila Sp members, buttonhead (btd) and Sp1, during leg development. Sp1 plays a more prominent role controlling leg growth than btd. A regulatory function of Sp1 in Notch signaling was identified, and a genome wide transcriptome analysis was performed to identify other potential Sp1 target genes contributing to leg growth. The data suggest a mechanism by which the Sp factors control appendage growth through the Notch signaling (Cordoba, 2016).

Understanding the molecular mechanisms that control the specification and acquisition of the characteristic size and shape of organs is a fundamental question in biology. Of particular interest is the development of the appendages of vertebrates and arthropods, i.e., non-homologous structures that share a similar underlying genetic program to build them, a similarity that has been referred to as 'deep homology.' Some of the conserved genes include the Dll/Dlx genes, Hth/Meis and the family of Sp transcription factors. The Sp family is characterized by the presence of three highly conserved Cys2-His2-type zinc fingers and the presence of the Buttonhead (BTD) box just N-terminal of the zinc fingers (Cordoba, 2016).

Members of the Sp family have important functions during limb outgrowth in a range of species from beetles to mice. In vertebrates, Sp6, Sp8 and Sp9 are expressed in the limb bud and are necessary for Fgf8 expression and, therefore, for apical ectodermal ridge (AER) maintenance. Moreover, Sp6/Sp8 phenotypes have been related to the split-hand/foot malformation phenotype (SHFM) and, in the most severe cases, to amelia (the complete loss of the limb) (Cordoba, 2016).

In Drosophila, two members of this family, buttonhead (btd) and Sp1, are located next to each other on the chromosome and share similar expression patterns throughout development. Recently, another member of the family, Spps (Sp1-like factor for pairing sensitive-silencing) has been identified with no apparent specific function in appendage development. The phenotypic analysis of a btd loss-of-function allele and of a deletion that removes both btd and Sp1 led to the proposal that these genes have partially redundant roles during appendage development (Estella, 2010; Estella, 2003). However, the lack of a mutant for Sp1 has prevented the analysis of the specific contribution of this gene during development (Cordoba, 2016).

In Drosophila, leg development is initiated in the early embryo by the expression of the homeobox gene Distal-less (Dll) in a group of cells in each thoracic segment. Later on, Dll expression depends on the activity of the Decapentaplegic (Dpp) and Wingless (Wg) signaling pathways, which, together with btd and Sp1, restrict Dll expression to the presumptive leg territory. Therefore, the early elimination of btd and Sp1 completely abolishes leg formation and, in some cases, causes a leg-to-wing homeotic transformation (Estella, 2010). As the leg imaginal disc grows, a proximo-distal (PD) axis is formed by the differential expression of three leg gap genes, Dll, dachshund (dac) and homothorax (hth), which divides the leg into distal, medial and proximal domains, respectively (reviewed by Estella., 2012). Once these genes have been activated, their expression is maintained, in part through an autoregulatory mechanism, and no longer relies on Wg and Dpp. Meanwhile, the distal domain of the leg is further subdivided along the PD axis by the activity of the epidermal growth factor receptor (EGFR) signaling pathway through the activation of secondary PD targets such as aristaless (al), BarH1 (B-H1) or bric-a-brac (bab). During these stages, btd and Sp1 control the growth of the leg but are no longer required for Dll expression (Estella, 2010). How btd and Sp1 contribute to the shape and size of the leg and the identity of their downstream effector targets is unknown (Cordoba, 2016).

One important consequence of the PD territorial specification is the generation of developmental borders that activate organizing molecules to control the growth and pattern of the appendage. In the leg, PD subdivision is necessary to localize the expression of the Notch ligands Delta (Dl) and Serrate (Ser), which in turn activate the Notch pathway in concentric rings at the borders between presumptive leg segments. However, it is still unknown how Notch controls leg growth and how the localization of its ligands is regulated. The present study generated a specific Sp1 null mutant, which, in combination with the btd mutant and a deletion that removes both btd and Sp1, allow analysis of the individual contributions of these genes to leg development. This study finds that Sp1 plays a fundamental role during patterning and growth of the leg disc, and that this function is not compensated by btd. The growth-promoting function of Sp1 depends in part on the regulation of the expression of Ser and, therefore, on Notch activity. In addition, other candidate targets of Sp1 affecting leg growth and morphogenesis were identified. Intriguingly, some of these Sp1 potential downstream targets are ecdysone-responding genes. These results highlight a mechanism by which btd and Sp1 control the size and shape of the leg, in part through regulation of the Notch pathway (Cordoba, 2016).

The two Sp family members in Drosophila, Sp1 and btd, display a similar spatial and temporal expression pattern during embryonic and imaginal development. Previous work suggested that btd and Sp1 have partially redundant functions during development. However, the lack of an Sp1 mutant has prevented the detailed analysis of the individual contributions of each gene. This study has generated an Sp1 null mutant that allowed elucidation unambiguously of the individual contributions of each of these genes to leg development (Cordoba, 2016).

Appendage formation starts in early embryos by the activation of Dll (through its early enhancer, Dll-304), btd and Sp1 by Wg, and their expression is repressed posteriorly by the abdominal Hox genes. Some hours later, there is a molecular switch from the early Dll enhancer (Dll-304) to the late enhancer (Dll-LT) to keep Dll expression throughout the embryo-larvae transition restricted to the cells that will form the leg. At this developmental stage, Sp1 and btd play redundant roles in Dll activation, as only the elimination of both genes suppresses Dll expression and Dll-LT activity in the leg primordia. Once Dll expression is activated in the leg disc by the combined action of Wg, Dpp and Btd/Sp1, its expression is maintained in part through an autoregulatory mechanism. At this time point, during second instar, btd and Sp1 are co-opted to control the growth of the leg. The leg phenotype of Sp1 and btd single mutants demonstrates the divergent contributions of each gene to leg growth. Removing btd from the entire leg only slightly affects the growth of proximo-medial segments, whereas loss of Sp1 causes dramatic growth defects along the entire leg. The different phenotypes of Sp1 and btd mutant legs could be a consequence of their distinct expression pattern along the leg PD axis, with btd being expressed more proximally than Sp1 (Cordoba, 2016).

The growth defects observed in Sp1 mutant legs are not due to gross defects in the localization of the different transcription factors that subdivide the leg along the PD axis, nor to defects in the expression of the EGFR ligand vn. By contrast, the results suggest a role for Sp1 in the regulation of the Notch ligand Ser. Notch pathway activation is necessary for the formation of the joints and the growth of the leg, and defects in these two processes were observed in Sp1 mutant legs. Moreover, the results demonstrate that Sp1 is necessary and sufficient for Ser expression at least in the distal domain of the leg and is therefore required for the correct activation of the Notch pathway. These results are consistent with the proposed role of Sp8 in allometric growth of the limbs in the beetle where the number of Ser-expressing rings is reduced in Sp8 knockdown animals (Cordoba, 2016).

The regulation of Ser expression is controlled by multiple CREs that direct its transcription in different developmental territories. Interestingly, although the wing and leg are morphologically different appendages and express a diverse combination of master regulators (e.g. Sp1 selects for leg identity whereas Vg determines wing fate), the same set of enhancers are accessible in both appendages, with the exception of the ones that control the expression of the master regulators themselves (McKay, 2013). These results imply that appendage-specific master regulators differentially interact with the same enhancers to generate a specific expression pattern in each appendage. The current analysis of Ser CREs identified a specific sequence that is active in the wing and in the leg. In the leg, this CRE reproduced Ser expression in the fourth tarsal segment and require the combined inputs of Sp1 and Ap. It is proposed that Sp1, in coordination with the other leg PD transcription factors, interacts with different Ser CREs to activate Ser expression in concentric rings in the leg. Meanwhile, given the same set of Ser CREs in the wing, the presence of a different combination of transcription factors regulate Ser expression in the characteristic 'wing pattern' (Cordoba, 2016).

Transcriptome analysis identified additional candidate Sp1 target genes that contribute to control the size and shape of the leg. Appendage elongation depends on the steroid hormone ecdysone through several of its effectors, such as Sb. Sb, as well as other genes related to the ecdysone pathway, were misregulated in Sp1 mutant discs. The characteristic change in cell shape that normally occurs during leg eversion does not happen correctly in these mutants. Other genes identified in this study are the Notch pathway targets dys and Poxn, which are both required for the correct development of the tarsal joints. dys and Poxn downregulation is consistent with Sp1 regulation of the Notch ligand Ser. Interestingly, the upregulation of the antenna-specific gene danr in Sp1 mutants might explain the partial transformation of the distal leg to antennal-like structures observed when two copies of Sp1 and one of btd are mutated. Interestingly, btd and Sp1 are only expressed in the antenna disc in a single ring corresponding to the second antennal segment whereas in the leg both genes are more broadly expressed. Consistent with this, misexpression of Sp1 in the antenna transforms the distal domain to leg-like structures, suggesting that different levels or expression domains of Sp1 helps distinguish between these two homologous appendages (Cordoba, 2016).

A considerable group of Hsp-related genes were downregulated in Sp1 mutant legs. Although their contribution to Drosophila leg development is unknown, downregulation of DnaJ-1, the Drosophila ortholog of the human HSP40, affects joint development and leg size, suggesting a potential role of these genes during leg morphogenesis (Cordoba, 2016).

An ancient common mechanism for the formation of outgrowths from the body wall has been suggested. Members of the Sp family are expressed and required for appendage growth in a range of species from Tribolium to mice (Beermann, 2004; Bell, 2003; Kawakami, 2004; Treichel, 2003). Consistent with the current results, knockdown of Sp8/Sp9 in the milkweed bug or the beetle generated dwarfed legs with fused segments that maintain the correct PD positional values (Beermann, 2004; Schaeper, 2009). As is the case for Drosophila Sp1 mutants, mouse Sp8-deficient embryos develop with truncated limbs. By contrast, loss of function of Sp6 results in milder phenotypes of limb syndactyly (Talamillo, 2010). A progressive reduction of the dose of Sp6 and Sp8 lead to increased severity of limb phenotypes from syndactyly to amelia, suggesting that these genes play partially redundant roles (Haro, 2014). This phenotypic analysis of Sp1 and btd are consistent with this model, in which Sp1 plays the predominant role in appendage growth and the complete elimination of btd and Sp1 together abolish leg formation. Therefore, Drosophila Sp1 mutants are phenotypically equivalent to vertebrate Sp8 mutants. In vertebrate Sp8 mutant limbs, Fgf8 expression is not maintained and a functional AER fails to form (Bell, 2003; Kawakami, 2004; Treichel, 2003). In Drosophila, FGF signaling does not seem to be involved in appendage development. Nevertheless, another receptor tyrosine kinase, EGFR, is activated at the tip of the leg and act as an organizer to regulate the PD patterning of the tarsus. The current results suggest that Sp1 acts in parallel with the EGFR pathway, as the ligand vn and EGFR target genes maintain their PD positional information in Sp1 mutant legs. However, a potential relationship between Sp1 and the EGFR pathway in later stages of leg development cannot be ruled out (Cordoba, 2016).

The results suggest that the Notch ligand Ser is a target of Sp1, and mediates in part the growth-promoting function of Sp1. Interestingly, members of the Notch pathway in vertebrates, including the Ser ortholog jagged 2 and notch 1 are expressed in the AER and regulate the size of the limb. It would be interesting to investigate further the possible relationship between Sp transcription factors and the Notch pathway in vertebrates, and test whether the functional relationship described in this work is also maintained throughout evolution (Cordoba, 2016).

A common set of DNA regulatory elements shapes Drosophila appendages

Animals have body parts made of similar cell types located at different axial positions, such as limbs. The identity and distinct morphology of each structure is often specified by the activity of different 'master regulator' transcription factors. Although similarities in gene expression have been observed between body parts made of similar cell types, how regulatory information in the genome is differentially utilized to create morphologically diverse structures in development is not known. This study used genome-wide open chromatin profiling to show that among the Drosophila appendages, the same DNA regulatory modules are accessible throughout the genome at a given stage of development, except at the loci encoding the master regulators themselves. In addition, open chromatin profiles change over developmental time, and these changes are coordinated between different appendages. It is proposed that master regulators create morphologically distinct structures by differentially influencing the function of the same set of DNA regulatory modules (McKay, 2013).

Given their diverse morphologies and transcription factor expression profiles, it was surprising to find that wing and leg imaginal discs also share very similar open chromatin profiles. Of the most pronounced open chromatin regions (the top 20%, 3,525 peaks), only 110 were differentially open. It is speculated that these few differences in open chromatin between wing and leg imaginal discs were important in determining morphological differences, as was the case with wing and haltere imaginal discs. Indeed, genes with open chromatin specific to the leg imaginal discs include Dll and Sp1, the master regulators of leg development. Similarly, genes with open regions specific to the dorsal imaginal discs (wing and haltere) include vg and blistered, transcription factors required for development of these appendages. Tests were performed to see whether these disc-specific open chromatin regions identified by FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) function as appendage-specific enhancers, and 6 of 7 accurately recapitulated gene expression in imaginal discs of late third-instar larvae. Similar to these observations from the embryonic time course, the presence of disc-specific open chromatin correlated with disc-specific enhancer activity—the cloned imaginal disc enhancers are active only in the imaginal discs in which they are accessible. For example, the VG01 enhancer identified by this study, which is open specifically in wing and haltere imaginal discs, recapitulates vg expression specifically in wing and haltere imaginal discs and is not active in leg imaginal discs. Together, these data demonstrate that genomic regions accessible for use in thoracic appendage imaginal discs are nearly identical, except at appendage master regulator gene loci (McKay, 2013).

This study addresses a long-standing question in developmental biology: how does a single genome give rise to a diversity of structures? The results indicate that the combination of transcription factors expressed in each thoracic appendage acts upon a shared set of enhancers to create different morphological outputs, rather than operating on a set of enhancers that is specific to each tissue. This conclusion is based upon the surprising observation that the open chromatin profiles of the developing appendages are nearly identical at a given developmental stage. Therefore, rather than each master regulator operating on a set of enhancers that is specific to each tissue, the master regulators instead have access to the same set of enhancers in different tissues, which they differentially regulate. This study also found that tissues composed of similar combinations of cell types have very similar open chromatin profiles, suggesting that a limited number of distinct open chromatin profiles may exist at a given stage of development, dependent on cell-type identity (McKay, 2013).

Different tissues were dissected from developing flies to compare their open chromatin profiles. These tissues are composed of different cell types, each with its own gene expression profile. The FAIRE data thus represent the average signal across all cells present in a sample. However, data from embryos and imaginal discs indicate that FAIRE is a very sensitive detector of functional DNA regulatory elements. For example, the Dll01 enhancer is active in 2-4 neurons of the leg imaginal disc; yet, the FAIRE signal at Dll01 is as strong as the Dll04 enhancer, which is active in hundreds of cells of the wing pouch. Thus, FAIRE may detect nearly all of the DNA regulatory elements that are in use among the cells of an imaginal disc. The study does not rule out the existence of DNA regulatory elements that are not marked by open chromatin or are otherwise not detected by FAIRE (McKay, 2013).

Despite this sensitivity, the approach does not identify which cells within the tissue have a particular open chromatin profile. For a given locus, it is possible that all cells in the tissue share a single open chromatin profile or that the FAIRE signal originates from only a subset of cells in which a given enhancer is active. Comparisons between eye-antennal discs, larval CNS, and thoracic discs suggest that the latter scenario is most likely, with open chromatin profiles among cells within a tissue shared by cells with similar identities at a given developmental stage (McKay, 2013).

The observation that halteres and wings share open chromatin profiles demonstrates that Hox proteins like Ubx can differentially interpret the DNA sequence within the same subset of enhancers to modify one structure into another. This is consistent with the idea that morphological differences are largely dependent on the precise location, duration, and magnitude of expression of similar genes, and it is further supported by the similarity in gene expression profiles observed between Drosophila appendages and observed between vertebrate limbs. However, that such dramatic differences in morphology could be achieved by using the same subset of DNA regulatory modules in different tissues genomewide was not known. The findings provide a molecular framework to support the hypothesis that Hox factors function as 'versatile generalists,' rather than stable binary switches. The similarity in open chromatin profiles between wings and legs suggests that this framework also extends to other classes of master regulators beyond the Hox genes. It is also noted that, like the Drosophila appendages, vertebrate limbs are composed of similar combinations of cell types that differ in their pattern of organization. Moreover, the Drosophila appendage master regulators share a common evolutionary origin with the master regulators of vertebrate limb development, suggesting that the concept of shared open chromatin profiles may also apply to human development (McKay, 2013).

The data suggest that open chromatin profiles vary both over time for a given lineage and between cell types at a given stage of development. Given the dramatic differences in the FAIRE landscape observed during embryogenesis and between the CNS and the appendage imaginal discs during larval stages, it appears as though the alteration of the chromatin landscape is especially important for specifying different cell types from a single genome. After cell-type specification, open chromatin profiles in the appendages continued to change as they proceeded toward terminal differentiation, suggesting that stage-specific functions require significant opening of new sites or the closing of existing sites. These findings contrast with those investigating hormone-induced changes in chromatin accessibility, in which the majority of open chromatin sites did not change after hormone treatment, including sites of de novo hormone-receptor binding. Thus, it may be that genome-wide remodeling of chromatin accessibility is reserved for the longer timescales and eventual permanence of developmental processes rather than the shorter timescales and transience of environmental responses (McKay, 2013).

Different combinations of 'master regulator' transcription factors, often termed selector genes, are expressed in the developing appendages. Selectors are thought to specify the identity of distinct regions of developing animals by regulating the expression of transcription factors, signaling pathway components, and other genes that act as effectors of identity. One property attributed to selectors to explain their unique power to specify identity during development is the ability to act as pioneer transcription factors. In such models, selectors are the first factors to bind target genes; once bound, selectors then create a permissive chromatin environment for other transcription factors to bind. The finding that the same set of enhancers are accessible for use in all three appendages, with the exception of the enhancers that control expression of the selector genes themselves and other primary determinants of appendage identity, suggests that the selectors expressed in each appendage do not absolutely control the chromatin accessibility profile; otherwise, the haltere chromatin profile (for example) would differ from that of the wing because of the expression of Ubx (McKay, 2013).

What then determines the appendage open chromatin profiles? Because open chromatin is likely a consequence of transcription factor binding, two nonexclusive models are possible. First, different combinations of transcription factors could specify the same open chromatin profiles. In this scenario, each appendage's selectors would bind to the same enhancers across the genome. For example, the wing selector Vg, with its DNA binding partner Sd, would bind the same enhancers in the wing as Dll and Sp1 bind in the leg. In the second model, transcription factors other than the selectors could specify the appendage open chromatin profiles. Selector genes are a small fraction of the total number of transcription factors expressed in the appendages. Many of the nonselector transcription factors are expressed at similar levels in each appendage, and thermodynamic models would predict them to bind the same enhancers. This model could also help to explain how the appendage open chromatin profiles coordinately change over developmental time despite the steady expression of the appendage selector genes during this same period. It is possible that stage-specific transcription factors determine which enhancers are accessible at a given stage of development. This would help to explain the temporal specificity of target genes observed for selectors such as Ubx. Recent work supports the role of hormone-dependent transcription factors in specifying the temporal identity of target genes in the developing appendages. Further experiments, including ChIP of the selectors from each of the appendages, will be required to determine the extent to which either of these models is correct (McKay, 2013).

This study has shown that binding of Ubx results in differential activity of enhancers in the haltere imaginal disc relative to the wing, despite equivalent accessibility of the enhancers in both discs, indicating that master regulators control morphogenesis by differentially regulating the activity of the same set of enhancers. It is likely that functional specificity of enhancers is achieved through multiple mechanisms. These include differential recruitment of coactivators and corepressors, modulation of binding specificity through interactions with cofactors, differential utilization of binding sites within a single enhancer, or regulation of binding dynamics through an altered chromatin context. This last mechanism would allow for epigenetic modifications early in development to affect subsequent gene regulatory events. For example, the activity of Ubx enhancers in the early embryo may control recruitment of Trithorax or Polycomb complexes to the PREs within the Ubx locus, which then maintain Ubx in the ON or OFF state at subsequent stages of development. Consistent with this model, Ubx enhancers active in the early embryo are only accessible in our 2-4 hr time point, whereas the accessibility of Ubx PREs varies little across developmental time or between tissues at a given developmental stage (McKay, 2013).

These results also have implications for the evolution of morphological diversity. Halteres and wings are considered to have a common evolutionary origin, but the relationship between insect wings and legs is unresolved. The observation that wings and legs share open chromatin profiles supports the hypothesis that wings and legs also share a common evolutionary origin in flies. Because legs appear in the fossil record before wings, the similarity in their open chromatin profiles suggests that the existing leg cis-regulatory network was co-opted for use in creation of dorsal appendages during insect evolution (McKay, 2013).

Sp1 modifies leg-to-wing transdetermination in Drosophila

During Drosophila development, the transcription factor Sp1 is necessary for proper leg growth and also to repress wing development. Tested here was the role of Sp1 during imaginal disc regeneration. Ubiquitous expression of wg induces a regeneration blastema in the dorsal aspect of the leg disc. Within this outgrowth, the wing selector gene vg is activated in some cells, changing their fate to wing identity in a process known as transdetermination. This report demonstratse that reducing the gene copy number of Sp1 significantly increases both the frequency and the area of transdetermination in regenerating leg discs. By examining the expression of known Sp1 target genes, it was also shown that the proximo-distal patterning gene dachshund is downregulated dorsally, leading to a break in its normal ring-shaped expression pattern. Transdetermination, as evidenced by Vg expression, is only observed when there is a broken ring of Dachshund expression. Combined, these studies establish a role for Sp1 in leg-to-wing transdetermination (Ing, 2013).

This report has characterized the expression patterns of genes in transdetermining leg discs. The experiments demonstrate that specific expression patterns of Wg, pMad, and Dac are needed for TD to occur (see Model of the prerequisites for leg-to-wing TD). Leg-to-wing TD only occurs when non-symmetrical Wg overlaps with broad strong pMad expression. This interaction correlates with a broken Dac ring, which is necessary for activation of Vg. Further investigations of genes involved in leg-to-wing TD should also examine the specific expression patterns of other genes that may be a prerequisite for TD (Ing, 2013).

Previous work has identified many genes involved in leg-to-wing TD, and this study has shown that Sp1 is a strong enhancer of wg-induced TD. Interestingly, the Sp1 ortholog, Sp8, has been shown as a leg determinant in vertebrate species (Beermann, 2004). For example, Sp8 null mice have truncated legs and fail to develop specific leg structures (Bell, 2003). This shows that Sp8 plays a role in both leg development and in determining leg identity. In addition, reducing Sp8 gene copy number truncates leg outgrowth in mice in a dose-dependent manner (Bell, 2003). In Drosophila, Sp1 is a recessive lethal and arrests development at different stages. More severe phenotypes are observed in animals that differentiate adult structures but do not eclose to flies. Such pharate adults were tested for appendage truncation and it was observed that reducing Sp1 gene copy number does not increase leg truncation. Moreover, there is evidence that the Wnt signaling pathway in developing chicks regulates Sp8 and not Sp9, the vertebrate equivalent of Drosophila btd (Kawakami, 2004). In Drosophila, the Wg signaling pathway plays a pervasive role in leg regeneration. This study provides evidence that Sp1 might also function during regeneration, but it remains to be seen if Sp8 also plays a role vertebrate limb regeneration (Ing, 2013).

Non-redundant selector and growth-promoting functions of two sister genes, buttonhead and Sp1, in Drosophila leg development

The radically distinct morphologies of arthropod and tetrapod legs argue that these appendages do not share a common evolutionary origin. Yet, despite dramatic differences in morphology, it has been known for some time that transcription factors encoded by the Distalless (Dll)/Dlx gene family play a critical role in the development of both structures. This study shows that a second transcription factor family encoded by the Sp8 gene family, previously implicated in vertebrate limb development, also plays an early and fundamental role in arthropod leg development. By simultaneously removing the function of two Sp8 orthologs, buttonhead (btd) and Sp1, during Drosophila embryogenesis, adult leg development was found to be completely abolished. Remarkably, in the absence of these factors, transformations from ventral to dorsal appendage identities are observed, suggesting that adult dorsal fates become derepressed when ventral fates are eliminated. Further, Sp1 was shown to play a much more important role in ventral appendage specification than btd, and Sp1 lies genetically upstream of Dll. In addition to these selector-like gene functions, Sp1 and btd are also required during larval stages for the growth of the leg. Vertebrate Sp8 can rescue many of the functions of the Drosophila genes, arguing that these activities have been conserved, despite more than 500 million years of independent evolution. These observations suggest that an ancient Sp8/Dlx gene cassette was used in an early metazoan for primitive limb-like outgrowths and that this cassette was co-opted multiple times for appendage formation in multiple animal phyla (Estella, 2010).

Prior to this study, understanding of the roles that btd and Sp1 play in ventral appendage development in Drosophila was largely derived from ectopic expression experiments showing that btd could induce ectopic leg development when expressed in dorsal imaginal discs. In addition, based on a large deficiency that removes >50 genes, it was suggested that these genes may function upstream of Dll in ventral appendage specification. What was lacking in this previous study was the ability to specifically analyze the functions of these genes, both in embryogenesis and during adult development, using loss-of-function null alleles. Using a newly derived deficiency, together with rescue experiments, this study showed that these Zn-finger transcription factors play non-redundant roles in ventral appendage development. Moreover, for all of the readouts examined in this study (leg allocation, leg growth, proliferation, and PD axis formation) btd plays a much more minor or no role compared to Sp1. Early, Sp1, but not btd, is required to define the group of cells that will give rise to the legs and perhaps additional ventral body structures as well. Thus, Sp1 is a selector-like gene for the entire ventral appendage. Later in development, both genes are required for the proper growth of the leg, although to very different degrees. It was also shown that vertebrate Sp8 retains both the selector and growth-promoting functions, suggesting that there has been a remarkable amount of functional conservation between the vertebrate and fly genes (Estella, 2010).

During larval development, it was found that Sp1 is required for the proper growth of the entire leg, from the coxa through the tarsus. In contrast, btd plays a much more limited role in the tibia and femur. At this stage, neither gene is required for leg identity, nor are they required for the development of ventral body structures that arise from the most proximal cells in the leg imaginal disc. These 'late' phenotypes are consistent with the expression patterns of these genes in the third instar leg imaginal discs, where they appear to mark the entire presumptive leg, but not more proximal cells. This is interesting, because prior to these observations there were no markers that distinguished between the hth-expressing cells that give rise to the coxa from the hth-expressing cells that give rise to the ventral body wall. Dll, for example, is expressed in the cells that give rise to the distal tibia and tarsus, and lineage tracing with the Dll-LT element marks the entire telopodite (trochanter, femur, tibia, and tarsus). The addition of the btd and Sp1 expression patterns and mutant phenotypes to previously characterized PD genes therefore adds an important demarcation that distinguishes leg from body fates (Estella, 2010).

This analysis also reveals dramatic differences in the post-embryonic functions of btd and Sp1. Specifically, most of the growth phenotypes observed when both genes are removed can be phenocopied by knocking down only Sp1. In contrast, btd^XG81 clones (or btd^XA clones) have no phenotypes in the antenna, and, in the leg, result in only partial fusions between the femur and tibia. Thus, Sp1, not btd, plays an important and non-redundant function in ventral appendage development at this stage (Estella, 2010).

Selector and selector-like genes have the property that they specify an entire organ or body part. The classic example is engrailed (en) which 'selects' posterior compartment identities in Drosophila. Another example is eyeless (ey), which is both necessary and sufficient for eye development in Drosophila. In the leg, previous work highlighted the role of Dll in ventral appendage specification. In the absence of Dll, the distal portion of the leg fails to develop, while dorsal appendages remain wild type. Moreover, ectopic expression of Dll can induce distal legs to develop in dorsal positions. Taken together, these observations suggested that Dll is a selector-like gene for the distal leg (Estella, 2010).

Despite the requirement for Dll in leg development, it has been known for sometime that the ventral appendage primordia form in the absence of Dll. Moreover, homeotic transformations are not observed in the absence of Dll. Thus, Dll cannot be considered a selector-like gene for the entire ventral appendage. These observations raise the question of what factor or factors initially specify the cells that will give rise to the ventral appendage. It is proposed that Sp1 fulfills this selector-like role (Estella, 2010).

The suggestion that Sp1 is a selector-like gene for the entire ventral appendage stems in part from the observation that when the function of this gene is removed early in development, ∼10% of the animals have dramatic transformations of ventral structures to dorsal structures. In many of these cases, both wing and notum tissue were observed developing in ventral positions. Molecularly, Dll and dac expression is lost in transformed leg discs, and ectopic expression of vg and eyg, two markers for the dorsal appendages, are observed instead. The expression of Dll-304, which is traditionally been considered a marker for the ventral appendage, in Df(btd,Sp1) embryos may seem at odds with the idea that Sp1 is required for the initial specification of leg fates. However, fate-mapping studies show that Dll-304-expressing cells give rise to both the ventral (leg) and dorsal (wing and haltere) appendages. Thus, Dll-304 cannot be considered a ventral marker, and its activity in Df(btd,Sp1) embryos only confirms the establishment of appendage primordia without ventral or dorsal identity (Estella, 2010).

In sum, the striking transformations of fate seen in Df(btd,Sp1) animals suggest that Sp1 promotes ventral fates, both the entire leg and ventral body wall, and that in the absence of this gene, dorsal fates are de-repressed. This change in developmental fate is analogous to other classical homeotic transformations, for example, when the leg is transformed to antenna in the absence of Antennapedia (Antp). Note that btd null clones made at the same early time in development only result in mild growth defects, but legs are still generated. Thus, btd is not required for this function. However, because an Sp1 null allele (btd+) is not currently available, we cannot at this time be completely certain that btd plays no role in this process (Estella, 2010).

Because wing development is normally limited to T2, it was unexpected to observe leg to wing transformations in the T1 and, to a lesser extent, T3 segments. One potential explanation for this violation of antero-posterior identity is due to the timing of clone induction. Although the Hox genes are responsible for determining the segmental identities of the dorsal appendages, it may be that they are deployed at different times in the ventral and dorsal primordia in the different thoracic segments. If this is the case, then the resulting transformations may be very sensitive to the time they were generated and to their segmental origins. It is also worth noting that the wing primordia and T2 identity can be generated in the absence of Hox input. Thus, wing fates, as opposed to haltere or humeral (dorsal T1) fates, represent a Hox-free default state, which may predominate in these aberrant developmental situations (Estella, 2010).

Together with previous studies, these findings allow a presentation of a more complete view of ventral appendage specification, which is brocken down into three main phases. In the first phase, Sp1, btd, and Dll (via it's early Dll-304 enhancer) are initially activated in parallel in a ventral domain in each thoracic hemisegment of stage 11 embryos. The activation of all three genes is dependent on Wg signaling. This early, Dll-304-driven expression of Dll does not require either btd or Sp1. This initial group of cells is fated to give rise to both the entire ventral and dorsal thoracic imaginal discs, in other words, the entire adult thorax. In the second phase, which begins at stage 14, Dll-304 is no longer active and Dll is controlled by late-acting enhancers such as Dll-LT, which is activated by Wg and Dpp signaling. Interestingly, as shown in this study, these late-acting Dll enhancers also require Sp1, but not btd, thus placing Sp1 genetically upstream of Dll. At this stage, the Dll+ cells will only give rise to the leg telopodite. Sp1 is also required for telopodite formation but is carrying out at least two additional functions. One is that, unlike Dll, Sp1 is required to specify more proximal leg segments (the coxapodite). Second, the ventral to dorsal homeotic transformations described above suggest that Sp1 is also required to repress dorsal fates. Finally, in the third phase, Dll begins to autoactivate it's expression and no longer depends on Wg and Dpp inputs. At this stage, Dll also no longer requires Sp1 to be expressed. Instead of working through Dll, btd and Sp1 continue to play a critical role in leg development but now work in parallel to Dll to promote the growth of the entire leg. Thus, the specification of the ventral primordia depends on a feed-forward logic in which Sp1 activates late embryonic Dll expression followed by a phase in which both btd and Sp1 contribute to appendage growth in parallel to Dll (Estella, 2010).

Besides having a PD axis, arthropod and vertebrate appendage morphologies have little in common. Moreover, the developmental logic of limb formation in Drosophila is very different from that of vertebrate limb development. In flies, Hedgehog signaling induces two antagonistic secondary signals, Dpp and Wg, which in turn establish the PD axis by activating genes such as Dll and dac. In vertebrate limb development, Sonic hedgehog induces the activity of fibroblast growth factor-like molecules such as FGF8 in the ectoderm, which drives the proliferation of the underlying mesenchyme and the nested expression of Hox genes to create a PD axis. Despite these differences, it is striking that multiple vertebrate orthologs of both Sp1 and Dll are expressed during vertebrate limb development. In addition, orthologs of both hth and exd (Meis and pbx, respectively) are expressed in the proximal domain of the developing mouse limb. Although the existence of multiple Dll and Sp1 orthologs (Dlx1/Dlx2/Dlx5/Dlx6 and Sp8/Sp9, respectively) makes it much more challenging to assess their functions in detail, the available data demonstrate that, as in flies, both sets of genes are critical for vertebrate limb development. The current results, illustrating that vertebrate Sp8 can rescue many of the Sp1 and btd loss of function phenotypes in Drosophila, support the idea that appendage development in these two phyla represents a case of 'deep homology'. Interestingly, that orthologs of both Sp1 and Dll gene families are used in both phyla argue that, for appendage development, the functions of these transcription factors have been much more conserved than those of the signaling pathways used in limb development. The same conclusion holds for eye development where the transcription factors, more than the deployment of specific signaling pathways, have been conserved over vast evolutionary distances. These observations imply that, once established, transcription factor networks may be very stable, while the organization of signaling pathway networks may be much more plastic and easily modified to accommodate radically distinct morphologies (Estella, 2010).

The role of buttonhead and Sp1 in the development of the ventral imaginal discs

The related genes buttonhead (btd) and Drosophila Sp1 (the Drosophila homolog of the human SP1 gene) encode zinc-finger transcription factors known to play a developmental role in the formation of the Drosophila head segments and the mechanosensory larval organs. A novel function of btd and Sp1 is reported: they induce the formation and are required for the growth of the ventral imaginal discs. They act as activators of the headcase (hdc) and Distal-less (Dll) genes, which allocate the cells of the disc primordia. The requirement for btd and Sp1 persists during the development of ventral discs: inactivation by RNA interference results in a strong reduction of the size of legs and antennae. Ectopic expression of btd in the dorsal imaginal discs (eyes, wings and halteres) results in the formation of the corresponding ventral structures (antennae and legs). However, these structures are not patterned by the morphogenetic signals present in the dorsal discs; the cells expressing btd generate their own signalling system, including the establishment of a sharp boundary of engrailed expression, and the local activation of the wingless and decapentaplegic genes. Thus, the Btd product has the capacity to induce the activity of the entire genetic network necessary for ventral imaginal discs development. It is proposed that this property is a reflection of the initial function of the btd/Sp1 genes that consists of establishing the fate of the ventral disc primordia and determining their pattern and growth (Estella, 2003).

In a search for genes with restricted expression in the adult cuticle, the MD808 Gal4 line was found to direct expression in the ventral derivatives of the adult body; proboscis, antennae, legs and genitalia. In the abdomen and analia no clear expression was discerned. It was also noticed that the insertion was located in the first chromosome and associated with a lethal mutation. The mutant larvae showed a head phenotype resembling that described for mutants at the btd gene: loss of antennal organ and the ventral arms of the cephalopharyngeal skeleton, and complementation analysis indicated that the chromosome carrying the insert contained a mutation at btd. The expression pattern found in MD808/UAS-lacZ embryos was also similar to that reported for btd, suggesting that the Gal4 insertion was located at this gene. In addition, the imaginal expression of MD808 and of btd was largely coincident (Estella, 2003).

Further to the genetic analysis and the expression data, DNA fragments at the insertion site were cloned to map the position of the P-element on the genome. It is located 753 bp 5' of the btd gene. The related gene Sp1 is immediately adjacent. It is likely that btd and Sp1 have originated by a tandem duplication of a primordial btd-like gene (Estella, 2003).

In early embryos btd is expressed in the head region, but by the extended germ band stage the expression domain has expanded to the ventral region of cephalic, thoracic and abdominal segments. During germ band retraction most of the abdominal and thoracic expression is lost, except in derivatives of the peripheral nervous system and the primordia of the imaginal discs. Sp1 is not expressed in early embryos, but from stage 11 onwards it shows the same pattern as btd (Estella, 2003).

Special attention was paid to the btd/Sp1 expression domain in the thoracic imaginal discs primordia, as it may suggest a novel function related to imaginal development. Double labelling with Dll and btd probes indicates that btd precedes Dll expression, but by stage 12 the two genes are co-expressed in a group of thoracic cells. However, the Dll domain is smaller and is included within the btd/Sp1 domain: there are cells expressing btd that do not show Dll activity, although all the cells expressing Dll express btd (Estella, 2003).

The ventral disc primordia include not only cells expressing Dll but also other cells containing expression of escargot (esg) and hdc, markers of the diploid cells that form the imaginal primordia. In late embryonic stages, esg is expressed in a ring domain surrounding the Dll-expressing cells and hdc is expressed in a similar pattern. Double label experiments were carried out with btd, hdc and esg probes; the expression of the two latter genes overlaps with that of btd (and with Sp1) in the thoracic disc primordia (Estella, 2003).

The overlap of the btd and of esg domains indicates that btd is also expressed in the hth domain, which is coincident with that of esg. As the hth/esg domain marks the precursor cells of the proximal region of the adult leg the embryonic expression data indicate that btd and Sp1 are active in the entire primordia of the ventral adult structures, including the distal and the proximal parts (Estella, 2003).

In the mature antennal disc, btd expression is restricted mostly to the region corresponding to the second antennal segment, where it co-localizes with both Dll and hth. In the leg disc btd also overlaps in part with Dll and with hth. The latter result is significant, for the expression of Dll and hth define two major genetic domains, which are kept apart by antagonistic interactions. The fact that btd is expressed in the two domains suggests that its regulation and function is independent from the interactions between the two domains. This observation is consistent with the results obtained in embryos and suggests that the btd domain includes the precursors of the whole ventral thoracic region from the beginning of development (Estella, 2003).

This work demonstrates a novel and also redundant function of btd and Sp1: they are responsible for the formation of the ventral imaginal discs by activating the genetic network necessary for their development. Furthermore, Btd protein retains the capacity of inducing the entire ventral genetic network during the larval period. It is proposed that the activation of btd/Sp1 is the crucial event in the determination of the ventral structures of the adult fly (Estella, 2003).

This argument is based on the finding that btd and Sp1 appear to mediate all events connected with the formation of the ventral discs. The discussion deals with the leg disc, but there is evidence that antennal primordium also requires btd. Moreover, the genital primordium is lacking in Df(1)C52 embryos, suggesting that this disc is also under the same control. Most of the experiments concern the function of btd but given the expression and functional similarities between the two genes, it is assumed that Sp1 fulfils the same or a very similar role. Therefore, btd/Sp1 will be considered to carry out a single function (Estella, 2003).

One crucial feature is that btd is an upstream activator of Dll and hdc, which are considered developmental markers of disc primordia: (1) btd expression precedes that of Dll and of hdc; (2) the btd expression domain includes those of Dll and hdc; (3) in btd mutants, Dll and hdc activity is much reduced, and completely absent in Df(1)C52 embryos; (4) ectopic btd function induces ectopic activation of Dll and hdc (Estella, 2003).

The role of btd in embryogenesis can be illustrated in the light of the models of Dll regulation. Dll is activated by wg and its expression modulated by the EGF spitz and by dpp, whereas it is repressed in the abdominal segments by the BX-C genes. The current experiments suggest that Dll regulation is mediated by btd: in wg mutants there is no btd expression and hence neither Dll nor hdc activity. In dpp mutant embryos, btd expands to the dorsal region resembling the effect on Dll. In Ubx^- embryos there is an additional group of cells in the first abdominal segment expressing btd; the same cells that also express Dll in those embryos. The interpretation of the role of btd mediating Dll regulation by Ubx is complicated by previous observations showing direct repression of Dll by the Ubx protein. It is possible that Ubx regulates Dll both directly and by controlling btd activity (Estella, 2003).

It is proposed that the localization of btd/Sp1 activity to a group of ventral cells is a major event in the specification of adult structures. btd and Sp1 are activated in response to spatial cues from Wg, Dpp, EGF and BX-C, and in turn their function induces the activity of the genes necessary for ventral imaginal development (Estella, 2003).

This hypothesis is strongly supported by the results obtained inducing ectopic btd activity in the dorsal discs; just the presence of the Btd product alone is sufficient to bring about ventral disc development. In the wing and the haltere discs, Btd induces a transformation into leg, whereas in the eye it induces antennal development. This indicates that it specifies ventral disc identity jointly with other factors, e.g., the Hox genes, possibly through the activation of subsidiary genes such as Dll, known to contribute to ventral appendage identity in combination with Hox genes (Estella, 2003).

The requirement for btd and Sp1 activity appears to be restricted only to the ventral discs, even during the early phases of the thoracic disc primordia. In this context it is worth considering the observation that in Df(1)C52 embryos there is esg expression in the wing and haltere disc primordia, even though it is absent in the leg discs. Thus, the wing and haltere discs are formed in the absence of btd and Sp1. Because in these embryos there is an almost complete lack of Dll expression, this observation raises the question of the origin of the dorsal thoracic discs, which are currently considered to derive from the original ventral primordium, formed by cells expressing Dll. Although some of the original group of ventral cells may contribute to the dorsal disc primordia, the data suggest that there may be cells recruited to form the dorsal discs that do not originate in the initial ventral primordium. Accordingly, it is worth considering that in the absence of Dll activity the leg and wing discs are formed, although the leg only differentiates proximal disc derivatives. Thus, the activity of Dll cannot be considered a reliable marker for imaginal discs (Estella, 2003).

RNA interference experiments also indicate that both btd and Sp1 are required for the growth of the antennal and leg discs. When the two gene functions are reduced simultaneously, leg segments fuse and there is an overall reduction in the size of antennae and legs. The reduction of growth affects the proximal and distal regions of the appendage, and assigns a role to the expression observed in the imaginal discs. The two genes are able to perform this function on their own, for the inactivation of only one is not sufficient to impair growth. This conclusion is also supported by the observation that mutant btd clones do not have any effect; they still possess Sp1 activity, which is sufficient for normal development. At this point the mechanism by which btd/Sp1 may affect growth is not known (Estella, 2003).

One particularly significant result about the mode of action of btd comes from the analysis of the ectopic leg patterns observed with ectopic btb expression in the wing and halteres. The clones of cells ectopically expressing btd tend to recapitulate the complete development of leg and antennal discs. For example, the whole genetic network necessary to make a leg appears to be activated. btd induces the activity of hth, dac and Dll, the domains of which account for the entire disc. Furthermore, hth, dac and Dll are activated in a spatially discriminated manner. The formation of the dac and Dll domains is dependent on signalling from Wg and Dpp, although they require different signal thresholds. In one clone, for example hth is expressed only in the peripheral region, resembling the normal expression in the leg disc; in another clone the discriminate expressions of dac and Dll define three distinct regions. The formation of the dac and Dll domains is dependent on signalling from Wg and Dpp, although they require different signal thresholds, but the hth domain is independent from Wg and Dpp (Estella, 2003).

The generation of distinct hth, dac and Dll domains within the clones suggested that btd-expressing cells in the wing and haltere generate their own signalling process. Indeed, within these clones there is local activation of en, the transcription factor that initiates Hh/Wg/Dpp signalling in imaginal discs. btd-expressing clones also acquire wg and dpp activity in subsets of cells. It is probably in the boundary of en-expressing with non expressing cells where the Wg and Dpp signals are generated de novo; subsequently, their diffusion initiates the same patterning mechanism which operates during normal leg development. The result of this process is that the hth, dac and Dll genes are expressed in different domains contributing to form leg patterns containing DV and PD axes. One question for which there is no clear answer is how the initial asymmetry is generated, so that a few cells within the group gain (or lose) en activity. The cells expressing en within the clones are those closer to the posterior compartment cells. It is conceivable that there might be an external signal, perhaps mediated by Hh, which triggers the initial asymmetry (Estella, 2003).

The ability of cells expressing btd to build their own patterning mechanism is also indicated by the observation that inducing btd activity in different parts of the wing disc results in the production of similar sets of leg pattern elements. For example, in MD743/UAS-btd and omb-Gal4/UAS-btd flies, btd is induced in different, non-overlapping wing regions, and yet all leg pattern elements are produced in both genotypes. Thus, the effect of btd is by and large independent of the position where it is induced, e.g., it does not depend on local positional signals (Estella, 2003).

A relevant issue is whether the ability of the Btd product to induce the formation of the full array of ventral structures has a functional significance in normal development. This property may be a faithful reflection of the original btd/Sp1 function: the activation of the developmental program to build the ventral adult patterns. btd/Sp1 function can be envisaged as follows. During the embryonic period, the conjunction of several regulatory factors (Wg, Dpp, EGF, Hox genes) allows activation of btd/Sp1 in a group of cells in each thoracic segment (it is assumed that a similar process takes place in the head). These cells become the precursors of the ventral imaginal discs and will eventually form the ventral thorax of the adult -- these include the trunk (the hth domain) and appendage (the Dll domain) regions. The activity of btd/Sp1 is instrumental in segregating these ventral discs precursors from those of the larval epidermis and determining their imaginal fate. It is involved in specifying their segment identity (in collaboration with the Hox genes) and in establishing their pattern and growth. To achieve the latter role btd/Sp1 induces the production of the growth signals Wg and Dpp, probably in response to localized activation of en and subsequent signalling by hedgehog (hh) (Estella, 2003).

A problem with this model is that in normal development all the genes involved, hth, en, hh, wg and dpp, are expressed in embryos prior to btd/Sp1. Why should a new round of activation be necessary? Although a totally satisfactory answer can not be provided, it is noted that clones of btd-expressing cells in wing or haltere lose their memory of en expression. Those that originated in the A compartment had no previous en expression, but gained it in some cells. Conversely, all cells in P compartment clones contained en activity but some lose it. The best explanation for this unexpected behavior is that btd provokes a 'naïve' cell state in which the previous commitment for en activity is lost. Later, en activity is re-established. This phenomenon may reflect the process that occurs in normal development. The initial btd/Sp1 domain may not inherit the previous developmental commitments and has to build a new developmental program. It is worth considering that the btd/Sp1function appears to determine ventral imaginal fate as different from larval fate. This is a major developmental decision, which may require de novo establishment of the genetic system responsible for pattern and growth. A key aspect of this would be the localized activation of en in part of the btd/Sp1 domain. It is speculated that there might be a short-range signal, perhaps Hh, emanating from nearby en-expressing embryonic cells, that acts as an inducer in the btd/Sp1 primordium. There is evidence that Hh can induce en activity (Estella, 2003).

Functions of Sp1 orthologs in other species

Sp6 and Sp8 transcription factors control AER formation and dorsal-ventral patterning in limb development

The formation and maintenance of the apical ectodermal ridge (AER) is critical for the outgrowth and patterning of the vertebrate limb. The induction of the AER is a complex process that relies on integrated interactions among the Fgf, Wnt, and Bmp signaling pathways that operate within the ectoderm and between the ectoderm and the mesoderm of the early limb bud. The transcription factors Sp6 and Sp8 are expressed in the limb ectoderm and AER during limb development. Sp6 mutant mice display a mild syndactyly phenotype while Sp8 mutants exhibit severe limb truncations. Both mutants show defects in AER maturation and in dorsal-ventral patterning. To gain further insights into the role Sp6 and Sp8 play in limb development, mice were produced lacking both Sp6 and Sp8 activity in the limb ectoderm. Remarkably, the elimination or significant reduction in Sp6;Sp8 gene dosage leads to tetra-amelia; initial budding occurs, but neither Fgf8 nor En1 are activated. Mutants bearing a single functional allele of Sp8 (Sp6-/-;Sp8+/-) exhibit a split-hand/foot malformation phenotype with double dorsal digit tips probably due to an irregular and immature AER that is not maintained in the center of the bud and on the abnormal expansion of Wnt7a expression to the ventral ectoderm. These data are compatible with Sp6 and Sp8 working together and in a dose-dependent manner as indispensable mediators of Wnt/βcatenin and Bmp signaling in the limb ectoderm. It is suggested that the function of these factors links proximal-distal and dorsal-ventral patterning (Haro, 2016).

Delineating a conserved genetic cassette promoting outgrowth of body appendages

The acquisition of the external genitalia allowed mammals to cope with terrestrial-specific reproductive needs for internal fertilization, and thus it represents one of the most fundamental steps in evolution towards a life on land. How genitalia evolved remains obscure, and the key to understanding this process may lie in the developmental genetics that underpins the early establishment of the genital primordium, the genital tubercle (GT). Development of the GT is similar to that of the limb, which requires precise regulation from a distal signaling epithelium. However, whether outgrowth of the GT and limbs is mediated by common instructive signals remains unknown. In this study, comprehensive genetic approaches were used to interrogate the signaling cascade involved in GT formation in comparison with limb formation. The FGF ligand responsible for GT development was shown to be FGF8 expressed in the cloacal endoderm. It was further shown that forced Fgf8 expression can rescue limb and GT reduction in embryos deficient in WNT signaling activity. These studies show that the regulation of Fgf8 by the canonical WNT signaling pathway is mediated in part by the transcription factor SP8. Sp8 mutants elicit appendage defects mirroring WNT and FGF mutants, and abolishing Sp8 attenuates ectopic appendage development caused by a gain-of-function beta-catenin mutation. These observations indicate that a conserved WNT-SP8-FGF8 genetic cassette is employed by both appendages for promoting outgrowth, and suggest a deep homology shared by the limb and external genitalia (Lin, 2013).

A conserved function of the zinc finger transcription factor Sp8/9 in allometric appendage growth in the milkweed bug Oncopeltus fasciatus

The genes encoding the closely related zinc finger transcription factors Buttonhead (Btd) and D-Sp1 are expressed in the developing limb primordia of Drosophila melanogaster and are required for normal growth of the legs. The D-Sp1 homolog of the red flour beetle Tribolium castaneum, Sp8 (appropriately termed Sp8/9), is also required for the proper growth of the leg segments. This study reports on the isolation and functional study of the Sp8/9 gene from the milkweed bug Oncopeltus fasciatus. Sp8/9 is expressed in the developing appendages throughout development, and the downregulation of Sp8/9 via RNAi leads to antennae, rostrum, and legs with shortened and fused segments. This supports a conserved role of Sp8/9 in allometric leg segment growth. However, all leg segments including the claws are present and the expression of the leg genes Distal-less, dachshund, and homothorax are proportionally normal, thus providing no evidence for a role of Sp8/9 in appendage specification (Schaeper, 2009).

The Sp8 zinc-finger transcription factor is involved in allometric growth of the limbs in the beetle Tribolium castaneum

Members of the Sp gene family are involved in a variety of developmental processes in both vertebrates and invertebrates. This study identified the ortholog of the Drosophila Sp-1 gene in the red flour beetle Tribolium castaneum, termed T-Sp8 because of its close phylogenetic relationship to the vertebrate Sp8 genes. During early embryogenesis, T-Sp8 is seen in segmental stripes. During later stages, TSp8 is dynamically expressed in the limb buds of the Tribolium embryo. At the beginning of bud formation, TSp8 is uniformly expressed in all body appendages. As the limbs elongate, a ring pattern develops sequentially and the expression profile at the end of embryogenesis correlates with the final length of the appendage. In limbs that do not grow out like the labrum and the labium, T-Sp8 expression remains uniform, whereas a two-ring pattern develops in the longer antennae and the maxillae. In the legs that elongate even further, four rings of T-Sp8 expression can be seen at the end of leg development. The role of T-Sp8 for appendage development was tested using RNAi. Upon injection of double stranded T-Sp8 RNA, larvae develop with dwarfed appendages. Affected T-Sp8(RNAi) legs were tested for the presence of medial and distal positional values using the expression marker genes dachshund and Distal-less, respectively. The results show that a dwarfed TSp8(RNAi) leg consists of proximal, medial and distal parts and argues against T-Sp8 being a leg gap gene. Based on the differential expression pattern of T-Sp8 in the appendages of the head and the thorax and the RNAi phenotype, it is hypothesised that T-Sp8 is involved in the regulation of limb-length in relation to body size - a process called allometric growth (Beermann, 2004).

Sp8 and Sp9, two closely related buttonhead-like transcription factors, regulate Fgf8 expression and limb outgrowth in vertebrate embryos

Initiation and maintenance of signaling centers is a key issue during embryonic development. The apical ectodermal ridge, a specialized epithelial structure and source of Fgf8, is a pivotal signaling center for limb outgrowth. Two closely related buttonhead-like zinc-finger transcription factors, Sp8 and Sp9, are expressed in the AER, and regulate Fgf8 expression and limb outgrowth. Embryological and genetic analyses have revealed that Sp8 and Sp9 are ectodermal targets of Fgf10 signaling from the mesenchyme. This study also found that Wnt/beta-catenin signaling positively regulates Sp8, but not Sp9. Overexpression functional analyses in chick unveiled their role as positive regulators of Fgf8 expression. Moreover, a dominant-negative approach in chick and knockdown analysis with morpholinos in zebrafish revealed their requirement for Fgf8 expression and limb outgrowth, and further indicate that they have a coordinated action on Fgf8 expression. This study demonstrates that Sp8 and Sp9, via Fgf8, are involved in mediating the actions of Fgf10 and Wnt/β-catenin signaling during vertebrate limb outgrowth (Kawakami, 2004).

Sp8 is crucial for limb outgrowth and neuropore closure

This report describes the developmental expression and function of Sp8, a member of the Sp family of zinc finger transcription factors, and provide evidence that the legless transgene insertional mutant is a hypomorphic allele of the Sp8 gene. Sp8 is expressed during embryogenesis in the forming apical ectodermal ridge (AER), restricted regions of the central nervous system, and tail bud. Targeted deletion of the Sp8 gene gives a striking phenotype, with severe truncation of both forelimbs and hindlimbs, absent tail, as well as defects in anterior and posterior neuropore closure leading to exencephaly and spina bifida. Outgrowth of the limb depends on formation of the AER, a signaling center that forms at the limb bud apex. In Sp8 mutants, the AER precursor cells are induced and initially express multiple appropriate marker genes, but expression of these genes is not maintained and progression to a mature AER is blocked. These observations indicate that Sp8 functions downstream of Wnt3, Fgf10, and Bmpr1a in the signaling cascade that mediates AER formation (Bell, 2003).

Search PubMed for articles about Drosophila Sp1

Beermann, A., Aranda, M. and Schroder, R. (2004). The Sp8 zinc-finger transcription factor is involved in allometric growth of the limbs in the beetle Tribolium castaneum. Development 131: 733-742. PubMed ID: 14724124

Bell, S. M., Schreiner, C. M., Waclaw, R. R., Campbell, K., Potter, S. S. and Scott, W. J. (2003). Sp8 is crucial for limb outgrowth and neuropore closure. Proc Natl Acad Sci U S A 100: 12195-12200. PubMed ID: 14526104

Cordoba, S., Requena, D., Jory, A., Saiz, A. and Estella, C. (2016). The evolutionary conserved transcription factor Sp1 controls appendage growth through Notch signaling. Development 143(19):3623-3631. PubMed ID: 27578786

Estella, C., Rieckhof, G., Calleja, M. and Morata, G. (2003). The role of buttonhead and Sp1 in the development of the ventral imaginal discs of Drosophila. Development 130: 5929-5941. PubMed ID: 14561634

Estella, C. and Mann, R. S. (2010). Non-redundant selector and growth-promoting functions of two sister genes, buttonhead and Sp1, in Drosophila leg development. PLoS Genet 6: e1001001. PubMed ID: 20585625

Estella, C., Herrer, I., Moreno-Moya, J. M., Quinonero, A., Martinez, S., Pellicer, A. and Simon, C. (2012). miRNA signature and Dicer requirement during human endometrial stromal decidualization in vitro. PLoS One 7: e41080. PubMed ID: 22911744

Haro, E., Delgado, I., Junco, M., Yamada, Y., Mansouri, A., Oberg, K. C. and Ros, M. A. (2014). Sp6 and Sp8 transcription factors control AER formation and dorsal-ventral patterning in limb development. PLoS Genet 10: e1004468. PubMed ID: 25166858

Ing, T., Tseng, A., Sustar, A. and Schubiger, G. (2013). Sp1 modifies leg-to-wing transdetermination in Drosophila. Dev Biol 373(2): 290-299. PubMed ID: 23165292

Kawakami, Y., Esteban, C. R., Matsui, T., Rodriguez-Leon, J., Kato, S. and Izpisua Belmonte, J. C. (2004). Sp8 and Sp9, two closely related buttonhead-like transcription factors, regulate Fgf8 expression and limb outgrowth in vertebrate embryos. Development 131: 4763-4774. PubMed ID: 15358670

Lin, C., Yin, Y., Bell, S. M., Veith, G. M., Chen, H., Huh, S. H., Ornitz, D. M. and Ma, L. (2013). Delineating a conserved genetic cassette promoting outgrowth of body appendages. PLoS Genet 9(1): e1003231. PubMed ID: 23358455

McKay, D. J. and Lieb, J. D. (2013). A common set of DNA regulatory elements shapes Drosophila appendages. Dev Cell 27: 306-318. PubMed ID: 24229644

Schaeper, N. D., Prpic, N. M. and Wimmer, E. A. (2009). A conserved function of the zinc finger transcription factor Sp8/9 in allometric appendage growth in the milkweed bug Oncopeltus fasciatus. Dev Genes Evol 219: 427-435. PubMed ID: 19760183

Talamillo, A., Delgado, I., Nakamura, T., de-Vega, S., Yoshitomi, Y., Unda, F., Birchmeier, W., Yamada, Y. and Ros, M. A. (2010). Role of Epiprofin, a zinc-finger transcription factor, in limb development. Dev Biol 337: 363-374. PubMed ID: 19913006

Treichel, D., Schock, F., Jackle, H., Gruss, P. and Mansouri, A. (2003). mBtd is required to maintain signaling during murine limb development. Genes Dev 17: 2630-2635. PubMed ID: 14597661

date revised: 18 December 2016

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