• Cephalic furrow or fold - the division between head and thorax
• Procephalic lobe - the pregnathal cephalic territory prior to segmentation
• Acron - the unsegmented preoral part of the head
• Atrium - Opening and most anterior portion of the foregut
• Gnathal - refering to the mouth appendage
• Stomodeum - the anterior ectodermal part of the gut
• Labella or labial palps - fleshy lobes that terminate the proboscus - the terminal part of the labium
• Proboscus - the sucking organ
• Optic placode - distinctive patch of cells that will form the eye-antennal disc
• Cephalopharyngeal apparatus - larval feeding organ consisting of mouth hooks, H Piece, medial tooth and cephalopharyngeal skeleton

This region, referred to as the anterior brain/eye anlage, gives rise to both the visual system and the protocerebrum. The anlage splits up into three main domains: the head midline ectoderm, protocerebral neurectoderm and visual primordium. Similar to their vertebrate counterparts, Hh and Dpp play an important role in the partitioning of the anterior brain/eye anlage. Dpp is secreted in the dorsal midline of the head. Lowering Dpp levels (in dpp heterozygotes or hypomorphic alleles) results in a 'cyclops' phenotype, where mid-dorsal head epidermis is transformed into dorsolateral structures, i.e. eye/optic lobe tissue, which causes a continuous visual primordium across the dorsal midline. Absence of Dpp results in the transformation of both dorsomedial and dorsolateral structures into brain neuroblasts. Regulatory genes that are required for eye/optic lobe fate, including sine oculis (so) and eyes absent (eya), are turned on in their respective domains by Dpp. The gene zerknuellt (zen), which is expressed in response to peak levels of Dpp in the dorsal midline, secondarily represses so and eya in the dorsomedial domain. Hh and its receptor/inhibitor, Patched (Ptc), are expressed in a transverse stripe along the posterior boundary of the eye field. Hh triggers the expression of determinants for larval eye (atonal) and adult eye (eyeless) in those cells of the eye field that are close to the Hh source. Eya and So, which are induced by Dpp, are epistatic to the Hh signal. Loss of Ptc, as well as overexpression of Hh, results in the ectopic induction of larval eye tissue in the dorsal midline (cyclopia). The similarities between vertebrate systems and Drosophila are discussed with regard to the fate map of the anterior brain/eye anlage, and its partitioning by Dpp and Hh signaling (Chang, 2001).

At the onset of gastrulation, the anlage that gives rise to the anterior brain (protocerebrum) and the eye, roughly defined by the expression of otd, extends from the cephalic furrow to the anlage of the foregut. In the dorsoventral axis, the anlage crosses the dorsal midline; laterally it reaches to ~50% of egg diameter where it is bounded by the ventral neurectoderm. During gastrulation and germband elongation, the anlage splits up into different components that can be recognized morphologically and with the help of molecular markers. Three main domains, the head midline ectoderm, protocerebral neurectoderm and the visual primordium, can be distinguished (Chang, 2001).

A narrow strip straddling the dorsal midline gives rise to the medial portion of the head epidermis. In the acephalic larva, these cells (and most other cells of the head epidermis) are folded inside the animal to form the dorsal pouch (Chang, 2001).

The lateral part of the head neurectoderm produces the neuroblasts that form the central protocerebrum, the major compartment of the insect brain that includes associative centers such as the mushroom bodies and central complex. A narrow domain within the dorsomedial protocerebrum is the anlage of the so-called pars intercerebralis, which contains clusters of neuroendocrine cells producing various neuropeptides. The neuroendocrine neurons project their axons in a peripheral nerve that leaves the brain and reaches the corpora cardiaca, a neurohemal organ located close to the heart. The pars intercerebralis-corpora cardiaca system is highly reminiscent of the vertebrate hypothalamus-pituitary axis, and this similarity extends to the embryonic origin of the corpora cardiaca. Thus, the corpora cardiaca arise as invaginations from the foregut. Their embryonic origin has been well documented in Manduca sexta; in Drosophila, the corpora cardiaca, along with precursors of the stomatogastric (i.e. autonomic) nervous system, also invaginate from the foregut (Chang, 2001).

The visual primordium, defined molecularly by the expression of so, is wedged in between the midline ectoderm and the protocerebral neurectoderm in the posterior head. During gastrulation and germband extension, cells of the visual primordium move laterally and are subdivided into the larval and adult eye primordia and the inner and outer optic lobe. The optic lobe and larval eye form a triangular placode that invaginates. The posterior lip of this invagination, marked by the expression of FasII, represents the primordium of the lamina and medulla, and gives rise to the lobula complex. The larval eye, or Bolwig's organ, labeled by FasII and mAb22C10, develops at the lateralmost tip of the optic lobe placode. The cells that will become the eye imaginal disc (adult eye) are anterior and dorsal to the optic lobe placode and can be recognized by the expression of eyeless (Chang, 2001).

Dpp expression and function were followed using an in situ RNA probe and an antibody against phosphorylated MAD protein (anti-pMAD), respectively. The patterns revealed by both markers in the embryonic head match closely, supporting the notion that dpp itself is a target of Dpp signaling. Dpp is expressed at the blastoderm stage in the entire dorsal half of the trunk and head of the embryo. Subsequently, the level of dpp and pMAD is elevated in a narrower dorsomedial stripe that includes the eye field. With the onset of gastrulation throughout the early extended germband stage (stages 7-10), dpp disappears from most of the head, except for an anterior domain in the anlage of the foregut, and a narrow posterior domain bordering the visual primordium posteriorly. This domain is contiguous with a dpp-expressing domain in the dorsal ectoderm of the trunk. During the late extended germband (stage 11) there appears a mid-dorsal domain of dpp expression in the posterior head, overlapping with prospective head epidermis. In addition, laterally, dpp appears in a small discrete spot in the antennal segment, immediately adjacent to the visual primordium. Based on this expression pattern, it is anticipated that the distribution of the Dpp protein in the head may be complex, and may shift during development from a dorsoventral gradient (early phase) over a posteroanterior gradient (intermediate phase) to a local point source (late phase) (Chang, 2001).

In the trunk, the effect of Dpp is inhibited in the ventral ectoderm by the Chordin homolog Sog and the transcriptional repressor Brk. Since the spatial control of the Dpp gradient in the head is likely to be influenced by the same players, the expression pattern of these genes in the embryonic head was investigated. At the blastoderm stage, sog and brk are expressed in the ventral half of the embryo along the entire anteroposterior axis. During gastrulation, expression in the head gradually spreads dorsally. At the extended germband stage sog and brk expression at a low level covers the protocerebral neurectoderm. sog disappears from the head during stage 11, while brk remains on somewhat longer. Note that the dorsal expression of sog and brk comes on later than the downturn of Dpp, which is complete with the onset of gastrulation. This suggests that the repression of dpp in the dorsal head is effected by factors in addition to Sog and Brk. Support for this hypothesis comes from the observation that in brk;sog double mutants, dpp expression does not expand into the protocerebral ectoderm, although it does cover most of the ventral ectoderm (Chang, 2001).

Loss, reduction and overexpression of Dpp in the head ectoderm results in a phenotype that can be most easily interpreted by assuming that similar to what has been postulated for the trunk, there is a graded requirement for Dpp at dorsomedial and dorsolateral levels. Reduction of Dpp function, as seen in the dpp hypomorph dpp^E87, or embryos lacking sog, results in a highly characteristic 'cyclops' phenotype. The dorsal epidermis that normally forms the dorsal pouch is absent, as evidenced by the loss of expression of the gene race that normally appears in the amnioserosa and dorsomedial head epidermis. Head epidermis is replaced by ectopic optic lobe and larval eye tissue which are exposed at the surface because head involution fails to occur. The pattern of protocerebral neuroblasts, visualized by anti-Sna antibody, is unchanged in dpp^E87, unlike the situation in dpp-null embryos where neuroblast levels are strongly increased. These findings imply that, similar to the amnioserosa of the trunk, the epidermal midline ectoderm of the head requires the highest levels of Dpp. Reduction of Dpp results in the transformation of the midline to dorsolateral structures that, in the head, are represented by the visual primordium (Chang, 2001).

A different and much more severe phenotype results from the total absence of Dpp. As in dpp hypomorphs, head midline epidermis does not form; however, instead of dorsolateral fates replacing the head midline fates, both midline and dorsolateral regions exhibit characteristics of lateral neurectoderm. Optic lobe and Bolwig's organ are absent. Neuroblasts are formed in realms of the head midline and visual system. To what dorsoventral level does the fate of the ectopic neural tissue correspond? The neurectoderm of the head gives rise to neuroblasts at ventrolateral levels (tritocerebrum and deuterocerebrum), as well as dorsolateral levels (protocerebrum). Based on the expression pattern of the markers ey, FasII and ind, it is concluded that the ectopic neuroblasts in dpp^- embryos appear to be of dorsolateral provenance. Thus, ind is normally expressed in the stage 9 wild-type embryo in a small dorsolateral cluster that gives rise to several protocerebral neuroblasts, as well as the anterior lip of the optic lobe. In dpp^-, ind-expressing cells are displaced to the dorsal midline (Chang, 2001).

The ubiquitously expressed driver line daughterless (da)-Gal4 was used to express UAS-dpp. This Gal4 line is not expressed in the blastoderm but comes on with gastrulation. Correspondingly, the resulting changes in cell fate in the head and trunk are relatively mild and can be best described in terms of a ubiquitously raised base level of Dpp, superimposed on the regular gradient of endogenous Dpp. Mid-dorsal structures, including the amnioserosa and head epidermis, were much wider than in wild type. Dorsolateral structures, including the visual primordium, are relatively normal in size and shape, but are shifted to lateral or ventrolateral levels. Ventral tissues are partially missing (Chang, 2001).

Overexpression of dpp by using the heat-inducible driver line hs-Gal4 results in a phenotype very similar to the one described for da-Gal4-driven UAS-dpp. Applying 2 hour heat pulses at different stages of development supports the idea that the phenocritical period of Dpp action is around the onset of gastrulation. Thus, a high number of embryos heat pulsed between 3 and 5 hours post fertilization show the characteristic dorsalization phenotype described above. Later heat pulses had no effect on head patterning (Chang, 2001).

The above described phenotypic effects observed in mid- and late-stage mutant embryos indicate that dorsal epidermal and visual system fates, in particular those of the posterior optic lobe and larval eye, are not expressed in dpp loss of function. It is likely that these abnormalities are the result of changes in early head gene expression. This was followed in detail by assaying the expression of several regulatory genes known to be required for the normal development of the visual primordium, including otd, tll, so and eya in dpp-null mutants:

1. otd is normally expressed in a wide domain that spans the dorsal midline and encompasses the entire dorsal head ectoderm. In normal development, its expression is turned off in the head midline (the head epidermis precursors) and in the part of the visual primordium forming the posterior optic lobe and larval eye. In dpp mutants, expression persists in the entire dorsal head ectoderm until stage 11. Expression then becomes patchy as many cells undergo apoptotic cell death (Chang, 2001).
2. tll appears in the protocerebral ectoderm, including the head midline ectoderm. Only later does expression spread to cover part of the visual primordium. In embryos that lack Dpp, expression is expanded from the beginning to include the entire dorsal head. As for otd, expression also persists in the head midline ectoderm (Chang, 2001).
3. so is expressed in a transverse stripe spanning the dorsal midline. This unpaired domain defines the eye field. Around gastrulation, so expression ceases in the dorsal midline and becomes restricted to the bilateral visual primordia. In addition to the visual system, so appears in the anlage of the stomatogastric nervous system (SNS) and head mesoderm. In a dpp-null fly, so is never expressed in the anlage of the visual system, although expression in the SNS and head mesoderm is unchanged (Chang, 2001).
4. eya is normally expressed in a complex pattern that essentially consists of three domains located in the anlage of the SNS, the anterior protocerebrum and the anlage of the visual system. In dpp-null embryos, eya expression in the primordia of the visual system and SNS is absent from the beginning. The anterior protocerebral expression is narrowed (Chang, 2001).

The observed downregulation of head gap genes and early eye genes in the dorsal midline is an indirect effect of Dpp mediated by the Dpp target zerknüllt (zen). Previous studies have demonstrated that high levels of Dpp in the dorsal midline upregulate and focus the expression of zen in the amnioserosa and, further anteriorly, in the dorsomedial head epidermis. An RNA in situ probe revealed the expression of zen in the early eye field of a stage 5-7 embryo. Assaying the expression of head gap and early eye genes in a zen-null mutant background demonstrates that Zen acts as a repressor of these genes. Whereas in wild type, after an initial unpaired expression straddling the dorsal midline, tll, so and eya are turned off in the dorsal midline, they continue to be expressed in this domain in a zen mutant. At later stages, lack of zen results in a cyclops phenotype (Chang, 2001).

Hh is expressed in metameric stripes that coincide with the posterior compartment of each segment. In the head, hh expression in the stage 5-7 embryo forms a wide stripe in front of the cephalic furrow. This stripe, which crosses the dorsal midline, includes the future antennal segment and posterior part of the visual anlage. As germ band extension proceeds, hh expression disappears from the dorsal midline and two separate bands are parceled out (antennal stripe, pre-antennal or occular stripe). The pre-antennal stripe overlaps with the lateral boundary of the visual primordium. Towards the late extended germband stage, the Hh head domain decreases in size and expression level. During stage 11 and early 12, only a small cluster of cells corresponding to the precursors of the larval eye located laterally in the visual primordium remain hh positive (Chang, 2001).

Hh signaling is negatively regulated by Ptc, a membrane linked protein that, by binding to Hh ligand, becomes inactivated in cells receiving high levels of Hh. Ptc expression in the head resembles hh expression at an early stage. A wide antennal/pre-antennal stripe traverses the head in front of the cephalic furrow. During germband extension, this domain splits into two stripes. At the late extended germ band stage, ptc remains expressed in a large domain that corresponds to the anterior optic lobe (Chang, 2001).

Loss of hh results in a strong reduction of the head midline epidermis, a reduction in the size of the brain and optic lobe, and the total absence of the larval and adult eye primordium. Temperature-sensitive shift experiments of hh^ts2 embryos indicate that the phenocritical period for Hh function in Bolwig's organ development is between 4 and 7 hours. Aside from the larval eye, the primordium of the compound eye, which is marked from stage 12 onward by the expression of eyeless (ey), is also affected by the loss of hh. Heatshock induced overexpression of hh, as well as loss of ptc, causes an increase in larval eye neurons and optic lobe precursors. Interestingly, ectopic Hh activity is able to induce optic lobe and Bolwig's organ tissue in the head midline and thereby generate a cyclops phenotype similar to the condition described above for partial reduction of dpp. Applying heatshocks at different times of development indicates that the phenocritical period for the Hh induced cyclops is early, between 2.5 and 5 hours. Thus, heat pulses administered during this time cause fusion of the optic lobe and, at a lower frequency, of the larval eye without significantly increasing the number of optic lobe and larval eye cells. By contrast, later heat pulses (after 5 hours) lead to larval eye/optic lobe hyperplasia but no concomitant cyclops phenotype (Chang, 2001).

The finding that both loss of Hh and Dpp cause the absence of visual structures, and ectopic expression of Hh and partial loss of Dpp cause transformation of head midline epidermis into visual primordium, begs the question of how the two signaling pathways interact. In Drosophila compound eye development, hh expression is required to turn on dpp expression. To establish whether a regulatory relationship exists between Hh and Dpp signaling, the expression of dpp and pMAD was examined in the background of hh loss of function, as well as hh, ptc and Cubitus interruptus (Ci) expression in the background of dpp loss of function. Cells in which Dpp signaling is activated can be visualized by an antibody against phosphorylated MAD (pMAD) protein. Dpp RNA expression and pMAD are normal in a stage 5-9 hh-null background, indicating that Hh is not required to activate Dpp signaling in the embryonic head (Chang, 2001).

The expression of hh and ptc is normal in early embryos mutant for dpp. Since ptc is a downstream target of Hh signaling, this result strongly suggests that Dpp signaling is not required to activate the Hh cascade. To show more directly whether this cascade is interrupted, the antibody AbN, which recognizes both the full-length Ci protein and the cleaved repressor form (Ci75) was used in the background of a dpp-null mutation. According to the present model, Hh function consists of preventing the cleavage of the Ci protein to generate the repressor form, which is able to enter the nucleus and inhibit transcription of target genes such as ato and/or hh. In a mutation of Ci that produces only the repressor form or in eye clones that lack hh, a higher level of Ci can be detected in the cells. In dpp-null embryos, cytoplasmic Ci signal in the visual primordium of stage 7 embryos is at the same level as in wild type, indicating that Dpp is not required for Hh signal to go through. However, it should be conceded that it is very difficult to quantify, in embryonic tissues as opposed to cultured cells, expression levels using the Ci antibodies available, which leaves open the possibility that Dpp might have a quantitative effect of on the strength of the Hh signal reaching the nucleus (Chang, 2001).

Taken together, these findings suggest that no direct interaction exists between Hh and Dpp signaling, and that the antagonistic effect of Hh and Dpp on the formation of visual structures is most probably based upon an indirect interaction between the two signaling pathways that involves the expression of the eye genes so and eya (Chang, 2001).

These results suggest that, similar to its expression in the trunk, Dpp forms a gradient that traverses the anterior brain/eye field from dorsal to ventral. In the trunk, Dpp is restricted by the maternal morphogen Dorsal to the dorsal half of the embryo. Ventrally, the Dorsal morphogen turns on the Chordin homolog sog, as well as a transcriptional repressor of Dpp-activated genes, brinker (brk). Highest levels of Dpp at a mid-dorsal level turn on or stabilize target genes such as zen, which commit cells to amnioserosa fate. Moderate Dpp levels activate pannier and other targets that specify dorsolateral fates (non-neural epidermis, tracheae). A second BMP homolog, Screw, is required with Dpp for mid-dorsal fates. The activity of sog and brk inhibits Dpp and Screw in the ventral ectoderm, thereby allowing the expression of proneural genes and the subsequent neuralization in this domain. Paradoxically, Sog potentiates Dpp function mid-dorsally (Chang, 2001).

In the head region, highest levels of Dpp are required to promote mid dorsal fates (head epidermis, analogous to amnioserosa in the trunk). The activation of screw is involved in this process, similar to its role in the dorsomedial trunk. Intermediate Dpp levels promote dorsolateral fates (visual primordium). Low levels of Dpp are reached in the protocerebral neurectoderm and are permissive for the formation of protocerebral neuroblasts. Several of the regulatory genes expressed in the anterior brain and eye field may be direct targets of Dpp signaling. The findings show that so, eya and omb are activated by Dpp in the visual primordium. These regulatory genes initiate the fate of visual structures, in particular larval eye and outer optic lobe. It has recently been shown that eya and so are also targets of Dpp signaling in the eye imaginal disc (Chang, 2001).

The secondary restriction of so (and other genes with bilateral expression domains developing from unpaired domains, including tll and otd) is effected by the Dpp target zen in the dorsal midline. This homeobox gene is expressed as a response to peak levels of Dpp in the dorsal midline, including amnioserosa and, in the head of the embryo, in the dorsomedial head epidermis primordium. Loss of zen, similar to reduction of Dpp, results in the absence of amnioserosa and head epidermis, and a cyclops phenotype (Chang, 2001).

Hh is positively required for the visual system. Loss of this gene causes the absence of the larval eye, as well as the adult eye primordium. This phenotype is reminiscent of the later requirement of Hh for the initiation of cell differentiation in the larval eye imaginal disc. Increased expression of Hh, as well as absence of the inhibitor of Hh function, Ptc, results in a cyclops phenotype (Chang, 2001).

In view of these results, it is speculated that the interaction between Dpp and Hh is indirect and requires the function of so, eya and possibly other 'early eye genes' -- according to this model, Dpp activates so and eya in the eye field. Slightly later, expression of so and eya is lost dorsomedially, due to repression by Zen at this level. In a second step, the expression of Hh (which comes on later than Dpp) triggers larval eye fate in cells close to the Hh source. The response of a cell to Hh, that is, its expression of ato, depends on its previously expressing so and eya. Finally, Ptc inhibits the range of Hh action, similar to its alleged function in the trunk and imaginal discs (Chang, 2001).

A model is proposed to explain the phenotypes resulting from manipulating Dpp, Hh and Ptc expression:

1. In wild type, Hh can activate larval eye only in cells expressing so and eya. No larval eye develops in the dorsal midline because so is down regulated in this region rapidly, and Hh 'has no opportunity' to overcome the ptc mediated inhibition and induce visual system at an early stage when so is still present in the dorsal midline (Chang, 2001).
2. In ptc^-, Hh is able to induce larval eye fate in the dorsal midline because it is not inhibited at the early stage when so is still expressed dorsomedially (Chang, 2001).
3. Heatshock-induced Hh expression at an early stage (stage 5; around 3 hours) has the same effect, overcoming the ptc-mediated inhibition and inducing larval eye dorsomedially (Chang, 2001).
4. If the level of Dpp is reduced (in dpp null heterozygote, or dpp hypomorph), so and eya are stably expressed in the dorsal midline, since zen, which normally inhibits the early eye genes, is not expressed. As a result Hh can induce larval eye dorsomedially (Chang, 2001).
5. In the cyclops phenotype that results from reduction of Dpp, the visual primordium develops as a double crested placode that spans the dorsal midline. In this placode, the posterior crest is formed by larval eye cells, in line with the tenet that Hh induces larval eye fate in the cells next to the Hh source (posteriorly). The anterior crest, which is further away from the Hh source, constitutes posterior optic lobe (Chang, 2001).
6. In the cyclops phenotype induced by loss of Ptc or overexpression of Hh, larval eye cells are increased in number, compared with the Dpp reduction induced cyclops. At the same time, posterior optic lobe cells are reduced in number (Chang, 2001).

The topology in which different derivatives of the anterior brain anlage are laid out in the early embryo exhibits considerable similarity to that of vertebrates. To appreciate this similarity, one needs to remember that the neurectoderm of insects does not invaginate. As a result, early embryonic tissues located in the dorsal midline (i.e. the head midline ectoderm) of the fly embryo remain where they are, i.e. mid-dorsally, whereas in vertebrates, they form the ventral midline of the neural tube. This inverse topology may explain in part why dorsomedial structures in Drosophila share several functional and molecular similarities with the ventral forebrain in vertebrates. For example, both give rise to neuroendocrine centers (the pars intercerebralis of the insect brain, hypothalamus of vertebrates). In both vertebrates and insects, cells that start out as epithelial placodes in the foregut anlage anteriorly adjacent to the eye field, form neurohemal structures (anterior pituitary in vertebrates, corpora cardiaca in insects) that become innervated by the neuroendocrine neurons derived from the midventral/mid-dorsal brain. The topological similarity between the eye field in Drosophila and vertebrates extends to the location of the eye. In both systems, the eye maps close to the midline and genetic manipulations affecting the midline result in the fusion of the eyes (cyclopia) (Chang, 2001).

The dorsal location of the eye field and protocerebral neurectoderm in Drosophila, as well as all extant arthropods, is not easy to reconcile with the hypothesis that the chordate body plan is derived from an arthropod/annelid-like ancestor whose dorsoventral axis is reversed, although it does not directly contradict this idea. Thus, eye field and protocerebral ectoderm of ancestral arthropods might have actually occupied a ventral position in front of the stomodeum, and subsequently shifted dorsally. However, given that no comparative-structural or fossil evidence exists for such a shift, an alternative hypothesis can be offered: the CNS of the ancestor of chordates (deuterostomes) and arthropods/annelids (protostomes) may have been restricted to the head of the animal where also sensory receptors (eyes, statocysts, chemoreceptors) are concentrated. In support of this view, nerve cells in many groups of platyhelminths, in particular Acoels (considered as the sister group of bilaterians according to recent molecular-phylogeneitc data), are exclusively derived from the anterior pole of the embryo. From this primitive anterior ganglion of the bilaterian ancestor, the protocerebrum/eye field of present day bilaterians is directly derived, with no change in dorsoventral axis. In the trunk region, which originally lacked central neurons, a central nervous system was 'added' that followed different patterns during evolution. In the line leading to higher protostomes, ganglia located ventrally were added, whereas a dorsal trunk neurectoderm formed in chordates (Chang, 2001).

Irrespective of which of the two aforementioned hypotheses regarding topology of the neural fate map will turn out to be correct, the high degree of conservation of signaling pathways and regulatory genes controlling the patterning of the fate map in Drosophila and vertebrates emphasizes how 'close' the body plans manifested during early embryogenesis still are. Dpp/BMP and Hh/Shh signaling are centrally involved in head patterning in both systems, and could have exerted this role already in the bilaterian ancestor. However, it is also true that the impact of Dpp and Hh signaling on midline and eye structures seems very different in chordates and arthropods, which makes the independent recruitment of the two signaling pathways into head patterning in these phyla a distinct possibility. In chordates, loss of Hh results in a cyclops phenotype and holoprosencephaly, since high levels of Hh are required for hypothalamus and optic stalk. Hh positively regulates Pax2, a regulatory gene expressed in and required for the optic stalk. In the Drosophila embryo, excess function of Hh causes cyclopia. Moreover, Hh has a positive effect on the Pax6 homolog, eyeless; ey expression requires the presence of the Hh signal (Chang, 2001).

The effect of BMPs/Dpp on early eye formation maybe more similar than the role of Shh/Hh signaling. In Drosophila, both at the early embryonic and larval stage, Dpp promotes eye formation and differentiation. Vertebrate BMPs are expressed in the dorsal neural tube and are required for dorsal cell fates in the spinal cord, brain and eye. In mouse, BMP2, BMP4, BMP5, BMP6, BMP6 and BMP7 are expressed in the dorsal telencephalon, a region that gives rise to the choroid plexus and dorsomedial walls of the cerebral cortex (hippocampus) and diencephalon. At a later stage, BMP7 is also expressed in the retina. Mice homozygous for BMP2 and BMP4 die long before fate changes in the forebrain can be scored. BMP7 homozygotes show a late embryonic phenotype that includes degeneration of the retina (Chang, 2001).

When comparing the expression pattern of conserved regulatory genes, such as otd, tll, so and many others in anterior brain and eye development of fruit flies and vertebrates, one is also struck by the high number of similarities. These similarities indicate that the bilaterian ancestor might have possessed a head in which photoreceptors, various brain structures and neuroendocrine cells were laid out in a way reminiscent of the pattern found in present day taxa. This obviously does not imply the existence of complex organs, such as the eye, pituitary or brain structures. What it does imply is that the bilaterian ancestor had an anterior ‘neurectoderm’ in which clusters of cells with the basic properties of photoreceptors, pigment cells, neuroendocrine cells or central neurons were positioned in a pattern reminiscent of the modern pattern formed by the progenitors of these structures in different animals. During evolution, these cell types diversified further and became shaped by morphogenetic movements into more complex organs. For example, in the chordates (including urochordates and cephalochordates), the anterior neurectoderm invaginated to form a tube that included all cells with the fate of photoreceptors, pigment cells and target neurons. In vertebrates these cells then evaginated as the optic cup, induced lens and other structures from the outer ectoderm and formed an eye. In the evolutionary line leading to arthropods, cells with the fate of photoreceptors and pigment cells were separated at an early developmental stage from cells destined to become optic target neurons. The former remained in the outer ectoderm and became organized into a compound eye, while the latter delaminated along with other neural stem cells to form the brain. The stage is set for comparative studies of eye morphogenesis and gene expression that will elucidate in more detail how a simple visual system changed into the various types of eyes that can be observed in extant animal groups (Chang, 2001).

The central neuroendocrine system in the Drosophila brain includes two centers, the pars intercerebralis (PI) and pars lateralis (PL). The PI and PL contain neurosecretory cells (NSCs) which project their axons to the ring gland, a complex of peripheral endocrine glands flanking the aorta. This paper presents a developmental and genetic study of the PI and PL. The PI and PL are derived from adjacent neurectodermal placodes in the dorso-medial head. The placodes invaginate during late embryogenesis and become attached to the brain primordium. The PI placode and its derivatives express the homeobox gene Dchx1 and can be followed until the late pupal stage. NSCs labeled by the expression of Drosophila insulin-like peptide (Dilp), FMRF, and myomodulin form part of the Dchx1 expressing PI domain. NSCs of the PL can be followed throughout development by their expression of the adhesion molecule FasII. Decapentaplegic (Dpp), secreted along the dorsal midline of the early embryo, inhibits the formation of the PI and PL placodes; loss of the signal results in an unpaired, enlarged placodeal ectoderm. The other early activated signaling pathway, EGFR, is positively required for the maintenance of the PI placode. Of the dorso-medially expressed head gap genes, only tailless (tll) is required for the specification of the PI. Absence of the corpora cardiaca, the endocrine gland innervated by neurosecretory cells of the PI and PL, does not affect the formation of the PI/PL, indicating that inductive stimuli from their target tissue are not essential for early PI/PL development (de Velasco, 2007).

The insect neuroendocrine system consists of several populations of neurosecretory cells (NSCs) with peripheral axons terminating in contact with specialized neurohemal glands where the neurohormones are released. The majority of NSCs are found in the dorso-medial protocerebrum, the so-called pars intercerebralis (PI) and pars lateralis (PL). These NSCs project their axons towards a set of small glands, the corpora cardiaca (CC), and corpora allata (CA). In Drosophila, the CC and CA, along with a third neuroendocrine gland, the prothoracic gland (PTG), are fused into a single complex, the ring gland, which surrounds the anterior tip of the aorta. The PI-PL/ring gland complex of insects has been repeatedly compared to the hypothalamus-pituitary axis in vertebrates, based on clear similarities between the two, anatomically and functionally (i.e., their shared role in energy metabolism, growth, water retention, and reproduction). Previous studies of the insect neuroendocrine system have focused on the neurosecretory cells of the PI and PL; however, not much is known about the different types of non-secretory PI neurons, and even less information exists about the development of this important part of the insect brain. This paper focused on the formation of the PI during Drosophila embryonic and larval development (de Velasco, 2007).

The PI is histologically recognized as the unpaired antero-medial domain of the protocerebral cortex that is located anterior of the calyces of the mushroom bodies and dorsal of the central complex. Beside innervating the neuroendocrine glands and thereby acting as the uppermost center of endocrine release, the neurites of PI neurons are structurally and functionally integrated into the medial compartments of the protocerebrum. Some of the cells of the PI were shown to play a role in various behaviors, including locomotor activity and flight behavior. Moreover, an early role of certain PI cells as pioneer neurons during the formation of protocerebral axon tracts has also been reported (de Velasco, 2007).

The extremely limited information about the development of the PI stems mainly from studies on grasshopper and Drosophila. For grasshopper, the dorso-medial subpopulation of brain neuroblasts, measuring approximately 20 in number, was tentatively assigned to the formation of the PI, although detailed lineage studies have not yet been carried out. Three groups of cells that form part of the adult PI have been followed in more detail from embryonic stages onward. These include (part of) the NSCs, a group of unpaired cells derived from a cell called the 'dorsal midline progenitor', and a small set of early differentiating neurons expressing the antigen TERM-1 that act as pioneers of the brain commissure (de Velasco, 2007).

Cells situated along the antero-dorso-medial edge of the population of brain neuroblasts can be assumed to produce neuronal offspring that gets incorporated into the PI, although specific lineages have not yet been followed. The neurectoderm of the so-called 'head midline', which gives rise to at least part of (if not the entire) PI was described as a specialized region with morphological and molecular similarities to the ventral (trunk) midline (mesectoderm). Both trunk and head midline domains contain neuronal progenitors that do not delaminate as individual cells like the neuroblasts of the lateral neurectoderm, but that invaginate as an elongated furrow (ventral midline) or as several separate placodes (head midline). Furthermore, both trunk and head midline require the activity of EGFR signaling for survival and fate specification. Another molecular characteristic shared by the midline of the trunk and head is the extended expression of the neurogenic genes of the E(spl) complex. These genes are activated in the neurectoderm (in general) by Notch signaling, and are responsible for inhibiting proneural gene expression, thereby mediating the lateral inhibition process that delimits the number of neuroblasts delaminating at any given position from the neurectoderm. At the stage when no more neuroblasts are born, Notch signaling and the expression of E(spl) transcripts cease. This happens around stage 11 in the lateral neurectoderm of the trunk and the head. However, in the midline domains, expression continues far beyond that stage, up until stage 14. This finding was interpreted to indicate a temporally extended neurogenic potential of the midline cells. In other words, neurectodermal cells along the head midline are still neurogenic at a stage when other ectodermal cells are already on their way to become epidermal cells (de Velasco, 2007).

A comprehensive developmental study of the Drosophila pars intercerebralis and pars lateralis has been undertaken with the aim of understanding their embryonic origin from the dorsal head midline, developmental morphogenesis, and architecture. The PI, marked by its continued expression of the homeobox gene Dchx1, is derived from a placode located anteriorly in the neurectoderm of the dorso-medial head. Three additional placodes are situated posterior to the PI placode; one, marked by the expression of FasII, gives rise to a cluster of neurons that form the pars lateralis (PL), which represents the second domain within the brain that contains neurosecretory cells). The remaining two placodes express the homeobox gene drx) and become part of the protocerebum surrounding the PI and PL. This study further investigated the role in PI development of early acting signaling pathways (Dpp, EGFR) and transcription factors expressed in the dorso-medial head neurectoderm [tailless (tll), ventral nerve cord defective (vnd), single minded (sim); orthodenticle (otd)] and in the corpora cardiaca [sine oculis (so), glass (gl), forkhead (fkh)] in the PI placode. The findings provide a developmental genetic framework for the study of the Drosophila central neuroendocrine system (de Velasco, 2007).

Pars intercerebralis: structure and development during the postembryonic phase: To analyze the PI in Drosophila larvae, pupae, and adult, brains were used in which GFP was driven by Chat-Gal4, which is expressed in most differentiating neurons, including their axons. This staining visualized most, if not all neuronal cell bodies and their proximal axons that comprise the PI. The PI of the adult and 3 day pupa appears as an unpaired cluster of cell bodies filling the cleft between the brain hemispheres. At most positions along the antero-posterior axis, it is well demarcated from the lateral cortex by a glial lamella. The following will distinguish the anterior PI (PIa), located around the medial lobes of the mushroom body, from the central PI (PIc; dorsal to the central complex) and posterior PI (in between central complex and protocerebral bridge). Most PIa neurons are small and weakly Chat-GFP-positive; in addition, one can distinguish a subset of large, strongly Chat-GFP-positive somata with axons that pass through the superior medial protocerebrum and form the NccI nerve that projects to the ring gland. Based on their trajectory, these large neurons comprise the neurosecretory cells of the PI. The central PI (PIc) is comprised of neuronal somata that form a rather homogenous population in regard to size and Chat-GFP expression levels. Axons of PIc neurons turn laterally and appear to branch in the posterior part of the superior-medial protocerebrum (called DP compartment in the larva). Neurons of the posterior PI (PIp) are small and densely packed; many PIp neurons form axons that fasciculate in a fiber tract projecting straight ventrally and then anteriorly into the fan-shaped body. One cannot define a clear boundary between the PIp and the laterally/posteriorly adjacent protocerebral cortex (de Velasco, 2007).

It is clear from the above said that the PI is formed by neurons that contribute to many different brain circuits, and that much work is needed to elucidate the exact structure and function of these cells. Do all of these cells share a common origin? In other words, can one define one or more specific neural lineages whose cells exclusively contribute to the PI? And is there an ontogenetic relationship between the PI and the PL, the second cluster of neurosecretory cells that, without specific markers, cannot be recognized within the brain of the larva, pupa or adult? To approach these questions, the development of the PI was followed backward in time. In the early pupa and larva, all components of the PI defined in the previous section for the late pupa and adult can be recognized; however, they do not form an unpaired cluster in the brain midline, but are split into bilaterally symmetric clusters. Thus, as an anatomical entity, the pars intercerebralis does not exist in the larva, but evolves during the pupal period (de Velasco, 2007).

In the one day-old pupa, cells with the characteristics of the anterior PI (strongly Chat-GFP-positive neurons located dorsal of the medial lobes) form axons that branch in the DA and CA compartments and continue peripherally as the NccI nerve (Ncc stands for nervus corporis cardiaci). In the central PI, large, strongly Chat-GFP-positive cells cover the developing ellipsoid body and fan-shaped body. The same picture presents itself in the wandering larva. Besides their large size and high expression level of Chat-Gal4, neurons of the PIa and PIc are distinguished from the lateral cortex by lacking any Neurotactin expression. Neurotactin expression defines a distinct, transient stage in neural differentiation. Thus, all primary neuroblasts and neurons of the late embryonic brain express neurotactin, to then lose it in the early larva. When neuroblasts become reactivated and produce secondary neurons, these also express Neurotactin until mid-pupal stages. The fact that the larval PIa/c has no Neurotactin-positive cells says that this domain consists only of primary neurons, or is characterized by a distinct mode of Neurotactin expression that sets it apart from other neurons. In either case, it serves as a suitable global marker of the larval PIa/c. Using the neuron-specific marker Elav supports the view that the PIa/c consists mainly of primary neurons which can be distinguished from undifferentiated secondary neurons by their large cell and nuclear size: anti-Elav reveals large neurons in the PIa/c which contrast sharply from the small secondary neurons of the laterally adjacent cortex (de Velasco, 2007).

As expected from its adult and late pupal morphology, the posterior PI (PIp) is difficult to define with any confidence in the larval brain. Neurons in the posterior-medial cortex, around the emerging commissure of the lateral horn which served as a topological landmark for the PIp at later stages, have axons directed anteriorly towards the central complex primordium. These neurons, which should include the cells comprising the later PI, belong to several lineages of secondary neurons of the DPM group. Without more specific markers, it is not possible to ascertain exactly which DPM lineages produce the posterior PI, and whether these lineages contribute only to the PI, or to laterally adjacent parts of the brain as well (de Velasco, 2007).

Definition of the PIa/c as a expression domain of the homeobox gene Dchx: The vertebrate gene Chx10 (also called Vsx) appears in the anlage of the forebrain during neurulation and is later expressed and required for neurons of the retina. The Drosophila genome contains two closely associated Chx10 homologs, Dchx1 and Dchx2. Their expression profile shows similarities to the expression of their vertebrate counterparts. Dchx1 appears in the early anlage of the visual system (the optic lobe placode/optic anlage of the late embryo and larva), as well as in differentiating neurons of the optic lobe. In addition, Dchx1 is expressed in a dorso-medial domain of the brain that overlaps with the PIa/c, and at least in part, the PIc as defined above. Thus, large Dchx-positive primary neurons are located anterior and dorsal of the medial lobe of the mushroom body and are flanked by the secondary lineages of the DAM and DPMl groups, respectively. Based on these criteria, the Dchx-positive neurons are recognized as the larval PIa/c as described above. Dchx-positive primary neurons of the PIa/c can be followed backward in time through early larval stages. Posterior to the group of large PIa/c neurons, a cluster of small Dchx-positive cells are found that overlap with the group of DPMm lineages. It is speculated that these neurons represent the primordium of the posterior PI. As expected, the Dchx-positive secondary neurons of the PIp are born in the late larva, when DPM lineages (along with most other lineages of the central brain) proliferate. In early larval and embryonic brains, only few scattered Dchx-positive cells appear in the postero-medial brain (de Velasco, 2007).

The expression of GFP driven by a Dchx promoter construct visualizes the projection pattern of the Dchx-positive neurons populating the PI. Most, if not all of these cells conform to a relatively simple commissural pattern, whereby axons cross in the anterior part of the supraesophageal commissure. Axonal and dendritic branches form a dense plexus that fills out the DA and CA compartments. A subset of PI neurons, notably those that co-express Drosophila insulin-like peptide (Dilp) form axons that leave the dorso-medial brain and project to the ring gland, as well as the subesophageal ganglion/tritocerebrum (de Velasco, 2007).

Neurosecretory cells targeting the ring gland lie within the PIa/c: The larval PI includes most, if not all, of the neurosecretory cells that project towards the corpora cardiaca/corpora allata through the NccI. Larval brains were labeled with antisera against Dilp, FMRFamide, and myomodulin. In all of these experiments, peptidergic neurons with axons to the ring gland were located in the Dchx-positive/neurotactin-negative PIa/c domain. Peptidergic axons branch in the CA/DA compartments, cross the midline, and then extend posterior, passing underneath the supraesophageal commissure. This nerve connection constitutes the NccI nerve. The axonal marker FasII reveals the NccI has already developed in the late embryo. A second connection between the dorso-lateral protocerebrum and the ring gland, called NccII, also expresses FasII from embryonic stages onward. The FasII-positive neurons that give rise to the NccII are not part of the PI, but instead turn out to represent the Pars lateralis (PL), which occupies a position in the dorsal brain cortex, anteriorly adjacent to the calyx of the mushroom body. The NccII root emitted by the PL forms a conspicuous tract of the larval brain that passes the calyx and peduncle medially before reaching the medial edge of the protocerebrum where it joins the NccI on its way to the ring gland (de Velasco, 2007).

The PI and PL are derived from a series of neuroepithelial placodes in the embryonic head: The Dchx-expressing cells of the PIa/c can be followed into the early embryonic period when they form a narrow cluster of approximately 40-50 cells in the antero-medial procephalon, right behind the furrow that separates the procephalon from the clypeolabrum. Two similar-sized domains are labeled by FasII and the Drosophila Rx homolog, Drx. Together, these three markers define linearly arranged, non-overlapping domains along the dorsal midline of the procephalon. The Dchx domain will be called 'embryonic PIa/c'. During later embryogenesis, a global morphogenetic movement shifts all of these domains posteriorly; at the same time, the FasII and Drx-positive domains also move laterally. Dchx remains strongly expressed in a cohesive cluster that develops into the larval PIa/c. It is currently unclear whether the secondary neuroblast(s) that later give rise to the neurons of the PIp are derivatives of the PIa/c, or whether they are recruited from outside this domain. In late embryos, faintly Dchx-positive neurons and neuroblasts can be recognized outside the PIa/c (not shown) (de Velasco, 2007).

Cells of the FasII-positive domain give rise to the NccII and therefore constitute the embryonic primordium of the PL. In the late embryo, shortly before the somata of these neurons lose FasII expression, one can follow a FasII-positive axon tract from the FasII cluster towards the primordium of the ring gland. This tract represents the embryonic NccII, given that it exhibits the same topographical characteristics as the NccII of the larva (point of origin in postero-lateral protocerebrum; passing the mushroom body towards medially; joining nascent NccI towards ring gland) (de Velasco, 2007).

The PI, PL and Drx domains visible in the mid-stage embryo (stages 11-13) form neuroepithelial placodes that split by invagination from the procephalic ectoderm. This mode of neurogenesis differs from the mechanism of delamination that produces all other brain neuroblasts within the surrounding neurectoderm. The use of anti-Crumbs (anti-Crb) as a marker for apical membrane domains of epithelial cells reveals a stereotypic pattern of small invaginations in the dorso-medial procephalon of stage 12/13 embryos. The anterior invagination ('1') is formed/surrounded by Dchx-positive cells and corresponds to the PIa/c; the second, intermediate invagination ('2') lies in the FasII-positive PL, and two posterior invaginations, one medially ('3'), one further laterally ('4'), flank the Drx domain. During stage 13/14, the invaginations pinch off the surface ectoderm and form small vesicles in between the outer epithelium (the nascent head epidermis) and the brain surface (de Velasco, 2007).

Two additional markers, a reporter construct of the E(spl)m5 gene and the neuronal differentiation marker Elav, were used to further characterize the placodeally derived PI/PL and Drx domain. The 'dorsal head midline', a domain that, along with the midline of the trunk, is set apart from the lateral neurectoderm by the prolonged expression of the Notch target E(spl), as well as the expression of/dependence on EGFR signaling. Triggered by Notch activation, E(spl) is expressed in the neurectoderm as long as this layer is 'active', i.e., produces neuroblasts. The phenomenon of prolonged E(spl) expression was interpreted as an indication for an extended period of neurogenic potential (de Velasco, 2007).

The previously defined dorsal head midline coincides with the PIa/c and PL, as evident from the double labeling experiments using the E(spl)-m5 reporter, anti-FasII and anti-Crb. E(spl)m5 is expressed in these domains throughout late embryogenesis into the early larval period. Furthermore, double-labeling with anti-Elav demonstrates that neuronal differentiation begins relatively late in these domains. Throughout stages 12, 13 and much of 14, the E(spl)m5-positive domains remain Elav-negative; Elav signal comes up faintly during stage 15, and only in the stage 16 embryo do most neurons of these domains express Elav (de Velasco, 2007).

The placodeally derived, Crb-positive vesicles remain visible at the brain surface until late embryogenesis (stage 16). Subsequently, the cells lose Crb expression; it is assumedd that the intermediate and posterior vesicles ('2-4') convert into neurons that become incorporated in the brain, although this needs to be definitively shown. The anterior pair of vesicles ('1') appears to become incorporated into the corpora allata, the dorsal most part of the ring gland. The early development of this endocrine gland which secretes juvenile hormone during larval stages has not been clearly documented for Drosophila. In other insects, ectodermal placodes that invaginate from the ectoderm of gnathal segments were observed to give rise to both prothoracic glands (source of ecdysone) and corpora allata. The present data, which need to be substantiated by additional markers, indicate that at least part of the CA is derived from the PIa/c. Thus, in the late embryo, the anterior vesicles, still expressing Crb and E(spl)m5, approach each other and eventually fuse in the midline, forming a cluster of cells that moves posteriorly, behind the level of the brain commissure, and becomes incorporated into the dorsal part of the ring gland (de Velasco, 2007).

Genetic specification of the PI: The dorsal procephalic ectoderm that gives rise to the PI is patterned by several signaling pathways, in particular the Dpp and DER pathway. Furthermore, the head gap genes tailless (tll) and orthodenticle (otd) are expressed in the dorsal procephalon. The Tll expression domain includes the PI/PL placodes; these placodes, on the other hand, are not part of the Otd domain. The midline determinant Single minded (Sim) is expressed faintly from stage 13 onward in a subset of cells in the PI primordium. Ventral nerve cord defective (Vnd) is expressed in a longitudinal medial domain of the ventral neurectoderm flanking the mesectoderm. The paramedian stripe of Vnd expression continues in the head, but ends laterally/ventrally of the PI/PL (de Velasco, 2007).

Loss of Dpp results in the absence of the dorso-medial head epidermis that in wild-type separates the bilateral PI anlagen from each other. The PI anlagen are enlarged and fused in the dorsal midline. In DER mutants, Dchx expression is virtually absent, supporting the previously reported finding that the dorso-medial procephalic ectoderm ('dorsal head midline') requires DER signaling. Dchx is also eliminated in tll mutants, but is present, if possibly reduced, in embryos mutant for otd and other head gap genes (de Velasco, 2007).

Aside from these genes, which are expressed in the PI anlage, the possibility was tested of inductive interactions between the PI and neighboring tissues, in particular the foregut and stomatogastric nervous system (which transiently contacts the dorso-medial procephalic ectoderm) and the corpora cardiaca, which receive axons from the PI derived neurons. To remove the foregut, a null mutation was used in the fkh gene; the stomatogastric nervous system was eliminated by a mutation in so, and the corpora cardiaca by a mutation in gl. The results indicate that none of these genetic manipulations grossly affects the formation of the PI. The gl null mutation survives until the late larval period, which gave an the opportunity to analyze the structure and innervation of the ring gland that lacks the corpora cardiaca. Both Dilp and FasII-positive axons reach the ring gland and send axons towards the corpora allata, the dorsal part of the ring gland, which is unaffected in gl mutants. The only abnormality in ring gland innervation was the defasciculation and aberrant projection of FasII and FMRFamide-positive axons. In particular, FasII-positive axons frequently followed Dilp fibers onto the dorsal vessel, a behavior not observed in wild-type. The number of FMRFamide-positive axons reaching the corpora allata was reduced (de Velasco, 2007).

Definition of the pars intercerebralis and pars lateralis in Drosophila: According to the classical anatomical definition that applies to adult insect brains, one can recognize the PI as an unpaired cluster of neuronal cell bodies located along the dorsal midline of the brain. In its anterior and intermediate part (PIa/c), the pars intercerebralis is clearly set apart from the adjacent lateral cortex by a glial lamella; at posterior levels, the boundary between the PI and neighboring cortex domains is fluid. The PI is comprised of several hundreds of neuronal cell bodies that include as a relatively small minority large NSCs with axonal projections to the ring gland. The PI as an unpaired midline structure appears first during the late pupal phase. Before that stage, cells of the PI form bilateral clusters in the dorso-medial cortex of both brain hemispheres. Two criteria allowed recognition the PI at these stages and follow it backward into the embryonic period. One was the expression of the homeobox gene Dchx1, the other the idiosyncratic proliferatory properties of the PI primordium. Dchx1 is expressed in bilateral placodes in the antero-medial neurectoderm of the early embryonic head. These placodes form the early primordium of the PI. In the late embryo, they move interiorly and become part of the dorso-medial brain cortex. During later larval stages, the PI primordium, aside from the continued expression of Dchx1, sets itself apart from the lateral cortex by the absence of stem cell-like neuroblasts producing secondary lineages. Instead, the PI primordium appears to grow, at a rather slow rate, by symmetric cell division. During metamorphosis, the cortex and neuropile of the brain hemispheres fuse, giving rise to the unpaired median PI and central complex (de Velasco, 2007).

As often in development, it is difficult to state with any certainty whether the boundaries of the PI as defined in the adult coincide precisely with those visible earlier. In other words, it cannot be presently stated with any certainty that the sharp PI boundary defined, in the adult and late pupal brain, by glial septa coincides with the Dchx-positive cluster of the embryo and larva. It can be said, however, that many, if not all, of neurosecretory cells of the classically defined PI fall within the Dchx-expressing cell cluster. This was demonstrated in this study for Dilp, FMRFamide, and myomodulin; it can be said confidently that the NSCs of the PI expressing other peptides will also be included within the Dchx-positive domain (de Velasco, 2007).

The pars lateralis (PL) has been defined as a cluster of NSCs that lie outside the PI, and whose peripherally projecting axons form the NccII. The NccII can be recognized from early embryonic stages onward by its expression of the adhesion molecule FasII. The FasII-positive cells that give rise to the NccII, and that therefore should be considered as the primordium of the PL, are derived from a neurectodermal placode located posteriorly adjacent to the placode that forms the PI. It may be significant that both PI and PL, the centers including neurosecretory cells, are derived from placodeal neurectoderm (de Velasco, 2007).

A third set of placodes appear in the dorso-medial protocerebral neurectoderm, posterior to the FasII-positive PL placode. The posterior placodes, at least partially, overlap with the main expression domain of the homeobox gene Drx. Drx-positive cells forming within this region can be followed into the larval stage. They spread out over a relatively large area of the dorso-posterior cortex. The expression of Drx may be significant given the fact that the vertebrate homolog of this gene is expressed and required in the primordium of the hypothalamus. However, in Drosophila, the relationship of the posterior placodes and their Drx-positive derivatives to the PI/PL is not clear. It is possible that non-ring gland-associated NSCs are derived from it; in addition, the drx-positive neurons may be functionally and anatomically closely connected to the PI/PL (de Velasco, 2007).

Origin of PI/PL from neurectodermal placodes: This paper has shown that the PI/PL originate as placodes from the dorso-medial neurectoderm of the head, in a way that is similar to the formation of the optic lobe. In all of these cases, small domains of the neurectoderm are seen which, during stages 10 or early 11 of development, adopt the shape of placodes, with cells elongating in the apico-basal axis and expressing a higher level of apical markers such as Crb at their apical surface. Eventually, all of these placodes invaginate and sever their connection to the ectoderm several hours after their initial appearance (stages 12-13). Subsequently, cells of the placodes lose their epithelial phenotype and directly turn into neural cells (as in the case of the PI placode, or the SNS placodes), or give rise to neuroblasts (as in the case of the optic lobe). In addition, during the interval between their first appearance and invagination, the placodes give rise to 'early neural progenitors' which delaminate from the surface and move inside. For example, the optic lobe placode gives rise to at least four neuroblasts that delaminate during stage 11 and then proliferate, like all other neuroblasts, in a stem cell-like manner. Similarly, individual neurons delaminate from the SNS placodes before these structures invaginate. It is considered likely that the dorso-medial placodes described in this study also give rise to several neuroblasts (de Velasco, 2007).

Most neurons of the insect brain are formed as part of fixed lineages, each lineage being produced by a stem cell-like neuroblast. Neuroblasts delaminate as individual cells, or small clusters of cells, from the neurectoderm, leaving behind other cells that then become specified and differentiate as epidermal cells. This peculiar mode of neural cell birth and proliferation is a derived feature found in insects and many crustaceans; it is not present in taxa considered basal in the arthropods, and taxa outside the arthropods. Early neurogenesis in chelicerates, myriapods and chilopods have recently been analyzed, and led to the interesting discovery that in these animals, the neurectoderm produces a large array of small placodes which subsequently invaginate and (after some additional rounds of mitosis) turn into the neurons and glial cells of the ventral nerve cord and brain. In terms of number and pattern, the placodes are comparable to the array of neuroblasts in insects, leading to the speculation that one might be able to define homologies between individual placodes and neuroblasts. To go a step further, one could speculate that at the root of arthropods, the neurectoderm was subdivided into a mosaic of small domains, each of which invaginated as a placode to then give rise to a specific part of the CNS. In time, this mode of neurogenesis was supplanted by the 'invention' of stem cell-like neuroblasts: instead of the entire placode invaginating, a single (or a few) cell(s) was selected from the placode at an early stage which then delaminated and continued to proliferate in an asymmetrical, stem cell-like manner. If this interpretation of neuroblasts vs. placodes among arthropods is correct, one would have to conclude that the occurrence of placodes along the ventral midline and head midline (as well the stomatogastric nervous system and optic lobe) of insects represents the phylogenetically older mode of neurogenesis. Likewise (and this notion is of course even more speculative), one could argue that molecular mechanisms at work in these placodes or the function of brain parts derived from them is phylogenetically more ancient compared to structures developing from neuroblasts. The same rationale has traditionally been put forward to argue that the 'fringe domains' of the cerebral cortex, including the archicortex (hippocampus) and paleocortex (entorhinal cortex) constitute the phylogenetically older regions of the mammalian brain (de Velasco, 2007).

Pars intercerebralis and hypothalamus: Similarities between the neuroendocrine system of vertebrates and arthropods on the structural, functional and developmental level have been emphasized in many previous studies. In both vertebrates and arthropods, the highest command center of the neuroendocrine system is comprised of groups of NSCs located in the brain; these cells, besides innervating brain centers and thereby influencing neural circuits as 'neuromodulators', send their axons to peripheral neurohemal glands in which the hormones produced by the NSCs are stored and released. In vertebrates, neurosecretory cells are located in the hypothalamus. The endocrine gland they act upon is the pituitary. The corresponding structures in arthropods would be the PI/PL and their peripheral targets, the CC/CA, respectively. The main hormone produced by the CC is adipokinetic hormone (AKH) that mobilizes lipids and carbohydrates from the fat body. AKH shares common functions with the vertebrate hormone glucagon that is produced in endocrine cells of the pancreas, as well as peptidergic neurons in the brain. AKH also shows some sequence similarity with the N-terminus of glucagons. Similar and possibly homologous to the relationship between Drosophila insulin-like peptides and AKH, the function of glucagon in vertebrates is antagonized by insulin. Insulin itself is expressed like glucagon in the endocrine pancreas, but a whole family of insulin-like growth factors is found in hypothalamic NSCs. Other neuropeptides found in NSCs of hypothalamus and PI alike are FMRFamides and tachykinins. Also, the sequence similarity between vertebrate CRF and insect CRF-like diuretic hormone deserves attention in this context. Here a scenario might be considered in which ancestrally a peptide directly exerted a diuretic effect, a condition maintained in the arthropod line of evolution; in the line of evolution leading up to chordates, other hormones (ACTH, aldosterone) were 'interpolated' between the original peptide (CRF) and the action on excretory cells (de Velasco, 2007).

Developmental similarities between vertebrate hypothalamus and arthropod PI are also strong. The anlagen of the pituitary and hypothalamus are neighboring structures within the anterior neural plate. Cells that will give rise to the anterior lobe of the pituitary (adenohypophysis) are anteriorly adjacent to the cells which will become the hypothalamus and the posterior pituitary. Numerous signals were found to be involved in delimiting the anlage of the neuroendocrine system within the anterior neural plate; they include Sonic hedgehog (Shh), members of the bone morphogenetic protein family (BMP7), and fibroblast growth factor family (FGF4/8). Among the molecular determinants that are switched on by these signaling pathways are the homeobox genes six3/6 and Rx, the paired-box genes pax6 and Nkx2.1/2, the PAS-bHLH gene sim1, and the orphan nuclear receptor Tlx. Genes acting further downstream in determining specific hypothalamic cell fates are POU III-related homeobox genes Brn-1, 2, and 4. It has been shown that loss-of-function mutant mice lacking Brn-2 do not develop part of the hypothalamus; Sim1 knock-out mutations in mice cause a similar phenotype to that one for Brn-2 (de Velasco, 2007 and references therein).

This study has shown that homologs of three of the transcription factors expressed in the anlage of the vertebrate hypothalamus also appear in or adjacent to the anlage of the Drosophila PI/PL: the Nkx2.1/2 homolog vnd, the Sim1 homolog sim, and the Rx homolog Drx. In addition, the Six3/6 homolog optix also appears to overlap with the PI/PL of the st.11 embryo. The role of these Drosophila genes in PI development awaits further study. A previous paper (DeVelasco, 2004; see Embryonic development of the corpus cardiacum, a component of the ring gland) has shown that the Six1 homolog sine oculis (so) is expressed and required for the formation of the CC and the ontogenetically closely related stomatogastric nervous system. In contrast, counterpart(s) of Dchx1, the gene that is expressed strongly and continuously throughout Drosophila PI development, apparently do not play a role in neuroendocrine development in vertebrates. Detailed expression studies of Chx10/Vsx1 documented that this gene is expressed in numerous tissues outside of the retina, notably the ventral hindbrain, the diencephalic-mesencephalic boundary, and the epithalamus. However, neither expression nor function of Chx10/Vsx1 in the ventral diencephalon has been reported (de Velasco, 2007).

In conclusion, this study presents evidence for a number of conserved properties in the way the progenitors of the central neuroendocrine system in vertebrate and Drosophila embryos are spatially laid out, and employ cassettes of signaling pathways and fate determinants. One may speculate that there existed in the common bilaterian ancestor a simple anterior brain with which sensory afferents and groups of neurosecretory cells were associated. These cells might have played pivotal roles in feeding behavior (olfactory/gustatory perception of food sources; feed back information from the intestinal tract and body cavity regarding the degree of urgency of feeding) and reproductive behavior, and could have evolved into the much more complex neuroendocrine systems that is found in today's highly derived bilateria, such as insects and vertebrates (de Velasco, 2007).

This study provides a genetic model of adult mouthpart development in Tribolium castaneum based on 13 genes. While previous studies have examined patterning in species with derived mouthpart morphologies, T. castaneum retains the pleisomorphic, mandibulate state of insect mouthparts. These results demonstrate the conservation of many gene functions in the maxilla and labium, relative to the legs, thus supporting the interpretation of novel gene functions in groups with derived mouthpart morphology as indicative of their specialized morphogenetic roles in those species. Mandibulate mouthparts such as those of T. castaneum include medial maxillary and labial endites, and the current data are consistent with hypotheses of reiteration in the PD axis and specification by Notch signaling, but rule out a direct role for dac in branch generation or patterning at metamorphosis. Additionally these results demonstrate that a regulator of distal leg development, Krn, which encodes an EGF ligand, is required for normal mandible elongation. This finding underscores the complex relationship between homology at the levels of anatomy and developmental patterning (Angelini, 2012).

Angelini, D. R., Smith, F. W., Aspiras, A. C., Kikuchi, M. and Jockusch, E. L. (2012). Patterning of the adult mandibulate mouthparts in the red flour beetle, Tribolium castaneum. Genetics 190(2): 639-54. PubMed Citation: 22135350

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De Velasco, B., Shen, J., Go, S. and Hartenstein, V. (2004). Embryonic development of the Drosophila corpus cardiacum, a neuroendocrine gland with similarity to the vertebrate pituitary, is controlled by sine oculis and glass. Dev. Biol. 274(2): 280-94. PubMed Citation: 15385159

de Velasco, B., Erclik, T., Shy, D., Sclafani, J., Lipshitz, H., McInnes, R. and Hartenstein, V. (2007). Specification and development of the pars intercerebralis and pars lateralis, neuroendocrine command centers in the Drosophila brain. Dev. Biol. 302(1): 309-23. PubMed citation: 17070515

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Suzuki, Y., Squires, D. C. and Riddiford, L. M. (2009). Larval leg integrity is maintained by Distalless and is required for proper timing of metamorphosis in the flour beetle, Tribolium castaneum. Dev. Biol. 326: 60-67. PubMed Citation: 19022238

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Genes involved in head morphogenesis

Genes involved in organ development

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