Neuron,
Vol. 9, 1025-1039, December,
1992, Copyright
0 1992 by Cell Press
Formation of the Drosophila larval Photoreceptor O rgan and Its Neuronal Differentiation Require Continuous l&i”/ Gene Activity Dietmar Schmucker, Heike Taubert, and Herbert Rickle Max-Planck-lnstitut fur Biophysikalische Chemie Abteilung Molekulare Entwicklungsbiologie Am FaBberg W-3488 Gottingen Federal Republic of Germany
Summary The Drosophila segmentation gene Kriippd (Kr) is rede ployed to play a critical role for the establishment of the larval visual system. Using reporter gene expression conducted by a specific Kr cis-acting element, we were able to trace back the origin of the larval photoreceptor organ, the Bolwig organ, to a single progenitor neuron and toexamine Krfunction in Bolwigorgan development when KP activity is absent from embryos due to specific mutations or reduced by neuron-specific and temporally restricted Kr antisense RNA expression. Our results show that Kr is required for neurons to differentiate into Bolwig organs, for fasciculation of the Bolwig nerve, and for this nerve to follow a specific pathway toward the synaptic targets in the larval brain. The transcription factor encoded by Kr is likely to regulate surface molecules necessary for neuronal cell adhesion and recognition in the developing larval visual system. Introduction The visual system of the adult Drosophila fly, compound eyes and three ocelli (Heisenberg and Wolf, 1984, is preceded by a larval photoreceptor system consisting of a pair of the so-called Bolwig organs (Bolwig, 1946). Each of them is composed of only 12 tightly clustered photoreceptor cells (Steller et al., 1987; Pollock and Benzer, 1988) resembling the rhabdomeric structures found in evolutionarily more primitive insect species (Melzer and Paulus, 1989). The cell bodies of the Bolwig photoreceptor neurons can be observed at midstage of embryogenesis, and they undergo stereotypical movements duringthe process of head involution (Steller et al., 1987). Concurrently, their neurons extend axons that fasciculate to form the Bolwig nerve that navigates posteriorly to enter theoptic lobe primordium. Someof theaxons contact specific cells of the optic lobe anlage; the others are thought to grow further and synapse in the developing brain (Tix et al., 1989), thereby forming the larval visual system. The stereotypical projection pathway of the Bolwig nerve is not preceded by any other nerve. Thus, the Bolwig nerve has to pioneer its specific way through a variety of tissues before it reaches the synaptic target ceils in the brain. This implies that the Bolwig nerve must have the capability to select a particular axonal
pathway likely to be guided by specific landmark cells or substances. Pathfinding of nerves involves the distalmost region of the axon, the growth cone, which can distinguish one group of cells and/or neurons from another. Based on the analyses of growth cone specificity, Raper et al. (1983a, 1983b, 1984) proposed that neighboring axon pathways or attracting tissue must be differentially labeled by surface recognition molecules that allow growth cones to distinguish among them, a notion called the labeled pathway hypothesis. The involvement of cell adhesion molecules serving as functional cell labels along specific axonal pathways in the developing central nervous system of the Drosophila embryo has recently been demonstrated (Harrelson and Goodman, 1988; Grenningloh et al., 1991). Furthermore, different adhesive or chemotropic factors that stimulate axonal outgrowth in vitro have been characterized in several organisms (for review see Jessell, 1988). However, such factors or cellular interactions necessary for the guidance of the Bolwig nerve through both neuronal and nonneuronal tissue to its synaptic partners are unknown. The pioneer function of the Bolwig nerve that eventually establishes the initial neuronal connection between the Bolwig organ and the embryonic brain is not only important for the processing of the perceived information by the larval brain, but also for proper innervation of the adult brain by the sensory neurons of the compound eye, which forms from an imaginal disc in third instar larvae (Steller et al., 1987). Initially, these neurons followthe pathway of the Bolwig nerve until they reach the optic lobe primordium (Steller et al., 1987). When this initial contact is stabilized, the Bolwig organs become histolyzed, and their nerves degenerate (Pollock and Benzer, 1988). The dependence of the adult visual system on the neuronal connections pioneered by the Bolwig nerve becomes obvious in mutants for the disconnected (disco) gene. Although the disco gene is not expressed in the Bolwig organ or its progenitor cells (Lee et al., 1991), mutations in the disco gene cause severe misrouting of the Bolwig nerves and frequent termination in ectopic locations (Steller et al., 1987). In these instances, the adult retinula photoreceptor neurons fail to project normally as well, and the optic lobes degenerate (Steller et al., 1987). These findings demonstrate that the initial contacts between the Bolwig nerve and the brain are essential for the developing adult visual system. Two genes coding for the transcription factor g/ass (g/) (Moses et al., 1989) and the cell adhesion molecule chaoptin (Van Vactor et al., 1988) are known to be expressed specifically in photoreceptor cells. Mutant analysis has shown that the absence of each of the two components causes abnormal photoreceptor development, and their activity is required for normal Bolwig organ formation (Van Vactor et al., 1988; Moses
Neuron 1026
et al., 1989). gl encodes a zinc finger-type protein regulating the expression of chaoptin (Moses et al., 1989) and of the photopigment rhodopsin Rhl in the larval photoreceptor neurons (Pollack and Benzer, 1988). In embryos that carry a strong gl mutation, the normal Bolwig organs are replaced by loosely clustered neurons with abnormal axonal projections (Moses et al., 1989). It was recently noted that Kriippel (Kr), a member of the gap class of segmentation genes, is expressed prominently in clusters of cells that are likely to represent the Bolwig organs (Gaul et al., 1987). In view of the expression of other segmentation genes, such as the pair-rule genes fushi tarazu and evenskipped, and their requirement for specifying the neural identity in defined subsets of cells in the developing central nervous system (Doe et al., 1988a, 1988b), we wanted to know whether segmentation genes might have cell commitment function in two distinct developmental processes, segmentation and neurogenesis, in general and which role Kr plays in the process of larval photoreceptor development in particular. W e first identified the Kr cis-acting sequences conducting Bolwig organ-specific gene expression. By use of reporter gene expression controlled by this Kr control element, we were then able to identify and to follow Bolwig organ progenitor cells and the pathway of the Bolwig nerve throughout development in wild-type and Kr mutant embryos and in embryos in which Kr’ activity had been reduced temporally or in a cell-specific manner. W e found that in contrast to the in the cenfunctions of fushi tarazu and even-skipped tral nervous system, Kr does not determine the neuronal identity of Bolwig organ progenitor cells. Instead, Kr’ activity acts thereafter to regulate subsequent steps of organ formation, fasciculation of axons, axonal extension, and target recognition processes. Our results show that both Krand the previously identified gl gene are independent components of the genetic circuitry leading to Bolwig organ formation, and both components are required for axonal guidance andlor adherence along the stereotypical Bolwig nerve pathway, which can be defined by specific landmarks.
W e have recently noted Kr expression in cell clusters that by morphological criteria correspond to the Bolwig organs in the head region of the late embryo (Gaul et al., 1987). To substantiate this observation, we performed double staining experiments using anti-Kr protein antibodies in combination with diagnostic monoclonal antibodies directed against acell surface molecule common to many neurons (monoclonal antibody22ClO[MAb22ClO]; Fujitaet al.,1982)oragainst the photoreceptor cell-specific membrane protein chaoptin (MAb 24810; Zipursky et al., 1984; Van Vactor et al., 1988). By this, we found that Kr is expressed not only in the Bolwig organ but also throughout all stages of its development, starting from a single Kr-
expressingcell (FigureslA-lD).Thiscell showsaxonal outgrowth with agrowth cone and expresses the MAb 2200 antigen (Figures IB-IE). This indicates that it has already adopted the neuronal fate when Krexpression is first seen. To establish the function that the segmentation gene Kr may exert in the process of larval visual system formation, we examined Bolwig organ development in wild-type embryos and in embryos with altered Kr’ activities. For this analysis, we constructed a cell-specific marker gene composed of the Kr cis-acting element (the Bolwig organ element; see Experimental Procedures) able to direct bacterial /acZ reporter gene expression in the developing Bolwig organs of transgenic embryos. Expression from this specific cell marker gene and the development of the Bolwig organs allowed us to study the development of the Bolwig organs from a single pair of neurons and to follow axonal processes due to the presence of B-galactosidase in the cytoplasm of the Bolwig organ neurons. Each of the two Kr-expressing neurons that later become part of a Bolwig organ is located in a mediolateral position to either side of the procephalic lobe of g-to IO-hr-old wild-type embryos (Figures IA and IB). On each side, the axon(s) of the first Kr-expressing neuron(s) elongates in a dorsal-posterior direction (Figures ID and IE). The contact cell at the pharynx (ccp) cell, which is stained by MAb 22ClO at stage 15 (Campos-Ortega and Hartenstein, 1985) (for details see Figure I), demarcates the position of the first turn of the axon in a ventrolateral direction (Figure 1; see also Figure 3 and Figure 6). A second morphological landmark is the posterior edge of the procephalic lobe. The contact of the nerve with this landmark can be seen at the time when a tight cluster of additional Kr-expressing neurons appears in close proximity to the first Kr-expressing cell body (Figures IC and ID). Since we have not observed this or any other Krexpressing cells in division, we assume that the additional Kr-expressing neurons are recruited from the surrounding cells rather than derived through division of Kr-expressing cells. The axons of these neurons add to the pioneering axon, and thefasciculated nerve continues to elongate along the brain hemisphere toward the optic lobe precursor cells (see Tix et al., 1989) (FigureslFandlG). W h e n thetipofthenerveisfound to be in contact with cells at the surface of the optic lobe anlagen, it changes its orientation at stage 15 (Campos-Ortega and Hartenstein, 1985) and grows almost perpendicularly to the surface, taking a straight route into the central brain region (see Figure 38). The synaptic partners of the Bolwig nerve in the central brain region are not yet identified. A detailed description and a schematic drawing summarizing the development of the larval visual system in wild-type embryos are given in Figure 1. larval Visual System Development Requires Kr+ Activity To elucidate the function of Kr in the Bolwig
organ,
Krijppel 1027
Function
in the Larval Visual System
we examined their development in embryos homozygous for Kr alleles of various strengths. As summarized in Table 1, a number of specific defects can be distinguished in Kr mutant embryos by morphological criteria and by the ability of Bolwig nerves to reach the different landmarks described for wild-type development. The most severe defects were observed in embryos carrying the loss-of-function allele Krl (a Kr gene deletion; see Preiss et al., 1985). In the absenceof Kr+activity, embryos lack the Bolwig organs, and in their places are loosely clustered groups of 2-6 neurons (Figures 2A-2D) that express the photoreceptorspecific cell adhesion molecule chaoptin (Table 1; Figure 2C). The extreme phenotype was observed in a portion of more than 20% of the embryos analyzed. In those embryos one of the two organs was missing, and the single organ rudiment consisted of no more than 3 cells. The majority of the embryos developed rudimentary organs with a maximum of 6 looselyclustered neurons. In rare cases in which a Bolwig nerve could be identified, it was never found to contact the ccp cells and always terminated in ectopic positions. In embryos homozygous for a Krallele of intermediate strength, such as KrV and KPz-125,the number of neurons forming the rudimentary Bolwig organs was also reduced to a maximum of 6. In KP2-lz homozygous mutant embryos, most of the neurons lack detectable axons (Figures 2G and 2H). In the few cases in which a misrouted nerve was observed, it never contacted the ccp cells, as observed in the Kr loss-offunction mutantsfseeabove). InKrVembryos,thecluster of neurons was split frequently, and each subcluster projected its own nerve. This phenomenon could not only be observed with homozygous mutant embryos, but also with a minor fraction of KP heterozygous embryos (Figures 2E and 2F). In contrast to Kr’ and KP2-125embryos, however, the Bolwig nerve in KP mutants reached the ccp landmark cell, but except for a few cases, the Bolwig nerve missed the optic lobe region. In these cases, the projection of the nerve along the brain hemisphere had an abnormal “wavy, shape, and the fasciculation of the nerve was interrupted. In contrast to wild-type embryos in which the nerve grows into thecentral brain region, the termination of nerves in Krv mutants occurred in a caudal position of the brain hemisphere, where severe defasciculation (“axonal sprouting”) takes place (Figure 3A). In embryos homozygous for the weak KP4 mutation, the Bolwig organ rudiment consists of a maximum of 8 neurons located in the wild-type position (compare Figure 11 and Figure 2D). The Bolwig nerve reaches both the ccp cell and the optic lobe anlagen. After having reached the latter position, the nerve defasciculates. In contrast to wild-type embryos in which a punctate pattern of termination can be observed, the KP2-14mutant Bolwig nerve splits into several branches (Figure 21). These results indicate that the formation of the Bolwig organ, fasciculation of axons, the neuronal path-
way, and synaptic targeting of the Bolwig nerve are conspicuously altered in the absence of the Kr wildtype gene product. In strong alleles, the formation of the Bolwig organ, fasciculation, and the early portion of the Bolwig nerve pathway are affected, while in mutants carrying the weak allele, the final targeting of the Bolwig nerve was the most obvious defect observed. Thus, the severity of the defects in the larval visual system of the different Kr mutants correlates well with the severity of the segment pattern defects caused by the corresponding alleles. It was therefore important to exclude the possibility that the defects observed in the larval visual system are of a secondary nature, since segmentation defects are known to cause secondary effects such as the abnormal head involution (Wieschaus et al., 1984). This possibility, however, appears unlikely, since minor defects (such as a slight reduction in the number of cells in the Bolwig organ and defective fasciculation and final targeting of the Bolwig nerve; Table 1 and Figures 2E and 2F) can be observed in the developing visual system of Kr heterozygous embryos that develop normal heads. To demonstrate unambiguously that the cellspecific perturbance of Kr+activity is the cause of mutant defects in the larval visual system, we performed rescue experiments involving organ-specific Kr+ gene expression in Kr mutant embryos, and we attempted to reduce specifically Kr’ activity in the neurons of Bolwig organs through the expression of Kr antisense RNA. Ceil Autonomous Kr+ Requirement in Bolwig Organ Precursors Rescue experiments were performed with a Kr minigene construct containing the Kr coding sequence and the Kr basal promoter under the control of the Bolwig organ element (BO) (BO-KrR minigene; see Experimental Procedures). After P element-mediated transformation, transgenic flies were crossed to KP flies in a way that Kr” homozygous embryos carried either one or two copies of the BO-KrR minigene. Such embryos show the KP mutant segmentation phenotype, but most aspects of the defects observed in the mutant larval visual system were rescued. The rescue includes an increase in Bolwig organ cells, fasciculation of the axons, and projection of the nerve to the optic lobe area in all KP mutant embryos containing the rescue construct. However, targeting in the correct brain region of some of these embryos was still abnormal, i.e., terminal sprouting of the Bolwig nerve occurs in a fashion similar to the defects observed in the mutants that carry the weak KP2-14allele (data not shown). An example of the rescue phenotype is shown in Figure 4. In complementary experiments we tried to reduce Kr’ activity by means of neuron-specific expression of Kr antisense RNA. For the production of Kr antisense RNA, we used a fusion gene construct containing the reverselyorientedKrcodingsequence(BO-Krasgene; see Experimental Procedures). Transgenic lines con-
Neuron 1028
Figure 1. Bolwig
Organ
Development
Visualized
by Reporter
Gene
Expression
and MAb 22ClO Staining
(A-K) Optical sections through wild-type embryos expressing B-galactosidase under the control of the Bolwig organ element to vis ualize Bolwig organ neurons specifically. Embryos were stained with anti-B-galactosidase antibodies (dark blue staining) and the ne uronspecific MAb 22ClO (bright brown staining). (A) Stage 12 embryo showing the first Bolwig organ neuron (arrow) located in a posterior ventral position of the procephalic lobe. MAb 22ClO weakly stains the first neurons and outgrowing axons of the peripheral nervous system at this stage.
Krijppel 1029
Function
in the Larval Visual System
taining the BO-Kras gene construct in different locations of the chromosomes had significantly reduced levels of Kr protein in the Bolwig organ neurons (see Experimental Procedures). The corresponding individuals were viable, but the developing visual system of such embryos was evidently impaired. The phenocopies produced by Kr antisense RNA showed in lower penetrance all the characteristics of defects of the larval visual system observed in the Kr mutants (Table 1; see examples in Figures 5A-5C). This finding and the defects observed in Kr heterozygous mutant embryos suggest that larval visual system development is critically dependent on the level of Kr+activity.
number of cells forming the Bolwig organ was higher, and the initial fasciculation of the axons appeared less affected than that observed with heat shock treatment at 9 hr. No effect on the Bolwig organ was observed in embryos that received heat shock treatment 12 hr after egg deposition, but defasciculation occurred in the terminalmost positions of the otherwise normally developed Bolwig nerve (Figure 6D). This result shows that the Bolwig organ of 12-hr-old embryos is no longer sensitive to hsp70-Kras gene expression, while the process of pathfinding, in particular the final synapsing in the brain, has not been completed at this stage of development (Table 1).
Continuous Kr+ Requirement for Axonal Guidance To determine the period when Kr+ activity is critically required during larval visual system development, we used heat shock-dependent Krantisense RNA expression to reduce the level of Kr+ activity by 30 min heat shock treatments of 9-, IO-, or IZhr-old embryos containing the “hsp70-Km” transgene (see Experimental Procedures). Severe defects in the fully developed visual system were observed in embryos that received heat shock treatment 9 hr after egg deposition. In these Kr antisense RNA-expressing embryos (see Experimental Procedures), the Bolwig organs were reduced to a variable number of 4-8 loosely connected neurons, and the nerves showed severe defasciculation (Table 1; Figures 6A-6C). Their axons terminate in different abnormal positions. In embryos that received the heat shock 10 hr after egg deposition, the
Complementary Functions of Kr and g/3 The Bolwig organ phenotype of Kr mutants strongly resembles the one recently described for gl mutants (Moses et al., 1989). Using /acZ expression conducted by the Bolwig organ element and anti-Kr protein antibody stainings in gl mutants, we observed expression both of Kr protein and B-galactosidase in the Bolwig organ precursors and in the loosely clustered neurons forming a Bolwig organ rudiment. This result excludes the possibility that gl is required for the control of Kr gene expression. Conversely, the chaoptin gene is expressed in Kr mutants (see above). Since gl activity is necessary to activate chaoptin gene expression (Moses et al., 1989), Kr+ activity is not required for chaoptin or for gl expression. The lack of gl and Kr gene activities, in Krlgl double mutants, resulted in the lack of storable Bolwig organs in most of these
(B) Posterior procephalic region of a stage 12 embryo showing a Bolwig organ progenitor neuron colabeled by anti-B-galactosidase and MAb 22ClO. Note that the growth cone (arrowhead) projects dorsally. The position of the Bolwig organ progenitor neuron is dorsal to the maxillary segment fms) and anterior to the dorsal ridge (dr) separated by the cephalic furrow (cf). Costaining with anti-Kr antibodies and anti-B-galactosidase antibodies revealed early Kr expression in those cells labeled by B-galactosidase staining (data not shown). (C) Stage 13 embryo showing additional B-galactosidaseexpressing cells, indicating a stepwise increase of the number of Bolwig organ neurons. Note additional MAb 22ClO staining ceils and axons in the periphery of the embryo, indicating that the peripheral nervous system has proceeded in development. (D and E) Posterior procephalic region of a stage 13 embryo. Note a cluster of 3-4 Bolwig organ progenitor cells (bo) in a position above the ventral organ fvo) of the antennomaxillary complex. Only one Bolwig organ progenitor neuron extends a pioneer axon (lower arrowhead in [D]) with a growth cone (upper arrowhead in [D]) projecting toward dorsal posterior. Note enhanced membrane staining of the Bolwig organ neurons due to the costaining with anti-Bgalactosidase antibodies and MAb 22ClO. Note also the dark, punctate staining pattern of growth cones (arrow in [El) that derive from the newly recruited Bolwig organ progenitor cells prior to their fasciculation with the pioneer axon (black arrowheads). (E) is composed of two separate photographs taken from slightly different optical sections of the same region of the embryo to show both the pioneer and successor axons in one focal plane. (F and G) Embryos before(F) and during (C] the process of head involution, indicating that the Bolwig organ has moved from a medialposterior head position (see [Cl and [I]) to a dorsal-anterior position (white arrows). Note the initial projection of the Bolwig nerve and a sharp turn in its terminal region (arrowhead) at a position where the nerve leaves the brain surface to grow inside. (H) Stage 15 embryo showing that the Bolwig organ (bo) is located next to the dorsal organ (do) of the antennomaxillary complex, close to its final position at stage 16. (I and K) Dorsal views of almost fully developed embryos. The nervous system is labeled with MAb 22ClO staining. Note the position of the Bolwig organ (bo) located in a posterior position of the antennomaxillary complex (amc), as revealed by B-galactosidase expression and counterstaining with MAb 22ClO (K). In the optical section shown, the projection of the Bolwig nerve is visible from the organ until it contacts the ccp cell marked by an arrow. For details see text and Table 1. (I) Schematic representation of Bolwig organ development and Bolwig nerve projection through midstages of embryogenesis. Stage 12-16 embryos (top to bottom) presented in lateral (left) and dorsal (right) views. Note that this representation does not imply a continuous growth of the Bolwig nerve as its initial target; the brain undergoes developmental changes as well. Thus, it may well be that the initial contact between optic lobe precursor cells and the Bolwig nerve is established early, and they undergo movements in different directions leading to the stereotypical pathway described in the text. Abbreviations: br, brain; pm, pharynx muscle; vc, ventral cord. Colors refer to antennomaxillary complex (blue), Bolwig organ (red), and optic lobe anlagen (green). Orientation of embryos is anterior (left) and dorsal (up). (A-H), lateral views; (I and K), dorsal views. For staging of embryos see Campos-Ortega and Hartenstein (1985).
Figure 2. Abnormal
Larval Visual
System
Development
in Kr Mutant
Embryos
Optical sections through Kr mutant embryos (stage 16; Campos-Ortega and Hartenstein, 1985) showing abnormal larval visual system development. Embryos were stained with either the neuron-specific MAb 22ClO antibodies (A, D, H) or the photoreceptor cell-specific MAb 24810 (B and C) or anti-B-galactosidase antibodies in cases in which the mutants were crossed with a line expressing the /acZ reporter gene in the Bolwig organ (E-G and I). (A-C) Kr’ homozygous embryos showing that the Bolwig nerve is severely misrouted; note the abnormal turn (marked by small arrow) of the nerve and its projection toward the periphery (arrowhead), thereby missing its normal targets in the brain (asterisks). (B) Bolwig nerve shown to split into distinct branches (arrow). (C)A cluster of 4 Bolwig organ neurons and a single neuron that is detached from the cluster. For details of the Kr’ mutant phenotype of the larval visual system see text and Table 1.
Kriippel 1031
Function
in the Larval Visual System
Figure 3. Abnormal
Targeting
of the Bolwig
Nerve
in Kr Mutants
Enlarged optical sections through the brain region of wild-type (A and B) and Kr mutant (C and D) embryos (stage 15 in [A] and [Cl and stage 16 in [B] and [D]; Campos-Ortega and Hartenstein, 1985) showing a comparison of normal (A and B) and abnormal (C and D) targeting of the Bolwig nerve. Embryos were stained with either MAb 22ClO (A-D) or with a combination of MAb 22ClO and MAb anti-crumbs (A, B, and D). Note that during stage 15 (A) the Bolwig nerve (arrowhead) contacts a specific location of the brain surface (arrow), the optic lobe precursor cells (see text and Tix et al., 1989, for details), from where the nerve (arrowhead] grows perpendicular to the surface toward the central brain (B). The lumen of the optic lobe anlagen (asterisks) is labeled by anti-crumbs antibodies CTepass et al., 1990). No arborization of the nerve as shown in Figure 21, Figure 38, and Figure 3C can be observed. In Kr” mutant embryos (C] the Bolwig nerve (arrowhead) appears broad and branches into several distinct termini (arrows) that are different from wild-type (A and B). The dark, out-of-focus staining in (C) shows the antennomaxillary complex (am). In the KP-” embryo (D) the Bolwig nerve (arrowhead) reaches the area of the optic lobe anlagen. In contrast to wild-type the Bolwig nerve fails to project ventrally to the lumen of the optic lobe anlagen labeled by anti-crumbs antibodies (Tepass et al., 1990), and in contrast to wild-type the nerve is split into two distinct branches (arrows). For details of Bolwig nerve development in wild-type and Kr mutant embryos see Figure 1 and Figure 2.
(D-F] Morphological defects in the larval visual system of heterozygous Kr’ (D) and Krv (E and F) embryos. (D) Misrouted Bolwig nerve; the nerve (arrowhead) is growing perpendicular to the normal direction in the anterior portion of the brain surface. The mutant nerve fails to reach the area of the optic lobe anlagen in the caudal brain part (asterisk) where the wild-type nerve would leave the surface to grow inside the brain. In Kr” heterozygous embryos (E and F) a dispersed array of cells from the Bolwig organ is observed (see text and Table 1). (G and H) KPyz5 homozygous embryos showing clusters containing 4 (G) and 6 (H) Bolwig organ neurons. Note that a fasciculated Bolwig nerve could not be observed in such embryos. (I) KP” homozygous embryos showing that the defasciculation of the Bolwig nerve (arrowhead and arrows pointing to three distinct branches) occurs at the brain surface close to the optic lobe anlagen where the wild-type nerve normally would grow inside.
NC?UKXl 1032
Figure 4. Rescue of the Larval Visual System Phenotype by Neuron-Specific Kr Expression in Kr Mutant Embryos (A)Stage16KrYmutantembryostainedwith MAb 22ClO. Note that such embryos have lost MAb 22ClO antigen-expressing cells in a central region of the embryo (white bar), in an area strongly affected by the lack of Krsegmentationgenefunction.TheBolwig organ of this embryo is rescued through the activity of BO-KrR, which is expressed specifically in the Bolwig organ neurons. The enlarged head region (B) shows that the Bolwig organ (BO) has the same size and shape as in wild-type embryos, and the cells are tightly clustered. BO axons fasciculate and project in a dorsoposterior position, where they contact the ccp cell (arrowhead). Note that fasciculation (arrow) and, in particular, the contact to the ccp cell were never observed in KP mutant embryos (see text and Table 1).
embryos and in Bolwig organ rudiments composed of a few clustered neurons in the others. These results indicate that during Bolwig organ development, the activities of both gland Kr are regulated and required independently of one another. Since both genes encode zinc finger-type transcription factors, they appear to regulate different target genes, as indicated by the presence or absence of chaoptin expression in Kr and gl mutants and by the additive strength of the single mutant effects observed in embryos lacking gl and Kr gene activities at the same time. We also examined the projection of Bolwig nerves in gl mutants and in KrVgldouble mutant embryos. In gl mutant embryos, the pioneer axon of the single precursor neuron elongates in the normal direction (Figure 7). In contrast to both wild-type and Kr mutant embryos, the successor axons do not fasciculate with the pioneer axon, and they appear to project randomly (Figure 7). This observation shows that gl is required for the directed outgrowth of the axons and for their contact with the pioneer axon. These processes appear to be independent of Kr gene function (see above). After having reached the posterior edge of the procephalic lobe, the Bolwig nerve becomes misrouted in gl mutants, and it has never been found to contact the correct target area (see above). In KPI gl double mutants, the Bolwig nerve could not be identified. In summary, these results indicate that
both gl and Kr are independently required for Bolwig organ formation and for the Bolwig nerve to form and project normally. However, neither gl nor Kractivities are required for thedirected outgrowth of the pioneer axon of the first Kr-expressing neuron. In the subsequently added neurons, gl might be required for directed outgrowth of their axons and for their initial fasciculation with the pioneer axon. Kr, in contrast, is not essential for these initial steps of Bolwig nerve formation, but for the establishment or maintenance of nerve projection pathways along the landmarks described and, in particular, for the maintenance of fasciculation of the Bolwig nerve after the initial fasciculation had occurred. Furthermore, Kractivity is essential for the final targeting in the central brain. Alternatively, our findings could also be consistent with a model that the target genes of Kr and glare the same, but some of these targets are more strongly dependent on one of these transcription factors than the others. Discussion The Drosophila segmentation gene Kr is redeployed for the control of morphogenetic events in the development of the larval light sensory system. In the Bolwig neurons, its expression is controlled by a specific cis-acting element. This element is sufficient to confer
Figure 5. Kr Antisense
RNA Expression
Causes
Phenocopies
of the Kr Mutant
Larval Visual
System
Phenotype
Wild-type embryos containing the BO-Kras transgene stained with MAb 22ClO. In such embryos (A and B) the Bolwig reach its target area, marked by an asterisk (A), and shows an abnormal splitting (arrowheads [A and B]). (B) Furthermore show a strong disruption of the Bolwig organ (C). Note that there are only 2-3 neurons (big white arrow) and two distinct a and b) that are evidently misrouted. One (a) is projecting toward anterior; the other is growing in a circle.
Figure 6. Kr Phenocopies Embryos
in the Larval Visual
System
Produced
by Heat Shock-Induced
Kr Antisense
RNA Expression
nerve failed to such embryos nerves (arrows
in Wild-Type
1985) stained with MAb 22ClO (A-D) and Optical sections through wild-type stage 16 embryos (Campos-Ortega and Hartenstein, B-galactosidase (A and B). Kr antisense RNA expression was induced by a 30 min heat shock of 9-hr- (A and B), IO-hr- (C), and 12-hr- (D) old embryos (see Experimental Procedures). (A-C) Single cells (small arrow) are detaching from the Bolwig organ (large arrow), and the nerve (B) fails to reach the area where it normally grows into the brain (asterisk). Note the local thickening of the Bolwig nerve in different positions along the nerve (arrows in [B]), indicating a region of defasciculation as seen in the enlarged anterior region of the embryo (C). Note the splitting of the nerve in two separate branches (arrowhead) that terminate in different positions (small arrows). (D) Embryo that received heat shock after 12 hr of development. No defects could be observed in the Bolwig organ (BO), but the termini of the Bolwig nerves (BN) are split in two branches projecting in opposite directions (arrowheads). To show the projection of the entire Bolwig nerve, two photographs (separated by the dotted line) from slightly different focal planes were mounted.
NeUKMl 1034
Table 1. Developmental
Events during
Larval Visual
Genotype Event
Determination of neuronal cell fate Outgrowth of the pioneering axon Bolwig nerve Fasciculation Contact to optic lobe anlagen Passive stretching lngrowth into central brain and targeting Bolwig organ Final cell number Movement to final position
Formation
in Wild-Type
and Kr Mutant
Embryos
Kr Antisense
Embryos
RNA Expression
Kr’
Kr’ T-
iF
KP T
KrE&‘25 Kr62-‘4 -Kr 62.125 Kr 62.14
++++
++++
++++
++++
++++
ii++
++++
++++
++++
++++
+++
++++
++++
++++
+++a
++++
++++
++++
++++
+++a
+ +
+ +++
+ ++
++ +++
+ ++
+++ +++
+ ++
++ +++
c ++++
++ ii+
b +
+++ +++
+++ +
++++ +++
+++ +
++++ +
+++ ++
+++ ++
++++ ++
++++ ++
O-6 b
B-12 ++++
Vol. 9, 1025-1039, December,
1992, Copyright
0 1992 by Cell Press
Formation of the Drosophila larval Photoreceptor O rgan and Its Neuronal Differentiation Require Continuous l&i”/ Gene Activity Dietmar Schmucker, Heike Taubert, and Herbert Rickle Max-Planck-lnstitut fur Biophysikalische Chemie Abteilung Molekulare Entwicklungsbiologie Am FaBberg W-3488 Gottingen Federal Republic of Germany
Summary The Drosophila segmentation gene Kriippd (Kr) is rede ployed to play a critical role for the establishment of the larval visual system. Using reporter gene expression conducted by a specific Kr cis-acting element, we were able to trace back the origin of the larval photoreceptor organ, the Bolwig organ, to a single progenitor neuron and toexamine Krfunction in Bolwigorgan development when KP activity is absent from embryos due to specific mutations or reduced by neuron-specific and temporally restricted Kr antisense RNA expression. Our results show that Kr is required for neurons to differentiate into Bolwig organs, for fasciculation of the Bolwig nerve, and for this nerve to follow a specific pathway toward the synaptic targets in the larval brain. The transcription factor encoded by Kr is likely to regulate surface molecules necessary for neuronal cell adhesion and recognition in the developing larval visual system. Introduction The visual system of the adult Drosophila fly, compound eyes and three ocelli (Heisenberg and Wolf, 1984, is preceded by a larval photoreceptor system consisting of a pair of the so-called Bolwig organs (Bolwig, 1946). Each of them is composed of only 12 tightly clustered photoreceptor cells (Steller et al., 1987; Pollock and Benzer, 1988) resembling the rhabdomeric structures found in evolutionarily more primitive insect species (Melzer and Paulus, 1989). The cell bodies of the Bolwig photoreceptor neurons can be observed at midstage of embryogenesis, and they undergo stereotypical movements duringthe process of head involution (Steller et al., 1987). Concurrently, their neurons extend axons that fasciculate to form the Bolwig nerve that navigates posteriorly to enter theoptic lobe primordium. Someof theaxons contact specific cells of the optic lobe anlage; the others are thought to grow further and synapse in the developing brain (Tix et al., 1989), thereby forming the larval visual system. The stereotypical projection pathway of the Bolwig nerve is not preceded by any other nerve. Thus, the Bolwig nerve has to pioneer its specific way through a variety of tissues before it reaches the synaptic target ceils in the brain. This implies that the Bolwig nerve must have the capability to select a particular axonal
pathway likely to be guided by specific landmark cells or substances. Pathfinding of nerves involves the distalmost region of the axon, the growth cone, which can distinguish one group of cells and/or neurons from another. Based on the analyses of growth cone specificity, Raper et al. (1983a, 1983b, 1984) proposed that neighboring axon pathways or attracting tissue must be differentially labeled by surface recognition molecules that allow growth cones to distinguish among them, a notion called the labeled pathway hypothesis. The involvement of cell adhesion molecules serving as functional cell labels along specific axonal pathways in the developing central nervous system of the Drosophila embryo has recently been demonstrated (Harrelson and Goodman, 1988; Grenningloh et al., 1991). Furthermore, different adhesive or chemotropic factors that stimulate axonal outgrowth in vitro have been characterized in several organisms (for review see Jessell, 1988). However, such factors or cellular interactions necessary for the guidance of the Bolwig nerve through both neuronal and nonneuronal tissue to its synaptic partners are unknown. The pioneer function of the Bolwig nerve that eventually establishes the initial neuronal connection between the Bolwig organ and the embryonic brain is not only important for the processing of the perceived information by the larval brain, but also for proper innervation of the adult brain by the sensory neurons of the compound eye, which forms from an imaginal disc in third instar larvae (Steller et al., 1987). Initially, these neurons followthe pathway of the Bolwig nerve until they reach the optic lobe primordium (Steller et al., 1987). When this initial contact is stabilized, the Bolwig organs become histolyzed, and their nerves degenerate (Pollock and Benzer, 1988). The dependence of the adult visual system on the neuronal connections pioneered by the Bolwig nerve becomes obvious in mutants for the disconnected (disco) gene. Although the disco gene is not expressed in the Bolwig organ or its progenitor cells (Lee et al., 1991), mutations in the disco gene cause severe misrouting of the Bolwig nerves and frequent termination in ectopic locations (Steller et al., 1987). In these instances, the adult retinula photoreceptor neurons fail to project normally as well, and the optic lobes degenerate (Steller et al., 1987). These findings demonstrate that the initial contacts between the Bolwig nerve and the brain are essential for the developing adult visual system. Two genes coding for the transcription factor g/ass (g/) (Moses et al., 1989) and the cell adhesion molecule chaoptin (Van Vactor et al., 1988) are known to be expressed specifically in photoreceptor cells. Mutant analysis has shown that the absence of each of the two components causes abnormal photoreceptor development, and their activity is required for normal Bolwig organ formation (Van Vactor et al., 1988; Moses
Neuron 1026
et al., 1989). gl encodes a zinc finger-type protein regulating the expression of chaoptin (Moses et al., 1989) and of the photopigment rhodopsin Rhl in the larval photoreceptor neurons (Pollack and Benzer, 1988). In embryos that carry a strong gl mutation, the normal Bolwig organs are replaced by loosely clustered neurons with abnormal axonal projections (Moses et al., 1989). It was recently noted that Kriippel (Kr), a member of the gap class of segmentation genes, is expressed prominently in clusters of cells that are likely to represent the Bolwig organs (Gaul et al., 1987). In view of the expression of other segmentation genes, such as the pair-rule genes fushi tarazu and evenskipped, and their requirement for specifying the neural identity in defined subsets of cells in the developing central nervous system (Doe et al., 1988a, 1988b), we wanted to know whether segmentation genes might have cell commitment function in two distinct developmental processes, segmentation and neurogenesis, in general and which role Kr plays in the process of larval photoreceptor development in particular. W e first identified the Kr cis-acting sequences conducting Bolwig organ-specific gene expression. By use of reporter gene expression controlled by this Kr control element, we were then able to identify and to follow Bolwig organ progenitor cells and the pathway of the Bolwig nerve throughout development in wild-type and Kr mutant embryos and in embryos in which Kr’ activity had been reduced temporally or in a cell-specific manner. W e found that in contrast to the in the cenfunctions of fushi tarazu and even-skipped tral nervous system, Kr does not determine the neuronal identity of Bolwig organ progenitor cells. Instead, Kr’ activity acts thereafter to regulate subsequent steps of organ formation, fasciculation of axons, axonal extension, and target recognition processes. Our results show that both Krand the previously identified gl gene are independent components of the genetic circuitry leading to Bolwig organ formation, and both components are required for axonal guidance andlor adherence along the stereotypical Bolwig nerve pathway, which can be defined by specific landmarks.
W e have recently noted Kr expression in cell clusters that by morphological criteria correspond to the Bolwig organs in the head region of the late embryo (Gaul et al., 1987). To substantiate this observation, we performed double staining experiments using anti-Kr protein antibodies in combination with diagnostic monoclonal antibodies directed against acell surface molecule common to many neurons (monoclonal antibody22ClO[MAb22ClO]; Fujitaet al.,1982)oragainst the photoreceptor cell-specific membrane protein chaoptin (MAb 24810; Zipursky et al., 1984; Van Vactor et al., 1988). By this, we found that Kr is expressed not only in the Bolwig organ but also throughout all stages of its development, starting from a single Kr-
expressingcell (FigureslA-lD).Thiscell showsaxonal outgrowth with agrowth cone and expresses the MAb 2200 antigen (Figures IB-IE). This indicates that it has already adopted the neuronal fate when Krexpression is first seen. To establish the function that the segmentation gene Kr may exert in the process of larval visual system formation, we examined Bolwig organ development in wild-type embryos and in embryos with altered Kr’ activities. For this analysis, we constructed a cell-specific marker gene composed of the Kr cis-acting element (the Bolwig organ element; see Experimental Procedures) able to direct bacterial /acZ reporter gene expression in the developing Bolwig organs of transgenic embryos. Expression from this specific cell marker gene and the development of the Bolwig organs allowed us to study the development of the Bolwig organs from a single pair of neurons and to follow axonal processes due to the presence of B-galactosidase in the cytoplasm of the Bolwig organ neurons. Each of the two Kr-expressing neurons that later become part of a Bolwig organ is located in a mediolateral position to either side of the procephalic lobe of g-to IO-hr-old wild-type embryos (Figures IA and IB). On each side, the axon(s) of the first Kr-expressing neuron(s) elongates in a dorsal-posterior direction (Figures ID and IE). The contact cell at the pharynx (ccp) cell, which is stained by MAb 22ClO at stage 15 (Campos-Ortega and Hartenstein, 1985) (for details see Figure I), demarcates the position of the first turn of the axon in a ventrolateral direction (Figure 1; see also Figure 3 and Figure 6). A second morphological landmark is the posterior edge of the procephalic lobe. The contact of the nerve with this landmark can be seen at the time when a tight cluster of additional Kr-expressing neurons appears in close proximity to the first Kr-expressing cell body (Figures IC and ID). Since we have not observed this or any other Krexpressing cells in division, we assume that the additional Kr-expressing neurons are recruited from the surrounding cells rather than derived through division of Kr-expressing cells. The axons of these neurons add to the pioneering axon, and thefasciculated nerve continues to elongate along the brain hemisphere toward the optic lobe precursor cells (see Tix et al., 1989) (FigureslFandlG). W h e n thetipofthenerveisfound to be in contact with cells at the surface of the optic lobe anlagen, it changes its orientation at stage 15 (Campos-Ortega and Hartenstein, 1985) and grows almost perpendicularly to the surface, taking a straight route into the central brain region (see Figure 38). The synaptic partners of the Bolwig nerve in the central brain region are not yet identified. A detailed description and a schematic drawing summarizing the development of the larval visual system in wild-type embryos are given in Figure 1. larval Visual System Development Requires Kr+ Activity To elucidate the function of Kr in the Bolwig
organ,
Krijppel 1027
Function
in the Larval Visual System
we examined their development in embryos homozygous for Kr alleles of various strengths. As summarized in Table 1, a number of specific defects can be distinguished in Kr mutant embryos by morphological criteria and by the ability of Bolwig nerves to reach the different landmarks described for wild-type development. The most severe defects were observed in embryos carrying the loss-of-function allele Krl (a Kr gene deletion; see Preiss et al., 1985). In the absenceof Kr+activity, embryos lack the Bolwig organs, and in their places are loosely clustered groups of 2-6 neurons (Figures 2A-2D) that express the photoreceptorspecific cell adhesion molecule chaoptin (Table 1; Figure 2C). The extreme phenotype was observed in a portion of more than 20% of the embryos analyzed. In those embryos one of the two organs was missing, and the single organ rudiment consisted of no more than 3 cells. The majority of the embryos developed rudimentary organs with a maximum of 6 looselyclustered neurons. In rare cases in which a Bolwig nerve could be identified, it was never found to contact the ccp cells and always terminated in ectopic positions. In embryos homozygous for a Krallele of intermediate strength, such as KrV and KPz-125,the number of neurons forming the rudimentary Bolwig organs was also reduced to a maximum of 6. In KP2-lz homozygous mutant embryos, most of the neurons lack detectable axons (Figures 2G and 2H). In the few cases in which a misrouted nerve was observed, it never contacted the ccp cells, as observed in the Kr loss-offunction mutantsfseeabove). InKrVembryos,thecluster of neurons was split frequently, and each subcluster projected its own nerve. This phenomenon could not only be observed with homozygous mutant embryos, but also with a minor fraction of KP heterozygous embryos (Figures 2E and 2F). In contrast to Kr’ and KP2-125embryos, however, the Bolwig nerve in KP mutants reached the ccp landmark cell, but except for a few cases, the Bolwig nerve missed the optic lobe region. In these cases, the projection of the nerve along the brain hemisphere had an abnormal “wavy, shape, and the fasciculation of the nerve was interrupted. In contrast to wild-type embryos in which the nerve grows into thecentral brain region, the termination of nerves in Krv mutants occurred in a caudal position of the brain hemisphere, where severe defasciculation (“axonal sprouting”) takes place (Figure 3A). In embryos homozygous for the weak KP4 mutation, the Bolwig organ rudiment consists of a maximum of 8 neurons located in the wild-type position (compare Figure 11 and Figure 2D). The Bolwig nerve reaches both the ccp cell and the optic lobe anlagen. After having reached the latter position, the nerve defasciculates. In contrast to wild-type embryos in which a punctate pattern of termination can be observed, the KP2-14mutant Bolwig nerve splits into several branches (Figure 21). These results indicate that the formation of the Bolwig organ, fasciculation of axons, the neuronal path-
way, and synaptic targeting of the Bolwig nerve are conspicuously altered in the absence of the Kr wildtype gene product. In strong alleles, the formation of the Bolwig organ, fasciculation, and the early portion of the Bolwig nerve pathway are affected, while in mutants carrying the weak allele, the final targeting of the Bolwig nerve was the most obvious defect observed. Thus, the severity of the defects in the larval visual system of the different Kr mutants correlates well with the severity of the segment pattern defects caused by the corresponding alleles. It was therefore important to exclude the possibility that the defects observed in the larval visual system are of a secondary nature, since segmentation defects are known to cause secondary effects such as the abnormal head involution (Wieschaus et al., 1984). This possibility, however, appears unlikely, since minor defects (such as a slight reduction in the number of cells in the Bolwig organ and defective fasciculation and final targeting of the Bolwig nerve; Table 1 and Figures 2E and 2F) can be observed in the developing visual system of Kr heterozygous embryos that develop normal heads. To demonstrate unambiguously that the cellspecific perturbance of Kr+activity is the cause of mutant defects in the larval visual system, we performed rescue experiments involving organ-specific Kr+ gene expression in Kr mutant embryos, and we attempted to reduce specifically Kr’ activity in the neurons of Bolwig organs through the expression of Kr antisense RNA. Ceil Autonomous Kr+ Requirement in Bolwig Organ Precursors Rescue experiments were performed with a Kr minigene construct containing the Kr coding sequence and the Kr basal promoter under the control of the Bolwig organ element (BO) (BO-KrR minigene; see Experimental Procedures). After P element-mediated transformation, transgenic flies were crossed to KP flies in a way that Kr” homozygous embryos carried either one or two copies of the BO-KrR minigene. Such embryos show the KP mutant segmentation phenotype, but most aspects of the defects observed in the mutant larval visual system were rescued. The rescue includes an increase in Bolwig organ cells, fasciculation of the axons, and projection of the nerve to the optic lobe area in all KP mutant embryos containing the rescue construct. However, targeting in the correct brain region of some of these embryos was still abnormal, i.e., terminal sprouting of the Bolwig nerve occurs in a fashion similar to the defects observed in the mutants that carry the weak KP2-14allele (data not shown). An example of the rescue phenotype is shown in Figure 4. In complementary experiments we tried to reduce Kr’ activity by means of neuron-specific expression of Kr antisense RNA. For the production of Kr antisense RNA, we used a fusion gene construct containing the reverselyorientedKrcodingsequence(BO-Krasgene; see Experimental Procedures). Transgenic lines con-
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Figure 1. Bolwig
Organ
Development
Visualized
by Reporter
Gene
Expression
and MAb 22ClO Staining
(A-K) Optical sections through wild-type embryos expressing B-galactosidase under the control of the Bolwig organ element to vis ualize Bolwig organ neurons specifically. Embryos were stained with anti-B-galactosidase antibodies (dark blue staining) and the ne uronspecific MAb 22ClO (bright brown staining). (A) Stage 12 embryo showing the first Bolwig organ neuron (arrow) located in a posterior ventral position of the procephalic lobe. MAb 22ClO weakly stains the first neurons and outgrowing axons of the peripheral nervous system at this stage.
Krijppel 1029
Function
in the Larval Visual System
taining the BO-Kras gene construct in different locations of the chromosomes had significantly reduced levels of Kr protein in the Bolwig organ neurons (see Experimental Procedures). The corresponding individuals were viable, but the developing visual system of such embryos was evidently impaired. The phenocopies produced by Kr antisense RNA showed in lower penetrance all the characteristics of defects of the larval visual system observed in the Kr mutants (Table 1; see examples in Figures 5A-5C). This finding and the defects observed in Kr heterozygous mutant embryos suggest that larval visual system development is critically dependent on the level of Kr+activity.
number of cells forming the Bolwig organ was higher, and the initial fasciculation of the axons appeared less affected than that observed with heat shock treatment at 9 hr. No effect on the Bolwig organ was observed in embryos that received heat shock treatment 12 hr after egg deposition, but defasciculation occurred in the terminalmost positions of the otherwise normally developed Bolwig nerve (Figure 6D). This result shows that the Bolwig organ of 12-hr-old embryos is no longer sensitive to hsp70-Kras gene expression, while the process of pathfinding, in particular the final synapsing in the brain, has not been completed at this stage of development (Table 1).
Continuous Kr+ Requirement for Axonal Guidance To determine the period when Kr+ activity is critically required during larval visual system development, we used heat shock-dependent Krantisense RNA expression to reduce the level of Kr+ activity by 30 min heat shock treatments of 9-, IO-, or IZhr-old embryos containing the “hsp70-Km” transgene (see Experimental Procedures). Severe defects in the fully developed visual system were observed in embryos that received heat shock treatment 9 hr after egg deposition. In these Kr antisense RNA-expressing embryos (see Experimental Procedures), the Bolwig organs were reduced to a variable number of 4-8 loosely connected neurons, and the nerves showed severe defasciculation (Table 1; Figures 6A-6C). Their axons terminate in different abnormal positions. In embryos that received the heat shock 10 hr after egg deposition, the
Complementary Functions of Kr and g/3 The Bolwig organ phenotype of Kr mutants strongly resembles the one recently described for gl mutants (Moses et al., 1989). Using /acZ expression conducted by the Bolwig organ element and anti-Kr protein antibody stainings in gl mutants, we observed expression both of Kr protein and B-galactosidase in the Bolwig organ precursors and in the loosely clustered neurons forming a Bolwig organ rudiment. This result excludes the possibility that gl is required for the control of Kr gene expression. Conversely, the chaoptin gene is expressed in Kr mutants (see above). Since gl activity is necessary to activate chaoptin gene expression (Moses et al., 1989), Kr+ activity is not required for chaoptin or for gl expression. The lack of gl and Kr gene activities, in Krlgl double mutants, resulted in the lack of storable Bolwig organs in most of these
(B) Posterior procephalic region of a stage 12 embryo showing a Bolwig organ progenitor neuron colabeled by anti-B-galactosidase and MAb 22ClO. Note that the growth cone (arrowhead) projects dorsally. The position of the Bolwig organ progenitor neuron is dorsal to the maxillary segment fms) and anterior to the dorsal ridge (dr) separated by the cephalic furrow (cf). Costaining with anti-Kr antibodies and anti-B-galactosidase antibodies revealed early Kr expression in those cells labeled by B-galactosidase staining (data not shown). (C) Stage 13 embryo showing additional B-galactosidaseexpressing cells, indicating a stepwise increase of the number of Bolwig organ neurons. Note additional MAb 22ClO staining ceils and axons in the periphery of the embryo, indicating that the peripheral nervous system has proceeded in development. (D and E) Posterior procephalic region of a stage 13 embryo. Note a cluster of 3-4 Bolwig organ progenitor cells (bo) in a position above the ventral organ fvo) of the antennomaxillary complex. Only one Bolwig organ progenitor neuron extends a pioneer axon (lower arrowhead in [D]) with a growth cone (upper arrowhead in [D]) projecting toward dorsal posterior. Note enhanced membrane staining of the Bolwig organ neurons due to the costaining with anti-Bgalactosidase antibodies and MAb 22ClO. Note also the dark, punctate staining pattern of growth cones (arrow in [El) that derive from the newly recruited Bolwig organ progenitor cells prior to their fasciculation with the pioneer axon (black arrowheads). (E) is composed of two separate photographs taken from slightly different optical sections of the same region of the embryo to show both the pioneer and successor axons in one focal plane. (F and G) Embryos before(F) and during (C] the process of head involution, indicating that the Bolwig organ has moved from a medialposterior head position (see [Cl and [I]) to a dorsal-anterior position (white arrows). Note the initial projection of the Bolwig nerve and a sharp turn in its terminal region (arrowhead) at a position where the nerve leaves the brain surface to grow inside. (H) Stage 15 embryo showing that the Bolwig organ (bo) is located next to the dorsal organ (do) of the antennomaxillary complex, close to its final position at stage 16. (I and K) Dorsal views of almost fully developed embryos. The nervous system is labeled with MAb 22ClO staining. Note the position of the Bolwig organ (bo) located in a posterior position of the antennomaxillary complex (amc), as revealed by B-galactosidase expression and counterstaining with MAb 22ClO (K). In the optical section shown, the projection of the Bolwig nerve is visible from the organ until it contacts the ccp cell marked by an arrow. For details see text and Table 1. (I) Schematic representation of Bolwig organ development and Bolwig nerve projection through midstages of embryogenesis. Stage 12-16 embryos (top to bottom) presented in lateral (left) and dorsal (right) views. Note that this representation does not imply a continuous growth of the Bolwig nerve as its initial target; the brain undergoes developmental changes as well. Thus, it may well be that the initial contact between optic lobe precursor cells and the Bolwig nerve is established early, and they undergo movements in different directions leading to the stereotypical pathway described in the text. Abbreviations: br, brain; pm, pharynx muscle; vc, ventral cord. Colors refer to antennomaxillary complex (blue), Bolwig organ (red), and optic lobe anlagen (green). Orientation of embryos is anterior (left) and dorsal (up). (A-H), lateral views; (I and K), dorsal views. For staging of embryos see Campos-Ortega and Hartenstein (1985).
Figure 2. Abnormal
Larval Visual
System
Development
in Kr Mutant
Embryos
Optical sections through Kr mutant embryos (stage 16; Campos-Ortega and Hartenstein, 1985) showing abnormal larval visual system development. Embryos were stained with either the neuron-specific MAb 22ClO antibodies (A, D, H) or the photoreceptor cell-specific MAb 24810 (B and C) or anti-B-galactosidase antibodies in cases in which the mutants were crossed with a line expressing the /acZ reporter gene in the Bolwig organ (E-G and I). (A-C) Kr’ homozygous embryos showing that the Bolwig nerve is severely misrouted; note the abnormal turn (marked by small arrow) of the nerve and its projection toward the periphery (arrowhead), thereby missing its normal targets in the brain (asterisks). (B) Bolwig nerve shown to split into distinct branches (arrow). (C)A cluster of 4 Bolwig organ neurons and a single neuron that is detached from the cluster. For details of the Kr’ mutant phenotype of the larval visual system see text and Table 1.
Kriippel 1031
Function
in the Larval Visual System
Figure 3. Abnormal
Targeting
of the Bolwig
Nerve
in Kr Mutants
Enlarged optical sections through the brain region of wild-type (A and B) and Kr mutant (C and D) embryos (stage 15 in [A] and [Cl and stage 16 in [B] and [D]; Campos-Ortega and Hartenstein, 1985) showing a comparison of normal (A and B) and abnormal (C and D) targeting of the Bolwig nerve. Embryos were stained with either MAb 22ClO (A-D) or with a combination of MAb 22ClO and MAb anti-crumbs (A, B, and D). Note that during stage 15 (A) the Bolwig nerve (arrowhead) contacts a specific location of the brain surface (arrow), the optic lobe precursor cells (see text and Tix et al., 1989, for details), from where the nerve (arrowhead] grows perpendicular to the surface toward the central brain (B). The lumen of the optic lobe anlagen (asterisks) is labeled by anti-crumbs antibodies CTepass et al., 1990). No arborization of the nerve as shown in Figure 21, Figure 38, and Figure 3C can be observed. In Kr” mutant embryos (C] the Bolwig nerve (arrowhead) appears broad and branches into several distinct termini (arrows) that are different from wild-type (A and B). The dark, out-of-focus staining in (C) shows the antennomaxillary complex (am). In the KP-” embryo (D) the Bolwig nerve (arrowhead) reaches the area of the optic lobe anlagen. In contrast to wild-type the Bolwig nerve fails to project ventrally to the lumen of the optic lobe anlagen labeled by anti-crumbs antibodies (Tepass et al., 1990), and in contrast to wild-type the nerve is split into two distinct branches (arrows). For details of Bolwig nerve development in wild-type and Kr mutant embryos see Figure 1 and Figure 2.
(D-F] Morphological defects in the larval visual system of heterozygous Kr’ (D) and Krv (E and F) embryos. (D) Misrouted Bolwig nerve; the nerve (arrowhead) is growing perpendicular to the normal direction in the anterior portion of the brain surface. The mutant nerve fails to reach the area of the optic lobe anlagen in the caudal brain part (asterisk) where the wild-type nerve would leave the surface to grow inside the brain. In Kr” heterozygous embryos (E and F) a dispersed array of cells from the Bolwig organ is observed (see text and Table 1). (G and H) KPyz5 homozygous embryos showing clusters containing 4 (G) and 6 (H) Bolwig organ neurons. Note that a fasciculated Bolwig nerve could not be observed in such embryos. (I) KP” homozygous embryos showing that the defasciculation of the Bolwig nerve (arrowhead and arrows pointing to three distinct branches) occurs at the brain surface close to the optic lobe anlagen where the wild-type nerve normally would grow inside.
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Figure 4. Rescue of the Larval Visual System Phenotype by Neuron-Specific Kr Expression in Kr Mutant Embryos (A)Stage16KrYmutantembryostainedwith MAb 22ClO. Note that such embryos have lost MAb 22ClO antigen-expressing cells in a central region of the embryo (white bar), in an area strongly affected by the lack of Krsegmentationgenefunction.TheBolwig organ of this embryo is rescued through the activity of BO-KrR, which is expressed specifically in the Bolwig organ neurons. The enlarged head region (B) shows that the Bolwig organ (BO) has the same size and shape as in wild-type embryos, and the cells are tightly clustered. BO axons fasciculate and project in a dorsoposterior position, where they contact the ccp cell (arrowhead). Note that fasciculation (arrow) and, in particular, the contact to the ccp cell were never observed in KP mutant embryos (see text and Table 1).
embryos and in Bolwig organ rudiments composed of a few clustered neurons in the others. These results indicate that during Bolwig organ development, the activities of both gland Kr are regulated and required independently of one another. Since both genes encode zinc finger-type transcription factors, they appear to regulate different target genes, as indicated by the presence or absence of chaoptin expression in Kr and gl mutants and by the additive strength of the single mutant effects observed in embryos lacking gl and Kr gene activities at the same time. We also examined the projection of Bolwig nerves in gl mutants and in KrVgldouble mutant embryos. In gl mutant embryos, the pioneer axon of the single precursor neuron elongates in the normal direction (Figure 7). In contrast to both wild-type and Kr mutant embryos, the successor axons do not fasciculate with the pioneer axon, and they appear to project randomly (Figure 7). This observation shows that gl is required for the directed outgrowth of the axons and for their contact with the pioneer axon. These processes appear to be independent of Kr gene function (see above). After having reached the posterior edge of the procephalic lobe, the Bolwig nerve becomes misrouted in gl mutants, and it has never been found to contact the correct target area (see above). In KPI gl double mutants, the Bolwig nerve could not be identified. In summary, these results indicate that
both gl and Kr are independently required for Bolwig organ formation and for the Bolwig nerve to form and project normally. However, neither gl nor Kractivities are required for thedirected outgrowth of the pioneer axon of the first Kr-expressing neuron. In the subsequently added neurons, gl might be required for directed outgrowth of their axons and for their initial fasciculation with the pioneer axon. Kr, in contrast, is not essential for these initial steps of Bolwig nerve formation, but for the establishment or maintenance of nerve projection pathways along the landmarks described and, in particular, for the maintenance of fasciculation of the Bolwig nerve after the initial fasciculation had occurred. Furthermore, Kractivity is essential for the final targeting in the central brain. Alternatively, our findings could also be consistent with a model that the target genes of Kr and glare the same, but some of these targets are more strongly dependent on one of these transcription factors than the others. Discussion The Drosophila segmentation gene Kr is redeployed for the control of morphogenetic events in the development of the larval light sensory system. In the Bolwig neurons, its expression is controlled by a specific cis-acting element. This element is sufficient to confer
Figure 5. Kr Antisense
RNA Expression
Causes
Phenocopies
of the Kr Mutant
Larval Visual
System
Phenotype
Wild-type embryos containing the BO-Kras transgene stained with MAb 22ClO. In such embryos (A and B) the Bolwig reach its target area, marked by an asterisk (A), and shows an abnormal splitting (arrowheads [A and B]). (B) Furthermore show a strong disruption of the Bolwig organ (C). Note that there are only 2-3 neurons (big white arrow) and two distinct a and b) that are evidently misrouted. One (a) is projecting toward anterior; the other is growing in a circle.
Figure 6. Kr Phenocopies Embryos
in the Larval Visual
System
Produced
by Heat Shock-Induced
Kr Antisense
RNA Expression
nerve failed to such embryos nerves (arrows
in Wild-Type
1985) stained with MAb 22ClO (A-D) and Optical sections through wild-type stage 16 embryos (Campos-Ortega and Hartenstein, B-galactosidase (A and B). Kr antisense RNA expression was induced by a 30 min heat shock of 9-hr- (A and B), IO-hr- (C), and 12-hr- (D) old embryos (see Experimental Procedures). (A-C) Single cells (small arrow) are detaching from the Bolwig organ (large arrow), and the nerve (B) fails to reach the area where it normally grows into the brain (asterisk). Note the local thickening of the Bolwig nerve in different positions along the nerve (arrows in [B]), indicating a region of defasciculation as seen in the enlarged anterior region of the embryo (C). Note the splitting of the nerve in two separate branches (arrowhead) that terminate in different positions (small arrows). (D) Embryo that received heat shock after 12 hr of development. No defects could be observed in the Bolwig organ (BO), but the termini of the Bolwig nerves (BN) are split in two branches projecting in opposite directions (arrowheads). To show the projection of the entire Bolwig nerve, two photographs (separated by the dotted line) from slightly different focal planes were mounted.
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Table 1. Developmental
Events during
Larval Visual
Genotype Event
Determination of neuronal cell fate Outgrowth of the pioneering axon Bolwig nerve Fasciculation Contact to optic lobe anlagen Passive stretching lngrowth into central brain and targeting Bolwig organ Final cell number Movement to final position
Formation
in Wild-Type
and Kr Mutant
Embryos
Kr Antisense
Embryos
RNA Expression
Kr’
Kr’ T-
iF
KP T
KrE&‘25 Kr62-‘4 -Kr 62.125 Kr 62.14
++++
++++
++++
++++
++++
ii++
++++
++++
++++
++++
+++
++++
++++
++++
+++a
++++
++++
++++
++++
+++a
+ +
+ +++
+ ++
++ +++
+ ++
+++ +++
+ ++
++ +++
c ++++
++ ii+
b +
+++ +++
+++ +
++++ +++
+++ +
++++ +
+++ ++
+++ ++
++++ ++
++++ ++
O-6 b
B-12 ++++
