InteractiveFly: GeneBrief

Zn finger homeodomain 2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The Drosophila wing imaginal disc gives rise to three main regions along the proximodistal axis of the dorsal mesothoracic segment: the notum, proximal wing, and wing blade. Development of the wing blade requires the Notch and Wingless signalling pathways to activate vestigial at the dorsoventral boundary. However, in the proximal wing, Wingless activates a different subset of genes, e.g., homothorax. This raises the question of how the downstream response to Wingless signalling differentiates between proximal and distal fate specification. A temporally dynamic response to Wingless signalling is shown to sequentially elaborate the proximodistal axis. In the second instar, Wingless activates genes involved in proximal wing development; later in the third instar, Wingless acts to direct the differentiation of the distal wing blade. The expression of a novel marker for proximal wing fate, Zn finger homeodomain 2 (zfh-2), is initially activated by Wingless throughout the 'wing primordium', but later is repressed by the activity of Vestigial and Nubbin, which together define a more distal domain. Thus, activation of a distal developmental program is antagonistic to previously established proximal fate. In addition, Wingless is required early to establish proximal fate, but later when Wingless activates distal differentiation, development of proximal fate becomes independent of Wingless signalling. Since P-element insertions in the zfh-2 gene result in a revertable proximal wing deletion phenotype, it appears that zfh-2 activity is required for correct proximal wing development. These data are consistent with a model in which Wingless first establishes a proximal appendage fate over notum, then the downstream response changes to direct the differentiation of a more distal fate over proximal. Thus, the proximodistal domains are patterned in sequence and show a distal dominance (Whitworth, 2003).

The Drosophila wing imaginal disc gives rise to the structures of the dorsal mesothoracic segment. This is subdivided into three main regions: the notum, the wing blade, and the proximal wing and hinge. The wing is attached to the thorax via a complex joint comprising a small portion of the appendage, the hinge, which consists of several interlocking sclerites and plates. The wing blade tapers toward the body, forming a short, narrow region that is attached at the hinge. This region shall be referred to as the proximal wing since it is morphologically and mechanically distinct from the hinge itself. Fate mapping of the late third instar imaginal disc has determined that the central portion, the wing pouch, develops as wing blade, a ring surrounding the wing pouch develops as proximal wing and hinge, and the large dorsal territory and a narrow ventral domain form the notum and ventral pleura (Whitworth, 2003).

Previous studies have attempted to follow the development of the proximal part of the wing by analysis of genes that have some expression in the proximal region of the wing disc, e.g., wg or nub, or by the exclusion of markers for notum and wing fates, e.g., teashirt (tsh) and vg, respectively. The identification and analysis is described of a novel marker for proximal wing fate that specifically demarcates the whole of the developing proximal wing tissue, the zinc-finger homeodomain gene zfh-2 (Fortini, 1991). In third larval instar (L3) wing discs, Wg is expressed in a stripe along the D/V boundary, forming the wing margin, and in two concentric rings around the wing pouch. In the adult wing, expression of a wg-lacZ reporter indicates that the two rings of wg delimit the proximal wing. The inner (distal) ring runs from the medial costa, through the humeral crossvein to the alula, and the outer (proximal) ring runs from the proximal end of the proximal costa to the axillary cord (Whitworth, 2003).

In both L3 wing discs and adult wings, Zfh-2 is expressed in a domain that completely overlaps the rings of Wg expression. In L3 wing discs, Zfh-2 does not extend either proximally into the notum or distally into the wing pouch. These observations indicate that, in late stages, Zfh-2 is specifically expressed throughout the developing proximal wing and therefore may be used as a useful marker for proximal wing fate. To determine the extent of coexpression of Zfh-2 and Wg, early L2 discs were examined with anti- Zfh-2 and anti-Wg antisera. Zfh-2 is expressed at this stage in a pattern that directly overlaps with Wg. As development proceeds, Zfh-2 quickly expands to cover the whole of the ventral portion of the wing disc, accompanying the expansion of the Wg domain (Whitworth, 2003).

Taken together, these observations suggest that, at the beginning of L2, the wing imaginal disc is divided into the presumptive notum and the appendage or 'wing' primordium, and that the wing primordium is undifferentiated with respect to the proximodistal axis. This is supported by reports that tsh is also expressed throughout L2 wing discs, but is later restricted to the presumptive notum and hinge regions, and also that vgQE is not yet activated to differentiate the wing pouch. From the dynamic expression pattern of the proximal wing marker zfh-2, it appears that the elaboration of distal elements within the disc, marked by the disappearance of Zfh-2 and concomitant activation of the vgQE, is initiated at the start of L3 at the center of the wing disc where the A/P and D/V boundaries intersect. This suggests that the proximal wing and wing pouch differentiate sequentially, the distal wing pouch being induced later than the already established proximal wing. The early expression of zfh-2 indicates that it is a specific marker for proximal fate (Whitworth, 2003).

At the beginning of the second larval instar, the wing imaginal disc expresses markers of proximal fate, hth and tsh, in the entire anlage. During early L2, the expression of wg and zfh-2 is initiated in an anterior-ventral wedge pattern. The data indicate that Wg function is required to activate zfh-2 expression at this stage, since early removal of Wg function leads to a simultaneous loss of zfh-2 expression. As development proceeds, wg and zfh-2 expression rapidly expands filling the whole of the ventral portion of the wing disc by the end of the second instar. Concomitant with the expansion of wg and zfh-2, both hth and tsh become repressed in the ventral portion of the disc. This transition appears to mark the first P-D differentiation of the wing disc into appendage and notum. However, since zfh-2 is expressed in the entire wing anlage at this time, it is believed that the appendage has not differentiated proximal wing and blade. Around the L2-L3 transition, the wing blade markers nub and vgQE are activated by the combined activity of the Wg and N signalling pathways. Nub and Vg, acting together or independently, repress zfh-2 expression in the center of the disc. This marks the second phase of P-D elaboration where the appendage anlage is split into proximal wing and blade. It is noted that, at this time, hth and tsh remain coexpressed in the notum, where zfh-2 is not expressed. The pattern of zfh-2 expression at this stage suggests that it is still influenced by Wg signalling since it remains restricted to areas of high Wg expression. During L3, the division of the wing disc into three distinct domains is maintained and refined as the individual domains undergo their characteristic patterning. At this time, Hth and Wg are upregulated in the proximal wing anlage, where their activities are interdependent, while zfh-2 expression persists but becomes independent of Wg activity (Whitworth, 2003).

These data further support a qualitative difference in the activity of Wg in the proximal wing compared with wing blade. In addition to the activation of different effectors, previous investigations have shown that ectopic Wg expression in the proximal wing causes large overgrowth of proximal tissue, but similar overexpression in the wing blade produces no overgrowth. This indicates that a different mitogenic response to Wg signalling is activated in the wing pouch compared with proximal wing (Whitworth, 2003).

Therefore, these observations suggest a model in which the wing disc is sequentially partitioned in a proximal to distal direction: notum, proximal wing, and finally wing blade. This view of temporal specification of PD identities is supported by transplantation experiments where L2 wing disc fragments can only differentiate proximal wing structures, whereas L3 disc fragments can produce wing blade elements. In support of the more general applicability of these findings, a study of PD patterning in the Drosophila leg has shown that Wg and Dpp act early to establish the PD axis, but later are not required. These data appear strikingly similar to the results presented in this study and suggest an important common mechanism for PD axis elaboration that has previously been unappreciated. This investigation also serves to emphasize the importance of considering the development of the imaginal disc as an extremely dynamic field, with respect to rapid changes in both size and patterning (Whitworth, 2003).

Specification of individual adult motor neuron morphologies by combinatorial transcription factor code

How the highly stereotyped morphologies of individual neurons are genetically specified is not well understood. This study identified six transcription factors (TFs; Ems, Zfh1, Pb, Zfh2, Pros and Toy) expressed in a combinatorial manner in seven post-mitotic adult leg motor neurons (MNs) that are derived from a single neuroblast in Drosophila. Unlike TFs expressed in mitotically active neuroblasts, these TFs do not regulate each other's expression. Removing the activity of a single TF resulted in specific morphological defects, including muscle targeting and dendritic arborization, and in a highly specific walking defect in adult flies. In contrast, when the expression of multiple TFs was modified, nearly complete transformations in MN morphologies were generated. These results show that the morphological characteristics of a single neuron are dictated by a combinatorial code of morphology TFs (mTFs). mTFs function at a previously unidentified regulatory tier downstream of factors acting in the NB but independently of factors that act in terminally differentiated neurons (Enriquez, 2015).

Neurons are the most morphologically diverse cell types in the animal kingdom, providing animals with the means to sense their environment and move in response. In Drosophila, neurons are generated by neuroblasts (NBs), specialized stem cells dedicated to the generation of neurons and glia. As they divide, NBs express a temporal sequence of transcription factors (TFs) that contribute to the generation of neuronal diversity. For example, in the embryonic ventral nerve cord (VNC), most NBs express a sequence of five TFs (Hunchback, Krüppel, Pdm1/Pdm2, Castor, and Grainyhead), while in medulla NBs and intermediate neural progenitors of the Drosophila larval brain a different series of TFs have been described. In vertebrates, analogous strategies are probably used by neural stem cells, e.g., in the cerebral cortex and retina, suggesting that this regulatory logic is evolutionarily conserved. Nevertheless, although temporally expressed NB TFs play an important role in generating diversity, this strategy cannot be sufficient to explain the vast array of morphologically distinct neurons present in nervous systems. For example, in the Drosophila optic lobe there is estimated to be ~40,000 neurons, classified into ~70 morphologically distinct types, each making unique connections within the fly's visual circuitry neurons (Enriquez, 2015).

A second class of TFs has been proposed to specify subtypes of neurons. For example, in the vertebrate spinal cord, all motor neurons (MNs) express a common set of TFs at the progenitor stage (Olig2, Nkx6.1/6.2, and Pax6) and a different set of TFs after they become post-mitotic (Hb9, Islet1/2, and Lhx3). Hox6 at brachial and Hox10 at lumbar levels further distinguish MNs that target muscles in the limbs instead of body wall muscles. Subsequently, limb-targeting MNs are further refined into pools, where all MNs in a single pool target the same muscle. Each pool is molecularly defined by the expression of pool-specific TFs, including a unique combination of Hox TFs. In Drosophila embryos, subclasses of MNs are also specified by unique combinations of TFs: evenskipped (eve) and grain are expressed in six MNs that target dorsal body wall, and Hb9, Nkx6, Islet, Lim3, and Olig2 are required for ventral-targeting MNs. However, each neuronal subtype defined by these TFs includes multiple morphologically distinct neurons, leaving open the question of how individual neuronal morphologies are specified neurons (Enriquez, 2015).

A third class of TFs suggested to be important for neuronal identity is encoded by terminal selector genes. Initially defined in C. elegans, these factors maintain a neuron's terminally differentiated characteristics by, for example, regulating genes required for the production of a particular neurotransmitter or neuropeptide. Consequently, these TFs must be expressed throughout the lifetime of a terminally differentiated neuron. Notably, as with neurons that are from the same subtype, neurons that share terminal characteristics, and are therefore likely to share the same terminal selector TFs, can have distinct morphological identities. For example, in C. elegans two terminal selector TFs, Mec-3 and Unc-86, function together to maintain the expression of genes required for a mechanosensory fate in six morphologically distinct touch sensitive neurons neurons (Enriquez, 2015).

In contrast to the logic revealed by these three classes of TFs, very little is known about how individual neurons, each with their own stereotyped dendritic arbors and synaptic targets, obtain their specific morphological characteristics. This paper addresses this question by focusing on how individual MNs that target the adult legs of Drosophila obtain their morphological identities. The adult leg MNs of Drosophila offer several advantages for understanding the genetic specification of neuronal morphology. For one, all 11 NB lineages that generate the ~50 leg-targeting MNs in each hemisegment have been defined. More than two-thirds of these MNs are derived from only two lineages, Lin A (also called Lin 15) and Lin B (also called Lin 24), which produce 28 and 7 MNs, respectively, during the second and third larval stages. Second, each leg-targeting MN has been morphologically characterized-both dendrites and axons-at the single-cell level. In the adult VNC, the leg MN cell bodies in each thoracic hemisegment (T1, T2, and T3) are clustered together. Each MN extends a highly stereotyped array of dendrites into a dense neuropil within the VNC and a single axon into the ipsilateral leg, where it forms synapses onto one of 14 muscles in one of four leg segments: coxa (Co), trochanter (Tr), femur (Fe), and tibia (Ti). Not only does each MN target a specific region of a muscle, the pattern of dendritic arbors of each MN is also stereotyped and correlates with axon targeting. The tight correlation between axon targeting and dendritic morphology has been referred to as a myotopic map. The stereotyped morphology exhibited by each MN suggests that it is under precise genetic control that is essential to its function neurons (Enriquez, 2015).

This study demonstrates that individual post-mitotic MNs express a unique combination of TFs that endows them with their specific morphological properties. Focus was placed on Lin B, which generates seven MNs, and six TFs were identified that can account for most of the morphological diversity within this lineage. Interestingly, these TFs do not cross-regulate each other and are not required for other attributes of MN identity, such as their choice of neurotransmitter (glutamine) or whether their axons target muscles in the periphery, i.e., they remain terminally differentiated leg motor neurons. Consistent with the existence of a combinatorial code, when two or three, but not individual, TFs were simultaneously manipulated nearly complete transformations in morphology were observed. However, removing the function of a single TF, which is expressed in only three Lin B MNs, resulted in a highly specific walking defect that suggests a dedicated role for these neurons in fast walking. Together, these findings reveal the existence of a regulatory step downstream of temporal NB factors in which combinations of morphology TFs (mTFs) control individual neuron morphologies, while leaving other terminal characteristics of neuronal identity unaffected neurons (Enriquez, 2015).

Inherent in the concept of a combinatorial TF code is the idea that removing or ectopically expressing a single TF will only generate a transformation of fate when a different wild-type code is generated. Consistent with this notion, only when the expression of two or three mTFs were simultaneously manipulated was it possible to partially mimic a distinct mTF code and, as a result, transform the identity of one Lin B MN into another. In contrast, manipulating single TFs typically resulted in aberrant or neo-codes that are not observed in wild-type flies. For example, removing pb function from Lin B resulted in two MNs with a code (Ems+Zfh1) and MN morphology that are not observed in wild-type Lin A and Lin B lineages. Analogously, ectopic Pb expression in Lin A, which normally does not express this TF, generated aberrant codes and MN morphologies. This latter experiment was particularly informative because although Pb redirected a subset of Lin A dendrites to grow in an anterior region of the neuropil, it did not alter the ability of these dendrites to cross the midline. Thus, the dendrites of these MNs had characteristics of both Pb-expressing Lin B MNs (occupying an antero-ventral region) and Pb-non-expressing Lin A MNs (competence to cross the midline). Axon targeting of these MNs was also aberrant: although they still targeted leg muscles, Pb-expressing Lin A MNs frequently terminated in the coxa, which is not a normal characteristic of Pb-expressing Lin B MNs or of any Lin A MN. These observations suggest that the final morphological identity of a neuron is a consequence of multiple TFs executing functions that comprise a complete morphological signature. Some functions, such as the ability to occupy the antero-ventral region of the neuropil, can be directed by a single TF (e.g., Pb), while other functions, such as the ability to accurately target the distal femur, require multiple TFs (e.g., Pb+Ems). Further, because it was possible to generate MNs that have both Lin B and Lin A morphological characteristics, hte results argue against the idea that there are lineage-specific mTFs shared by all progeny derived from the same lineage. Instead, the data are more consistent with the idea that the final morphological identity of an MN depends on its mTF code neurons (Enriquez, 2015).

Drosophila NBs, and perhaps vertebrate neural stem cells, express a series of TFs that change over time and have therefore been referred to as temporal TFs. For Lin B, the sequence of these factors is unknown, in part because the Lin B NB is not easily identified in the second-instar larval VNC, the time at which it is generating MNs. Nevertheless, each MN derived from Lin B and Lin A has a stereotyped birth order, consistent with the idea that temporal TFs play an important role in directing the identities of MNs derived from these lineages and, therefore, the mTFs they express. For Lin B, this birth order is Co1->Tr1->Fe1->Tr2->Co2->Co3->Co4. Interestingly, according to the mTF code proposed in this study, each of these MNs differs by at most two mTFs in any successive step. For example, Tr1 has the code [Zfh1, Ems, Pb, Zfh2] while Fe1, the next MN to be born, has the code [Zfh1, Ems, Pb]. Thus, it is posited that the sequence of temporal TFs acting in the NB is responsible for directing each successive change in mTF expression in postmitotic MNs (e.g., in the Tr1->Fe1 step, repression of zfh2). Although a link between temporal TFs and TFs expressed in postmitotic neurons has been proposed in Drosophila, the role of these TFs in conferring neuron morphologies is not known. Further, there may be additional diversity-generating mechanisms in lineages that produce many more neurons than the seven MNs generated by Lin B. One additional source of diversity may come from NB identity TFs, which distinguish lineages based on their position. Such spatial information could in principle allow the same temporal TFs to regulate different sets of mTFs in different NB lineages. It is also likely that differences in the levels of some mTFs may contribute to neuronal identities. Consistent with this idea, the levels of Zfh2 and Pros differ in the Lin B MNs expressing these TFs, differences that are consistent in all three thoracic segments and between animals. Further, Zfh1 levels vary between Lin B MNs and its levels control the amount of terminal axon branching. Previous studies also demonstrated that TF levels are important for neuron morphology, including Antp in adult leg MNs derived from Lin A and Cut in the control of dendritic arborization complexity in multidendritic neurons. If the levels of mTFs are important, it may provide a partial explanation for why the transformations of morphological identity generated in this study with the MARCM technique, which cannot control levels, are typically only partially penetrant neurons (Enriquez, 2015).

Another distinction between temporal TFs and mTFs is that no evidence has been observed of cross-regulation between mTFs. In situations when mTFs were either removed (e.g., pb^-/-; ems^RNAi) or ectopically expressed (e.g., UAS-pb + UAS-ems) in postmitotic Lin B MARCM clones, the expression of the remaining mTFs was unchanged. In contrast, when an NB lineage is mutant for a temporal TF, the prior TF in the series typically continues to be expressed. These observations suggest that the choice of mTF expression is made in the NB and that once the postmitotic code is established, it is not further influenced by coexpressed mTFs neurons (Enriquez, 2015).

The data further suggest that mTFs are distinct from terminal selector TFs. In mutants for the mTFs studied here, the resulting neurons remain glutamatergic leg motor neurons: they continue to express VGlut, which encodes a vesicular glutamate transporter, expressed by all Drosophila MNs, and they still exit the VNC to target and synapse onto muscles in the adult legs. Thus, whereas terminal selector TFs maintain the terminal characteristics of fully differentiated neurons, mTFs are required transiently to execute functions required for each neuron's specific morphological characteristics. Together, it is suggested that the combined activities of terminal selector TFs and mTFs specify and maintain the complete identity of each post-mitotic neuron neurons (Enriquez, 2015).

Although the mTFs defined in this study, e.g., Ems, Pb, and Toy, do not fit the criteria for a terminal selector TF, it is plausible that some TFs function both as mTFs and terminal selector TFs. One example may be Apterous, a TF that is expressed in six interneurons in the thoracic embryonic segments and that functions with other TFs to control the terminal differentiation state of these neuropeptide-expressing neurons. In addition to the loss of neuropeptide expression, these neurons display axon pathfinding defects in the absence of apterous. Despite the potential for overlapping functions, it is conceptually valuable to consider the specification of neuronal morphologies as distinct from other terminal characteristics, as some mTFs regulate morphology without impacting these other attributes. It is also plausible that some of the TFs that have been previously designated as determinants of subtype identity may also be part of mTF codes. For example, eve is required for the identity of dorsally directed MNs inDrosophila embryogenesis, but the TFs required for distinguishing the individual morphologies of these neurons are not known. It may be that Eve is one component of the mTF code and that it functions together with other mTFs to dictate the specific morphologies of these neurons neurons (Enriquez, 2015).

Flies containing a single pb mutant Lin B clone exhibited a highly specific walking defect: when walking at high speed, these flies were significantly more unsteady compared to control flies. The restriction of this defect to high speeds suggests that the Pb-dependent characteristics of these MNs may be specifically required when the walking cycle is maximally engaged, raising the possibility that Tr1, Tr2, and Fe1 are analogous to so-called fast MNs described in other systems. Further, these data support the idea that the highly stereotyped morphology of these MNs is critical to the wild-type function of the motor circuit used for walking. In particular, the precise dendritic arborization pattern exhibited by these MNs, which is disrupted in the pb mutant, is likely to be essential for their function. Although it cannot be excluded that other pb-dependent functions contribute to this walking defect, these observations provide strong evidence that the myotopic map, in which MNs that target similar muscle types have similar dendritic arborization patterns, is important for the fly to execute specific adult behaviors neurons (Enriquez, 2015).

REGULATION

Transcriptional Regulation

eagle (eg) is expressed in neuroblasts and is involved in the fate determination of serotonergic neurons. Serotonin is an evolutionarily conserved neurotransmitter, found in both invertebrates and vertebrates, and involved in locomotor and behavioral roles. Serotonin is produced in descendents of neuroblast NB 7-3. NB7-3 expresses several genes including engrailed, huckebein, seven-up, pdm1 and eagle. Although eg is expressed in both lateral and medial NB 7-3 derived serotonin cells, the eg loss-of-function mutants often affect the development of only one serotonin cell from each pair. The two cells can be distinguished from one another by differential expression of zinc finger homeobox 2 (zfh-2). This dual domain transcription factor has been shown to bind to and activate the DDC gene (Lundell, 1992 and 1994). The differential expression of zfh-2 and of another gene, pdm-1, can be used to determine that the remaining serotonin positive single cell in eg mutants expresses markers characteristic of the more lateral serotonin cell. In a wild-type CNS, both zfh-2 and pdm1 are selectively expressed in the more lateral serotonin cell but not in the more medial cell. engrailed and eagle are expressed in both these serotonin cells. In eg mutants only the medial cell consistently fails to become a serotonin cell. Therefore, even though eg is normally expressed in both serotonin cells, the absence of Eg protein has a more dramatic effect on the fate of the more medial neuron. This important observation suggests that the lateral serotonin cell can maintain its fate in the absence of Eg (Lundell, 1998).

Analysis of gene expression in eg mutants shows that expression of zfh-2 and en is dependent on eg function but expression of pdm1 is independent of eg function. Loss of eg function appears to have no affect on the expression of pdm1. Clearly, the serotonin cell phenotype in eg mutants is not directly related to the expression of pdm1. Loss of eg function affects the expression of zfh-2 in the lateral serotonin neuron. Loss of eg function affects the expression of en in both serotonin neurons. Thus, eagle is necessary for the maintenance of both engrailed and zfh-2 expression in the serotonin neurons (Lundell, 1998).

The simplest explanation for the difference between the medial and lateral serotonin neurons is that the lateral cell contains a redundant mechanism that allows continued synthesis of serotonin in the absence of Eg protein. This redundant mechanism is not 100% efficient, since not all segments in an eg mutant CNS contain serotonin cells. Since zfh-2 but not pdm1, expression is affected in eg mutants, it is suggested that zfh-1 is a potential factor for this redundant pathway, which establishes eg-independent serotonin synthesis. In an eg-loss-of-function mutant, the loss of Ddc expression is always accompanied by the loss of en expression, but can occur independently in the two serotonin cells. In a hemisegment where both cells fail to express Ddc, neither cell shows en expression. In a hemisegment where only the lateral serotonin cells continues to express Ddc, this lateral cell shows en expression but the medial cell does not. It is concluded that the two serotonin cells have distinctive regulatory networks. In the medial cell, eagle is required for the serotonin fate, while in the lateral cell, engrailed and zhf-1 are required but eagle is not. It is shown that hypomorphic alleles of eagle can produce viable adults that have a dramatic reduction in the number of serotonin-producing neurons (Lundell, 1998).

The overlap between Zfh-2 and Wg throughout the larval stages suggests that zfh-2 may be activated by Wg signalling. In order to test this, the effect of ectopic Wg expression on zfh-2 expression was analyzed. dpp-GAL4 was used to drive the expression of a UAS-wg construct along the A/P boundary in all domains along the P/D axis. Under these conditions, Zfh-2 shows a broad expansion into the presumptive notum region but no ectopic expression in the wing pouch. This indicates that ectopic Wg can activate zfh-2 at a distance from its site of expression (Whitworth, 2003).

In the wild type proximal wing disc, Nub overlaps the Zfh-2 domain at the inner ring of Wg. This situation is recapitulated when ectopic Wg is driven by dpp-GAL4, since ectopic Nub is only detected in regions of high Wg expression. This indicates that ectopic Wg is inducing a response similar to the inner ring and suggests that the region expressing ectopic Zfh-2 is now differentiating as proximal wing (Whitworth, 2003).

To assess whether the cells expressing ectopic Zfh-2 have altered their fate, the expression of the notum marker Tsh was analyzed. Tsh is completely repressed throughout the region of ectopic Zfh-2, indicating that cells are no longer fated as notum. Since Wg is an important factor in the development of wing blade, the expression of a wing blade marker, Vg, was also examined. Vg shows no expansion into more proximal regions and is still restricted to the wing pouch (Whitworth, 2003).

These observations support the idea that Wg is able to direct the differentiation of proximal wing fate at the expense of notum. This can also be inferred from an examination of the phenotype of pharate adults of genotype dpp-GAL4/UAS-wg. An outgrowth of tissue is seen with characteristic proximal wing sclerites and a concomitant loss of macrochaete and scutellum normally associated with the notum (Whitworth, 2003).

This experiment to induce ectopic proximal wing was carried out in such a way that the ectopic Wg was expressed in a pattern that intersects the endogenous domain of Wg expression and results in a continuous region of Wg expression. The observed effects could therefore be interpreted as directed overgrowth of the endogenous proximal wing and not differentiation of proximal wing de novo. This is supported by observations that ectopic expression of Wg in the proximal wing anlagen causes disc overgrowth and consequently overgrows proximal wing tissue. In view of this, attempts were made to reproduce the effects of ectopic Wg in a manner that was discontinuous with the endogenous wg domain. To achieve this, clones of wg-expressing cells were induced that were contained entirely within the notal region, outside of the endogenous proximal wing. To prevent diffusion of ectopic Wg, a construct (UAS-Nrt-flu-wg) was used that directs the expression of a membrane-tethered form of Wg marked with a Flu epitope tag. The colocalization of Nrt-Wg bound to the cell surface and GAL4-expressing cells confirms two things: (1) that the only cells expressing GAL4 induce expression of the UAS construct; (2) that the Flu epitope marker is not detectable beyond the site of expression, indicating that the Flu/Wg hybrid molecule is membrane-bound and not detectably diffusible. When stained to reveal Zfh-2, it can be clearly see that Zfh-2 is induced at a distance of several cell diameters from the site of Nrt- Flu-Wg expression, producing a large zone of Zfh-2-expressing cells surrounded by an epithelial fold. This observation is surprising considering that Wg protein is believed to be tethered to the cell membrane. In the wing pouch, the same construct elicits a Wg signal response only in the expressing cell and its immediate neighbors. Although the nature of the long-range induction cannot be explained at present, this does confirm that ectopically expressed Wg is able to induce the expression of Zfh-2 and therefore drive differentiation of proximal wing fate (Whitworth, 2003).

Taken together, these results show Wg is sufficient to direct the differentiation of proximal wing fate. Furthermore, Wg can only induce ectopic Zfh-2, and thereby proximal wing fate, in the more proximal notum tissue and not in the more distal wing pouch (Whitworth, 2003).

To determine whether Wg is required for zfh-2 expression and how this changes through development, a number of methods were employed to remove Wg function at different developmental stages. The temperature-sensitive allele wg^IL114 was used in trans to a wg-lacZ insertion line to create a conditional null mutant. When larvae were moved to the restrictive temperature just prior to L2, Zfh-2 expression was no longer detected in the wing primordium. This indicates that Wg function is required at least for initiation of zfh-2 expression in the L2 wing disc. Wg signal transduction can also be antagonized by the expression of a dominant negative TCF (DN-TCF), a component of the Wg signalling pathway. dpp-GAL4 was used to drive expression of DNTCF along the A/P boundary from early larval stages. zfh-2 fails to be activated in the presence of DN-TCF, even into L3. This further supports the findings that Wg signal transduction is absolutely required for initiation of zfh-2 expression during L2 (Whitworth, 2003).

However, when Wg signalling is removed later in L3, under all experimental conditions tested, no effect on zfh-2 expression is seen. Large wg null clones or the expression of a dominant negative form of wg during L3 shows no detectable reduction in Zfh-2 levels. Similarly, no loss of Zfh-2 is observed with clonal expression of DN-TCF. This shows that, after activation in the L2, Wg activity is no longer required during L3 for the maintenance of zfh-2 expression (Whitworth, 2003).

Taken together, these results show that the regulation of zfh-2 by Wg is temporally dynamic. Although Wg is required early to activate zfh-2, when both are extensively coexpressed, Wg appears not to be required later to maintain zfh-2 expression. This raises the possibility that, once activated, zfh-2 might regulate its own expression by an unknown mechanism. This interpretation would also mean that the downstream response to Wg signal is temporally dynamic, since it appears that one set of genes, e.g., those required to determine proximal wing fate, is activated early and later becomes independent of Wg, and then another set of genes is in turn activated, e.g., those delimiting the wing blade (Whitworth, 2003).

Ectopic expression of Wg can induce zfh-2 only in regions outside of the wing pouch. This suggests that some factor has a repressive effect on zfh-2 in the pouch that cannot be overcome by Wg activation. Genes fundamental to wing blade development may be responsible for this repression. Since Vg expression is restricted to the presumptive wing blade and is required for wing blade development, the effects of ectopic expression of vg on the proximal wing region were examined. Using dpp-GAL4 to direct expression of vg along the A/P boundary represses zfh-2 in the proximal wing region. Endogenous wg expression, monitored with the wg-lacZ reporter, also shows complete repression at the point of intersection. Conversely, in vg1 mutant discs, the Zfh-2 expression domain is expanded into the remnant of the wing pouch and shows a greater overlap with Nub expression than in the wild type. In vg1 discs, much of the wing pouch anlagen fails to develop, and this is accompanied by complete loss of Wg expression at the wing margin; however, the two rings of Wg delimiting the proximal wing are maintained. This suggests that derepression of the zfh-2 domain into the pouch region is not caused by ectopic Wg activity (Whitworth, 2003).

Since the loss of vg does not result in complete derepression of zfh-2, it suggests that another repressor must be acting with vg. Nub is also required for wing blade development. Hypomorphic nub alleles display a severely reduced wing phenotype and a transformation of distal structures into proximal ones. nub² discs show a complete loss of the inner ring of Wg and an expansion of Wg expression at the wing margin. In nub² mutant discs, Zfh-2 expression is expanded into the wing pouch, along the line of the wing margin. This indicates two things: (1) that Nub normally acts to repress zfh-2 expression, and thus proximal wing fate, within the wing pouch, and (2) that ectopic zfh-2 is induced where Wg is expressed. Therefore, in an environment of reduced Nub, it can be predicted that ectopic Wg would be able to induce ectopic Zfh-2. To test this, ectopic Wg was expressed in a nub mutant background. As in the nub² background, Zfh-2 is ectopically induced in the wing pouch along the wing margin . In addition, Zfh-2 can now be detected in the wing pouch along the line of dpp-GAL4, where high levels of Wg are ectopically expressed. This demonstrates that, in an environment of reduced Nub, Zfh-2 expression can be induced wherever Wg is expressed and is no longer restricted from the pouch. It is noted that, whereas Wg expression is expanded at the wing margin in nub discs, where ectopic Wg is induced in a nub background, endogenous Wg is expressed normally at the wing margin; however, the reason for this is unknown (Whitworth, 2003).

In nub discs, vg expression is unaffected, but vg is upregulated by high levels of ectopic Wg. Thus, it appears that the increased levels of Vg are not sufficient to repress Zfh-2 in the absence of Nub when Wg is present at high levels. However, further from the source of ectopic Wg, Zfh-2 is not induced in the nub background, and presumably here, Vg alone can repress Zfh-2. Taken together, these data suggest that zfh-2 expression is regulated by a balance between activation by Wg and repression by a combination of Nub and Vg, acting together or independently. The loss of either Nub or Vg is enough to cause only a partial derepression of zfh-2 in the wing pouch, indicating that alone neither Nub nor Vg is sufficient to completely repress proximal wing fate. However, their combined action, as is the case in the wild type, is able to completely repress zfh-2 expression in the wing pouch. Thus, these factors act to restrict zfh-2 expression to the periphery of the wing disc, thereby defining the distal limit of the proximal wing primordium (Whitworth, 2003).

Recent work has indicated that the homeobox gene homothorax (hth) is required for the correct development of the proximal wing by both upregulating Wg expression in the proximal wing and limiting the area of wing blade differentiation. Since loss of Hth function in the proximal wing leads to a dramatic reduction in the level of Wg expression, attempts were made to determine whether Hth is also required for regulation of Zfh-2 expression. In hth^- clones, neither the expression pattern nor the level of Zfh-2 is altered compared with neighboring wild type tissue. This is consistent with the observation that late removal of wg does not affect the expression of zfh-2. Similarly, ectopic expression of Hth shows no effect on zfh-2 expression. These data suggest that Hth does not play a role in establishing or regulating the determination of proximal wing fate, since no change in the expression of Zfh-2 was observed. Thus, it appears that the prime functions of Hth in the proximal wing are to maintain Wg expression and define the limits of the wing pouch (Whitworth, 2003).

Targets of Activity

A 40-bp upstream regulatory region of the DOPA decarboxylase gene (Ddc), that is important for cell-specific expression in the Drosophila CNS, has been investigated. This region contains two redundant elements which when simultaneously mutated result in lowered DDC expression in serotonin neurons. A protein binding site within one of these elements has been uncovered, and a factor has been cloned that binds to the site. This factor is the product of the zfh-2 gene, a complex homeodomain/zinc finger protein previously identified by binding to an opsin regulatory element. The in vivo profile of Zfh-2 in the larval CNS shows intriguing overlap with DDC in specific serotonin and dopamine neurons. Zfh-2 is related to a human transcription factor ATBF1. The multiple homeodomain and zinc finger motifs in these two proteins show a similar linear arrangement that implies coordinate action among the motifs. In addition, the homology defines a new homeodomain subtype (Lundell, 1992).

A targeted genetic screen identifies crucial players in the specification of the Drosophila abdominal Capaergic neurons

The central nervous system contains a wide variety of neuronal subclasses generated by neural progenitors. The achievement of a unique neural fate is the consequence of a sequence of early and increasingly restricted regulatory events, which culminates in the expression of a specific genetic combinatorial code that confers individual characteristics to the differentiated cell. How the earlier regulatory events influence post-mitotic cell fate decisions is beginning to be understood in the Drosophila NB 5-6 lineage. However, it remains unknown to what extent these events operate in other lineages. To better understand this issue, a very highly specific marker was used that identifies a small subset of abdominal cells expressing the Drosophila neuropeptide Capa: the ABCA neurons. The data support the birth of the ABCA neurons from NB 5-3 in a cas temporal window in the abdominal segments A2-A4. Moreover, it was shown that the ABCA neuron has an ABCA-sibling cell which dies by apoptosis. Surprisingly, both cells are also generated in the abdominal segments A5-A7, although they undergo apoptosis before expressing Capa. In addition, a targeted genetic screen was performed to identify players involved in ABCA specification. It was found that the ABCA fate requires zfh2, grain, Grunge and hedgehog genes. Finally, it was shown that the NB 5-3 generates other subtype of Capa-expressing cells (SECAs) in the third subesophageal segment, which are born during a pdm/cas temporal window, and have different genetic requirements for their specification (Gabilondo, 2011).

The findings strongly suggest that the Capaergic abdominal ABCA neuron arises from NB 5-3. This conclusion is based on the expression in ABCA cells of gsb, wg and unpg, and the absence of the markers lbe(K) and hkb. However, even though gsb expression is known to be maintained specifically in the lineage of rows 5 and 6 NBs, whether expression of the other genetic markers used to identify NBs at stage 11 changes late in embryogenesis remains unanswered. Nonetheless, the specific combination of NB markers found in ABCA cells and their position in the hemineuromere are consistent with their birth from NB 5-3. Previous accounts showed that this NB gives rise to a lineage of 9–15 cells. Additionally, observations derived from studies in which PCD was blocked showed that NB 5-3 can potentially produce a large lineage (ranging from 19 to 27 cells), suggesting that it could generate 13 or 14 GMCs. The lack of a NB 5-3 specific-lineage marker prevented resolution of its complete lineage, and thus determining the birth order of the ABCA cell (Gabilondo, 2011).

Recent findings on the NB 5-6 and NB 5-5 demonstrate that cas and grh act together as critical temporal genes to specify peptidergic cell fates at the end of these lineages. cas mutants lack ABCA cells and Cas is expressed in these cells, while the normal pattern of ABCA cells is found in grh mutants, and Grh is not present in ABCA neurons. These data strongly support the birth of ABCA cells in a cas-only temporal window. This is different from the subesophageal Capaergic SECA cells, which while also arising from NB 5-3, show a reduction in cell number in both pdm and cas mutants, demonstrating birth at a mixed pdm/cas temporal window. Previous studies in other lineages have shown that when a temporal gene is mis-expressed, all progeny cells posterior to that temporal window can be transformed to the specific fate born at that particular temporal window. However, cas mis-expression failed at inducing ectopic ABCA cells, suggesting that cas in necessary but not sufficient to specify the ABCA fate (Gabilondo, 2011).

Programmed cell death (PCD) is a basic process in normal development. The results suggest that the ABCA and its sibling are equivalent cells committed to achieve the ABCA fate. First, it was shown that the ABCA-sibling cell dies by apoptosis, but produces an ABCA-like Capaergic neuron if PCD is inhibited. Second, when PCD is blocked, NB 5-3 also produces a GMC generating two ABCA-like Capaergic cells in the A5–A7 segments. These data indicate that a segment-specific mechanism prevents death of the ABCA cells in A2–A4 neuromeres. Segment specific cell death has been previously reported for the NB 5-3 lineage, and detailed studies on segment-specific apoptosis of other lineages have shown that this process is under homeotic control. In addition, the results show a different timing in the PCD undergone by the ABCA sibling and the ABCA cells born in A5–A7. This interpretation is based on the differential effect of p35 expression when cas-Gal4 or elav-Gal4 drivers were used. Although elav-Gal4 is transiently express in NBs and GMCs, robust and maintained driver expression commences in differentiating neurons. In contrast, cas expression starts in the NB and is maintained in the GMC and neuronal progeny. Therefore, the finding that death of the ABCA-sibling cell can be prevented by directing p35 with cas-Gal4, but not with elav-Gal4, suggests that the death of the ABCA sibling occurs earlier in development than the death undergone by the ABCA cells in A5–A7 segments (Gabilondo, 2011).

In the ABLK/LK peptidergic fate (derived from the NB 5-5), activation of Notch (N) signaling in the peptidergic cell prevents its death, while its sibling, N^OFF cell undergoes apoptosis. On the contrary, in the EW3/Crz peptidergic fate (derived from the NB 7-3), silencing of N signaling is essential for the neuron survival, and therefore for it proper specification. The current results are in accordance with the last scenario, in which the ABCA cell is N^OFF, and it sibling, which undergoes apoptosis, is N^ON. Therefore, Notch signaling must be switch off for the proper specification of the ABCA neuron (Gabilondo, 2011).

To search for genes involved in specification of the ABCA neural fate, a reduced set of mutants was screened of genes that are expressed in the embryonic CNS at stage 11, a time at which distinctly defined sublineages are being generated from all active NBs. Even though this method will certainly overlook important genes, the results reveal that it is in fact a very effective way to find genes involved in specification of a particular neural fate. Indeed, the ratio of success has been very satisfactory: 33.3% of the genes analyzed display a significant phenotype. Moreover, the set of identified genes could be further expanded by, for example, searching in interactome databases, and performing the subsequent screen on those putative interactors (Gabilondo, 2011).

It is assumed that the specification of a concrete cellular fate requires the combination of several transcription factors, namely a genetic combinatorial code. Recently, a detailed combinatorial code has been reported for three neuropeptidergic fates: ap4/FMRFa, ap1/Nplp1 and ABLK/Lk. However, very little is known about the specification of the rest of the 30 peptidergical fates. This study has identified several genes involved in the specification of the ABCA fate, which fit into three categories. First, genes were found whose loss-of-function produces a relevant increase of the number of ABCA cells. Most remarkable are the klu and rn phenotypes, which consist of duplications of the ABCA cells. These phenotypes suggest that these two transcription factors repress the ABCA fate in other neural cells (or/and NBs/GMCs). Interestingly, the normal phenotype of nab mutants indicates that, contrary to its mode of action in the wing, Rn does not work with the transcription cofactor Nab in this context (Gabilondo, 2011).

Second, genes were found whose loss-of-function produces a significant decrease of the number of ABCA neurons. In this category, the zhf2, ftz and grain phenotypes stand out. The effects of mutations on ftz are in agreement with its early role in segmentation: ftz is a pair-rule segmentation gene that defines even-numbered parasegments in the early embryo, and absence of ABCA cells was found in the A3 segment in ftz mutants. However, zfh2 and grain seem to be part of the specific combinatorial code of the ABCA cells. The Drosophila GATA transcription factor Grain has been reported to be involved in the specification of other cell fates, such as the aCC motoneuron fate. Based on its expression, the zinc finger homeodomain protein zfh2 has been proposed to mediate specification of the serotoninergic fate, but this has not been further demonstrated. Interestingly, during wing formation, zfh2 is required for establishing proximo-distal domains in the wing disc, and it does so partly by repressing gene activation by Rn. The opposite phenotypes that was observed in rn and zfh2 mutants suggest that similar interactions occur during ABCA specification. Analyses aimed to test this hypothesis are currently being performed (Gabilondo, 2011).

Third, two genes were found whose loss-of-function abolishes the ABCA fate: Grunge and hh. Grunge encodes a member of the Atrophin family of transcriptional co-repressors that plays multiple roles during Drosophila development. Taken together, studies from C. elegans to mammals suggest that Atrophin proteins function as transcriptional co-repressors that shuttle between nucleus and cytoplasm to transduce extracellular signals, and that they are part of a complex gene regulatory network that governs cell fate in various developmental contexts. Similarly, Hh is an extracellular signaling molecule essential for the proper patterning and development of tissues in metazoan organisms. It is noteworthy that two genes implicated in extracellular signaling pathways, Grunge and hh, are absolutely required for ABCA fate. Further studies will be needed to identify at which step/s they exert their actions, and to unravel possible interactions between them and with other players of the combinatorial code for ABCA specification (Gabilondo, 2011).

DEVELOPMENTAL BIOLOGY

Embryonic

The zfh-2 gene displays a limited expression pattern, largely restricted to the CNS of late embryos (Lai, 1991).

Serotonin has been implicated as a stimulatory modulator of locomotion in invertebrates. Although eagle is expressed in lateral and medial serotonin cells, the eg loss of-function mutants often affect the development of only one serotonin cell of each pair. Would the development of one specific cell in each pair be consistently affected? In grasshopper, the two sister cells have slightly different growth patterns and projections, but these are impossible to discern in Drosophila. The two cells can be distinguished from each other by differential expression of zfh-2 (Lundell, 1994). In a wild-type CNS, both zfh-2 and pdm-1 are selectively expressed in the more lateral of two serotonin cells but not in the more medial cell. Both antigens are also expressed in the midline dopamine cells and by the entire midline. The remaining single serotonin cells in eg mutants consistently express both zfh-2 and pdm-1, characteristic of the more lateral serotonin cell (Lundell, 1998).

The Dichaete gene of Drosophila encodes a group B Sox protein related to mammalian Sox1, -2, and -3. Like these proteins, Dichate is widely and dynamically expressed throughout embryogenesis. In order to unravel new Dichaete functions, the organization of the Dichaete gene was characterized using a combination of regulatory mutant alleles and reporter gene constructs. Dichaete expression is tightly controlled during embryonic development by a complex of regulatory elements distributed over 25 kb downstream and 3 kb upstream of the transcription unit. A series of regulatory alleles which affect tissue-specific domains of Dichaete were used to demonstrate that Dichaete has functions in addition to those during segmentation and midline development that have been previously described. (1) Dichaete has functions in the developing brain. A specific group of neural cells in the tritocerebrum fails to develop correctly in the absence of Dichaete, as revealed by reduced expression of labial, zfh-2, wingless, and engrailed. (2) Dichaete is required for the correct differentiation of the hindgut. The Dichaete requirement in hindgut morphogenesis is achieved, in part, via regulation of dpp, since ectopically supplied dpp can rescue Dichaete phenotypes in the hindgut. Taken together, there are now four distinct in vivo functions described for Dichaete that can be used as models for context-dependent comparative studies of Sox function (Sanchez-Soriano, 2000).

To look for defects in the development of Dichaete mutant brains the expression of Zfh-2, a transcription factor expressed in many neuronal lineages, was examined. In comparison to the wild type, in D^r8 stage 16 mutant embryos, a reduction in Zfh-2-positive is seen in cells in the posterior part of the brain, most likely the tritocerebrum. Similarly, ming-LacZ expression, another marker abundant in the CNS, is reduced in this region at stage 12 and 16 when analyzed in null and D^r8 mutant backgrounds. However, anti-Fasciclin II stainings of wild-type and mutant embryos reveal no detectable differences in the location or arrangement of the major brain commissures and longitudinal connectives. Thus, despite widespread expression, Dichaete requirement is mainly restricted to a specific region of the embryonic brain and has no apparent function in the development of the major identified axon tracts in the brain (Sanchez-Soriano, 2000).

Larval

A GAL4 insertion within the zfh-2 transcription unit, MS209, (zfh-2^MS209) and antisera against Zfh-2 have been used to monitor the expression of zfh-2. In both L3 wing discs and adult wings, Zfh-2 is expressed in a domain that completely overlaps the rings of Wg expression. In L3 wing discs, Zfh-2 does not extend either proximally into the notum or distally into the wing pouch. These observations indicate that, in late stages, Zfh-2 is specifically expressed throughout the developing proximal wing and therefore may be used as a useful marker for proximal wing fate (Whitworth, 2003).

wg expression, monitored by a lacZ reporter, is initiated in the early second instar (L2) in an approximately anterior-ventral domain. A lacZ reporter driven by zfh-2^MS209 is expressed in a very similar pattern. To determine the extent of coexpression of Zfh-2 and Wg, early L2 discs were examined with anti- Zfh-2 and anti-Wg antisera. Zfh-2 is expressed at this stage in a pattern that directly overlaps with Wg. It is apparent that, although the wg-lacZ reporter gene shows wg expression induced in a narrow domain, the protein can be detected at some distance outside of this region. This is a measure of the mobility of Wg protein. Consistent with this, Zfh-2 nuclear expression is at high levels in the wedge-like domain of wg-lacZ, but is also detectable away from this region at lower levels. As development proceeds, Zfh-2 quickly expands to cover the whole of the ventral portion of the wing disc, accompanying the expansion of the Wg domain. The expression of Wg at this stage is proposed to determine the differentiation of the presumptive 'wing primordium' as opposed to notum. However, since Zfh-2 is also widely expressed at this time, it suggests that the 'wing primordium' has not been further subdivided into proximal or distal domains. At the onset of L3, Zfh-2 begins to decline in the center of the disc, suggesting differentiation of more distal fates here. At this time, Wg is still expressed throughout the 'wing primordium' but becomes upregulated at the D/V boundary, where it plays a central role in defining the wing margin. This is also the time when the vg quadrant enhancer (vgQE) is activated on either side of the D/V boundary, marking the establishment of the wing pouch. During mid-L3, the pattern of Wg expression is refined further, becoming upregulated at the periphery of the wing pouch and at the D/V boundary, the presumptive wing margin. Zfh-2 is also refined and is now only present in a ring around the wing pouch overlapping the rings of Wg (Whitworth, 2003).

EVOLUTIONARY HOMOLOGS

ATBF1 is a transcription factor containing four homeodomains and 17 zinc fingers. Since the Drosophila homolog, ZFH-2, is implicated in neurogenesis, ATBF1 expression was examined in developing mouse brain and in P19 mouse embryonal carcinoma cells during differentiation. Pre- and post-natal mouse brains express high levels of ATBF1 mRNA, but the adult brain contains only a small amount of ATBF1 transcripts. In P19 cells, ATBF1 transcripts are undetectable before differentiation; however, 1 day after induction of neuronal differentiation with retinoic acid, ATBF1 mRNA is expressed at a high level. This increased level reaches a maximum on the 4th day and then declines. No comparable level of ATBF1 mRNA is expressed when P19 cells are treated with dimethyl sulfoxide to induce muscle cells. These temporal patterns of ATBF1 expression in vivo and in vitro suggest that ATBF1 may play a role in neuronal differentiation (Ido, 1994).

zfh-4 has been identified as a new member of the zinc finger-homeodomain (zfh) family of transcription factors. Zfh-4 expression is prominent in developing muscle and brain. In both tissues, zfh-4 RNA levels are highest embryonically, then decrease gradually to barely detectable levels in adults. In myogenic cell lines, far more zfh-4 is expressed in proliferating myoblasts than in myotubes, suggesting a cellular basis for the developmental regulation observed in vivo. In contrast, zfh-4 RNA in brain is more abundant in postmitotic cells of the marginal zone than in proliferating cells of the ventricular zone. Within the brain, zfh-4 RNA is regionally localized: expression is highest in midbrain, readily detectable in hindbrain, and very low in cerebral cortex. Its patterns of expression, and its homology to known DNA binding proteins, support the idea that zfh-4 may be a regulator of gene expression in developing brain and muscle (Kostich, 1995).

The human ATBF1-B gene encodes a 306-kDa protein containing 4 homeodomains and 18 zinc fingers including one pseudo zinc finger motif. A second ATBF1 cDNA, 12 kilobase pairs long, termed ATBF1-A, has been cloned. The deduced ATBF1-A protein is 404 kDa in size and differs from ATBF1-B by a 920-amino acid extention at the N terminus. Analysis of 5'-genomic sequences shows that the 5'-noncoding sequences specific to ATBF1-A and ATBF1-B transcripts are contained in distinct exons that can splice to a downstream exon common to the ATBF1-A and ATBF1-B mRNAs. The expression of ATBF1-A transcripts increases to high levels when P19 and NT2/D1 cells are treated with retinoic acid to induce neuronal differentiation. Preferential expression of ATBF1-A transcripts is also observed in developing mouse brain. Transient transfection assays show that the 5.5-kilobase pair sequence upstream of the ATBF1-A-specific exon (exon 2) supports expression of the linked chloramphenicol acetyltransferase gene in neuronal cells derived from P19 cells but not in undifferentiated P19 or in F9 cells, which do not differentiate into neurons. These results show that ATBF1-A and ATBF1-B transcripts are generated by alternative promoter usage combined with alternative splicing and that the ATBF1-A-specific promoter is activated during neuronal differentiation (Miura, 1995).

A mouse ATBF1 cDNA has been isolated that is 12-kb long and capable of encoding a 406-kDa protein containing four homeodomains and 23 zinc-finger motifs. Mouse ATBF1 is 94% homologous to the human ATBF1-A transcription factor. Northern blot and RNase protection analysis have shown that levels of ATBF1 transcripts are low in adult mouse tissues, but high in developing brain, consistent with a role for ATBF1 in neuronal differentiation (Ido, 1996).

Using the yeast two-hybrid system, the transcription factor ATBF1 was identified as v-Myb- and c-Myb-binding protein. Deletion mutagenesis revealed amino acids 2484-2520 in human ATBF1 and 279-300 in v-Myb as regions required for in vitro binding of both proteins. Further experiments identified leucines Leu325 and Leu332 of the Myb leucine zipper motif as additional amino acid residues important for efficient ATBF1-Myb interaction in vitro. In co-transfection experiments, the full-length ATBF1 was found to form in vivo complexes with v-Myb and inhibit v-Myb transcriptional activity. Both ATBF1 2484-2520 and Myb 279-300 regions are required for the inhibitory effect. Finally, the chicken ATBF1 was identified, showing a high degree of amino acid sequence homology with human and murine proteins. These data reveal Myb proteins as the first ATBF1 partners detected so far and identify amino acids 279-300 in v-Myb as a novel protein-protein interaction interface through which Myb transcriptional activity can be regulated (Kaspar, 1999).

The mouse zfh-4 cDNA is 12 kb long and capable of encoding a 3,550-amino acid protein containing four homeodomains and 22 zinc fingers including two pseudo zinc finger motifs. The mouse ZFH-4 is 51% homologous to the mouse ATBF1 and 23% to the Drosophila ZFH-2. The homeodomain and zinc finger regions are highly conserved between ZFH-4 and ATBF1 except that one zinc finger is missing in ZFH-4. Analysis of partial genomic sequences shows that the mouse zfh-4 and ATBF1 genes are similar in exon-intron organization. RT-PCR analysis of zfh-4 transcripts in adult mouse tissues shows that zfh-4 expression is low but reproducibly detectable in brain, heart, lung and muscle. In these mouse tissues, ATBF1 transcripts were poorly amplified by PCR under the conditions where zfh-4 transcripts were amplified, suggesting that the expression of zfh-4 mRNA is higher than that of ATBF1 mRNA. Other comparative analysis suggests functional similarities and dissimilarities between ZFH-4 and ATBF1 (Sakata, 2000).

The ATBF1 gene encodes two protein isoforms, the 404-kDa ATBF1-A, possessing four homeodomains and 23 zinc fingers, and the 306-kDa ATBF1-B, lacking a 920-amino acid N-terminal region of ATBF1-A which contains 5 zinc fingers. In vitro, ATBF1-A is expressed in proliferating C2C12 myoblasts, but its expression levels decrease upon induction of myogenic differentiation in low serum medium. Forced expression of ATBF1-A in C2C12 cells results in repression of MyoD and myogenin expression and elevation of Id3 and cyclin D1 expression, leading to inhibition of myogenic differentiation in low serum. In contrast, transfection of C2C12 cells with the ATBF1-B isoform leads to an acceleration of myogenic differentiation, as indicated by an earlier onset of myosin heavy chain expression and formation of a higher percentage of multinucleated myotubes. The fourth homeodomain of ATBF1-A binds to an AT-rich element adjacent to the E1 E-box of the muscle regulatory factor 4 promoter mediating transcriptional repression. The ATBF1-A-specific N-terminal region possesses general transcription repressor activity. These results suggest that ATBF1-A plays a role in the maintenance of the undifferentiated myoblast state, and its down-regulation is a prerequisite to initiate terminal differentiation of C2C12 cells (Berry, 2001).

Ptosis is defined as drooping of the upper eyelid and can impair full visual acuity. It occurs in a number of forms including congenital bilateral isolated ptosis, which may be familial and for which two linkage groups are known on chromosomes 1p32-34.1 and Xq24-27.1. The analysis is described of the chromosome breakpoints in a patient with congenital bilateral isolated ptosis and a de novo balanced translocation 46,XY,t(1;8)(p34.3;q21.12). The 1p breakpoint lies ~13 Mb distal to the previously reported linkage locus at 1p32-1p34.1 and does not disrupt a coding sequence, whereas the chromosome 8 breakpoint disrupts a gene homologous to the mouse zfh-4 gene. Murine zfh-4 codes for a zinc finger homeodomain protein and is a transcription factor expressed in both muscle and nerve tissue. Human ZFH-4 is therefore a candidate gene for congenital bilateral isolated ptosis (McMullan, 2002).

The ATBF1 gene encodes transcription factors containing four homeodomains and multiple zinc finger motifs. However, the gene products have yet to be identified and the role remains unknown in vivo. In this study, an antiserum was raised for ATBF1; high levels of expression of ATBF1 were found in developing rat brain. Western and Northern blot analyses detected a 400 kDa protein and 12.5 kb mRNA in developing rat brain, respectively; both corresponding to ATBF1-A but not the B isoform. The protein is highly expressed in the midbrain and diencephalon and mRNA is highly expressed in the brainstem, mostly in embryo and neonatal brain. Immunohistochemistry identified postmitotic neurons in the brainstem as the major site of ATBF1 expression, and the expression levels vary depending on age of and location in the brain. Expression is transient and weak in the precursor cells at early neurogenesis. ATBF1 decreases postnatally, but remains in mature neurons, including those expressing DOPA decarboxylase (DDC). High levels of ATBF1 are expressed in precursor cells in accordance with neurogenesis and are continued to the mature neurons in specific areas such as the inferior colliculus. Expression is not significant from precursor cells to mature neurons in the cerebral cortex and hippocampus. ATBF1 and its Drosophila homolog, Zfh-2, are known to regulate cell differentiation and proliferation via the interaction with either of the basic helix-loop-helix transcription factors, c-myb, or the DDC gene. Together with these reported functions the expression features detected in this study suggest that ATBF1 may participate in the regulation of neuronal cell maturation or region-specific central nervous system differentiation (Ishii, 2003).

Characterization of nuclear localization and SUMOylation of the ATBF1 transcription factor in epithelial cells

ATBF1/ZFHX3 is a large transcription factor that functions in development, tumorigenesis and other biological processes. ATBF1 is normally localized in the nucleus, but is often mislocalized in the cytoplasm in cancer cells. The mechanism underlying the mislocalization of ATBF1 is unknown. This study analyzed the nuclear localization of ATBF1 and found that ectopically expressed ATBF1 formed nuclear body (NB)-like dots in the nucleus, some of which indeed physically associated with promyelocytic leukemia (PML) NBs. A 3-amino acid motif, KRK2615-2617, was defined as the nuclear localization signal (NLS) for ATBF1. Interestingly, diffusely distributed nuclear SUMO1 proteins were sequestered into ATBF1 dots, which could be related to ATBF1's physical association with PML NBs, known SUMOylation hotspots. Furthermore, ATBF1 itself was SUMOylated. ATBF1 SUMOylation occurred at more than 3 lysine residues including K2349, K2806 and K3258 and was nuclear specific. Finally, the PIAS3 SUMO1 E3 ligase, which interacts with ATBF1 directly, diminished rather than enhanced ATBF1 SUMOylation, preventing the co-localization of ATBF1 with SUMO1 in the nucleus. These findings suggest that nuclear localization and SUMOylation are important for the transcription factor function of ATBF1, and that ATBF1 could cooperate with PML NBs to regulate protein SUMOylation in different biological processes (Sun, 2014).

REFERENCES

Search PubMed for articles about Drosophila Zn finger homeodomain 2

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Enriquez, J., Venkatasubramanian, L., Baek, M., Peterson, M., Aghayeva, U. and Mann, R. S. (2015). Specification of individual adult motor neuron morphologies by combinatorial transcription factor codes. Neuron 86(4):955-70. PubMed ID: 25959734

Fortini, M. E., Lai, Z. C., Rubin, G. M. (1991). The Drosophila zfh-1 and zfh-2 genes encode novel proteins containing both zinc-finger and homeodomain motifs. Mech. Dev. 34: 113-122. 1680376

Gabilondo, H., et al. (2011). A targeted genetic screen identifies crucial players in the specification of the Drosophila abdominal Capaergic neurons. Mech. Dev. 128(3-4): 208-21. PubMed Citation: 21236339

Hashimoto, T., et al. (1992). A new family of homeobox genes encoding multiple homeodomain and zinc finger proteins. Mech. Dev. 39: 125-126. 1362648

Ido, A., Miura, Y. and Tamaoki, T. (1994}. Activation of ATBF1, a multiple-homeodomain zinc-finger gene, during neuronal differentiation of murine embryonal carcinoma cells. Dev. Biol. 163(1): 184-7. 8174773

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date revised: 25 October 2023

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