empty spiracles

DEVELOPMENTAL BIOLOGY

Embryonic

A 2.4 kb RNA corresponding to the EMS transcript is expressed from cellular blastoderm throughout all embryonic and larval stages. During the cellular blastoderm stage ems is expressed in the developing head in a single anterior band, overlapping the domain of Deformed. Later in development this site can be identified as just anterior to and extending along the whole cephalic furrow. This location correlates with EMS function as an anterior gap gene, expressed in the preantennal, antennal and intercalary segments [Images] and required for the development of the antennal sense organ, the optic lobe and parts of the head skeleton. Later still ems is expressed segmentally in the clypeolabrum, the lateral regions of the procephalic lobe, and in three gnathal, as well as three thoracic and 10 abdominal segments. Expression is confined to lateral regions of each segment, where the tracheal pits form and lateral neuroblasts originate, as well as in the posterior spiracles. This late expression partially correlates with defects seen in the tracheal tree of ems null embryos ( Dalton, 1989 and Walldorf, 1992).

Early tailless expression (blastoderm stage) covers the anlage of the entire brain. Beginning approximately with the onset of gastrulation, an anterior-dorsal region with a high expression level (called HL domain) can be distinguished from a posterior-ventral domain expressing tll at a somewhat lower level. The HL domain coincides with part of the central and anterior protocerebral neurectoderm. The low expression level LL domain covers the remaining part of the protocerebral neuroectoderm. orthodenticle is expressed in a circumferential domain of the cellular blastoderm but during gastrulation becomes restricted to a domain that encompasses most of the protocerebral neurectoderm and an adjacent part of the deuterocerebral neurectoderm. All neurobasts segregating from this domain transiently express otd during stages 10 and 11. buttonhead is initially expressed in a wide domain including the anlagen of the antennal, intercalary and mandibular segments, as well as the acron. With the beginning of gastrulation, expression disappears from most of the procephalon, except for small domains of the posterior part of the deuterocerebral and tritocerebral neurectoderm and a dorsoanterior patch that partially overlaps with the dorsoanterior protocerebrum. Both the late deutocerebral and tritocerebral btd domains contain few, if any neuroblasts. empty spiracles is in an asymmetric circumferential domain of the cellular blastoderm. During gastrulation, this pattern resolves into two stripes that occupy anterior portions of the deuterocerebral neuroectoderm and the mandibular metamere, respectively. In addition, a small circular domain corresponding to the tritocerebral neurectoderm appears ventral to the deuterocerebral stripe (Younossi-Hartenstein, 1997).

Loss of tll function results in the absence of all protocerebral neuroblasts and loss of all four coherent domains of Fas II expression in the protocerebrum. Also missing is the optic lobe. orthodenticle functions in a domain that includes a large part of the protocerebrum and a smaller part of the adjacent deuterocerebrum. Loss of otd results in loss of protocerebral P1, P2 and P4 coherent domains of Fas II expression. Also missing is a nerve that carries axons from the antennal organ. In buttonhead mutation the D/T cluster is missing; consequently a cervical connection is missing that normally sends nerves to the labral sensory organ, the hypopharyngeal sensory organ and the stomatogastric nervous system (Younossi-Hartenstein, 1997).

The Drosophila brain develops from the procephalic neurogenic region of the ectoderm. About 100 neural precursor cells (neuroblasts) delaminate from this region on either side in a reproducible spatiotemporal pattern. Neuroblast maps have been prepared from different stages of the early embryo (stages 9, 10 and 11, when the entire population of neuroblasts has formed), in which about 40 molecular markers representing the expression patterns of 34 different genes are linked to individual neuroblasts. In particular, a detailed description is presented of the spatiotemporal patterns of expression in the procephalic neuroectoderm and in the neuroblast layer of the gap genes empty spiracles, hunchback, huckebein, sloppy paired 1 and tailless; the homeotic gene labial; the early eye genes dachshund, eyeless and twin of eyeless; and several other marker genes (including castor, pdm1, fasciclin 2, klumpfuss, ladybird, runt and unplugged). Based on the combination of genes expressed, each brain neuroblast acquires a unique identity, and it is possible to follow the fate of individual neuroblasts through early neurogenesis. Furthermore, despite the highly derived patterns of expression in the procephalic segments, the co-expression of specific molecular markers discloses the existence of serially homologous neuroblasts in neuromeres of the ventral nerve cord and the brain. Taking into consideration that all brain neuroblasts are now assigned to particular neuromeres and individually identified by their unique gene expression, and that the genes found to be expressed are likely candidates for controlling the development of the respective neuroblasts, these data provide a basic framework for studying the mechanisms leading to pattern and cell diversity in the Drosophila brain, and for addressing those mechanisms that make the brain different from the truncal CNS (Urbach, 2003).

The cephalic gap genes are expressed in large domains of the procephalon and play a crucial role not only in the patterning of the peripheral ectoderm, but also in regionalizing the brain primordium. The segmental organization of the Drosophila brain is based on the expression pattern of segment polarity and DV patterning genes. To see whether the cephalic gap genes respect the neuromeric boundaries segment polarity and DV patterning genes, and to provide a basis for studying their potential role in the formation or specification of brain precursor cells, the expression was studied of orthodenticle, empty spiracles, sloppy paired 1, tailless, huckebein, and hunchback in the developing head ectoderm, as well as in the entire population of identified NBs during stages 9-11 (Urbach, 2003).

During early neurogenesis in the trunk, empty spiracles (ems) is metamerically expressed in lateral ectodermal patches. In the head, it acts as a gap gene, which is expressed in a circumferential procephalon domain in the early cellular blastoderm. During gastrulation this circumferential stripe dissolves into three smaller ectodermal domains between the anterior part of the mandibular segment and the posterior part of the ocular segment; these domains are not in segmental register. During further development, the third domain splits into a mandibular/intercalary and an antennal component. All these domains contribute NBs to the brain. In addition to ems expression in the intercalary and antennal segments, and the corresponding trito- and deutocerebral neuromers, ems expression was also detected in a small neuroectodermal region. Finally, a further ems patch is located in the dorsoanterior procephalic ectoderm ('dorsal patch'), which becomes part of the labral ectoderm and does not appear to give rise to brain NBs. Thus, from stage 9 onwards, part of the antennal/ocular ems domain overlaps with the En-positive hs, and from stage 10/11 onwards these genes are also found to be co-expressed in the en hs-derived protocerebral NBs Ppd5 and Ppd8 (although en and ems expression also partly overlaps in the trunk ectoderm, a co-expression of both genes in trunk NBs is never seen). In contrast to earlier observations, showing that most of the tritocerebral NBs are included in the ems-expressing domain, only the dorsal Td6 was identified as Ems-positive. Ems protein is detectable in clusters of brain cells until the end of embryogenesis (Urbach, 2003).

Taken together, among the cephalic gap genes, slp1 appears to respect segmental boundaries during early neurogenesis of the brain. By contrast, in the considered period of development (stage 9-11), the expression of ems, otd and tll does not seem to respect these borders, contradicting claims in previous reports. All three genes are expressed in NBs deriving from ectodermal domains that are part of two or three neighboring segments. For example, ems is expressed in a small number of NBs comprising about six posterior ocular and four anterior deutocerebral NBs (all of which derive from the same ems domain, except Dv3 and Pcv5), and one tritocerebral NB. Accordingly, ems mutants show defects in the intercalary, antennal, and the ocular segment (e.g., the en hs is missing). Considering that ems is expressed in only a few trito- and deuto-cerebral NBs it is remarkable that ems mutants show a deletion of the trito- and deuto-cerebrum. An explanation for this could be that ems expression, which during earlier development covers the neuroectoderm of the respective segments, possibly confers specific identities to the arising trito- and deuto-cerebral NBs. The lack of these NBs might be responsible for the loss of NB-specific gene expression, and (secondarily) for the gross morphological defects seen in the ems mutant brain. A similar proposal has been made to explain the brain defects that occur in buttonhead (btd) mutants, although btd is not expressed in NBs of the corresponding brain regions (Urbach, 2003).

Specification of neuronal subtypes by different levels of Hunchback

During the development of the central nervous system, neural progenitors generate an enormous number of distinct types of neuron and glial cells by asymmetric division. Intrinsic genetic programs define the combinations of transcription factors that determine the fate of each cell, but the precise mechanisms by which all these factors are integrated at the level of individual cells are poorly understood. This study analyzed the specification of the neurons in the ventral nerve cord of Drosophila that express Crustacean cardioactive peptide (CCAP). There are two types of CCAP neurons: interneurons and efferent neurons. Both were found to be specified during the Hunchback temporal window of neuroblast 3-5, but are not sibling cells. Further, this temporal window generates two ganglion mother cells that give rise to four neurons, which can be identified by the expression of empty spiracles. The expression of Hunchback in the neuroblast increases over time, and evidence is provided that the absolute levels of Hunchback expression specify the two different CCAP neuronal fates (Moris-Sanz, 2014).

This study analyzed how CCAP-expressing neurons are specified. Evidence was obtained that both the the efferent subset of CCAP neurons (CCAP-ENs) and interneuron subset (CCAP-INs) of all embryonic segments are generated by NB3-5. The results also indicate that CCAP neurons are generated in the Hb temporal window, are not sibling cells and that the CCAP-ENs are generated first followed by the CCAP-INs. Although the Hb temporal window in NB3-5 generates two GMCs that can be distinguished by the expression of Pdm in GMC1, Pdm does not seem to play any role in the specification of these neurons, as no phenotype was observed in pdm mutants (Moris-Sanz, 2014).

These findings raised the question of how these two neuronal fates are generated, and the results that are presented in this study suggest that different levels of Hb expression specify them. The evidence for this is as follows. First, Hb expression in NB3-5 increases over time from stage 9 to early stage 11, then its expression quickly fades, coinciding with the reported expression of Svp, which is known to close the Hb temporal window. During this time window, NB3-5 divides twice and generates four neurons. Second, overexpression of high levels of Hb using a pan-NB driver extends the IN fate. Third, in an hb hypomorphic condition CCAP-INs are lost or converted into ENs, as monitored by the expression of Dac and the presence of axons that exit the ganglion (Moris-Sanz, 2014).

This mechanism for generating distinct neuronal fates is different from that proposed for subdividing the Cas temporal window in NB5-6, which involves two sequential feed-forward loops and several genes to define the fates of four cells (Ap1-4) that are sequentially generated and form the Apterous (Ap) cluster of neurons. However, the mechanism that was proposed is very similar to the role that the grh gene plays in the Ap cluster, since Grh expression increases gradually over time from Ap1 to Ap4, and overexpression of Grh converts all four Ap neurons into Ap4 (Moris-Sanz, 2014).

In addition to the different levels of Hb expression observed in NB3-5, it was found that CCAP-ENs and CCAP-INs express low and high levels of Hb, respectively, and overexpression of Hb in postmitotic cells convert the ENs into INs. These observations raise the question of how a high level of Hb expression in the NB leads to a high level of expression in the neuron. A recent analysis of the hb regulatory region revealed a specific postmitotic enhancer, so it would be tempting to propose that this enhancer is only activated in neurons that are generated by a NB expressing a high level of Hb. However, no expression of this enhancer was detected in any of the CCAP neurons, and overexpression of Hb in the NB did not lead to activation of the enhancer in neurons. Therefore, further work is needed to identify the mechanism by which only a subset of the neurons generated in the Hb temporal window expresses a high level of Hb and how this is translated into different neuronal fates (Moris-Sanz, 2014).

CCAP-INs express a high level of Hb and do not express Dac, and upon Hb overexpression the expression of Dac is lost in many, although not all, cells. This could place dac as a direct target of Hb. Analysis of dac cis-regulatory domains indicates the presence of a 5.8 kb domain in the first intron that, when placed in a Gal4 vector, was sufficient to drive GFP expression in vivo in many neurons of late embryos . A preliminary analysis of the sequence of this domain suggests the presence of conserved regions and putative Hb binding sites. Further analysis will be required to confirm the presence and elucidate the function of such sequences (Moris-Sanz, 2014).

Ikaros (or Ikzf1), a mouse ortholog of Hb, is expressed in all early retinal progenitor cells (RPCs) of the developing retina. Its expression in RPCs is necessary and sufficient to confer the competence to generate early-born neurons. These and other observations suggest that, as in the Drosophila CNS, cell-intrinsic mechanisms act in the RPC to control temporal competence. Ikaros is expressed in the early RPCs that give rise to several cell types, namely horizontal, amacrine and gangion cells; however, it is unclear whether distinct levels of Ikaros expression are responsible for the production of these different cell types (Moris-Sanz, 2014).

In the early embryo, different concentrations of Hb seem to elicit different cellular responses. At low concentrations, Hb monomers function as activators, whereas at high concentrations they form dimers that either repress transcription or block activation. Analysis of the Hb protein has led to the identification of two conserved domains: a DNA-binding domain and a dimerization domain. More recently, it has been shown that, in CNS development, Hb repressor function is required to maintain early NB competence and to specify early-born neuronal identity. These results are compatible with the evidence presented in this study that it is the absolute level of Hb in a NB that determines whether it is expressed in the postmitotic progeny and so specifies the different neuronal subtypes (Moris-Sanz, 2014).

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).

Effects of Mutation or Deletion

Structural and functional comparisons between ems and other embryonic patterning genes of Drosophila suggest that ems acts, in part, as a homeotic selector gene, specifying the identity of some of the most anterior head segments. Mutant embryos lacking EMS protein have severe patterning defects in the anterior head and are missing tracheal structures, including the filzkörper, which are normally developed by the eighth abdominal segment (Dalton, 1989).

ems is required for the development of specific brain segments in Drosophila. The embryonic brain is composed of three segmental neuromeres. The orthodenticle gene is expressed predominantly in the anterior neuromere; expression of ems is restricted to the two posterior neuromeres. Mutation of otd eliminates the first (protocerebral) brain neuromere. Mutation of ems eliminates the second (deutocerebral) and third (tritocerebral) neuromeres. otd is also necessary for development of the dorsal protocerebrum of the adult brain (Hirth, 1995).

Cell lineage-specific expression and function of the empty spiracles gene in adult brain development of Drosophila melanogaster

The empty spiracles (ems) gene, encoding a homeodomain transcription factor, is a member of the cephalic gap gene family that acts in early specification of the anterior neuroectoderm in the embryonic brain of Drosophila. ems is also expressed in the mature adult brain in the lineage-restricted clonal progeny of a single neuroblast in each brain hemisphere. These ems-expressing neuronal cells are located ventral to the antennal lobes and project a fascicle to the superior medial protocerebrum. All adult-specific secondary neurons in this lineage persistently express ems during postembryonic larval development and continue to do so throughout metamorphosis and into the adult. Mosaic-based MARCM mutant analysis and genetic rescue experiments demonstrate that ems function is autonomously required for the correct number of cells in the persistently expressing adult-specific lineage. Moreover, they indicate that ems is also required cell autonomously for the formation of the correct projections in this specific lineage. This analysis of ems expression and function reveals novel and unexpected roles of a cephalic gap gene in translating lineage information into cell number control and projection specificity in an individual clonal unit of the adult brain (Lichtneckert, 2007).

During postembryonic development of the Drosophila brain, expression of the ems gene is observed in eight neuroblast lineages per hemisphere. In seven of these, ems expression is transient and disappears during pupal development. This cessation of expression during metamorphosis could be related to the dynamic pattern of ems expression within each lineage. Thus, during larval development of these lineages, ems expression appears limited to the neuroblast and its recently generated progeny, suggesting that expression in the progeny may be transient. This type of dynamic expression could explain the fading out of the Ems signal in the seven lineages once their neuroblasts stop proliferation at the early pupal stage (Lichtneckert, 2007).

By contrast, in the eighth neuroblast lineage, ems expression is persistent. During larval development the neuroblast and all its adult-specific progeny express ems; this expression continues throughout metamorphosis and into the adult in all postmitotic cells of the EM lineage. The mechanisms responsible for the maintenance of ems expression in the adult-specific cells of the EM lineage are currently unknown. However, there is some evidence that ems is also expressed and maintained in the primary neurons of the EM lineage generated during embryogenesis. In all postembryonic stages and in the adult, approximately 30 ems-expressing neurons are closely associated with the early born, adult-specific neurons of the EM clone. These neurons are not generated postembryonically, and their number does not change significantly during postembryonic development. This suggests that the mechanisms responsible for the persistence of ems expression in the EM lineage may operate in all cells of the lineage, embryonic and postembryonic (Lichtneckert, 2007).

During early embryogenesis, ems is expressed in a total of eleven neuroblasts per embryonic brain hemisphere. An unambiguous link between these embryonic brain neuroblasts and the eight postembryonic ems-expressing neuroblasts has not yet been established. If the persistent expression of ems is a unique feature of the EM lineage, it should be possible to trace this lineage back into embryonic stages and identify its embryonic neuroblast of origin. For the remaining seven postembryonic ems-expressing neuroblasts, this may be more difficult and require a combination of molecular markers and neuroanatomical lineage mapping (Lichtneckert, 2007).

The postembryonic expression of ems in the fly brain has interesting parallels to the expression of the Emx1 and Emx2 genes in the mammalian brain. In addition to early expression in the neural plate, the Emx1 gene is expressed in many differentiating and mature neurons of the murine cortex. Brain-specific expression of Emx2 appears to be more transient in later stages and in the adult brain seems to be restricted to neural stem cells. Thus, spatially restricted persistent and transient expression patterns are observed for the ems/Emx genes in neural progenitors and in neurons during brain development and maturation in flies and mice (Lichtneckert, 2007).

For mutant analysis of ems function focus was placed on the EM lineage and clonal techniques were used to ensure that the secondary adult-specific neurons are mutant from the time of their birth onwards. Two lineage-specific mutant phenotypes are apparent in these loss-of-function experiments. The number of adult-specific neurons is reduced and projection defects occur in mutant clones. Both phenotypes are cell autonomous, and both can be fully restored in genetic rescue experiments. Moreover, both mutant phenotypes are seen in larval stages and persist in the adult brain. These findings implicate the ems transcription factor in translating lineage information into neuronal cell number control and neurite projection specificity (Lichtneckert, 2007).

There are several possible explanations for the 50% reduction in cell number observed in ems mutant EM clones. First, proliferation of the mutant neuroblast might cease due to cell cycle arrest or to premature neuroblast death. This seems unlikely, because proliferating neuroblasts can be identified in larval ems mutant clones based on expression of specific markers. Second, cell division of ganglion mother cells might be suppressed in favour of a direct differentiation of each neuroblast progeny into a single neuron, resulting in a total clone size reduction of 50%. This also appears unlikely, since GMCs expressing a cell proliferation marker can be identified repeatedly in mutant clones, indicating that they divide normally to produce two daughter cells. Third, the time window of proliferative activity or the proliferation rate of the persistent neuroblast is shortened in ems mutants. While this possibility cannot be ruled out, it also appears unlikely for the following three reasons: first, mutant and wild-type clones contain a similar number of cells at 48 hours ALH, suggesting that the proliferation rate is not affected at this stage; second, BrdU-incorporation studies reveal no difference in mitotic activity at late larval stage brains of wild-type versus ems mutant clones; third, the percentage of neuroblasts expressing the mitotic marker H3p at late larval stages was comparable for wild-type and ems mutant clones. The final explanation for the marked reduction in cell number seen in mutant clones is that postmitotic cells die due to apoptosis. This possibility is supported by two observations: (1) late larval ems mutant EM clones contain apoptotic cells, as assayed by the apoptosis marker cleaved Caspase 3 and (2) blockage of cell death in the ems mutant lineage through a pancaspase inhibitor results in significant restoration of the clonal cell number to a value comparable to that observed in the wild type. Based on these findings, it is posited that ems is required in the adult-specific EM lineage for survival of clonal postmitotic progeny (Lichtneckert, 2007).

Two types of neurite projection defects are observed in ems mutant EM lineages. (1) In the adult brain of all ems mutants, short aberrant projections extend from the cell bodies in a misdirected manner into adjacent neuropile. Misdirected projections of this type are also present in the larval ems mutant EM lineages. This suggests that ems is already required during larval stages to prevent the formation of these misprojections. Whether the aberrant projections formed in the larva persist into the adult, or whether misprojections of this type are continuously formed (and retracted) during metamorphosis and in the adult, is currently not known. However, the fact that neurite projections, albeit short and ectopic, are formed in all mutant EM clones implies that the ems gene is not required for process outgrowth per se. Rather, the ems gene appears to be required to prevent the formation of misdirected processes, suggesting a role of the gene in neuronal pathfinding (Lichtneckert, 2007).

(2) Another projection defect is observed in the adult brain in approximately half the ems mutant EM lineages. It consists in the complete absence of the fascicle projecting to the superior medial protocerebrum. This projection phenotype in the adult has a corresponding projection phenotype in the larva, in that the primary neurite bundle is missing in approximately half the mutant lineages. These observations suggest that the formation of the primary neurite bundle during larval development might be a prerequisite for the process extension to adult-specific targets during metamorphosis; this would indicate a larval requirement of ems for neurite fascicle formation (Lichtneckert, 2007).

Both projection phenotypes seen in mutant neuroblast clones, short ectopic neurite projections and the absence of the fascicle to the protocerebrum, are also apparent in ems mutant single cell clones of the larval brain. Given that all other cells in the lineage, including the EM neuroblast, are wild-type-like in these experiments, this finding indicates that individual postmitotic neurons of the EM lineage have a cell-autonomous requirement for the ems gene in order to form correct projections in larval brain development (Lichtneckert, 2007).

This analysis of ems function in the EM lineage demonstrates that homeobox transcription factors can influence adult brain architecture in a cell-autonomous and lineage-specific way. A lineage-specific, cell-autonomous requirement of other transcription factors in brain development has been shown for the olfactory projection neurons and for mushroom body neurons in Drosophila. Thus, increasing evidence indicates that key developmental control genes, which operate early in embryogenesis, also act later in a lineage-specific manner in controlling anatomical features of the adult Drosophila brain. It may be a general feature of brain development that developmental control genes implicated in early neurogenesis and patterning are re-expressed and have different roles in later embryogenesis and postembryonic brain development (Lichtneckert, 2007).

A comparison of the role of ems in Drosophila brain development with that of Emx1 and Emx2 in mammalian brain development is interesting, especially when the cortical phenotypes of Emx1/Emx2 double mutants are considered. The cortical surface area of Emx1/Emx2 double mutants is about half that of wild type, and the thickness of the preplate and cortical plate is reduced, suggesting that Emx genes regulate the numbers of cortical neurons. Moreover, Emx1/Emx2 double mutants have major defects in the pathfinding of most cortical axons, implying an important role for Emx genes in axonal pathfinding in the brain. Thus, mutant analyses in Drosophila and mouse suggest that loss of function of ems/Emx genes may result in comparable brain phenotypes, namely in reduction of neuronal cell number and in neurite projection defects. This in turn suggests that the morphological differentiation of brain architecture in both flies and mammals may involve conserved functions of orthologous ems/Emx homeobox genes, not only in the early embryo but also during later stages of brain development (Lichtneckert, 2007).

Ems and Nkx6 are central regulators in dorsoventral patterning of the Drosophila brain

In central nervous system development, the identity of neuroblasts critically depends on the precise spatial patterning of the neuroectoderm in the dorsoventral (DV) axis. This study has uncovered novel gene regulatory network underlying DV patterning in the Drosophila brain; the cephalic gap gene empty spiracles (ems) and the Nk6 homeobox gene (Nkx6) encode key regulators. The regulatory network implicates novel interactions between these and the evolutionarily conserved homeobox genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind) and muscle segment homeobox (msh). Msh cross-repressively interacts with Nkx6 to sustain the boundary between dorsal and intermediate neuroectoderm in the tritocerebrum (TC) and deutocerebrum (DC), and Vnd positively regulates Nkx6 by suppressing Msh. Remarkably, Ems is required to activate Nkx6, ind and msh in the TC and DC, whereas later Nkx6 and Ind act together to repress ems in the intermediate DC. Furthermore, the initially overlapping expression of Ems and Vnd in the ventral/intermediate TC and DC resolves into complementary expression patterns due to cross-repressive interaction. These results indicate that the anteroposterior patterning gene ems controls the expression of DV genes, and vice versa. In addition, in contrast to regulation in the ventral nerve cord, cross-inhibition between homeodomain factors (between Ems and Vnd, and between Nkx6 and Msh) is essential for the establishment and maintenance of discrete DV gene expression domains in the Drosophila brain. This resembles the mutually repressive relationship between pairs of homeodomain proteins that pattern the vertebrate neural tube in the DV axis (Seibert, 2009).

This study shows that the evolutionarily conserved homeodomain protein Ems is an integral component of the gene regulatory network that governs DV patterning in the posterior brain neuromeres, the TC and DC. This novel function is surprising because ems has hitherto been exclusively connected with patterning functions along the AP axis. It has been proposed that the combined activities of the gap genes ems, buttonhead and orthodenticle (ocelliless - FlyBase) generate head segments and that ems mutants exhibit defects in the formation of the intercalary and antennal segment as well as in the corresponding TC and DC in accordance with the early pattern of ems expression. ems probably also has a homeotic function in specifying aspects of intercalary segment identity. This study provides evidence that another crucial function of Ems is its cross-repressive interaction with Vnd. Previously, it was shown that vnd expression is dynamic and exhibits specific differences in the TC and DC. This study demonstrates that Ems is involved in the regulation of brain-specific differences in vnd expression, and that Vnd acts to repress ems in complementary parts of the TC and DC. These interactions help to refine the pattern into mutually exclusive domains at the onset of neurogenesis, which is important as both genes provide positional information that subsequently specifies the identity of individual brain NBs. Depending on the context, Vnd/Nkx2 can act as a transcriptional activator or repressor, as determined by physical interaction with the co-repressor Groucho, which enhances repression. Interestingly, it was observed that Ems also regulates the expression of two Nkx genes in an opposing manner: it represses vnd/Nkx2 but is necessary to activate Nkx6. The repressor function of Ems most likely also depends on Groucho; Ems has been reported to bind Groucho in vitro (Seibert, 2009).

In ems mutants, defects in proneural gene expression (lethal of scute and achaete) are restricted to NE regions where ems is normally expressed during early neurogenesis, leading to the loss of a subset of NBs in the TC and DC. This contrasts with the phenotype of the late embryonic ems mutant brain, which exhibits a severe reduction, or entire elimination, of the TC and DC, suggesting that the proper development of a larger NE domain and/or fraction of NBs in the TC and DC must be affected. However, in ems mutants the organization of the early procephalic NE appears normal until stages 9/10 and apoptosis is not detected. A possible explanation for the subsequent complete loss of TC and DC is that in ems mutants, vnd becomes derepressed in the ventral/intermediate NE of both neuromeres, and expression of msh, ind and Nkx6 is not activated. It has been shown that ectopic vnd prevents the expression of many NB identity genes. Indeed, the expression of a number of molecular markers has been reported to be absent in the ems mutant brain. It is therefore conceivable that in the TC and DC of ems mutants, as a consequence of lacking ems and ectopic vnd (and the absence of proneural gene activation), some NBs do not form. Additionally, owing to mis-specification of the NE (where neural identity gene expression is absent or altered), the other NBs and their progeny might still form but degenerate at later stages (Seibert, 2009).

It has been largely unclear how expression of Nkx6 is regulated in the brain NE, although Vnd has been suggested to act as a positive regulator. At the blastodermal stage, coexpression of ems and vnd is only observed in the intermediate and ventral NE of the TC and DC, which might account for early Nkx6 expression being limited to the respective NE in the brain and absent from the trunk. The data indicate that Ems and Vnd together facilitate the activation of Nkx6. Ems expression closely prefigures the domain of Nkx6 expression in the TC and DC, and together with the fact that Nkx6 is completely abolished in ems mutants, this suggests that Ems might act as a direct activator to regulate the extension of the Nkx6 domain along the AP axis. Vnd indirectly regulates the enlargement of the Nkx6 domain along the DV axis by repressing the Nkx6-repressor Msh. That DV patterning in the brain NE integrates AP signals is additionally supported by the fact that Ems is also necessary for activation of ind and msh, indicating that ems is a key regulator in DV patterning of the TC and DC. Evidence is also provided for a negative-feedback control in the DV regulatory network, in which Ems is needed to activate its own later-stage repressors, Nkx6 and Ind. Together, these data suggest not only that Ems regulates the expression of all DV genes (activating Nkx6, ind, msh and repressing vnd), but also that DV factors (Nkx6, Ind and Vnd) control expression of ems, indicating that integration of DV and AP patterning signals takes place at different levels in the DV genetic network (Seibert, 2009).

Nkx6 has been identified as specifically involved in DV patterning of the TC and DC. In addition to later suppression of ems (in concert with Ind), a further pivotal function of Nkx6 is to maintain the suppression of msh in the intermediate/ventral TC and DC that was initiated by Vnd. Since in both neuromeres the expression of Nkx6 starts before and persists longer than that of ind, and because msh is ventrally derepressed in Nkx6 but not in ind mutants, this implies that Nkx6 (but not Ind) is the major msh suppressor necessary to prevent intermediate/ventral NE and the descending NBs from adopting dorsal fates. Consequently, Nkx6 indirectly regulates the proper specification of brain NB identity by suppressing msh (and ems). Further experiments are required to show whether Nkx6 is also more directly involved in the fate specification of NBs and progeny cells in the brain, as has been shown in the VNC, where Nkx6 promotes the fate of ventrally projecting, and represses the fate of dorsally projecting, motoneurons (Seibert, 2009).

Additionally, cross-inhibitory interactions were observed between Nkx6 and Msh. It is assumed that this mutually repressive regulation in the TC and DC is necessary to stabilize the boundary between dorsal and intermediate NE, and to ensure the regionalized expression of msh and Nkx6 over time. It is likely that Nkx6 and Msh/Msx interact with the co-repressor Groucho to repress each other at the transcriptional level. Interestingly, aspects of the genetic interactions between Nkx6 and Msh/Msx seem to be evolutionarily conserved, since Msx1, which is expressed in the vertebrate midbrain and functions as a crucial determinant in the specification of dopamine neurons, represses Nkx6.1 in ventral midbrain dopaminergic progenitors of mice (Seibert, 2009).

It had not been shown until now that domains of DV gene expression in the Drosophila brain become established through cross-repressive regulation, and it is possible that such genetic interactions are more common than previously thought (e.g. Ind and Msh act as mutual inhibitors). This suggests that in the fly brain, cross-inhibition between pairs of homeodomain transcription factors is fundamental for establishing and maintaining DV neuroectodermal and corresponding stem cell domains. By contrast, in the NE of the VNC, where DV patterning is much better understood, cross-repressive interactions of homeobox genes are largely omitted. There, DV patterning is proposed to be conducted by a strict ventral-dominant hierarchy according to which ventral genes repress more-dorsal genes. However, one exception to the rule seems to be the cross-inhibitory interaction between Vnd and Ind. Interestingly, in the developing vertebrate neural tube, cross-repressive interactions of homeodomain proteins are common and indeed crucial for the establishment of discrete DV progenitor domains. This bears a marked resemblance to the mutually antagonistic relationship between pairs of homeodomain proteins that dorsoventrally pattern the fly brain (Seibert, 2009).

A predominant feature of the brain-specific DV genetic network described in this study, and a general design feature of gene regulatory networks, is the extensive use of transcriptional repression to regulate target gene expression in spatial and temporal dimensions. All factors involved in the network operate as repressors (except Ems, which may also serve as an activator), via mutual repression (between Ems and Vmd, and between Nkx6 and Msh), a double-negative mechanism (Vnd represses Msh, which represses Nkx6), and a negative-feedback loop (Ems is needed to activate Nkx6 and Ind, which in turn repress Ems). The spatial and temporal complexity of the regulatory interactions that have been deciphered implies similar complexity in the underlying cis-regulatory control of these factors. For example, the domain of msh expression is regulated by the input of at least two transcriptional repressors acting in subsequent time windows (Vnd early and Nkx6 late), and the input of at least three repressors regulates the dynamics of ems expression (Vnd early, Ind and Nkx6 late). The brain-specific DV patterning network probably comprises further genes in addition to those that have been identified, and it is likely that interactions with other putative regulators (e.g. Dorsal, Egfr, Dpp) will complement the present model. Altogether, these data provide the basis for a systematic comparison of the genetic processes underlying DV patterning of the brain between different animal taxa at the level of gene regulatory networks (Seibert, 2009).

The genetic factors considered in this study in the developing fly brain are expressed in similar NE domains from early embryonic stages onwards in the anterior neural plate in vertebrates. Emx2, for example, is expressed in the laterodorsal region, and Nkx2 genes in the ventral region, of the early vertebrate forebrain. At the four-somite stage (~E8), these two domains exhibit a common border, similar to that observed in Drosophila after Ems and Vnd have, through cross-repression, regulated their mutually exclusive expression domains. Moreover, whereas Msx genes are mainly expressed in dorsal regions of the posterior forebrain, midbrain and hindbrain, expression of Nkx6 genes is reported in more lateroventral regions, overlapping ventrally with the expression of Nkx2 genes. However, even though these patterns of gene expression exhibit certain similarities between insects and vertebrates, it remains to be shown whether their genetic interactions are also conserved (Seibert, 2009).

Andrioli, L. P., Digiampietri, L. A., de Barros, L. P. and Machado-Lima, A. (2012). Huckebein is part of a combinatorial repression code in the anterior blastoderm. Dev. Biol. 361(1): 177-85. PubMed Citation: 22027434

Aspöck, G., Ruvkun, G. and Bürglin. R. (2003). The Caenorhabditis elegans ems class homeobox gene ceh-2 is required for M3 pharynx motoneuron function. Development 130: 3369-3378. 12810585

Barbieri, A. M., et al. (2002). Vax2 inactivation in mouse determines alteration of the eye dorsal-ventral axis, misrouting of the optic fibres and eye coloboma. Development 129: 805-813. 11830579

Bell, E., et al. (2001). Dynamic domains of gene expression in the early avian forebrain. Dev. Bio. 236: 76-88. 11456445

Bishop, K. M., Goudreau, G. and O'Leary, D. D. (2000). Regulation of area identity in the mammalian neocortex by Emx2 and Pax6. Science 288(5464): 344-9. Medline abstract: 10764649

Bishop, K. M., Rubenstein, J. L. R. and O'Leary. D. D. M. (2002). Distinct actions of Emx1, Emx2, and Pax6 in regulating the specification of areas in the developing neocortex. J. Neurosci. 22(17): 7627-7638. 12196586

Bishop, K. M., Garel, S., Nakagawa, Y., Rubenstein, J. L. and O'Leary, D. D. (2003). Emx1 and Emx2 cooperate to regulate cortical size, lamination, neuronal differentiation, development of cortical efferents, and thalamocortical pathfinding. J. Comp. Neurol. 457: 345-360. Medline abstract: 12561075

Boncinelli, E., Gulisano, M. and Pannese, M. (1993). Conserved homeobox genes in the developing brain. C. R. Acad. Sci. III 316: 972-984. PubMed Citation: 7915635

Bordogna, W., et al. (2005). EMX homeobox genes regulate microphthalmia and alter melanocyte biology. Exp. Cell Res. 311(1): 27-38. Medline abstract: 16197942

Boyl, P. P., et al. (2001): Forebrain and midbrain development requires epiblast-restricted Otx2 translational control mediated by its 3' UTR. Development 128: 2989-3000. PubMed Citation: 11532921

Breiling, A., et al. (2001). General transcription factors bind promoters repressed by Polycomb group proteins. Nature 412: 651-655. 11493924

Capellini, T. D., et al. (2010). Scapula development is governed by genetic interactions of Pbx1 with its family members and with Emx2 via their cooperative control of Alx1. Development 137(15): 2559-69. PubMed Citation: 20627960

Castelli-Gair, J. (1998). The lines gene of Drosophila is required for specific functions of the Abdominal-B HOX protein. Development 125, 1269-1274. PubMed Citation: 9477325

Chiang, C., et al. (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383: 407-413. PubMed Citation: 8837770

Cohen, S.M. and Jürgens, G. (1990). Mediation of Drosophila head development by gap-like segmentation genes. Nature 346: 482-485. PubMed Citation: 1974035

Crozatier, M., et al. (1996). collier, a novel regulator of Drosophila head development, is expressed in a single mitotic domain. Curr. Biol. 6(6): 707-718. PubMed Citation: 8793297

Crozatier, M., et al. (1999). Head versus trunk patterning in the Drosophila embryo; collier requirement for formation of the intercalary segment. Development 126: 4385-4394. PubMed Citation: 10477305

Dalton, D., Chadwick, R. and McGinnis, W. (1989). Expression and embryonic function of empty spiracles: a Drosophila homeo box gene with two patterning functions on the anterior-posterior axis of the embryo. Genes Dev 3: 1940-56. PubMed Citation: 2576012

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

Fernandez, A. S., et al. (1998). Expression of the Emx-1 and Dlx-1 homeobox genes define three molecularly distinct domains in the telencephalon of mouse, chick, turtle and frog embryos: implications for the evolution of telencephalic subdivisions in amniotes. Development 125: 2099-2111. PubMed Citation: 9570774

Gall, A., et al. (2003). Glypican 4 modulates FGF signalling and regulates dorsoventral forebrain patterning in Xenopus embryos. Development 130: 4919-4929. 12930779

Galli, R., et al. (2002). Emx2 regulates the proliferation of stem cells of the adult mammalian central nervous system. Development 129: 1633-1644. 11923200

Galliot, B., et al. (2009). Origins of neurogenesis, a cnidarian view. Dev. Biol. 332: 2-24. PubMed Citation: 19465018

Gauchat, D., et al. (2000). Evolution of Antp-class genes and differential expression of Hydra Hox/paraHox genes in anterior patterning. Proc. Natl. Acad. Sci. 97: 4493-4498. PubMed Citation: 10781050

Goldstein, R. E., et al. (2005). An eh1-like motif in Odd-skipped mediates recruitment of Groucho and repression in vivo. Mol. Cell. Biol. 25(24): 10711-20. 16314497

Gont, L. K., et al. (1996). Overexpression of the homeobox gene Xnot-2 leads to notochord formation in Xenopus. Dev. Biol. 174: 174-178. PubMed Citation: 8626017

Grossniklaus, U., Cadigan, K. M. and Gehring, W. J. (1994). Three maternal coordinate systems cooperate in the patterning of the Drosophila head. Development 120: 3155-3171. PubMed Citation: 7720559

Groves, J. A., Gillman, C., DeLay, C. N. and Kroll, T. T. (2019). Identification of novel binding partners for transcription factor Emx2. Protein J. PubMed ID: 30628007

Gulisano, M., et al. (1996). Emx1 and Emx2 show different patterns of expression during proliferation and differentiation of the developing cerebral cortex in the mouse. Eur. J. Neurosci. 8(5): 1037-1050. PubMed Citation: 8743751

Hallonet, M., et al. (1998). Vax1 is a novel homeobox-containing gene expressed in the developing anterior ventral forebrain. Development 125(14): 2599-2610. PubMed Citation: 9636075

Halpern, M. E., et al. (1995). Cell-autonomous shift from axial to paraxial mesodermal development in zebrafish floating head mutants. Development 121: 4257-4264. PubMed Citation: 8575325

Hamasaki, T., Leingartner, A., Ringstedt, T. and O'Leary, D. D. (2004). EMX2 regulates sizes and positioning of the primary sensory and motor areas in neocortex by direct specification of cortical progenitors. Neuron 43(3): 359-72. 15294144

Hartmann, B., Reichert, H. and Walldorf, U. (2001). Interaction of gap genes in the Drosophila head: tailless regulates expression of empty spiracles in early embryonic patterning and brain development. Mech. Dev. 109(2): 161-72. 11731230

Hasegawa, Y., Takata, N., Okuda, S., Kawada, M., Eiraku, M. and Sasai, Y. (2016). Emergence of dorsal-ventral polarity in ESC-derived retinal tissue. Development 143(21): 3895-3906. PubMed ID: 27633992

Hassan, B, et al. (1997). Prospero is a panneural transcription factor that modulates homeodomain protein activity. Proc. Natl. Acad. Sci. 94(20): 10991-10996. PubMed Citation: 9380747

Hirth, F., (1995). Developmental defects in brain segmentation caused by mutations of the homeobox genes orthodenticle and empty spiracles in Drosophila. Neuron 15: 769-778. PubMed Citation: 7576627

Hirth, F., Hartmann, B. and Reichert, H. (1998). Homeotic gene action in embryonic brain development of Drosophila. Development 125: 1579-1589. PubMed Citation: 9521896

Hirth, F. (2010). On the origin and evolution of the tripartite brain. Brain Behav. Evol. 76(1): 3-10. PubMed Citation: 20926853

Hitoshi, S., et al. (2002). Neural stem cell lineages are regionally specified, but not committed, within distinct compartments of the developing brain. Development 129: 233-244. 11782416

Hong, S. M., et al. (2007). Reduced hippocampal neurogenesis and skill reaching performance in adult Emx1 mutant mice. Exp Neurol. [Epub ahead of print]. Medline abstract: 17490651

Hu, N. and Castelli-Gair, J. (1999). Study of the posterior spiracles of Drosophila as a model to understand the genetic and cellular mechanisms controlling morphogenesis. Dev. Biol. 214(1): 197-210. PubMed Citation: 10491268

Jones, B. and McGinnis, W. (1993). The regulation of empty spiracles by Abdominal-B mediates an abdominal segment identity function. Genes Dev 7: 229-40. PubMed Citation: 8094700

Kim, N., Min, K. W., Kang, K. H., Lee, E. J., Kim, H. T., Moon, K., Choi, J., Le, D., Lee, S. H. and Kim, J. W. (2014). Regulation of retinal axon growth by secreted Vax1 homeodomain protein. Elife 3: e02671. PubMed ID: 25201875

Li, H., Bishop, K. M. and O'Leary, D. D. (2006). Potential target genes of EMX2 include Odz/Ten-M and other gene families with implications for cortical patterning. Mol. Cell Neurosci. 33(2): 136-49. Medline abstract: 16919471

Lichtneckert, R., Bello, B. and Reichert, H. (2007). Cell lineage-specific expression and function of the empty spiracles gene in adult brain development of Drosophila melanogaster. Development 134(7): 1291-300. Medline abstract: 17314131

Lichtneckert, R., Nobs, L. and Reichert, H. (2008). empty spiracles is required for the development of olfactory projection neuron circuitry in Drosophila. Development 135: 2415-2424. PubMed Citation: 18550709

Ligon, K. L., et al. (2003). Loss of Emx2 function leads to ectopic expression of Wnt1 in the developing telencephalon and cortical dysplasia. Development 130: 2275-2287. 12668639

Lord, P. C., et al. (1995). Normal expression and the effects of ectopic expression of the Drosophila muscle segment homeobox (msh) gene suggest a role in differentiation and patterning of embryonic muscles. Dev Biol 171: 627-640. PubMed Citation: 7556942

Lowe, C. J., et al. (2003). Anteroposterior patterning in hemichordates and the origins of the chordate nervous system. Cell 113: 853-865. 12837244

Macías, A. and Morata, G. (1996). Functional hierarchy and phenotypic suppression among Drosophila homeotic genes: the labial and empty spiracles genes. EMBO J. 15: 334-343. PubMed Citation: 8617208

Mallamaci, A., et al. (2000). Area identity shifts in the early cerebral cortex of Emx2-/- mutant mice. Nature Neurosci. 3: 679-686. PubMed Citation: 10862700

Masai, I., et al. (1997). floating head and masterblind regulate neuronal patterning in the roof of the forebrain. Neuron 18: 43-57

Melby, A. E., Warga, R. M. and Kimmel, C. B. (1996). Specification of cell fates at the dorsal margin of the zebrafish gastrula. Development 122: 2225-2237

Merabet, S., et al. (2002). A green fluorescent protein reporter genetic screen that identifies modifiers of Hox gene function in the Drosophila embryo. Genetics 162: 189-202. 12242233

Miyamoto, N., et al. (1997). Defects of urogenital development in mice lacking Emx2. Development 124, 1653-1664

Mohler, J. (1995). Spatial regulation of segment polarity gene expression in the anterior terminal region of the Drosophila blastoderm embryo. Mech Dev 50: 151-161

Morita, T., et al. (1995). Differential expression of two zebrafish emx homeoprotein mRNAs in the developing brain. Neurosci. Lett. 198: 131-134

Moris-Sanz, M., Estacio-Gomez, A., Alvarez-Rivero, J. and Diaz-Benjumea, F. J. (2014). Specification of neuronal subtypes by different levels of Hunchback. Development 141: 4366-4374. PubMed ID: 25344076

Mui, S. H., et al. (2002). The homeodomain protein Vax2 patterns the dorsoventral and nasotemporal axes of the eye. Development 129: 797-804. 11830578

Mui, S. H., Kim, J. W., Lemke, G. and Bertuzzi, S. (2005). Vax genes ventralize the embryonic eye. Genes Dev. 19(10): 1249-59. 15905411

Muzio, L. and Mallamaci, A. (2003). Emx1, emx2 and pax6 in specification, regionalization and arealization of the cerebral cortex. Cereb. Cortex 13(6): 641-7. Medline abstract: 12764040

Nazar, A. P., Delgado, M. J. and Lavore, A. (2022). Empty-spiracles is maternally expressed and essential for neurodevelopment and early embryo determination in Rhodnius prolixus. Dev Biol 490: 144-154. PubMed ID: 35988717

Ochoa-Espinosa, A., Yu, D., Tsirigos, A., Struffi, P. and Small, S. (2009). Anterior-posterior positional information in the absence of a strong Bicoid gradient. Proc. Natl. Acad. Sci. 106(10): 3823-8. PubMed Citation: 19237583

Oda, I. and Saiga, H. (2001). Hremx, the ascidian homologue of ems/emx, is expressed in the anterior and posterior-lateral epidermis but not in the central nervous system during embryogenesis. Dev. Genes Evol. 211: 291-298. 11466524

Palsson, A., Wesolowska, N., Reynisdottir, S., Ludwig, M. Z. and Kreitman, M. (2014). Naturally occurring deletions of Hunchback binding sites in the Even-Skipped stripe 3+7 enhancer. PLoS One 9: e91924. PubMed ID: 24786295

Pannese, M., et al. (1998). The Xenopus emx genes identify presumptive dorsal telencephalon and are induced by head organizer signals. Mech. Dev. 73(1): 73-83

Pellegrini, M., et al. (1996). Dentate gyrus formation requires Emx2. Development 122: 3893-3898

Pellegrini, M., et al. (2001). Agenesis of the scapula in Emx2 homozygous mutants. Dev. Biol. 232: 149-156. PubMed ID: 11254354

Peterson, K. J., et al. (1999), Expression pattern of Brachyury and Not in the sea urchin: comparative implications for the origins of mesoderm in the basal deuterostomes. Dev. Biol. 207(2): 419-31

Schock, F., et al. (2000). Phenotypic suppression of empty spiracles is prevented by buttonhead. Nature 405: 351-354.

Schulte, D., et al. (1999). Misexpression of the Emx-related homeobox genes cVax and mVax2 ventralizes the retina and perturbs the retinotectal map. Neuron 24: 541-553.

Seibert, J., Volland, D. and Urbach. R. (2009). Ems and Nkx6 are central regulators in dorsoventral patterning of the Drosophila brain. Development 136(23): 3937-47. PubMed Citation: 19906861

Shimogori, T., et al. (2004). Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex, Development 131: 5639-5647. 15509764

Shinozaki, K., et al. (2002). Absence of Cajal-Retzius cells and subplate neurons associated with defects of tangential cell migration from ganglionic eminence in Emx1/2 double mutant cerebral cortex. Development 129: 3479-3492. 12091317

Shinozaki K, Yoshida M, Nakamura M, Aizawa S, Suda Y. (2004). Emx1 and Emx2 cooperate in initial phase of archipallium development. Mech. Dev. 121(5): 475-89. 15147765

Skottheim Honn, J., Johansson, L. and Rasmuson Lestander, A. (2016). Regulation of twin of eyeless during Drosophila development. Gene Expr Patterns. PubMed ID: 26976323

Spahn, P., Huelsmann, S., Rehorn, K. P., Mischke, S., Mayer, M., Casali, A. and Reuter, R. (2013). Multiple regulatory safeguards confine the expression of the GATA factor serpent to the hemocyte primordium within the Drosophila mesoderm. Dev Biol 386(1): 272-9. PubMed ID: 24360907

Strutt, H. and Paro, R. (1997). The polycomb group protein complex of Drosophila melanogaster has different compositions at different target genes. Mol. Cell. Biol. 17(12): 6773-6783

Suda, Y., et al. (2001). Emx2 directs the development of diencephalon in cooperation with Otx2. Development 128: 2433-2450. 11493561

Suda, Y., et al. (2001). Emx2 directs the development of diencephalon in cooperation with Otx2. Development 128: 2433-2450. 11493561

Suda, Y., et al. (2010). The same enhancer regulates the earliest Emx2 expression in caudal forebrain primordium, subsequent expression in dorsal telencephalon and later expression in the cortical ventricular zone. Development 137(17): 2939-49. PubMed Citation: 20667915

Taglialatela, P., et al. (2004). Compromised generation of GABAergic interneurons in the brains of Vax1^-/- mice. Development 131: 4239-4249. 15280216

Take-uchi, M., Clarke, J. D. W. and Wilson, S. W. (2003). Hedgehog signalling maintains the optic stalk-retinal interface through the regulation of Vax gene activity. Development 130: 955-968. 12538521

Talbot, W. S., et al. (1995). A homeobox gene essential for zebrafish notochord development. Nature 378: 150-157. PubMed Citation: 7477317

Taylor, H. S. (1998). A regulatory element of the empty spiracles homeobox gene is composed of three distinct conserved regions that bind regulatory proteins. Molec. Reprod. Dev. 49: 246-253. 9491376

Theil, T., et al. (1999). Gli3 is required for Emx gene expression during dorsal telencephalon development. Development 126: 3561-3571. Medline abstract: 10409502

Theil, T., et al. (2002). Wnt and Bmp signaling cooperatively regulate graded Emx2 expression in the dorsal telencephalon. Development 129: 3045-3054. 12070081

Urbach, R. and Technau, G. M. (2003). Molecular markers for identified neuroblasts in the developing brain of Drosophila. Development 130: 3621-3637. 12835380

Vacik, T., Stubbs, J. L. and Lemke, G. (2011). A novel mechanism for the transcriptional regulation of Wnt signaling in development. Genes Dev. 25(17): 1783-95. PubMed Citation: 21856776

von Frowein, J., Wizenmann, A. and Gotz, M. (2006). The transcription factors Emx1 and Emx2 suppress choroid plexus development and promote neuroepithelial cell fate. Dev. Biol. 296(1): 239-52. Medline abstract: 16793035

Wagner, G. P. (2007). The developmental genetics of homology. Nat. Rev. Genet. 8: 473-479. PubMed Citation: 17486120

Walldorf, U. and Gehring, W. J. (1992). Empty spiracles, a gap gene containing a homeobox involved in Drosophila head development. EMBO J 11: 2247-59. PubMed Citation: 1376248

Yoshida, M., et al. (1997). Emx1 and Emx2 functions in development of dorsal telencephalon. Development 124: 101-111. PubMed Citation: 9006071

Younossi-Hartenstein, A., et al. (1997). Control of early neurogenesis in the Drosophila brain by the head gap genes tll, otd, ems, and btd. Dev. Biol 182: 270-283. PubMed Citation: 9070327

Zembrzycki, A., et al. (2007). Genetic interplay between the transcription factors Sp8 and Emx2 in the patterning of the forebrain. Neural Devel. 2: 8. Medline abstract: 17470284

Date revised: 23 July 2023


  

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