fork head

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of fkh at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

fkh is expressed in most anterior and posterior regions of the embryo at the end of the syncytial stage. In the cellular blastoderm FKH is detected in both anterior and posterior terminal regions. The posterior region marks the amnioproctodael invagination. Posterior expression begins at stage 5. Anterior region transcripts spread into the primordium of the anterior midgut. At state 11 the anterior region of expression includes the invagination of stomodeum and the anterior midgut primordium. FKH transcripts are contained in the posterior midgut primordia and outbudding Malpighian tubules, as well as in salivary gland placodes and the periphery of the yolk sac (Weigel, 1989).

Postmitotic specification of Drosophila insulinergic neurons from pioneer neurons

Insulin and related peptides play important and conserved functions in growth and metabolism. Although Drosophila has proved useful for the genetic analysis of insulin functions, little is known about the transcription factors and cell lineages involved in insulin production. Within the embryonic central nervous system, the MP2 neuroblast divides once to generate a dMP2 neuron that initially functions as a pioneer, guiding the axons of other later-born embryonic neurons. Later during development, dMP2 neurons in anterior segments undergo apoptosis but their posterior counterparts persist. Surviving posterior dMP2 neurons no longer function in axonal scaffolding but differentiate into neuroendocrine cells that express insulin-like peptide 7 (Ilp7) and innervate the hindgut. The find that the postmitotic transition from pioneer to insulin-producing neuron is a multistep process requiring retrograde bone morphogenetic protein (BMP) signalling and four transcription factors: Abdominal-B, Hb9, Forkhead, and Dimmed. These five inputs contribute in a partially overlapping manner to combinatorial codes for dMP2 apoptosis, survival, and insulinergic differentiation. Ectopic reconstitution of this code is sufficient to activate Ilp7 expression in other postmitotic neurons. These studies reveal striking similarities between the transcription factors regulating insulin expression in insect neurons and mammalian pancreatic beta-cells (Miguel-Aliaga, 2008).

The observed death of some Drosophila pioneer neurons has been used to argue that their function is transient, but persistence in other cases suggested that, either they continue to play an axonal-scaffolding role, or that they adopt some other identity. The current findings resolve this long-standing issue by clearly demonstrating that, for dMP2 neurons, the axonal scaffolding function is only transient. After this role is no longer required, surviving dMP2 neurons become insulinergic and innervate the hindgut. The other known innervation of the Drosophila gut occurs much more anteriorly, in the foregut and anterior midgut, from neuronal cell bodies located in the peripheral ganglia of the stomatogastric nervous system. Unlike dMP2 neurons, however, the individual identities of the stomatogastric neurons and their cell lineages remain to be clearly defined. Thus, dMP2 neurons may provide a simple and well-characterised system for studies of the guidance cues involved in enteric innervation. Future studies, however, will be needed to determine the functions of Ilp7 in dMP2 neurons. It will be important to distinguish if this posterior neural source of insulin acts humorally to promote growth, like the more anterior brain mNSCs, or if it has more local effects in abdominal tissues. In this regard, the presence of Ilp7-expressing neurites in close proximity to the Ilp2-producing mNSCs is intriguing (Miguel-Aliaga, 2008).

The transition from pioneer to neuroendocrine neuron is not unique to dMP2 neurons, as Drosophila MP1 pioneer neurons also become neuropeptidergic at larval stages (Wheeler, 2006). In the grasshopper, segment-specific survival of pioneer neurons has also been reported, raising the possibility that they too may become neuroendocrine. Studies in other species, including vertebrates, will be needed to reveal the extent to which the linkage between pioneer and neuroendocrine functions is conserved. Identifying pioneer neurons with an 'ancestral' neuroendocrine identity in other phyla would lend further support to the proposal that pioneer neurons are highly conserved in evolution (Miguel-Aliaga, 2008).

Apoptosis of postmitotic neurons is a widespread feature of normal VNC development, but few developmental regulators of core pro-apoptotic genes such as grim, hid, and rpr have been identified. This study uncovers roles for Fkh and Hb9. Hb9, at least, appears linked to cell death in neurons other than dMP2: in Df(3L)H99 mutant embryos, where apoptosis is blocked, ectopic Hb9-positive RP motor neurons are observed in segments A7-A8. Hb9 is an important regulator of motor neuron identity in both Drosophila and vertebrates. Finding of a pro-apoptotic function for Hb9 in Drosophila, together with the neurotrophic requirement for motor neuron survival in vertebrates, raises the possibility that the same genetic programs specifying the identities of motor neurons also sensitize them for postmitotic editing via apoptosis (Miguel-Aliaga, 2008).

Fkh function in CNS development has not been characterized. Fkh is expressed in segmentally repeated clusters of midline neurons, including dMP2, vMP2, MP1 neurons, and the VUM interneurons. Within the MP2 lineage, Fkh is first expressed in the MP2 neuroblast at stage 9-10 and continues to be expressed in both the dMP2 and vMP2 daughters throughout embryonic and larval stages. In fkh mutants, 95% of anterior dMP2 neurons fail to undergo apoptosis, and 95.3% of posterior dMP2 neurons (and 100% of ectopic anterior counterparts) fail to express Ilp7. Both of these dramatic phenotypes could be rescued to near wild-type levels by reintroducing Fkh under odd-GAL4 regulation, indicating a cell-autonomous requirement for promoting dMP2 apoptosis and Ilp7 expression (Miguel-Aliaga, 2008).

Hb9 and Fkh expression in many neurons that do not die suggests a combinatorial mechanism for the control of developmental apoptosis. One possibility is that several transcription factors function in combination to activate the core pro-apoptotic genes. Given the proposed role for Foxa proteins in chromatin accessibility, Fkh expression in dMP2 neurons may render the promoters of core pro-apoptotic genes responsive to activation by Hb9. An alternative but not mutually exclusive mechanism involves individual transcription factors activating different pro-apoptotic genes such that a combination of these would then be required to trigger neuronal death. For example, Hb9 could be required for rpr/skl but not grim expression. Some support for this idea comes from the observation that loss of hb9 activity blocks rpr/skl-mediated death of dMP2 neurons but not the largely grim-dependent apoptosis of anterior MP1 neurons (Miguel-Aliaga, 2008).

An important conclusion from this study is that the combinatorial transcription factor code controlling apoptosis partially overlaps with that regulating insulinergic identity. Thus, Fkh and Hb9 are both essential components of the codes for anterior apoptosis and also Ilp7 expression, illustrating that these transcription factors play surprising dual roles as pro-apoptotic and pro-differentiation factors within the same neuronal subtype. Importantly, the results also show that the segment-specific Hox protein Abd-B acts as a postmitotic switch, converting the pro-apoptotic Fkh⁺ Hb9⁺ code into an insulinergic Fkh⁺ Hb9⁺ Abd-B⁺ code (Miguel-Aliaga, 2008).

Three Ilp7 regulators (Hb9, Abd-B, and Fkh) are expressed at least 12 h before Ilp7 is first activated: from the time when the MP2 neuroblast exits the cell cycle. In the case of Hb9, it was not possible to uncouple two temporally separable functions. Early postmitotic expression of Hb9 is important for its death-activating function, whereas later expression suffices for activating Ilp7. Similarly, the Hox protein Abd-B generates a segment-specific neuropeptide pattern via postmitotic regulation of posterior dMP2 survival and also Ilp7 activation. As vertebrate neuropeptides are also expressed in restricted neuronal populations within specific rostrocaudal domains, they may be similarly regulated by Hox survival/neuroendocrine inputs. In the case of Fkh, it is required for many different aspects of the progression from the early to the late postmitotic dMP2 fate. Fkh expression is restricted to VNC midline neurons and its vertebrate orthologue Foxa2 functions in the differentiation of the floor plate and ventral dopaminergic and serotonergic neurons (Ferri, 2007; Jacob, 2007; Norton, 2005). Thus, in both the Drosophila midline and its vertebrate counterpart, the floor plate, Fkh proteins play a conserved role in the differentiation of ventral neuronal subtypes (Miguel-Aliaga, 2008).

The other two dMP2 regulators identified in this study, Dimm and the BMP pathway, are switched on shortly before the onset of Ilp7 expression. The timing of onset of these two broad neuroendocrine regulators is likely to specify when Ilp7 is first activated, whereas the earlier factors Fkh, Hb9, and Abd-B may contribute more specifically to insulinergic identity. Together, the genetic and expression analyses in this study demonstrate that the combinatorial code of genetic inputs required for Ilp7 expression is assembled in a step-wise manner during postmitotic maturation. Importantly, this allows a subset of the components to be shared (such as Fkh and Hb9) between sequential neuronal programmes (survival and Ilp7 expression) without losing output specificity (Miguel-Aliaga, 2008).

Two observations from this study indicate that insulinergic combinatorial codes can vary from cell-to-cell and also from one Ilp to another. (1) None of the regulators of Ilp7 in dMP2 neurons appear to regulate it in DP neurons. (2) The dMP2 insulinergic code is sufficient to trigger ectopic expression of Ilp7 but not Ilp2 or other neuropeptides such as FMRFa. These findings suggest the existence of additional, as yet unidentified, insulinergic factors in DP neurons and also in the brain mNSCs where Ilp2 is expressed. Identification of the neural progenitor for these mNSCs (Wang, 2007) should facilitate characterization of the Ilp1/Ilp2/Ilp3/Ilp5 combinatorial codes and thus clarify the extent to which different insulinergic transcriptional programmes overlap (Miguel-Aliaga, 2008).

The finding that an Ilp7-expressing neuron derives from the MP2 lineage reveals that at least some insulinergic regulators are similar in insects and mammals. Three apparent similarities may not be very insulin-specific but reflect more general processes shared by neural and endocrine programmes in many species. (1) Notch signalling singles out the MP2 neuroblast and distinguishes its two progeny neurons, while in mammals, it limits pancreatic expression of the 'proneural' gene Ngn3 to prospective endocrine cells. (2) The survival and pro-Ilp7 functions mediated by Abd-B in the dMP2 neuron could also have their postmitotic counterparts in ß-cells, either mediated by related Hox genes or via another homeobox gene, Pdx-1, following its early input into pancreatic induction. (3) Nerfin-1 is required for dMP2 pioneer function (Kuzin, 2005), while its mammalian orthologue Insm1/IA1 is important for pancreatic ß-cell specification (Miguel-Aliaga, 2008).

Several more specific regulatory similarities exist between the insulinergic differentiation factors active in postmitotic dMP2 neurons. For example, the role of fkh in dMP2 neurosecretory differentiation described in this study is similar to the functions of HNF3b/Foxa2 in islet maturation and insulin secretion (Sund, 2001). In addition, mammalian Nkx2.2 is important for pancreatic ß-cell specification and is known to activate transcription of the insulin regulator Nkx6.1: an important late event in ß-cell differentiation. Intriguingly, the Drosophila orthologue of Nkx2.2, Vnd, is required for dMP2 formation. Drosophila Nkx6.1, the orthologue of mammalian Nkx6 (FlyBase name HGTX), is expressed by postmitotic dMP2 neurons, and it will be interesting to determine whether it too functions downstream of Vnd during Ilp7 regulation. Most strikingly, mammalian equivalents of two of the insulinergic inputs identified in this study, Hb9 and BMP signalling, are also required for several aspects of late ß-cell differentiation including the expression of Nkx6.1 and insulin. Together, these insect-mammalian comparisons provide evidence that, although the cell types involved look very different, some of the genetic circuitry regulating insulin is conserved between arthropods and chordates. This suggests that the power of fly genetics can now be harnessed to identify additional mammalian regulators of neuroendocrine cell fates and insulin expression (Miguel-Aliaga, 2008).

Effects of Mutation

The Drosophila Brachyury homolog brachyenteron (byn) is essential for the development of hindgut, anal pads and Malpighian tubules. byn is activated by the terminal gap gene tailless (tll) in a region of 0%-20% egg length of the syncytium (0% = posterior tip). With completion of cellularization, the byn expression becomes downregulated in the posteriormost cap of the embryo, which will later form the posterior midgut, by the terminal gap gene huckebein (hkb). Thus, the expression of byn is confined to a ring of cells from about 10%-20% egg length. The dorsal and the lateral aspects of that ring correspond to the proctodeum, from which the hindgut, the anal pads and the Malpighian tubules later develop. Intriguingly, hkb also determines the posterior extent of the ventral mesoderm primordium by repressing the mesodermal determinant snail (sna). This suggests that the ventralmost aspect of byn expression might comprise the posterior tip of the mesoderm primordium (Kusch, 1999).

The visceral musculature of the larval midgut of Drosophila has a lattice-type structure and consists of an inner stratum of circular fibers and an outer stratum of longitudinal fibers. The longitudinal fibers originate from the posterior tip of the mesoderm anlage, which has been termed the caudal visceral mesoderm (CVM). The CVM migrates in an orderly movement anteriorly and eventually forms an outer layer of longitudinal muscle fibers surrounding the midgut. The progenitors of a second tissue, the inner sheet of circular muscles of the midgut, are recruited from 11 parasegmentally arranged clusters of dorsal mesoderm in the trunk region and are therefore referred to as trunk visceral mesoderm (TVM) (Kusch, 1999).

In this study, the specification of the CVM has been investigated and particularly the role of the Drosophila Brachyury-homolog brachyenteron. Supported by fork head, brachyenteron mediates the early specification of the CVM along with zinc-finger homeodomain protein-1. This is the first function described for brachyenteron or fork head in the mesoderm of Drosophila. The mode of cooperation resembles the interaction of the Xenopus homologs Xbra and Pintallavis. Another function of brachyenteron is to establish the surface properties of the CVM cells, which are essential for their orderly migration along the trunk-derived visceral mesoderm. During this movement, the CVM cells, under the control of brachyenteron, induce the formation of one muscle/pericardial precursor cell in each parasegment. It is here proposed that the functions of brachyenteron in mesodermal development of Drosophila are comparable to the roles of the vertebrate Brachyury genes during gastrulation (Kusch, 1999).

During germband retraction and midgut closure, the progenitors of the the outer, longitudinally oriented fibers of the visceral mesoderm, the CVM, perform an ordered movement that can be subdivided into three phases. The first migratory phase starts at early germband retraction when the cells begin to move anteriorly from their position at the posterior tip of the mesodermal germ layer and split into two tightly packed, bilaterally symmetrical clusters on each side of the posterior midgut primordium. When these clusters have reached the anterior tip of the posterior midgut primordium, the cells detach from each other and disperse anteriorly as two rows along the germband, the second phase of the migration. During this movement, the cells are arranged along the dorsal and ventral edge of the midgut primordia and are in close contact with the band of progenitors of the circular muscle fibers. The band seems to serve as a migration substratum. During the last phase of the migration, which takes place as the midgut encloses the yolk, the progenitors of the longitudinal muscle fibers spread regularly over the underlying circular muscle fibers. The cells acquire a spindle shape, then stretch in an anteroposterior direction and form about 16-20 regularly spaced longitudinal muscle fibers. These fibers reach from the proventriculus to the midgut-hindgut transition where the ureters of the Malpighian tubules insert. The foregut and the hindgut lack any longitudinal muscles and are solely covered by the inner layer of circular muscles (Kusch, 1999).

The specification of the CVM and its fate were monitored by the detection of Byn protein or the expression of CVM-specific markers like croc-lacZ and cpo-lacZ. The initial byn expression at the posterior pole is regulated by tll and hkb. Thus it is likely that the CVM cells are specified under the control of the same genes. In fact, in hkb embryos, the size of the CVM primordium is enlarged and comprises more cells than normal. This corroborates the notion that the CVM primordium constitutes the most posteriorly located mesoderm primordium. tll expression reaches more anteriorly than the hkb domain and encompasses the primordia of the proctodeum and of the CVM. One would therefore expect that the formation of the CVM is entirely dependent on tll. Indeed, this is the case: the CVM is missing in tll mutant embryos. Part of the function of tll seems to be mediated by byn. In byn mutants, a significantly reduced number of CVM cells is seen, and these few cells form clusters that are less compact and migrate significantly slower than in wild type. Later, they fail to contact the TVM and do not distribute along the germband. During stage 11, most of the cells acquire a condensed appearance resembling apoptotic bodies. A high level of apoptosis is detected in the proctodeum of byn embryos as well as in the posteriormost mesoderm. By stage 13, cells with the properties of the CVM are not detectable any longer in the mutants and, as expected from this, the dissected midguts of byn embryos lack the outer, longitudinal muscle fibers (Kusch, 1999).

byn embryos show morphological aberrations at a time before the CVM begins to migrate anteriorly. The severely shortened hindgut causes a significant shift in the spatial relationship of the various primordia at the posterior region of the embryo and thereby might indirectly affect the migration of the CVM. In order to exclude such an indirect influence, byn embryos were generated that expressed byn in the CVM precursors, but not in the hindgut. In such embryos, the CVM survives and disperses virtually the same as in wild type along the TVM, whereas the proctodeum remains rudimentary as in ordinary byn mutants. These results demonstrate that the defective migration and the death of the CVM cannot be attributed to the disordered morphology of the posterior gut structures. It has therefore been concluded that byn in the mesoderm is essential for the adhesive and migratory properties of the CVM precursors. byn cannot be the only gene that mediates the function of tll in the specification and further development of the CVM since the lack of tll causes a far stronger phenotype than the lack of byn. In addition to byn, the gene fkh is known to act downstream of tll in the posterior gut. fkh is expressed in a large domain at the posterior pole that encompasses the byn expression domain including the ventral, mesodermal aspect. In fkh mutants, the CVM specification seems less impaired than in byn mutants: the number of CVM cells is initially quite normal. However, as in byn mutants, the cells fail to migrate along the germband although differentiation of the migration substratum, the TVM, is not affected. By stage 14, most of the CVM cells have been eliminated by apoptosis. On this level of analysis, fkh mutants resemble embryos homozygous for weak byn alleles. However, the phenotype of byn fkh double mutants shows that byn and fkh either have distinct functions in the specification of the CVM or act synergistically. In double mutants, no CVM cells are distinguishable, just as in tll mutants. Therefore, the function of tll in the specification of the CVM appears to be mediated by byn and fkh (Kusch, 1999).

Only the anterior and the posterior mesoderm are competent to be specified by byn as CVM, in conjunction with fkh. Therefore, at least one other gene must exist that confines the competence to form CVM to these two regions. A good candidate for this gene is zinc finger homeodomain protein-1 (zfh-1). At the blastoderm stage, zfh-1 is expressed in high levels in the terminal regions of the mesoderm including the primordium of the CVM. zfh-1 is essential for the migration of the CVM: in zfh-1 mutant embryos, CVM-specific gene expression such as croc-lacZ is deleted. From the restricted effects of ectopic byn /fkh, it has been proposed that the two genes are capable of specifying CVM development only in the region of high zfh-1 expression. zfh-1, byn and fkh act in parallel downstream of tll. High levels of caudal zfh-1, as with byn and fkh, are dependent on tll, and there is no crossregulation between zfh-1, byn and fkh (Kusch, 1999).

In vertebrates (deuterostomes), brain patterning depends on signals from adjacent tissues. For example, holoprosencephaly, the most common brain anomaly in humans, results from defects in signaling between the embryonic prechordal plate (consisting of the dorsal foregut endoderm and mesoderm) and the brain. Whether a similar mechanism of brain development occurs in the protostome Drosophila has been examined; the foregut and mesoderm have been found to act to pattern the fly embryonic brain. When the foregut and mesoderm of Drosophila are ablated, brain patterning is disrupted. The loss of Hedgehog expressed in the foregut appears to mediate this effect, as it does in vertebrates. One mechanism whereby these defects occur is a disruption of normal apoptosis in the brain. These data argue that the last common ancestor of protostomes and deuterostomes had a prototype of the brains present in modern animals, and also suggest that the foregut and mesoderm contributed to the patterning of this 'proto-brain'. They also argue that the foreguts of protostomes and deuterostomes, which have traditionally been assigned to different germ layers, are actually homologous (Page, 2002).

As the Drosophila foregut invaginates, it normally becomes ensheathed by visceral mesoderm. Thus, when the foregut is ablated, visceral mesoderm is displaced from its normal position adjacent to the brain. How much does the loss of mesoderm contribute to the brain phenotype seen in foregut ablated animals? Embryos lacking function of the NK-2 class transcription factor Tinman have defects in forming mesoderm around the foregut, as revealed using mesodermal markers as Fasciclin III expression, but do form foregut ectoderm. In 65% of Tinman loss-of-function embryos there were excess cells at the dorsal midline of b1; the area occupied by neuronal nuclei was increased when compared with wild type in this region of the brain, and the preoral brain commissure was abnormally thin (Page, 2002).

Why are there excess cells at the dorsal midline in foregut- and mesoderm-ablated embryos? During normal brain development, more neurons are born than will be present in the adult brain and apoptosis eliminates the excess cells. Defects in apoptosis could contribute to the observed defects in brain patterning by failing to remove excess cells. To see if apoptosis was perturbed when the foregut and mesoderm were ablated, Acridine Orange staining, which labels apoptotic cells, was carried out. In forkhead loss-of-function embryos, the pattern of apoptosis in the brain at the level of the preoral brain commissure was clearly different from wild type at late ES13. In the wild-type b1 neuromere, there were groups of apoptotic cells at the dorsomedial edges of the hemispheres. This correlates with previous observations regarding the expression of the apoptosis regulatory protein Reaper. In forkhead loss-of function embryos, which are defective in foregut development, there was a clear reduction in the number of these cells. Examination of tinman loss-of-function embryos, which are defective in mesodermal development, showed that removal of mesoderm results in a similar reduction in the number of apoptotic cells at the dorsal midline, thus suggesting that the mesoderm and possibly the foregut have an influence on the normal pattern of apoptosis in brain development (Page, 2002).

The results of foregut and mesoderm ablation experiments strongly suggest that the brain is patterned by induction from these tissues. Did ablation of these tissues remove inductive signals required for normal brain development? What molecular signals could be mediating this effect? In vertebrates, Hedgehog signaling originating from the prechordal plate functions in forebrain patterning. Thus, the Hedgehog pathway in Drosophila seemed a good place to begin to look for inductive signals involved in brain development. Null mutations in Drosophila Hedgehog result in a phenotype in 70% of embryos that strongly resembles the one seen in the foregut ablation experiments. In brain segment 1 (b1), the right and left hemispheres of the brain are joined at the midline or separated by an abnormally small space because of excess cells in this region, and the preoral brain commissure shows abnormal defasciculation. In addition, in b1 the frontal commissure is missing, and there is a significant decrease in the area occupied by neuronal nuclei and the number of glia. In b2-S3 (S3 is ventrolateral to the foregut), the longitudinal connectives are disrupted, and the area occupied by neuronal nuclei and the number of glia is significantly reduced, and the number of Fasciclin II-expressing neurons is reduced (Page, 2002).

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