The Interactive Fly

Genes involved in tissue and organ development

The Drosophila head

Structure of the head

Genetic determination of head segment identity

The role of cell death in head morphogenesis

Anatomical terms and images

The function of Dpp and Hh in partitioning the embryonic dorsal head neurectoderm

Specification and development of the pars intercerebralis and pars lateralis, neuroendocrine command centers in the Drosophila brain: The PI and PL are derived from adjacent neurectodermal placodes in the dorso-medial head

Patterning of the adult mandibulate mouthparts in the red flour beetle, Tribolium castaneum

Cell-autonomous and non-cell-autonomous function of Hox genes specify segmental neuroblast identity in the gnathal region of the embryonic CNS in Drosophila

Progressive derivation of serially homologous neuroblast lineages in the gnathal CNS of Drosophila

Patterned contractile forces promote epidermal spreading and regulate segment positioning during Drosophila head involution

Notch-dependent epithelial fold determines boundary formation between developmental fields in the Drosophila antenna

Drosophila Pax6 promotes development of the entire eye-antennal disc, thereby ensuring proper adult head formation

Wnt6 is required for maxillary palp formation in Drosophila


Genes active in the head



The structure of the head

It has been thought that there are seven head segments (technically called parasegments): four are pregnathal (anterior to the mouth), and three are gnathal [Images]. Behind the last head segment (labial) is the prothoracic segment (thoracic 1).

Based on an analysis of engrailed expression, the currently accepted model for head structure differs slightly from this model. The structure of the insect head was examined in the insect orders Diptera (Drosophila), Siphonaptera (the flea), Orthoptera (the cricket) and Hemiptera (milkweed bug). The results of this comparative embryology in conjunction with genetic experiments on Drosophila lead to the following conclusions: (1) The insect head is composed of six Engrailed accumulating segments, four postoral (intercalary, mandibular, maxillary and labial) and two preoral (ocular and antennal). The potential seventh and eighth segments (clypeus or labrum) do not accumulate Engrailed (The exception is Drosophila. Among the six insect orders studied, the Drosophila is the only one showing expression in the clypeolabrum). (2) The structure known as the dorsal ridge (associated with the eye-antennal disc) is not specific to the Diptera but is homologous to structures found in other insect orders. (3) A part of this structure is a single segment-like entity composed of labial and maxillary segment derivatives which produce the most anterior cuticle capable of taking a dorsal fate. The segments anterior to the maxillary segment produce only ventral structures. (4) As in Drosophila, the process of segmentation of the insect head is fundamentally different from the process of segmentation in the trunk. Whereas segmentation in the trunk is driven by gap and pair rule genes, in the head it is driven by head gap genes as well as engrailed, and wingless. (5) The pattern of Engrailed accumulation and its presumed role in the specification and development of head segments appears to be highly conserved while its role in other pattern formation events and tissue-specific expression is variable. An overview of the pattern of Engrailed accumulation in developing insect embryos provides a basis for discussion of the generality of the parasegment and the evolution of Engrailed patterns. It is concluded that the parasegment may not be the fundamental unit of pattern. The parasegment is clearly not the primary unit of homeotic gene expression (Rogers, 1996).

Pregnathal head segments (including brain segments) and their identifying numbers

Gnathal segments and their identifying numbers

The genetic determination of head segment identity

The head segments are defined early in embryonic development by the combined activity of more than a dozen genes. The first three segments are initially defined by the torso pathway, and then further subdivided by giant (defining the clypeolabral), tailless (defining the acron), and orthodenticle, empty spiracles, buttonhead and sloppy paired defining the antennal segments, with empty spiracles and buttonhead expressed in subdivisions (Finkelstein, 1991). Expression of labial with empty spiracles, sloppy paired, and buttonhead drives intercalary development. The first segment of the gnathal region, giving rise to the mouth appendage is the mandible. Deformed promotes mandibular development in combination with cap'n'collar, buttonhead and sloppy paired, but only in the absence of teashirt. Deformed expression along with spalt promotes development of maxillary structures. Sex combs reduced (along with spalt, giant and hunchback) drives labial development. Behind the labial segment is the prethoracic segment (T1). In conjunction with tsh, Scr drives prothoracic development (Mohler, 1995 and Finkelstein, 1991).

The head is regulated independently of the anterior midgut, that requires forkhead and huckebein. engrailed is expressed in the posterior of each of these segments, and wingless is expressed in the anterior (Schmidt-Ott, 1993), thus defining the borders of each segment.

The laterally symmetrical pregnathal region of the adult head is largely formed from the fusion of the two eye-antennal discs while the gnathal region is derived from labial discs. The dorsal region of the head, derived from the eye-antennal discs includes is occupied by a characteristic set of structural features that lie between the compound eyes. There are three morphological domains:

The ocellar cuticle contains the three ocelli, associated macrochaetes (bristles), and microchaetes. The ocellar domain is flanked by the ridged cuticle of the frons. The frons converges anterior to the ocelli such that it delineates a triangular area surrounding the ocellar region. The lateral subdivision of the dorsal head is the orbital region (Royet, 1995). The labial disc gives rise to the proboscus and the labial palps.

In the trunk of the Drosophila embryo, the segment polarity genes are initially activated by the pair-rule genes; later, the segment polarity genes maintain one another's expression through a complex network of cross-regulatory interactions. These interactions, which are critical to cell fate specification, are similar in each of the trunk segments. To determine whether segment polarity gene expression is established differently outside the trunk, the regulation of the genes hedgehog (hh), wingless (wg), and engrailed (en) was studied in each of the segments of the developing head. The cross-regulatory relationships among these genes, as well as their initial mode of activation in the anterior head are significantly different from those in the trunk. In addition, each head segment exhibits a unique network of segment polarity gene interactions. It is proposed that these segment-specific interactions evolved to specify the high degree of structural diversity required for head morphogenesis (Gallitano-Mendel, 1997).

The proposed interactions between hh, wg and en are described below.

1. The intercalary segment. In this cephalic segment, hh expression is en-independent. In addition, ptc mutations cause the loss of wg rather than ectopic wg expression The dependence of wg, en, and hh expression on ptc indicates a unique role for segment polarity genes in the intercalary segment. Unlike wg action in the trunk and gnathal segments, wg restricts rather than maintains en and hh expression in this segment. Finally, en expression, as it occurs in the trunk, depends on hh function. However, this dependence cannot be mediated through wg, since wg does not maintain en expression in the intercalary segment.

2. The antennal segment. As in the trunk, hh antennal expression depends on en, while wg expression requires hh. The requirement for hh is presumably mediated through ptc, which represses wg in this segment. Unlike in the trunk, wg restricts the expression domains of both en and hh. As in the intercalary segment, regulation of en by hh is wg- independent.

3. The ocular segment. In this segment, hh is en-independent and wg expression does not require hh. Although the wg domain (the head blob) does not expand in ptc mutant embryos, noncontiguous ectopic wg expression appears in its vicinity. Unlike its action in the trunk and the other head segments, wg is required to initiate en expression in the ocular segment. However, hh expression still expands in wg mutant embryos (as in the intercalary and antennal segment). As in the intercalary and antennal segments, regulation of en by hh does not depend on wg.

It is concluded that cross-regulatory interactions among the segment polarity genes in the anterior head are very different from those in the posterior head and trunk segments. The mode of patterning of the anterior head (the acron and cephalic segments) is thought to be more ancient than that of the posterior head (the gnathal segments). This distinction appears to be reflected in the segmentation mechanism used by certain present day short germ insects and primitive arthropods. In these organisms, the early germ band includes only the acron, cephalic segments, and tail. Gnathal and trunk segments are generated later in embryogenesis by a progressive budding process (Gallitano-Mendel, 1997).

The role of cell death in head morphogenesis

An analysis has been carried out of the correlation between the pattern of expression of reaper and morphogenetic movements affecting head development. The defects in head development resulting from the absence of apoptosis in embryos deficient for rpr have also been investigated. In the head, domains of high incidence of cell death as marked by expression of rpr correlate with regions where most morphogenetic movements occur; these regions are involved in formation of mouth structures, the internalization of neural progenitors, and head involution. Cellular events driving these movements are delamination, invagination, and intercalation, as well as disruption and reformation of contacts among epithelial cells. At the late blastoderm stage (stage 5/6), a transient low level expression of rpr is seen in stripes delimiting the anterior and posterior trunk. This diffuse expression subsides by the onset of gastrulation (stage 7) and is replaced by multiple strongly expressed foci in the head, as well as a few in the tail region. Patchy expression of rpr is seen in the anterior endoderm and head mesoderm during stages 7-10. These tissues give rise to the anterior midgut and hemocytes, respectively. Nassif (1998) provides detailed descriptions of six expression domains in the head, as follows:


  1. Gnathocephalon: A high level and complex pattern of rpr expression is observed in the three gnathal segments (mandibular, maxillary, and labial). These large, interconnected domains can be distinguished on the basis of time of onset and peak expression of rpr. The dorsal gnathal domain is located most dorsally, bordering the optic lobe; it shows expression of rpr first, during stages 10-11. During stage 11, the dorsal portions of the gnathal segments are dramatically reduced in size and fuse into a single lobe-like structure, the dorsal ridge. Later during stage 11, rpr expression is activated in three domains, shaped like inverted horseshoes, that outline the mandibular, maxillary, and labial lobes. During stage 12, a third domains of rpr expression, the ventral gnathal domain, is observed in the mid-ventral portion of the gnathal segments. Within this region, rpr expression is strongest in the maxillary lobe, and in a labial stripe that flanks the salivary placode, which is itself free of rpr expression (Nassif, 1998).
  2. The ventral procephalon: Anterior to the gnathocephalon, in the ventral procephalon, lie the antennal and intercalary segment, the antennal segment being found just dorsal to the intercalary segment. These two segments give rise to the antennal and hypopharyngeal lobes, respectively. A large focus of rpr expression, the ventral antennal domain, appears during stage 11 in the anterior-ventral antennal region. During late stage 11, this focus becomes prominent, appearing as an array of three highly expressing cell clusters arranged in a crescent. During stage 12, a second focus of rpr expression, the dorsal antennal domain, appears in the dorsal part of the antennal domain, adjacent to the gnathocephalon and coincident with the region where fusion between antennal lobe and gnathocephalon will occur. During stage 11, a stripe-like focus, appears that marks the boundary between the intercalary and antennal segments (Nassif, 1998).
  3. Stomatogastric nervous system: During early stage 11, a reaper focus appears transiently in the dorsal portion of the esophagus in a placode that will give rise to the stomatogastric nervous system. Expression in this focus fades and then later reappears, during stages 13 and 14, in the three SNS vesicles that have invaginated from the placode (Nassif, 1998).
  4. Clypeolabrum: Apart from the strongly expressing optic lobe focus in the posterior procephalon, the clypeolabrum is the most prominent domain of rpr expression in the early embryo. Expression within the clypeolabrum, typical of most strongly expressing foci, is mottled, with single or small groups of strongly expressing cells surrounded by cells with weaker expression. While expression throughout most of the clypeolabrum declines during stage 11, expression in a small domain remains until stage 13. Later, during stages 13 and 14, a more ventral portion of the labrum that will form the pharynx roof (epipharynx) shows a prominent focus of rpr expression. Cell death in the midline of the clypeolabrum contributes to the medial shift of the labral sensilla, which in wild type arise on either side of the clypeolabrum (Nassif, 1998).
  5. Medial procephalon: During stage 13, rpr expression begins in the dorsomedial procephalon. It is from here that neural progenitors segregate from the surface ectoderm in a "mass-delamination" movement that is distinct from the individual delamination movements of the majority of brain neuroblasts that occur at an earlier stage. Later, during stages 14-15, scattered and relatively weak rpr expression can be seen in the medial procephalon as it folds into the dorsal pouch. In rpr mutants, the number of vesicles associated with the dorsal surface of the brain is significantly increased (Nassif, 1998).
  6. Posterior procephalon: A large focus containing 30-50 rpr-expressing cells, designated optic lobe 1, is seen at the boundary between dorsal procephalon (future brain and optic lobe) and amnioserosa. Expression in OL1 is high during stages 7-10, a period of approximately 2 hours. During stage 11, while expression in OL1 declines, rpr expression is activated in two to three small groups of cells (which togethar are designated OL2) located slightly more anterior and medial to OL1. These OL2 cells lie at the border between dorsomedial brain and optic lobe. Finally, during stage 12, rpr is expressed in a large focus (OL3) that borders the invaginating optic lobe. Cell death is shown to play three morphogenetic functions in the development of the optic lobe: (1) reducing the number of cells, (2) facilitating the ventral shift of the optic lobe primordium, which normally occurs during early embryogenesis (and presumably involves major horizontal intercalation of cells in the optic lobe primordium), and (3) enabling the optic lobe primordium to separate from the surface epithelium following invagination (Nassif, 1998).

In all domains expressing rpr, each involving apoptosis, profound morphogenetic movements take place during embryogenesis. These include the following major processes:

The analysis of rpr-deficient embryos demonstrates that despite the widespread occurrence of apoptosis during normal head morphogenesis, many aspects of this process proceed in an apparently unperturbed manner even when cell death is blocked. In particular, movements that happen early during embryonic development and that are evolutionarily more ancient (e.g., formation of the dorsal ridge and the pharynx) take place almost normally in rpr-deficient embryos. Later events which are mostly associated with head involution (e.g., retraction of the clypeolabrum, formation of the dorsal pouch, fusion of lateral gnathal lobes) are evolutionarily more recent and fail to occur normally in rpr-deficient embryos (Nassif, 1998).

Head anatomical terms [Images]

The function of Dpp and Hh in partitioning the embryonic dorsal head neurectoderm

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 dppE87, 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 dppE87, 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 hhts2 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).

Specification and development of the pars intercerebralis and pars lateralis, neuroendocrine command centers in the Drosophila brain: The PI and PL are derived from adjacent neurectodermal placodes in the dorso-medial head

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/Visual system homeobox 1 ortholog 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).

Patterning of the adult mandibulate mouthparts in the red flour beetle, Tribolium castaneum

Specialized insect mouthparts, such as those of Drosophila, are derived from an ancestral mandibulate state, but little is known about the developmental genetics of mandibulate mouthparts. The metamorphic patterning of mandibulate mouthparts of the beetle Tribolium castaneum was studied RNA interference to deplete the expression of 13 genes involved in mouthpart patterning. These data were used to test three hypotheses related to mouthpart development and evolution. First, the prediction was tested that maxillary and labial palps are patterned using conserved components of the leg-patterning network. This hypothesis was strongly supported: depletion of Distal-less and dachshund led to distal and intermediate deletions of these structures while depletion of homothorax led to homeotic transformation of the proximal maxilla and labium, joint formation required the action of Notch signaling components and odd-skipped paralogs, and distal growth and patterning required epidermal growth factor (EGF) signaling. Additionally, depletion of abrupt or pdm/nubbin caused fusions of palp segments. Second, the hypotheses was tested for how adult endites, the inner branches of the maxillary and labial appendages, are formed at metamorphosis. The data reveal that Distal-less, Notch signaling components, and odd-skipped paralogs, but not dachshund, are required for metamorphosis of the maxillary endites. Endite development thus requires components of the limb proximal-distal axis patterning and joint segmentation networks. Finally, adult mandible development is considered in light of the gnathobasic hypothesis. Interestingly, while EGF activity is required for distal, but not proximal, patterning of other appendages, it is required for normal metamorphic growth of the mandibles (Angelini, 2012).

In D. melanogaster, Dll mutants lack maxillary structures and portions of the proboscis (i.e., labium), although Dll expression in the maxillary anlagen is weaker than in the leg or antennal discs. Paralleling the results for T. castaneum, in the horned beetle Onthophagus taurus distal regions of the adult mouthparts were deleted with larval Dll RNAi (Simonnet 2011). The embryonic and metamorphic functions of Dll in T. castaneum are also similar: the gene is required for the development of distal structures at both stages, and during embryogenesis Dll is expressed throughout the developing palps. Interestingly, removal of T. castaneum Dll expression earlier during larval life led to delayed metamorphosis, as well as changes in appendage morphology (Suzuki, 2009). Many insects delay molting after appendage loss to allow time for regeneration, and this dual role of Dll suggests a mechanism linking these processes (Angelini, 2012).

The data from T. castaneum provide evidence for a conserved gap gene role of dac during patterning of mouthparts and legs of this species. dachshund is not expressed in or required for development of the labial and maxillary anlagen of D. melanogaster. In T. castaneum embryos dac is expressed strongly in the proximal maxilla and part of the developing endite. Embryonic dac expression is weaker in the distal maxillary palp and the labium. The current data show a clear metamorphic requirement for dac in the intermediate regions of the maxillary and labial palps, as does a recent study of O. taurus (Simonnet, 2011). A function for dac in the development of an intermediate portion of the maxillary and labial appendages has so far only been observed in these two beetles, while data from two species with specialized mouthparts (the milkweed bug O. fasciatus and D. melanogaster) found that dac is not required for PD patterning of the mouthparts. Thus, comparative data from other species do not support the hypothesis that this mouthpart patterning role is ancestral. However, if mandibulate mouthparts evolved from leg-like structures similarities in the expression and function of genes patterning both legs and mouthparts are expected to be plesiomorphic. This hypothesis can be further tested by examining the role of dac in mouthpart development in additional insect orders, particularly those that retain mandibulate mouthparts, and in other arthropods (Angelini, 2012).

The effects of hth depletion are distinct in different species, but typically involve some degree of homeotic transformation. In D. melanogaster, hth is expressed in the labial discs, but without nuclear expression of its cofactor Extradenticle. Maxillary palps are retained in hth loss-of-function flies, but they may possess bristles typical of legs, indicating a partial proboscis-to-leg transformation. In the cricket Gryllus bimaculatus, which has mandibulate mouthparts, hth depletion causes transformation of proximal mouthpart structures towards antennal identity, with a loss of endites, while distal structures are transformed towards leg identity (Ronco, 2008). hth RNAi in T. castaneum transformed intermediate regions of the maxilla and labium towards distal mouthpart identity. Proximal regions also appeared transformed, but their identity could not be established, while distal regions appeared wild type. In the beetle O. taurus, proximal regions of the labium are transformed towards maxillary endite identity, but distal regions of the labium and the entire maxilla remain relatively unaffected (Angelini, 2012).

These results highlight the similarity between patterning of the maxilla, labium and legs in T. castaneum. Functional data from two species with highly derived mouthpart morphologies, D. melanogaster and the milkweed bug Oncopeltus fasciatus, suggest only limited similarity between mouthpart and leg patterning. One explanation for this low degree of conservation is that evolution of the ancestral patterning mechanism has occurred in concert with the functional and morphological diversification of these mouthparts. A correlation between generative mechanisms and structural morphology has been used as a common null hypothesis, although exceptions in which similar morphologies result from different developmental pathways are documented. Nevertheless, this hypothesis predicts that developmental patterning should be more highly conserved across appendage types in species that retain the ancestral mandibulate mouthpart morphology (Angelini, 2012).

The maxillary and labial palps are an interesting case of serial homology. Despite a difference in overall size, their shape and arrangement of sensillae are similar. The intermediate segments of each palp type are also similar, but differ in number, which suggests that segment number is regulated independently from other morphological traits. The RNAi depletion of pdm in T. castaneum caused the reduction and deletion of the third maxillary palp segment, producing a phenotype closely resembling the wildtype morphology of the labial palps. While a role for pdm in the labium cannot be excluded, the absence of observed labial phenotypes was significant compared to maxillary results. Therefore, it is hypothesized that the difference in the number of palp segments results from specific activation of pdm in the maxillary palp. Loss of function in the Hox gene Deformed during T. castaneum embryogenesis causes a transformation of the larval maxillae towards labial identity. Since Hox genes are the primary determinants of body segment identity, it is proposed that pdm is activated by Deformed, and repressed by the labial Hox gene Sex combs reduced. RNAi targeting pdm in another mandibulate insect, the cricket Acheta domesticus, generated defects in the antenna and legs, but no defects in the mouthparts, despite similar pdm expression in these appendages (Turchyn; 2011; Angelini, 2012).

Endites are a primitive component of arthropod appendages, and they are retained in insect mouthparts, as well as in the mouthparts and thoracic appendages of many crustaceans (Boxshall 2004). At least three hypotheses have been put forward for how endites are patterned, and these hypotheses are not mutually exclusive. The first hypothesis states that multiple PD axes result from redeployment of a PD axis patterning mechanism shared by palps and endites. A second hypothesis posits that endites and appendage segments form by the same mechanism, Notch-mediated in-folding of the cuticle. A third hypothesis states that dac expression initiates endite branching from the main appendage axis. The axis redeployment hypothesis predicts that depletion of genes involved in PD axis patterning will have similar effects on the development of palps and endites. Some support for this hypothesis comes from studies of endite morphogenesis and the expression and function of leg gap genes in the embryos of T. castaneum and the orthopteran Schistocerca americana, but not all data are consistent with it. The segmentation hypothesis predicts that endites will fail to differentiate if genes required for joints are depleted. This hypothesis was posed based on a comparative developmental study of segmented and phyllopodous crustacean limbs. Finally, the dac-mediated hypothesis predicts that depletion of dac will lead to reduced endites. This hypothesis emerged from the observation that dac expression is reiterated along the medial edges of larval endites in the crustacean Triops longicaudatus. Comparative expression data from the isopod Porcellio scaber are also consistent with the dac-mediated hypothesis (Angelini, 2012).

The current data are consistent with predictions of the axis redeployment and segmentation hypotheses but do not support a role for dac in endite metamorphosis. Adult endites were disrupted by depletion of Dll, Krn, the odd-related genes, and Notch signaling, and to a lesser degree hth. In the maxilla depletion of most of these genes led to the failure of the single larval endite to divide into two distinct branches, while in the labium, their depletion caused reduction of the ligula. Their requirement in the endites is consistent with the hypothesis that these structures are generated by redeploying appendage PD axis determinants. Depletion of Notch signaling components and the odd paralogs produced reductions and fusions between palp segments, between the palps and endites, and between the lacinia and galea. Thus, these data are compatible with both the hypothesis that a reiterated PD axis is used to pattern the endites and the hypothesis that endite formation is linked to joint formation. Normal endite development in dac-depleted specimens is inconsistent with the dac-mediated hypothesis (Angelini, 2012).

It is noteworthy that endite specification and the division of the single larval endite into the adult galea and lacinia appear to be separable functions. For example, Ser RNAi resulted in a single endite lobe with lacinia identity medially and galea identity laterally. In contrast, severe Dll RNAi individuals had a single endite that lacked also obvious lacinia identity (Angelini, 2012).

The mandibulate structure of Tribolium mouthparts is the pleisomorphic state for insects and is shared by a majority of insect orders. These mouthparts are characterized by robust mandibles, lacking segmentation. A classic debate in arthropod morphology concerns whether the mandibles of insects and myriapods are derived from a whole appendage or only from proximal appendage regions; the latter are called gnathobasic mandibles. Palps are retained on the mandibles of many crustaceans, making it clear that the biting regions of their mandibles are gnathobasic. Phylogenetic support for the gnathobasic hypothesis comes from phlyogenetic studies that place insects nested within crustaceans (Regier, 2010). The first developmental genetic support for the gnathobasic hypothesis came from the discovery that insect mandibles lack Dll expression. Furthermore, neither mutations in Dll nor its depletion through RNAi have been observed to alter mandible development in insects, including T. castaneum. This evidence has led to widespread acceptance of the gnathobasic hypothesis. Of the 13 genes depleted in this study, two (Krn and hth) produced results that would not be predicted by the most straightforward form of the gnathobasic hypothesis for mandible origins (Angelini, 2012).

Loss of EGF function in insects leads to distal appendage defects, including pretarsal or tarsal deletions. The role of EGF signaling in distal appendage regions is conserved in T. castaneum metamorphosis, since depletion of the EGF ligand Krn leads to reduction of the antennal flagellum, and maxillary and labial palps, as well as to deletion of the pretarsus and malformation of the tarsus. In light of the restriction of Krn’s role to distal appendage regions and regulation of distal EGF ligand expression by Dll in D. melanogaster, the gnathobasic hypothesis predicts that Krn should not be required for normal development of the mandible in T. castaneum. In contrast to this prediction, depletion of Krn produced a significant reduction in mandible length (Angelini, 2012).

The hypothesis of a gnathobasic mandible also predicts that hth depletion should produce effects in the mandible similar to those in the proximal regions of other appendage types. In T. castaneum, hth RNAi during metamorphosis caused homeotic transformation of proximal regions of the maxilla, labium and legs. However, the mandibles were not affected by hth depletion. In the beetle O. taurus, hth depletion slightly altered mandible shape, but also without apparent homeosis. In contrast, hth RNAi in embryos of the cricket G. bimaculatus transformed the mandible towards a leg-like structure distally and an antenna-like structure proximally, paralleling the transformation observed in other appendages. Because these results come from only two lineages and from different life stages, additional data are needed to determine whether a homeotic role for hth was present ancestrally in insect mandibles (Angelini, 2012).

These data must be weighed alongside other evidence bearing on the gnathobasic hypothesis. In T. castaneum, the lack of phenotypic effects on mandible metamorphosis of other genes in this study is consistent with the gnathobasic hypothesis. In particular, it was observed that mandible metamorphosis was normal following depletion of genes involved in distal growth and patterning or joint formation. Moreover, homology at one biological level, such as anatomy, does not preclude divergence at other levels, such as development. Nevertheless, since developmental genetic studies of Dll and other appendage-patterning genes have been used as strong support for the gnathobasic homology of the insect mandible, the findings of Krn function highlight the difficulties in establishing serial homology based solely on developmental data (Angelini, 2012).

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).

Patterning of the adult mandibulate mouthparts in the red flour beetle, Tribolium castaneum

Specialized insect mouthparts, such as those of Drosophila, are derived from an ancestral mandibulate state, but little is known about the developmental genetics of mandibulate mouthparts. The metamorphic patterning of mandibulate mouthparts of the beetle Tribolium castaneum was studied RNA interference to deplete the expression of 13 genes involved in mouthpart patterning. These data were used to test three hypotheses related to mouthpart development and evolution. First, the prediction was tested that maxillary and labial palps are patterned using conserved components of the leg-patterning network. This hypothesis was strongly supported: depletion of Distal-less and dachshund led to distal and intermediate deletions of these structures while depletion of homothorax led to homeotic transformation of the proximal maxilla and labium, joint formation required the action of Notch signaling components and odd-skipped paralogs, and distal growth and patterning required epidermal growth factor (EGF) signaling. Additionally, depletion of abrupt or pdm/nubbin caused fusions of palp segments. Second, the hypotheses was tested for how adult endites, the inner branches of the maxillary and labial appendages, are formed at metamorphosis. The data reveal that Distal-less, Notch signaling components, and odd-skipped paralogs, but not dachshund, are required for metamorphosis of the maxillary endites. Endite development thus requires components of the limb proximal-distal axis patterning and joint segmentation networks. Finally, adult mandible development is considered in light of the gnathobasic hypothesis. Interestingly, while EGF activity is required for distal, but not proximal, patterning of other appendages, it is required for normal metamorphic growth of the mandibles (Angelini, 2012).

In D. melanogaster, Dll mutants lack maxillary structures and portions of the proboscis (i.e., labium), although Dll expression in the maxillary anlagen is weaker than in the leg or antennal discs. Paralleling the results for T. castaneum, in the horned beetle Onthophagus taurus distal regions of the adult mouthparts were deleted with larval Dll RNAi (Simonnet 2011). The embryonic and metamorphic functions of Dll in T. castaneum are also similar: the gene is required for the development of distal structures at both stages, and during embryogenesis Dll is expressed throughout the developing palps. Interestingly, removal of T. castaneum Dll expression earlier during larval life led to delayed metamorphosis, as well as changes in appendage morphology (Suzuki, 2009). Many insects delay molting after appendage loss to allow time for regeneration, and this dual role of Dll suggests a mechanism linking these processes (Angelini, 2012).

The data from T. castaneum provide evidence for a conserved gap gene role of dac during patterning of mouthparts and legs of this species. dachshund is not expressed in or required for development of the labial and maxillary anlagen of D. melanogaster. In T. castaneum embryos dac is expressed strongly in the proximal maxilla and part of the developing endite. Embryonic dac expression is weaker in the distal maxillary palp and the labium. The current data show a clear metamorphic requirement for dac in the intermediate regions of the maxillary and labial palps, as does a recent study of O. taurus (Simonnet, 2011). A function for dac in the development of an intermediate portion of the maxillary and labial appendages has so far only been observed in these two beetles, while data from two species with specialized mouthparts (the milkweed bug O. fasciatus and D. melanogaster) found that dac is not required for PD patterning of the mouthparts. Thus, comparative data from other species do not support the hypothesis that this mouthpart patterning role is ancestral. However, if mandibulate mouthparts evolved from leg-like structures similarities in the expression and function of genes patterning both legs and mouthparts are expected to be plesiomorphic. This hypothesis can be further tested by examining the role of dac in mouthpart development in additional insect orders, particularly those that retain mandibulate mouthparts, and in other arthropods (Angelini, 2012).

The effects of hth depletion are distinct in different species, but typically involve some degree of homeotic transformation. In D. melanogaster, hth is expressed in the labial discs, but without nuclear expression of its cofactor Extradenticle. Maxillary palps are retained in hth loss-of-function flies, but they may possess bristles typical of legs, indicating a partial proboscis-to-leg transformation. In the cricket Gryllus bimaculatus, which has mandibulate mouthparts, hth depletion causes transformation of proximal mouthpart structures towards antennal identity, with a loss of endites, while distal structures are transformed towards leg identity (Ronco, 2008). hth RNAi in T. castaneum transformed intermediate regions of the maxilla and labium towards distal mouthpart identity. Proximal regions also appeared transformed, but their identity could not be established, while distal regions appeared wild type. In the beetle O. taurus, proximal regions of the labium are transformed towards maxillary endite identity, but distal regions of the labium and the entire maxilla remain relatively unaffected (Angelini, 2012).

These results highlight the similarity between patterning of the maxilla, labium and legs in T. castaneum. Functional data from two species with highly derived mouthpart morphologies, D. melanogaster and the milkweed bug Oncopeltus fasciatus, suggest only limited similarity between mouthpart and leg patterning. One explanation for this low degree of conservation is that evolution of the ancestral patterning mechanism has occurred in concert with the functional and morphological diversification of these mouthparts. A correlation between generative mechanisms and structural morphology has been used as a common null hypothesis, although exceptions in which similar morphologies result from different developmental pathways are documented. Nevertheless, this hypothesis predicts that developmental patterning should be more highly conserved across appendage types in species that retain the ancestral mandibulate mouthpart morphology (Angelini, 2012).

The maxillary and labial palps are an interesting case of serial homology. Despite a difference in overall size, their shape and arrangement of sensillae are similar. The intermediate segments of each palp type are also similar, but differ in number, which suggests that segment number is regulated independently from other morphological traits. The RNAi depletion of pdm in T. castaneum caused the reduction and deletion of the third maxillary palp segment, producing a phenotype closely resembling the wildtype morphology of the labial palps. While a role for pdm in the labium cannot be excluded, the absence of observed labial phenotypes was significant compared to maxillary results. Therefore, it is hypothesized that the difference in the number of palp segments results from specific activation of pdm in the maxillary palp. Loss of function in the Hox gene Deformed during T. castaneum embryogenesis causes a transformation of the larval maxillae towards labial identity. Since Hox genes are the primary determinants of body segment identity, it is proposed that pdm is activated by Deformed, and repressed by the labial Hox gene Sex combs reduced. RNAi targeting pdm in another mandibulate insect, the cricket Acheta domesticus, generated defects in the antenna and legs, but no defects in the mouthparts, despite similar pdm expression in these appendages (Turchyn; 2011; Angelini, 2012).

Endites are a primitive component of arthropod appendages, and they are retained in insect mouthparts, as well as in the mouthparts and thoracic appendages of many crustaceans (Boxshall 2004). At least three hypotheses have been put forward for how endites are patterned, and these hypotheses are not mutually exclusive. The first hypothesis states that multiple PD axes result from redeployment of a PD axis patterning mechanism shared by palps and endites. A second hypothesis posits that endites and appendage segments form by the same mechanism, Notch-mediated in-folding of the cuticle. A third hypothesis states that dac expression initiates endite branching from the main appendage axis. The axis redeployment hypothesis predicts that depletion of genes involved in PD axis patterning will have similar effects on the development of palps and endites. Some support for this hypothesis comes from studies of endite morphogenesis and the expression and function of leg gap genes in the embryos of T. castaneum and the orthopteran Schistocerca americana, but not all data are consistent with it. The segmentation hypothesis predicts that endites will fail to differentiate if genes required for joints are depleted. This hypothesis was posed based on a comparative developmental study of segmented and phyllopodous crustacean limbs. Finally, the dac-mediated hypothesis predicts that depletion of dac will lead to reduced endites. This hypothesis emerged from the observation that dac expression is reiterated along the medial edges of larval endites in the crustacean Triops longicaudatus. Comparative expression data from the isopod Porcellio scaber are also consistent with the dac-mediated hypothesis (Angelini, 2012).

The current data are consistent with predictions of the axis redeployment and segmentation hypotheses but do not support a role for dac in endite metamorphosis. Adult endites were disrupted by depletion of Dll, Krn, the odd-related genes, and Notch signaling, and to a lesser degree hth. In the maxilla depletion of most of these genes led to the failure of the single larval endite to divide into two distinct branches, while in the labium, their depletion caused reduction of the ligula. Their requirement in the endites is consistent with the hypothesis that these structures are generated by redeploying appendage PD axis determinants. Depletion of Notch signaling components and the odd paralogs produced reductions and fusions between palp segments, between the palps and endites, and between the lacinia and galea. Thus, these data are compatible with both the hypothesis that a reiterated PD axis is used to pattern the endites and the hypothesis that endite formation is linked to joint formation. Normal endite development in dac-depleted specimens is inconsistent with the dac-mediated hypothesis (Angelini, 2012).

It is noteworthy that endite specification and the division of the single larval endite into the adult galea and lacinia appear to be separable functions. For example, Ser RNAi resulted in a single endite lobe with lacinia identity medially and galea identity laterally. In contrast, severe Dll RNAi individuals had a single endite that lacked also obvious lacinia identity (Angelini, 2012).

The mandibulate structure of Tribolium mouthparts is the pleisomorphic state for insects and is shared by a majority of insect orders. These mouthparts are characterized by robust mandibles, lacking segmentation. A classic debate in arthropod morphology concerns whether the mandibles of insects and myriapods are derived from a whole appendage or only from proximal appendage regions; the latter are called gnathobasic mandibles. Palps are retained on the mandibles of many crustaceans, making it clear that the biting regions of their mandibles are gnathobasic. Phylogenetic support for the gnathobasic hypothesis comes from phlyogenetic studies that place insects nested within crustaceans (Regier, 2010). The first developmental genetic support for the gnathobasic hypothesis came from the discovery that insect mandibles lack Dll expression. Furthermore, neither mutations in Dll nor its depletion through RNAi have been observed to alter mandible development in insects, including T. castaneum. This evidence has led to widespread acceptance of the gnathobasic hypothesis. Of the 13 genes depleted in this study, two (Krn and hth) produced results that would not be predicted by the most straightforward form of the gnathobasic hypothesis for mandible origins (Angelini, 2012).

Loss of EGF function in insects leads to distal appendage defects, including pretarsal or tarsal deletions. The role of EGF signaling in distal appendage regions is conserved in T. castaneum metamorphosis, since depletion of the EGF ligand Krn leads to reduction of the antennal flagellum, and maxillary and labial palps, as well as to deletion of the pretarsus and malformation of the tarsus. In light of the restriction of Krn’s role to distal appendage regions and regulation of distal EGF ligand expression by Dll in D. melanogaster, the gnathobasic hypothesis predicts that Krn should not be required for normal development of the mandible in T. castaneum. In contrast to this prediction, depletion of Krn produced a significant reduction in mandible length (Angelini, 2012).

The hypothesis of a gnathobasic mandible also predicts that hth depletion should produce effects in the mandible similar to those in the proximal regions of other appendage types. In T. castaneum, hth RNAi during metamorphosis caused homeotic transformation of proximal regions of the maxilla, labium and legs. However, the mandibles were not affected by hth depletion. In the beetle O. taurus, hth depletion slightly altered mandible shape, but also without apparent homeosis. In contrast, hth RNAi in embryos of the cricket G. bimaculatus transformed the mandible towards a leg-like structure distally and an antenna-like structure proximally, paralleling the transformation observed in other appendages. Because these results come from only two lineages and from different life stages, additional data are needed to determine whether a homeotic role for hth was present ancestrally in insect mandibles (Angelini, 2012).

These data must be weighed alongside other evidence bearing on the gnathobasic hypothesis. In T. castaneum, the lack of phenotypic effects on mandible metamorphosis of other genes in this study is consistent with the gnathobasic hypothesis. In particular, it was observed that mandible metamorphosis was normal following depletion of genes involved in distal growth and patterning or joint formation. Moreover, homology at one biological level, such as anatomy, does not preclude divergence at other levels, such as development. Nevertheless, since developmental genetic studies of Dll and other appendage-patterning genes have been used as strong support for the gnathobasic homology of the insect mandible, the findings of Krn function highlight the difficulties in establishing serial homology based solely on developmental data (Angelini, 2012).

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).

Cell-autonomous and non-cell-autonomous function of Hox genes specify segmental neuroblast identity in the gnathal region of the embryonic CNS in Drosophila

In thoracic and abdominal segments of Drosophila, the expression pattern of Bithorax-Complex Hox genes is known to specify the segmental identity of neuroblasts (NB) prior to their delamination from the neuroectoderm. This study identified and characterized a set of serially homologous NB-lineages in the gnathal segments and used one of them (NB6-4 lineage) as a model to investigate the mechanism conferring segment-specific identities to gnathal NBs. It was shown that NB6-4 is primarily determined by the cell-autonomous function of the Hox gene Deformed (Dfd). Interestingly, however, it also requires a non-cell-autonomous function of labial and Antennapedia that are expressed in adjacent anterior or posterior compartments. The secreted molecule Amalgam (Ama) was identified as a downstream target of the Antennapedia-Complex Hox genes labial, Dfd, Sex combs reduced and Antennapedia. In conjunction with its receptor Neurotactin (Nrt) and the effector kinase Abelson tyrosine kinase (Abl), Ama is necessary in parallel to the cell-autonomous Dad pathway for the correct specification of the maxillary identity of NB6-4. Both pathways repress CyclinE (CycE) and loss of function of either of these pathways leads to a partial transformation (40%), whereas simultaneous mutation of both pathways leads to a complete transformation (100%) of NB6-4 segmental identity. Finally, the study provides genetic evidences, that the Ama-Nrt-Abl-pathway regulates CycE expression by altering the function of the Hippo effector Yorkie in embryonic NBs. The disclosure of a non-cell-autonomous influence of Hox genes on neural stem cells provides new insight into the process of segmental patterning in the developing CNS (Becker, 2016).

The Drosophila head consists of seven segments (4 pregnathal and 3 gnathal) all of which contribute neuromeres to the CNS. The brain is formed by approximately 100 NBs per hemisphere, which have been individually identified and assigned to specific pregnathal segments [The anterior pregnathal region (procephalon) is composed of the labral, ocular, antennal, intercalary segments, see Segment polarity and DV patterning gene expression reveals segmental organization of the Drosophila brain]. As judged from comparison of the combinatorial codes of marker gene expression only few brain NBs appear to be serially homologous to NBs in the thoracic/abdominal ventral nerve cord, reflecting the highly derived character of the brain neuromeres. The connecting tissue between brain and the thoracic VNC consists of three neuromeres formed by the gnathal head segments named mandibular (mad), maxillary (max) and labial (lab) segment, but the number and identity of the neural stem cells and their lineage composition in these segments is still unknown. Compared to the thoracic ground state the segmental sets of gnathal NBs might be reduced to different degrees, but are thought to be less derived compared to the brain NBs. Therefore, to fully understand segmental specification during central nervous system development, it is important to identify the neuroblasts and their lineages in these interconnecting segments (Becker, 2016).

Assuming that most NBs in the gnathal segments still share similarities to thoracic and abdominal NBs, this study sought serially homologous NB-lineages, which are suitable for genetic analyses. Using the molecular marker eagle (eg), which specifically labels four NB-lineages in thoracic/abdominal hemisegments this study identified three serial homologs (NB3-3, NB6-4 and NB7-3) in the gnathal region. To investigate the mechanisms conferring segmental identities, focus was placed on one of them, the NB6-4 lineage, which shows the most significant segment-specific modifications. The analysis reveals a primary role of the Antennapedia-Complex (Antp-C) Hox gene Deformed (Dfd) in cell-autonomously specifying the maxillary fate of NB6-4 (NB6-4max). Surprisingly, an additional, non-cell-autonomous function was uncovered of the Antp-C Hox genes labial (lab, expressed anterior to Dfd) and Antennapedia (Antp, expressed posterior to Dfd) in specifying NB6-4max. In a mini-screen for downstream effectors the secreted protein Amalgam (Ama) was identified as being positively regulated by lab, Dfd and Antp and negatively regulated by the Antp-C Hox gene Sex combs reduced (Scr). Loss of function of Ama and its receptor Neurotactin (Nrt) as well as the downstream effector kinase Abelson tyrosine kinase (Abl) lead to a transformation of NB6-4max similar to Dfd single mutants. Thus, in parallel to the cell-autonomous role of Dfd, a non-cell-autonomous function of Hox genes lab and Antp, mediated via the Ama-Nrt-Abl pathway, is necessary to specify NB6-4max identity. Disruption of either of these pathways leads to a partial misspecification of NB6-4max (approx. 40%), whereas simultaneous disruption of both pathways leads to a complete transformation (approx. 100%) of NB6-4max to a labial/thoracic identity. It was further shown that both pathways regulate the expression of the cell cycle gene CyclinE, which is necessary and sufficient to generate labial/thoracic NB6-4 identity. Whereas Dfd seems to directly repress CyclinE transcription (similar to AbdA/AbdB in the trunk), indications are provided that the Ama-Nrt-Abl pathway prevents CyclinE expression by altering the activity of the Hippo/Salvador/Warts pathway effector Yorkie (Yki) (Becker, 2016).

Along the anterior-posterior axis the CNS consists of segmental units (neuromeres) the composition of which is adapted to the functional requirements of the respective body parts. In Drosophila the CNS comprises 10 abdominal, three thoracic, three gnathal and four pregnathal (brain) neuromeres that are generated by stereotyped populations of neural stem cells (neuroblasts, NBs). The pattern of NBs in thoracic segments resembles the ground state while NB patterns in the other segments are derived to various degrees. Within each segment individual NBs are specified by positional information in the neuroectoderm. NBs delaminating from corresponding positions in different segments express similar sets of molecular markers, generate similar lineages, and are called serial homologs. However, for thoracic and abdominal neuromeres it has been shown that the composition of a number of serially homologous NB-lineages shows segment-specific differences. In the more derived gnathal and pregnathal head segments embryonic NB-lineages and the mechanisms of their segmental specification have not been analyzed so far (Becker, 2016).

Using the well-established molecular marker Eagle (Eg) which labels four embryonic NB-lineages (NB2-4, NB3-3, NB6-4, NB7-3) in all thoracic and most of the abdominal segments this study identified serially homologous lineages of NB3-3, NB6-4 and NB7-3 in gnathal segments. The embryonic NB7-3 lineage shows segmental differences as it comprises increasing cell numbers from mandibular (2 cells), maxillary (3 cells) to labial (3-5 cells) segments, while cell numbers are decreasing from T1-T2 (4 cells), T3-A7 (3 cells) to A8 (2-3 cells). Reduced cell numbers in the mandibular and maxillary NB7-3 lineages depend on Dfd and Scr function, respectively . While NB7-3 appeared in all three gnathal segments, NB3-3 and NB6-4 was only found in labial and maxillary segments, and NB2-4 was not found in any of them. Preliminary data suggest that the missing NBs are not generated in these segments, instead of being eliminated by apoptosis. For the terminal abdominal neuromeres (A9, A10) it has recently been shown that the formation of a set of NBs (including NB7-3) is inhibited by the Hox gene Abdominal-B. Similarly, in Dfd mutants the formation was observed of a NB with NB6-4 characteristics in mandibular segments (10%), in which it is never found in wild type (Becker, 2016).

Similar to the thoracic and abdominal segments NB6-4 showed dramatic differences between maxillary and labial segments. NB6-4max produces glial cells only (like abdominal NB6-4), whereas the labial homolog produces neurons in addition to glial cells (like thoracic NB6-4). The number of glial cells produced by the glioblasts NB6-4max (4 cells) and abdominal NB6-4 (2 cells) and by the neuroglioblasts NB6-4lab (3 glia) and thoracic NB6-4 (3 glia) is segment-specific(Becker, 2016).

Thus segment-specific differences among serially homologous lineages may concern types and/or numbers of specific progeny cells and may result from differential specification of NBs and their progeny, differential proliferation and/or differential cell death of particular progeny cells. It has been shown that the segment-specific modification of serially homologous lineages is under the control of Hox genes and that during neurogenesis Hox genes act on different levels, i.e. they act in a context-specific manner at different developmental stages and in different cells. In the thoracic/abdominal region segmental identity is conferred to NBs early in the neuroectoderm by cell-autonomous function of Hox genes of the Bithorax-Complex. This study used the NB6-4 lineage to clarify mechanisms of segmental specification in the gnathal segments (Becker, 2016).

In segments of the trunk, the action of Hox genes strictly follows the rule of the posterior prevalence concept: More posterior expressed Hox genes repress anterior Hox genes and thereby determine the segmental identities. In the gnathal segments this phenomenon was not observed on the level of the nervous system. Removing Hox genes of the Antp-C had no or only minor impact on the expression domain of other Antp-C Hox genes. Similar results were also obtained in a study that analyzed cross-regulation of Hox genes upon ectopic expression (Becker, 2016).

Moreover, it seems that at least in the case of the differences monitored between labial and maxillary segments Hox gene function has to be added to realize the more anterior fate. Antennapedia has no impact on NB6-4 identity in the labial segment, but specification of the maxillary NB6-4 requires the function of Deformed and Sex combs reduced. These two Hox genes are not repressed or activated by Antp. Also, cross-regulation between Dfd and Scr seems to be unlikely or is very weak since only mild effects were observed on the protein level and on the phenotypic penetrance. In principle Scr can repress Dfd, but it was suggested that this occurs only when products are in sufficient amounts. In NB6-4 Dfd and Scr are co-expressed, but Scr levels appear to be insufficient to repress Dfd. Dfd seems to be the major Hox gene that cell-autonomously confers the maxillary NB6-4 fate, since the loss of Dfd showed the highest transformation rate and, more importantly, ectopic expression of Dfd in thoracic segments leads to a robust transformation towards maxillary fate. Scr does not act redundantly since in double mutants Dfd/Scr no synergistic effect was observed. It might have a fine-tuning effect, as it was shown that Scr influences Ama by repressing its transcription, whereas all other Antp-C Hox genes seem to activate Ama. However, since only minor changes were found in cell identities and numbers in Scr LoF background, the role of Scr in NB6-4max stays enigmatic (Becker, 2016).

Surprisingly cell-autonomous Hox gene function was not the only mechanism that confers segmental identity in NB6-4max. Loss of Dfd showed an effect in approx. 43% of all segments. Moreover, mutations of the adjacently expressed Hox genes labial and Antennapedia in combination with Dfd LoF showed a dramatic increase in the transformation rate of NB6-4max. Their expression patterns on the mRNA and protein level were carefully studied in wild type and Hox mutant background. In no case were these genes found to be expressed in NB6-4max or in the neuroectodermal region from which NB6-4max delaminates. This indicates that labial and Antennapedia influence NB6-4max fate in a non-cell-autonomous manner. That Hox genes can act non-cell-autonomously on stem cells was recently shown in the male germ-line, were AbdB influences centrosome orientation and the proliferation rate through regulation of the ligand Boss in the Sevenless-pathway. In this study Antp-C Hox genes controled the expression of the secreted molecule Amalgam, which spreads to adjacent segments and ensures segmental specification of NB6-4max in a parallel mechanism to the cell-autonomous function of Dfd. Thus, this study provides first evidence for parallel non-cell-autonomous and cell-autonomous functions of Antp-C genes during neural stem cell specification in the developing CNS (Becker, 2016).

Abelson kinase (Abl) was shown to be required for proper development of the Drosophila embryonic nervous system. In neurons Abl interacts with proteins like Robo or Chickadee and influences the actin cytoskeleton in the growth cone to regulate axonogenesis and pathfinding. In this system it was also demonstrated that Ama and Nrt are dominant modifiers of the Abl phenotype. It is proposed that the interaction of secreted Ama and the membrane-bound Nrt regulates Abl function in NBs. This leads to the correct segmental specification of NB6-4max. Antp-C Hox genes lab, Antp and Dfd regulate the expression of Ama and in mutants for theses Hox genes expression of Ama is severely reduced, which leads to the transformation of NB6-4max due to missing Abl function and de-repression of the cell cycle gene CyclinE. That Abl can influence the expression of CyclinE was also demonstrated in a modifier-screen in the Drosophila eye, but the mechanism remained unclear. Genetic analysis now suggests that in NBs this might occur via the regulation of the highly conserved Hippo-Salvador-Warts pathway and its downstream transcriptional co-activator Yki, which is known to regulate CyclinE expression. The Hippo-Salvador-Warts pathway controls organ growth and cell proliferation in Drosophila and vertebrates but so far has not been implicated in embryonic NB development. This study observed Yki cytoplasmic localization in wild type NB6-4max prior to division suggesting the active Hippo pathway. Nuclear localization of Yki could not be detected in Abl mutants, the loss of Yki activity in the Abl mutant background leads to a significant reduction in the strength of the Abl single mutant phenotype showing their genetic interaction and therefore supporting the proposed model in which Abl influences Yki activity. Moreover, expression of constitutive active Yki also lead to the transformation of NB6-4max and phenotypes that were similar to those observed in Abl mutants. Attempts were made to assess how Abl might influence Yki activity. Work in vertebrates suggests that this could be at least on two levels: first, c-Abl was shown to directly phosphorylate and activate the vertebrate MST1 and MST2 (Hpo homologue) and the Drosophila Hpo on a conserved residue (Y81) and second, c-Abl can also phosphorylate YAP1, which changes its function to become pro-apoptotic. This analysis suggests that in NBs Abl might regulate Hpo, since changes were found in the stability of Salvador, which is used as a Hpo activity readout, but a parallel direct regulation of Yki could not be ruled out, since it was recently shown that other pathways like the AMPK/LKB1 pathway can directly influence Yki activity. Since severe over-proliferation was observed in Abl or lab/Dfd mutants, that have an impaired Ama-Nrt-Abl pathway, or upon overexpression of YkiCA, future studies need to elucidate whether and how the proto-oncogene Abl kinase and Hox genes act on growth and proliferation or even tumor initiation through regulation of the Hippo/Salvador/Warts pathway (Becker, 2016).

Progressive derivation of serially homologous neuroblast lineages in the gnathal CNS of Drosophila

Along the anterior-posterior axis the central nervous system is subdivided into segmental units (neuromeres) the composition of which is adapted to their region-specific functional requirements. In Drosophila melanogaster each neuromere is formed by a specific set of identified neural stem cells (neuroblasts, NBs). In the thoracic and anterior abdominal region of the embryonic ventral nerve cord segmental sets of NBs resemble the ground state (2nd thoracic segment, which does not require input of homeotic genes), and serial (segmental) homologs generate similar types of lineages. The three gnathal head segments form a transitional zone between the brain and the ventral nerve cord. It has been shown recently that although all NBs of this zone are serial homologs of NBs in more posterior segments, they progressively differ from the ground state in anterior direction (labial > maxillary > mandibular segment) with regard to numbers and expression profiles. To study the consequences of their derived characters the embryonic lineages of gnathal NBs were traced using the Flybow and DiI-labelling techniques. For a number of clonal types serial homology is rather clearly reflected by their morphology (location and projection patterns) and cell specific markers, despite of reproducible segment-specific differences. However, many lineages, particularly in the mandibular segment, show a degree of derivation that impedes their assignment to ground state serial homologs. These findings demonstrate that differences in gene expression profiles of gnathal NBs go along with anteriorly directed progressive derivation in the composition of their lineages. Furthermore, lineage sizes decrease from labial to mandibular segments, which in concert with decreasing NB-numbers lead to reduced volumes of gnathal neuromeres, most significantly in the mandibular segment (Rickert, 2018).

Patterned contractile forces promote epidermal spreading and regulate segment positioning during Drosophila head involution
Epithelial spreading is a fundamental mode of tissue rearrangement occurring during animal development and wound closure. It has been associated either with the collective migration of cells or with actomyosin-generated forces acting at the leading edge (LE) and pulling the epithelial tissue. During the process of Drosophila head involution (HI), the epidermis spreads anteriorly to envelope the head tissues and fully cover the embryo. This results in epidermal segments of equal width that will give rise to the different organs of the fly. This study performed a quantitative analysis of tissue spreading during HI. Combining high-resolution live microscopy with laser microsurgery and genetic perturbations, it was shown that epidermal movement is in part, but not solely, driven by a contractile actomyosin cable at the LE. Additional driving forces are generated within each segment by a gradient of actomyosin-based circumferential tension. Interfering with Hedgehog (Hh) signaling can modulate this gradient, thus suggesting the involvement of polarity genes in the regulation of HI. In particular, it was shown that disruption of these contractile forces alters segment widths and leads to a mispositioning of segments. Within the framework of a physical description, it was confirmed that given the geometry of the embryo, a patterned profile of active circumferential tensions can indeed generate propelling forces and control final segment position. This study thus unravels a mechanism by which patterned tensile forces can regulate spreading and positioning of epithelial tissues (Czerniak, 2016).

Notch-dependent epithelial fold determines boundary formation between developmental fields in the Drosophila antenna

Compartment boundary formation plays an important role in development by separating adjacent developmental fields. Drosophila imaginal discs have proven valuable for studying the mechanisms of boundary formation. This study examined the boundary separating the proximal A1 segment and the distal segments, defined respectively by Lim1 and Dll expression in the eye-antenna disc. Sharp segregation of the Lim1 and Dll expression domains precedes activation of Notch at the Dll/Lim1 interface. By repressing bantam miRNA and elevating the actin regulator Enabled, Notch signaling then induces actomyosin-dependent apical constriction and epithelial fold. Disruption of Notch signaling or the actomyosin network reduces apical constriction and epithelial fold, so that Dll and Lim1 cells become intermingled. These results demonstrate a new mechanism of boundary formation by actomyosin-dependent tissue folding, which provides a physical barrier to prevent mixing of cells from adjacent developmental fields (Ku, 2017).

This study attempted to unravel the molecular and cellular mechanisms of boundary formation in the Drosophila head. Focus was placed on the antennal A1 fold that separates the A1 and A2-Ar segments. The results showed that the expression of the selector genes Lim1 and Dll, which are expressed in A1 and A2-Ar, respectively, was sharply segregated. This step was followed by differential expression of Dl, Ser and Fng, as well as activation of N signaling at the interface between A1 and A2. N signaling then induced apical constriction and epithelial fold, possibly through repression of bantam to allow levels of the bantam target Ena to become elevated, with this latter inducing the actomyosin network. The actomyosin-dependent epithelial fold then provided a mechanical force to prevent cell mixing. When N signaling or actomyosin was disrupted, or when bantam was overexpressed, the epithelial fold was disrupted and Dll and Lim1 cells become mixed. Thus this study describes a clear temporal and causal sequence of events leading from selector gene expression to the establishment of a lineage-restricting boundary (Ku, 2017).

Sharp segregation of Dll/Lim1 expressions began before formation of the A1 fold, suggesting that fold formation is not the driving force for segregation of Dll/Lim1 expression. Instead, the fold functions to safeguard the segregated lineages from mixing. Whether Dll/Lim1 segregated expression is due to direct or indirect antagonism between the two proteins is not known (Ku, 2017).

Actomyosin-dependent apical constriction is an important mechanism for tissue morphogenesis in diverse developmental processes, e.g., gastrulation in vertebrates, neural closure and Drosophila gastrulation, as well as dorsal closure and formation of the ventral furrow and segmental groove in embryos. This study describes a new function of actomyosin, i.e., the formation of lineage-restricting boundaries via apical constriction during development (Ku, 2017).

This actomyosin-dependent epithelial fold provides a mechanism distinctly different from other known types of boundary formation. The cells at the A1 fold still undergo mitosis, suggesting that mitotic quiescence is not involved. Perhaps epithelial fold as a lineage barrier is needed in situations in which mitotic quiescence does not happen. Mechanically and physically, epithelial folds could serve as stronger barriers than intercellular cables when mitotic activity is not suppressed. The drastic and sustained morphological changes, including reduced apical area and cell volume, may be accompanied by increased cortical tension of cells along the A1 fold, with such high interfacial tension then preventing cell intermingling and ensuring Dll and Lim1 cell segregation. Although similar to actomyosin boundaries, the epithelial fold in the A1 boundary is distinctly different from the supracellular actomyosin cable structure in fly parasegmental borders, the wing D/V border, and the interrhombomeric boundaries of vertebrates. The adherens junction protein Echinoid, which is known to promote the formation of supracellular actomyosin cables, is not involved in A1 fold formation. Although actomyosin is enriched in a ring of cells in the A1 fold, it does not exert a centripetal force to close the ring, unlike the circumferential cable described in dorsal closure and wound healing (see review. In the A1 fold, the constricting cells become smaller in both their apical and basolateral domains, thus differing from ventral furrow cells where cell volume remains constant (Ku, 2017).

A tissue fold probably provides a strong physical or mechanical barrier to prevent cell mixing. In addition, whereas in a flat tissue where the boundary involves only one to two rows of cells, the tissue fold involves more cells engaging in cell-cell communication. The close apposition of cells within the fold may allow efficient signaling within a small volume. This may be an evolutionarily conserved mechanism for boundary formation that corresponds to stable morphological constrictions such as the joints in the antennae and leg segments (Ku, 2017).

Although N signaling has been reported to be involved in many developmental processes, a role in inducing actomyosin-dependent apical constriction and epithelial fold is a novel described function for N. For the A1 boundary, N activity is possibly mediated through repression of bantam and consequent upregulation of Ena. In the wing D/V boundary, N signaling is also mediated through bantam and Ena, but the outcome is formation of actomyosin cables, i.e., without apical constriction and epithelial fold [19]. Thus, the N/bantam/Ena pathway for tissue morphological changes is apparently context-dependent (Ku, 2017).

Tissue constriction also occurs later in joint formation of the legs and antennae. N activation also occurs in the joints of the leg disc and is required for joint formation. This role is conserved from holometabolous insects like the fruitfly Drosophila melanogaster and the red flour beetle Tribolium castaneum to the hemimetabolous cricket Gryllus bimaculatus. It is possible that for segmented structures that telescope out in the P/D axis, like the antennae, legs, proboscis and genitalia, N signaling is used to demarcate the boundaries between segments, which are characterized by tissue constriction. N-dependent epithelial fold morphogenesis has also been reported in mice cilia body development without affecting cell fate, suggesting that such N-dependent regulation in morphogenesis is evolutionarily-conserved (Ku, 2017).

It is proposed that N signaling is important in all boundaries that involve stable tissue morphogenesis. For those boundaries corresponding to stable morphological constrictions, e.g., the joints in insect appendages, N acts via actomyosin-mediated epithelial fold. The wing D/V boundary represents a different type of stable tissue morphogenesis. It becomes bent into the wing margin and involves N signaling via actomyosin cables, rather than apical constriction. In contrast, actomyosin-dependent apical constrictions do not involved N signaling and are involved in transient tissue morphogenesis, such as gastrulation in vertebrates, neural closure, Drosophila gastrulation, dorsal closure, as well as formation of the ventral furrow, eye disc morphogenetic furrow, and segmental groove in embryos (Ku, 2017).

N signaling is also involved in the boundary between new bud and the parent body of Hydra, where it is required for sharpening of the gene expression boundary and tissue constriction at the base of the bud [78]. Whether the role of N in these tissue constrictions is due to actomyosin-dependent apical constriction and epithelial fold is not known (Ku, 2017).

Boundaries may be established early in development. As the tissue grows in size through cell divisions and growth, boundary maintenance become essential. This study found that N activity is maintained by actomyosin, suggesting feedback regulation to stably maintain the boundary. Mechanical tension generated by actomyosin networks has been suggested to enhance actomyosin assembly in a feedback manner. Interestingly, the N-mediated wing A/P and D/V boundaries, which form actomyosin cables rather than tissue folds, did not exhibit such positive feedback regulation. Instead, the stability of the Drosophila wing D/V boundary is maintained by a complex gene regulatory network involving N, Wg, N ligands and Cut. Perhaps this is necessary for a boundary not involving tissue morphogenesis (Ku, 2017).

The segmented appendages of arthropods (antennae, legs, mouth parts) are homologous structures of common evolutionary origin. It has been proposed that the generalized arthropod appendage is composed of a proximal segment called the coxopodite and a distal segment called the telopodite, either of which can further develop into more segments. The coxopodite is believed to be an extension of the body wall, whereas the telopodite represents the true limb, and thus represents an evolutionary addition. Dll mutants lack all distal segments except for the coxa in legs and the A1 segment in antennae. Lineage tracing studies have shown that Dll-expressing cells contributed to all parts of the legs except the coxa. These results indicate that the leg coxa and antenna A1 segment correspond to the Dll-independent coxopodite, and that Dll is the selector gene for the telopodite. Therefore, the antennal A1 fold is the boundary between the coxopodite and telopodite. It is postulated that the same N-mediated epithelial fold mechanism also operates in the coxopodite/telopodite boundary of legs and other appendages (Ku, 2017).

Drosophila Pax6 promotes development of the entire eye-antennal disc, thereby ensuring proper adult head formation

Paired box 6 (Pax6) is considered to be the master control gene for eye development in all seeing animals studied so far. In vertebrates, it is required not only for lens/retina formation but also for the development of the CNS, olfactory system, and pancreas. Although Pax6 plays important roles in cell differentiation, proliferation, and patterning during the development of these systems, the underlying mechanism remains poorly understood. In the fruit fly, Drosophila melanogaster, Pax6 also functions in a range of tissues, including the eye and brain. This report describes the function of Pax6 in Drosophila eye-antennal disc development. Previous studies have suggested that the two fly Pax6 genes, eyeless (ey) and twin of eyeless (toy), initiate eye specification, whereas eyegone (eyg) and the Notch (N) pathway independently regulate cell proliferation. This study shows that Pax6 controls eye progenitor cell survival and proliferation through the activation of teashirt (tsh) and eyg, thereby indicating that Pax6 initiates both eye specification and proliferation. Although simultaneous loss of ey and toy during early eye-antennal disc development disrupts the development of all head structures derived from the eye-antennal disc, overexpression of N or tsh in the absence of Pax6 rescues only antennal and head epidermis development. Furthermore, overexpression of tsh induces a homeotic transformation of the fly head into thoracic structures. Taking these data together, this study demonstrates that Pax6 promotes development of the entire eye-antennal disc and that the retinal determination network works to repress alternative tissue fates, which ensures proper development of adult head structures (Zhu, 2017).

In contrast to vertebrates that have a single Pax6 gene, the Drosophila genome contains two Pax6 homologs, ey and toy. Both genes are expressed broadly throughout the entire eye-antennal disc but are later limited to a far more restricted domain within the undifferentiated cells of the eye field. Whereas most studies on Pax6 in the eye-antennal disc have focused on the developing compound eye, several reports have hinted at a role for both genes outside of the eye. However, the underlying mechanism of how Ey/Toy promote eye-antennal disc development has been elusive. This is, in part, because of the use of single Pax6 mutants to study development. The phenotypes associated with individual mutants are variable and often restricted to the eye. Several studies have suggested that Ey and Toy function redundantly to each other. This finding most likely explains the variability of phenotype severity and penetrance. Thus, the combined loss of both Ey/Toy may be a more accurate reflection of the effect that Pax6 loss has on Drosophila development. Indeed, this appears to be the case as it is reported that the combined loss of both ey and toy leads to the complete loss of all head structures that are derived from the eye antennal disc. This study attempted to determine the mechanism by which Ey/Toy support eye-antennal disc development (Zhu, 2017).

Previous studies in the fly eye proposed that Pax6 is concerned solely with eye specification, whereas Notch signaling and other retinal determination proteins, such as Eyg, Tsh, and Hth, control cell proliferation and tissue growth. This study proposes an alternate model in which Ey/Toy are in fact required for cell survival and proliferation in addition to eye specification. The data indicate that Ey/Toy regulate growth of the eye-antennal disc through Tsh, N/Eyg, and additional N-dependent proliferation promoting genes. It is proposed that on simultaneous removal of Ey and Toy the eye-antennal disc fails to develop, in part, because the expression of eyg and tsh is lost in complete absence of Pax6. Expression of tsh and activation of the N pathway are sufficient to restore tissue growth to the eye-antennal disc. Support for this model linking Ey/Toy to cell proliferation via Eyg and Tsh comes from studies showing that eyg loss-of-function mutants display a headless phenotype identical to that seen in the ey/toy double knockdowns, that cells lacking eyg do not survive in the eye disc, and overexpression of Tsh causes overproliferation (Zhu, 2017).

The results also show that the combined loss of Ey and Toy affects the number of cells that are in S and M phases of the cell cycle. This observation directly supports the model that Ey/Toy control growth of the eye-antennal disc and is consistent with studies in vertebrates that demonstrate roles for Pax6 in the proliferation of neural progenitors within the brain. Earlier studies observed cells undergoing apoptosis in Pax6 single-mutant eye-antennal discs and showed that blocking cell death alone can partially rescue the head defects of the eyD and toyhdl mutants. Although this study shows that retinal progenitor cells lacking both Pax6 proteins undergo even greater levels of apoptosis, blocking cell death does not restore the eye-antennal disc. What accounts for the differences in the two experiments? In the eyD and toyhdl rescue experiments, each genotype contained wild-type copies of the other Pax6 paralog, but this study has knocked down both Pax6 genes simultaneously. Another possible difference is that Pax6 levels are being reduced while the eyD and toyhdl mutants are likely functioning as dominant negatives. It is concluded from these results that a reduction in cell proliferation but not elevated apoptosis levels is the proximate cause for the complete loss of the eye-antennal disc (Zhu, 2017).

Although the activation of Tsh and the Notch pathway can restore antennal and head epidermal development, neither factor is capable of restoring eye development to the ey/toy double-knockdown discs. This is most likely because both Pax6 genes are also required for the specification of the eye. In particular, Ey/Toy are required for the activation of several other retinal determination genes, including so, eya, and dac. Thus, the results suggest that Notch signaling, Eyg, and Tsh can restore nonocular tissue growth to the eye field but cannot compensate for the Pax6 requirement in eye specification (Zhu, 2017).

Finally, the results using the double knockdown of ey/toy are consistent with the dosage effects that are seen in mammalian Pax6 mutants. Although mutations in ey have just eye defects, the combined loss of ey/toy lacks all head structures. Mice that are heterozygous for Pax6 mutations have small eyes, whereas those that are homozygous completely lack eyes, have severe CNS defects, and die prematurely. Similarly, human patients carrying a single mutant copy of Pax6 suffer from aniridia, whereas newborns that are homozygous for the mutant Pax6 allele have anophthalmia, microcephaly, and die very early as well. As a master control gene of eye development, Pax6 appears to initiate both retinal specification and proliferation. These data demonstrate that the functions of Ey and Toy in the eye-antennal disc are redundant and dependent upon gene dosage, thereby making the roles of Pax6 in the Drosophila similar to what is observed in vertebrates where Pax6 controls both specification and proliferation of the brain and retina in a dosage-sensitive manner (Zhu, 2017).

Wnt6 is required for maxillary palp formation in Drosophila

Wnt6 is an evolutionarily ancient member of the Wnt family. In Drosophila, Wnt6 loss-of-function animals have not yet been reported, hence information about fly Wnt6 function is lacking. In wing discs, Wnt6 is expressed at the dorsal/ventral boundary in a pattern similar to that of wingless, an important regulator of wing size. To test whether Wnt6 also contributes towards wing size regulation, Wnt6 knockout flies were generated. Wnt6 knockout flies are viable and have no obvious defect in wing size or planar cell polarity. Surprisingly, Wnt6 knockouts lack maxillary palps. Interestingly, Wnt6 is absent from the genome of hemipterans, correlating with the absence of maxillary palps in these insects. It is concluded Wnt6 is important for maxillary palp development in Drosophila, and phylogenetic analysis indicates that loss of Wnt6 may also have led to loss of maxillary palps on an evolutionary time scale (Doumpas, 2018).

Wnt6 appears to have a specific role during Drosophila development, promoting maxillary palp formation. This is surprising, given that Wnt6 is quite ancient and present in most bilaterians. One possible interpretation is that the Wnt6 function might be redundant in most parts of the animal, perhaps due to overlapping expression with wingless, whereas Wnt6 expression in the maxillary palp might have been acquired in insects in a non-redundant fashion. This specific function in promoting maxillary palp formation might serve as a useful tool for studying the contribution of maxillary palps to olfaction and behavior. The maxillary palp contribution is currently assayed by surgical removal of the palps, whereas this could now also be accomplished genetically (Doumpas, 2018).

The Wnt6 gene is located directly adjacent to the wingless gene, raising the possibility that it arose as a genomic duplication of wingless. Accordingly, Wnt6 expression overlaps with that of wingless in numerous places (Jason, 2001). One possible reason for the overlapping expression patterns could be that Wnt6 expression is induced by wingless signaling; however, the data suggest this is not the case. Instead, it is likely that they either share enhancer elements or that regulatory elements were also duplicated alongside the open reading frame. Since wingless and Wnt6 have similar expression patterns and presumably transcriptional regulation, and since the anti-Wingless monoclonal antibody 4D4, the most widely-used in the field to detect wingless, appears to cross-react with Wnt6, some caution might be warranted in interpreting results with this antibody (Doumpas, 2018).

Given that Wnt6 is able to induce canonical wingless signaling in S2 cells, it was surprising that Wnt6 is quite poor at inducing wingless signaling in the wing disc. Consistent with this observation, expression of UAS-Wnt6 with various GAL4 drivers such as patchedts-GAL4 (with GAL80ts) and nubbin-GAL4 cause pupal lethality; however, this does not yield obvious morphological defects in the resulting wings, suggesting that the lethality is likely due to expression in other parts of the body. In contrast, Wnt6 expression in the central nervous system, including the maxillary palp, induces obvious significant morphological effects. One possible explanation could be that a component required for Wnt6 signaling might be expressed at higher levels in the nervous system compared to wing discs (Doumpas, 2018).

The specific absence of Wnt6 from the aphid A. pisum and the plant lice D. citri, both belonging to the order Hemiptera, a group that has lost maxillary palps, suggests that Wnt6-loss could have been the underlying genetic alteration leading to this morphological change. In hemipterans, the mouthparts are modified to form a tube-like structure for piercing. The tube, formed by the labrum and labium, comprises piercing-sucking structures formed by the modified mandible and the maxilla. The evolutionary loss of the maxillary palps was one of many structural modifications leading to the specialized hemipteran mouthparts. The loss of the maxillary palps could have compromised the sense of smell in hemipteran ancestors, but this may have been compensated by the elaboration of sensory structures on the labium. The specific phenotype of the Wnt6 knock-out in Drosophila contrasts with the pleiotropic effects of other secreted signaling molecules including wingless. This means that the deletion of the gene, rather than tinkering with its regulatory regions, could have resulted in a subtle morphological change, the loss of the maxillary palp, contributing to the morphological evolution of the beak-like hemipteran mouthparts (Doumpas, 2018).

Although Wnt6 expression overlaps substantially with that of wingless, it appears to play a critical role in maxillary palp growth, but not wing growth. Phylogenetic analysis suggests that loss of Wnt6 also correlates with loss of maxillary palps on an evolutionary timescale (Doumpas, 2018).

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Genes involved in head morphogenesis

Genes involved in organ development

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