Embryonic development of the enteric nervous system of the grasshopperSchistocerca americana

THE JOURNAL OF COMPARATIVE NEUROLOGY 372581496 (1996)

Embryonic Development of the Enteric Nervous System of the Grasshopper

Schistocerca americaaa

M A R ~ A D. GANFORNINA, DIEGO SANCHEZ, AND MICHAEL J. BASTIANI Biology Department, University of Utah, Salt Lake City, Utah 84112

ABSTRACT The enteric nervous system (ENS) of the grasshopper Schistocerca americana is organized

into four ganglia located in the foregut (the dorsal unpaired frontal and hypocerebral ganglia, and the paired ingluvial ganglia), and two plexuses that innervate the foregut and midgut. A dorsomedial recurrent nerve and two lateral esophageal nerves connect the ganglia. The midgut plexus is arranged in four nerves running along the midgut surface. In this study, we have focused on the embryonic development of the grasshopper ENS; we have studied the proliferation pattern, morphogenesis, and some aspects of neuronal differentiation by using a number of specific molecular markers. The grasshopper ENS develops early in embryogenesis (25-30%) from three neurogenic zones (NZs) located on the roof of the stomodeum. These NZs slightly invaginate from an epithelial placode. The expression pattern of specific cell surface proteins and the analysis of the mitotic activity showed that NZs cells delaminate from the epithelium, become neuronal precursors, divide symmetrically, and then actively migrate to their final position in the enteric ganglia or plexuses. The grasshopper enteric ganglia are composed of mixed populations of cells from different NZs. The foregut and midgut plexuses are formed by the dispersal of cells from the developing hypocerebral and ingluvial ganglia. The main ENS nerves are pioneered by axons extending anteriorly from hypocerebral and ingluvial neurons. The insect ENS exhibits an enormous variation in design. Several features of the grasshopper program of neurogenesis and pattern of cell migration are compared to other insects, and some evolutionary implications are discussed.

Indexing terms: insect development, stomatogastric, neurogenesis, pathfinding, cell migration

o 1996 Wiley-Liss, Inc.

The enteric nervous system (ENS) is the subdivision of the insect nervous system that innervates the alimentary tract and other related structures. It is composed of a group of interconnected ganglia on the surface of the foregut and a network of neurons organized as a plexus on the surface of both the foregut and the midgut. The central nervous system (CNS) also contributes to the gut innervation. Anteriorly, a frontal ganglion is connected to the tritocerebrum by the paired frontal ganglion connectives. Caudally, the hindgut is innervated by nerves that come from the terminal abdominal ganglion in the CNS. In spite of these inputs, the ENS is largely autonomous in its functions concerning the control of feeding, peristaltic movements of the gut, moulting, and metabolism (see Penzlin, 1985 for a review).

The embryonic development of the ENS has been studied to different extents in several insect orders (Lal-Roonwal, 1937; Poulson, 1950; Okada, 1960; Miyakawa, 1974; Ko- bayashi a n d h d o , 1983). Recently, a more detailed analysis of the underlying cellular and molecular basis for ENS ontogenesis has been performed in two well-established

model insects, Manduca sexta (Copenhaver and Taghert, 1989a, 1989b, 1990, 1991; Copenhaver, 1993) and Dro- sophila melanogaster (Hartenstein et al., 1994; Gonzalez- Gaitan and Jackle, 1995). The enteric ganglia are formed from precursors that segregate from the dorsal epithelium of the stomodeum. Neurogenesis in the insect CNS and in the peripheral nervous system (PNS) results from the acquisition of competence of some ectodermal cells to become neuronal precursors, followed by the restriction of the number of precursor cells by a cell-cell interaction process called lateral inhibition. Two classes of gene prod- ucts, produced by the proneural and neurogenic genes, control these two phases of the neurogenic pathway (reviewed by Campos-Ortega, 1994; Goodman and Doe, 1993). In contrast, the ENS precursors develop from several cell

Accepted April 14,1996. D.S. and M.D.G. contributed equally to this work Address reprint requests to M.D. Ganfornina, Biology Department, 229

South Biology, University of Utah, Salt Lake City, UT 84112. E-mail: [email protected]

O 1996 WILEY-LISS, INC.

M.D. GANFORNINA ET AL. 582

clusters in the epithelium where every cell belonging to the cluster will adopt a neurogenic fate. These precursor cells generate neurons and some glial cells without an apparent lateral inhibition process. Other interesting features of ENS morphogenesis are the extensive migrations that neurons undergo prior to their differentiation, and the considerable variability in position, pattern of axonal projections, and other phenotypic traits such as neurotransmitter expression. This latter property contrasts with the substantial stereotypy observed in the CNS and PNS. Interestingly, these characteristics of the enteric neurogenic program resemble some aspects of vertebrate neurogenesis (Sidman and Rakic, 1973; Le Douarin, 1982; Austin and Cepko, 1990).

In this work, we have studied the ontogenesis of the ENS in the grasshopper Schistocerca americana. Although this orthopteran has been a fruitful model system to study the genesis, architecture, and function of the CNS and PNS (reviewed by Boyan and Ball, 1993; Sanchez et al., 1995b), limited information is available concerning the development of the ENS beyond a general description (Baden, 1936; Lal-Roonwal, 1937). We used immunohistochemis- try, labelling with a thymidine analogue, and dye iontopho- resis on identified nerve tracts to describe the neurogenic program, morphogenesis, and axon pathway formation in the grasshopper ENS.

MATERIAL AND METHODS Grasshopper (Schistocerca americana) embryos were

obtained from a colony maintained at 31°C and 60% humidity at the University of Utah. They were staged by percentage of embryonic development according to Bentley et al. (1979). The following monoclonal antibodies (mAbs) were used for immunocytochemistry: 3B11 anti-Fasciclin I (Bastiani et al., 1987), 8C6 anti-Fasciclin I1 (Bastiani et al., 1987), 6F8 anti-Semaphorin I (Kolodkin et al., 1992), 10E6 anti-Lazarillo (Ganfornina, Sanchez, and Bastiani, 1995), lCl0 anti-Lachesin (Karlstrom et al., 1993), 7F7 anti- REGA-1 (Carpenter and Bastiani, 1991), and 5A12 anti- Neuroglian (Bieber et al., 1989) (all of them generated against cell surface proteins of the grasshopper embryo except 5A12, which recognizes a surface epitope of Neuro- glian in Drosophila melanogaster and shows crossreactivity with a grasshopper antigen), and finally the mAb 8B7 that recognizes an axonal-specific intracellular antigen in the grasshopper CNS and PNS. The following serum antibodies (sAbs) were also used: anti-HRP as a general neuronal marker that recognizes a complex carbohydrate epitope (Snow et al., 1987) and anti-tubulin (Sigma).

Immunocytochemistry Antibody labelling was carried out essentially as de-

scribed in Shchez, Ganfornina, and Bastiani, (1995a). Embryos were removed from the egg case and dissected in Ringer’s solution (4.6 mM TES, pH 7.4, 150 mM NaC1, 3 mM KCl, 2 mM CaC12, 1 mM MgS04) with 6 mg/ml of glycine, fked in 4% paraformaldehyde in 0.1 M sodium phosphate, pH 7.2, and washed in 20 mM potassium phosphate, pH 7.2, 150 mM NaCl. Embryos were incubated for 1 hour in blocking solution: 50 mM Tris, pH 7.2, 350 mM NaCl (50 mM Tris-buffered saline [TBS]); 30 mg/ml bovine serum albumin (bovine serum albumin [BSA] frac- tion V, Sigma); 2% normal goat serum; and variable detergent concentrations (see below). Embryos were incu-

bated with the primary antibody at 4°C for 8-12 hours, then washed with 50 mM TBS, blocked again, exposed for 2 hours at room temperature to horseradish peroxidase (HRP)-conjugated goat anti-mouse I& (Jackson Immunore- search) diluted 1:500 in blocking solution, and washed again with 50 mM TBS with a last change to 0.1 M TBS (0.1 M Tris, pH 7.2,350 mM NaCl). Embryos were reacted with diaminobenzidine (1 mg/ml), glucose oxidase (3 mU/ml, type V), and p-D-glucose (2 mg/ml), washed in 0.1 M TBS, and cleared in glycerol. Differential interference contrast microscopy (DIC) was used to analyze the preparations. mAbs 3Bll,8C6,1C10, and 7F7 were used as ascites fluids diluted 1:200-1:500; mAbs 6F8, 10E6, 5A12, and 8B7 as hybridoma supernatant diluted 1: 1; and sAbs anti-HRP and anti-tubulin were diluted 1:200 in blocking solution. Sapo- nin (0.4%) was added to the blocking solution when using mAb 8B7, whereas no detergent was used for mAb 10E6. Triton X-100 (0.2%) was added to the blocking solution when using the rest of the antibodies. To study the pattern of expression in grasshopper embryos ranging from 60% of development to hatching, guts were dissected, and the standard protocol was followed by using longer incubation times with antibodies: 24 hours for the primary and 18-24 hours for the secondary antibody.

Analysis of mitotic activity by BrdU incorporation

Grasshopper embryos were dissected under sterile conditions and cultured in medium consisting of 50% Schneider’s Drosophila medium (Gibco-BRL), 49% minimum essential medium (a medium, Gibco-BRL), and 1% antibiotic- antimycotic solution (Sigma). Each embryo was incubated at 33°C in a microwell plate (Nunc) with 20 pl of prewarmed medium containing 5-bromo-2’-deoxyuridine (BrdU, Sigma). Several BrdU concentrations and incubation times were tested. The optimal conditions were 15 pg/ml of BrdU during 9 hours. After incubation, embryos were fixed in PEM (0.1 M Pipes, 2 mM MgS04, 1 mM EGTA, pH 7.0)-2% formaldehyde for 1 hour, washed in phosphate-buffered saline (PBS: 20 mM sodium phosphate, pH 7.4, 150 mM NaCl), and incubated in 2 N HCl for 30 minutes. After washes in PBS, embryos were blocked with PBS plus 0.4% Triton X-100 and 2% normal goat serum for 45 minutes and incubated at 4°C for 12 hours with a mAb against BrdU (Beckton Dickinson) diluted 1: lOO. After washes in PBS plus 0.025% Triton X-100 and blocking, an HRP-conjugated goat anti-mouse IgG (Jackson Immunoresearch) was added at 1:200, and incubation proceeded for 24 hours at room temperature. Development of HRP was performed as described above, and embryos were rinsed in PBS and mounted in glycerol.

DiI labellings Staged embryos were pinned on a slide, fixed for 15

minutes in 2% paraformaldehyde, and washed in Ringer’s solution for 30 minutes. A solution of 1% 1,l’-dioctadecyl- 3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI; Mo- lecular probes) in dimethyl sulfoxide (Sigma) was iontopho- retically applied through a glass micropipette onto the neuronal projections along the recurrent and esophageal nerves. After the dye application, the preparation was left at room temperature for 1 hour prior to observation on a Leitz fluorescence microscope.

STOMATOGASTRIC NERVOUS SYSTEM DEVELOPMENT 583

A

Fig. 1. A Drawing of a dorsal overview of a grasshopper embryo at 35% of development showing the stomodeum (with three neurogenic zones, 21-2~) and proctodeum. B: Dorsal view of a grasshopper gut at 80% of development showing the elements ofthe ENS. C: Schematics of the enteric ganglia and nerves in different insects. See text for details.

RESULTS Gut development and general morphology

of the ENS The digestive tract of insects is composed of three parts:

the foregut, midgut, and hindgut. Both foregut and hindgut originate early in development as part of the gastrulation process. In the grasshopper Schistocerca americana, a protostome, the foregut arises at 20% of development as an invagination called the stomodeum near the cephalic end of the embryo, whereas the hindgut develops at 25% from a caudal invagination, the proctodeum (Fig. 1A). Two cell layers of ectodermal and mesodermal origin are present in these invaginations. They will give rise to the foregut and hindgut epithelium and musculature, respectively. The ENS arises from three proliferative zones (Zl, Z2, and Z,) in the foregut epithelium. The midgut is laid out at 50% of development. After katatrepsis, the provisional dorsal clo- sure grows around the yolk forming a sac, the primordium of the midgut. The splanchnic mesoderm surrounds this sac and differentiates into muscle cells, whereas the expansion of the ectoderm of the stomodeum and proctodeum generates the midgut epithelium (Lal-Roonwal, 1937). At the midgut-foregut boundary six invaginations, the caeca, will appear around 65% of development.

C

grasshopper

f beetle -

f tobacco hornwom

fruit fly

F, foregut; C, caeca; M, midgut; H, hindgut; MP, malpighian tubules; fg, frontal ganglion; fgc, frontal ganglion connectives; hg, hypocerebral ganglion; ca, corpora allata; rn, recurrent nerve; ig, ingluvial ganglia; en, esophageal nerves; fp, foregut plexus; mp, midgut plexus; hn, hindgut nerves; p, plexus; pe, paraesophageal ganglion.

The ENS originates at the dorsomedial surface of the foregut. In Figure 1B we show a representation of the gut at 80% of development, when all the components of the mature ENS are already present (see also Fig. 7). The frontal ganglion is connected by the recurrent nerve with the hypocerebral ganglion from which two esophageal nerves arise to connect with the paired ingluvial ganglia. The foregut plexus is composed of neurons dispersed over the surface of the stomodeum. The midgut plexus is formed by neurons scattered along four main longitudinal pathways on the midgut surface.

The disposition of ganglia and nerves is remarkably diverse among different orders of insects (Fig. 1C; see also Bickley, 1942). A single unpaired frontal ganglion is fairly conserved in insects, with the exception of the fly Dro- sophila melanogaster, in which it splits in two lateral parts. The hypocerebral ganglion is more variable, with cases such as the moth Manduca sexta, in which it is very reduced and fused to the frontal ganglion (Copenhaver and Taghert, 1991), or Drosophila, in which two ganglia, hypocerebral and paraesophageal, appear caudal to the recurrent nerve (Campos-Ortega and Hartenstein, 1985; Hartenstein et al., 1994). However, the most distal part of the ENS shows the highest variability, with the absence of ingluvial ganglia (in Manduca and some beetles) or the presence of single (like in

584 M.D. GANFORNINA ET AL.

the dragonfly Libellula) or paired ingluvial ganglia (in Schistocerca and other orthopteroids), and a wide variety of design in the arrangement of the esophageal nerves and the plexus that innervates the foregut and midgut.

Epithelial foregut differentiation prior to the onset of neurogenesis

Early events of differentiation were observed in the grasshopper foregut epithelium before neurogenesis by using several antibody probes. The cell surface glycoprotein Fasciclin I is expressed at 22% of development in the epithelial cells at the tip of the stomodeum, which has recently invaginated into the body cavity, indicating the first signs of stomodeal cell differentiation (Fig. 2A). Other proteins like REGA-1 (Fig. 2B) and Fasciclin I1 (not shown) are expressed early in the stomodeal epithelium as a gradient. A higher expression of both proteins is observed at the distal region of the stomodeum, and a posterior boundary exists in the REGA-1 labeling (Fig. 2B, arrows) that excludes the Fasciclin I-expressing cells at the tip. Moreover, REGA-1 expression is more prominent at the basal surface of the epithelium. The early differential expression of these three surface molecules suggests the existence of axial information in the stomodeum that might help to allocate the developing neurogenic zones. Morpho- logical differences along the dorsoventral axis are also seen at early stages. By 23-24% (Fig. 2B) the epithelium at the dorsal midline has thickened as epithelial cells become more columnar, a process called placodal transformation that usually precedes invagination or evagination of epithelial sheets (Fristrom, 1988). These placodal cells eventually form a pseudostratified epithelium.

The next epithelial change is the appearance of three patches of Fasciclin I expression along the dorsal midline of the stomodeum at 25% of development (arrows in Fig. 2C). These regions are the prospective neurogenic zones (NZs). The cells in these regions are specifically marked by Fascic- lin I before any apparent change in cell morphology. These cells will subsequently undergo a neurogenic program of differentiation. We have named these zones Z1, Z2, and Z3 to meet the nomenclature proposed for Manduca (Copen- haver and Taghert, 1991). Between 25 and 30% the NZs start showing morphological changes. In a dorsal view of the stomodeum (Fig. 2D) the three zones have the appearance of “rosettes”; they were previously referred to as “clear zones” in grasshoppers (Baden, 1936). The caudal Z3 is the largest zone and the first to differentiate, closely followed by Z1 and Z2. At each NZ (Fig. 2E) cells delaminate from the epithelial sheet to become mesenchymal in a morphogenetic mechanism called ingression (Fristrom, 1988). This process is preceded by a constriction of the apical pole of the cells into long necks or endfeet (arrow in Fig. 2E) and a bulging of the basal pole toward the surface of the stomodeum facing the body cavity. Cells detach from their neighbors, retract their endfeet, and round out on top of each NZ where they initially accumulate. As ingression proceeds, cells are replaced by new ones from cell divisions of the surrounding epithelium. The newly ingressed cells keep expressing Fasciclin I, but at a lower level (arrowhead in Fig. 2E).

The ingression process at each NZ generates a slight invagination of the epithelium that is accompanied by specific changes in molecular expression (Fig. 2E-I). Super- imposed on the early gradient mentioned above, three gaps in the epithelial expression of REGA-1 are observed at the

basal surface of each NZ epithelium (arrows in Fig. 2G). Likewise, the cytoplasmic antigen recognized by mAb 8B7 is expressed by the stomodeal epithelial cells only at their basal pole, but this expression ceases at the NZs (Fig. 2F). A mAb generated against Drosophila Neuroglian specifically labels the endfeet of the ingressing cells converging at the center of the NZs (arrow in Fig. 2H). Furthermore, the cell surface molecule Semaphorin I is restricted to the apical surface of epithelial cells at the slight invagination of each NZ (Fig. 21). In summary, the restricted expression of Neuroglian and Semaphorin I and the loss of expression of REGA-1 and the 8B7 antigen accompany the morphogenetic changes of the ingressing cells in the NZs, suggesting that extensive molecular changes in both the cytoskeleton and the cell surface are taking place.

Proliferation, segregation, and neuronal differentiation in the neurogenic zones

Soon after the NZ cells have undergone the epithelial-to- mesenchymal transformation, they start to divide symmetrically. We observed divisions of cells located dorsally to each NZ when labelling with the anti-Lachesin mAb (Fig. 3A, arrow) and with serum anti-tubulin antibody (not shown). To further confirm these observations we studied the proliferation pattern by BrdU incorporation during inter- vals of 9 hours ( = 1.8% of development) in embryos ranging from 25% to 40% of development. Mitotically active cells are seen dispersed in the mesodermal and epithelial layers of the growing stomodeum, but the NZs in the epithelium are devoid of proliferative activity (not shown). A group of cells that incorporate BrdU is detected dorsal to each NZ at 30% of development (arrows in Fig. 3B). These cells have delaminated from the epithelium, and after one or a few rounds of mitotic divisions they stop dividing. After cell division these cells disperse, rapidly locate anteriorly to each NZ (arrowheads in Fig. 3B), and start expressing neuronal glycoproteins recognized by sAb anti-HRP, a specific marker for postmitotic neurons (arrowheads in Fig. 3C). Thus, the ingressed cells can be considered as neuronal precursors that proliferate after their segregation from the epithelium. However, neurons from the different NZs are not equivalent as deduced from the expression pattern of Lazarillo. This surface glycoprotein is differentially expressed by postmitotic neurons that appear to originate from Z1 and Z2 (arrowheads in Fig. 3D). Lazarillo could then be considered a lineage marker for neurons originating at Z1 and Zz, although a specific regulation of its expression by reasons other than lineage cannot be excluded. In summary, neurogenesis proceeds through the steps depicted in Figure 3E. Cells differentiate within the three epithelial regions defined early in development (1). These cells adopt a tear-like shape and the epithelium invaginates at each NZ (2). Subsequently, cells detach from the epithelium and become neuronal precursors that divide (3) and give rise to postmitotic neurons (4) that express different markers depending on the NZ they come from. Later, they start a migratory phase (5) that brings them to their final site of differentiation within the enteric nervous system (see below).

All the cells we detect at the NZs after ingression and division of precursor cells label with our neuronal markers. However, no specific marker allows us to identify if, at the same time or later in development, glial cells are also being generated as is the case for Manduca (Copenhaver, 1993). We have not detected neurons derived from structures

Fig. 2. Epithelial foregut differentiation and onset of neurogenic zones (NZ). All pictures are lateral views of the stomodeum unless noted. Dorsal is up and anterior is left. A: Fasciclin I is expressed early (22% of development) by the epithelial cells at the tip of the stomodeum (arrowhead). Loose cells around the epithelium are mesodermal in origin. B: REGA-1 is expressed in the stomodeal epithelium after 23-24% of development in a gradient-like fashion. The REGA-1 expression domain, shown here at 25%, excludes the cells at the tip of the stomodeum (arrows). At this stage cells at the dorsal midline of the stomodeum have become more columnar than those in the surrounding epithelium. C: Expression of Fasciclin I appears in three patches of epithelial cells at the dorsal midline (arrows) before the cells start to show the typical morphology of the future NZs (25% of development). Cells at the tip keep expressing Fasciclin I (arrowheads). D: View of the dorsal surface of a stomodeum at 30% of development. Between 27 and 30% the three NZs (Z,-Z3) become apparent. The cells in the NZs express Fasciclin I. E: Closer lateral view of Z3 labeled with mAb

anti-Fasciclin I (33% of development) to show the invagination of the epithelium, and the modified cell morphology: Endfeet anchor cells to the apical surface at the center of the invagination (large arrow), whereas nuclei and most of the cytoplasm have moved dorsally and protrude at the basal surface. After ingression from the epithelium the cells are lightly labeled by this mAb (arrowhead). Mesodermal cells (small arrow) present no labeling. F-I: Expression pattern of several molecules that correlate with NZs formation. F: mAb 8B7 labels an intracellular epitope at the basal region of epithelial cells leaving a gap (arrows) at each NZ. G: The epithelial expression of REGA-1 also excludes the NZs (boundaries of expression denoted by arrows). Al- though the protein appears throughout the epithelial cells membrane, it is more concentrated at their basal surface. H: mAb 5A12 against Drosophila Neuroglian recognizes an epitope expressed on the endfeet of NZ cells (arrow). I: Semaphorin I is expressed on the apical surface of the invaginating NZ cells (arrows). Scale bars = 30 bm.


Fig. 3. Neurogenesis at the NZs. Pattern of mitotic activity and early differentiation of postmitotic neurons. A Dorsal view of 2 3 labeled with mAb anti-Lachesin. Labeled cells that have detached from the epithelium are seen dividing on top of 2 3 (arrow). B: Lateral view of stomodeum after a 9-hour pulse of BrdU starting at 35% of development. Cells located dorsal to the three NZs (arrows) have divided during this period, whereas cells immediately anterior to them (arrowheads) have not incorporated BrdU. The localization of the NZs in the epithelium is marked by dashes in B-D. C: The delaminated cells located anteriorly to the NZs (arrowheads) are postmitotic neurons as revealed by labeling with sAb anti-HRP. D: Lazarillo is expressed by

postmitotic neurons arising from Z1 and Z2 (arrowheads), but neurons from Z3 never express it. E: Schematic drawing of the process of neurogenesis from the NZs. First, (1) cells that were part of an undifferentiated epithelium start expressing different surface proteins. (2) They change their shape, becoming tear-like with an endfoot attached to the apical surface. Later (3) , cells detach from the apical surface and become neuronal precursors that divide and (4) produce postmitotic neurons that turn on the expression of neuronal specific markers recognized hy sAb anti-HRP. Finally (5) , the neurons start migrating to reach their final position in the ENS. Scale bars = 30 pm in A, 50 km in B-D.

other than the NZs, as described in Manduca (Copenhaver and Taghert, 1991) and Drosophila (Hartenstein et al., 1994).

anteriorly (Fig. 4A). They advance while extending short processes directed anteriorly. This migration toward the anterior part of the stomodeum precedes axonogenesis and

Active neuronal migration gives origin to the enteric ganglia in the foregut

Postmitotic neurons are displaced from the NZs soon after their generation in a manner that suggests a direc- tional migration. We distinguish three migratory phases: an anterior migration to form the dorsal unpaired hypocere- bra1 and frontal ganglia, and two phases of lateral migration to form the bilaterally located ingluvial ganglia. The anterior migration starts at 31% and is first seen in Z1, where a pair of cells separate from the zone and move

occurs along the dorsomedial aspect of the Gomodeum, forming a columnar ridge three to four cell diameters wide (see Fig. 3C for a lateral view, and Fig. 4B for a dorsal view). This process is slightly different in the other two NZs. Neurons coming from Zz and 2 3 start their anterior migration, but they actively avoid crossing over the NZs they encounter, i.e., Z1 in the case of 2 2 neurons and Z2 in the case of Z3 neurons. Instead, these neurons surround the NZs leaving them as clear neuron-free zones (Fig. 4B). Only after axonogenesis has started are anteriorly migrating neurons able to cross over NZs and form a compact package


Fig. 4. Three rounds of cell migration give rise to the ganglia of the foregut. A-C: Anterior migration (dorsal views). D,E Lateral migration of neurons (lateral views). F-H Second round of lateral migration (dorsal views). A: First neuron migrating anteriorly from the package of cells produced at Z1 (31% of development, mAb anti-Lazarillo). Migra- tory neurons display short processes (arrowhead), but axonogenesis begins later in development. B: Neurons from the three NZs visualized with sAb anti-HRP (32% of development). Neurons from 2 3 and Zz initially seem to avoid crossing over the NZ anterior to them. Many neurons are seen instead anterior to Z1. They accumulate at the base of the stomodeum (arrowhead) where the frontal ganglion will form. No axonogenesis is detected yet. C: At a later stage (40% of development, sAb anti-HRP) no more avoidance of NZ surface is observed, and neurons form a compact package. Axons have emerged from many cells and converge to a point (arrowhead) to form a single dorsomedial bundle of axons directed anteriorly. D: Lateral migration is first seen at 38% of development (sAb anti-HRP). A few neurons are seen advancing laterally and leaving behind their axons (arrows), pioneering the esophageal nerve. E: More neurons join the lateral cluster by migration along the pioneer axons (arrowhead) and form an axon bundle (38-39% of development, sAb anti-HRP). F-H: At 40% of development the first round of lateral migration has finished, and two compact neuronal

of cells (Fig. 4C). The anteriorly directed neuronal migration organizes two main groups of cells that will form the hypocerebral ganglion at the level of Zz and the frontal ganglion at the anterior border of the stomodeum. But what is the contribution of the different NZs to the fully differentiated dorsal unpaired ganglia? All of the neurons in the frontal ganglion and a subset of them in the hypocerebral ganglion are labeled with the anti-Lazarillo mAb later in development. Comparison of this labeling with other markers expressed in the three NZs suggests that neurons arising from Z1 and Zz contribute to the frontal ganglion,

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clusters have formed laterally (ingluvial ganglia, ig) that connect with the dorsomedial cluster (hypocerebral ganglion) by the esophageal nerves (small arrows). Dashed lines indicate foregut-midgut boundary. F: New cells emerging from Z3 and labeled with mAb anti-Fasciclin I (45% of development) form a stripe that expands laterally toward the ingluvial ganglia (arrowheads). G: These cells divide (BrdU analysis in a 50% embryo) while dorsal to Z3. H: They are not labeled with sAb anti-HRP during their lateral migration (arrowheads, 53% of development). I: Measurements of stomodeal growth over development. The following distances were measured: a = total length of stomodeum (open circles), b = diameter of stomodeum at the level of Z3 (solid circles), c = Zl-base of stomodeum (open diamonds), d = ZzZl (open inverted triangles), e = Z3-Zz (open triangles), f = Z3-tip of stomodeum (open squares). Total longitudinal growth between 23% and 53% of development is approximately linear with a slope of 14.2 pm/l% of development. Most of this growth occurs between Z1 and the base of stomodeum (7.1 pm/l%), whereas the position of the three NZs with respect to each other or to the stomodeum tip does not vary greatly (slopes are d = 4.85 pm/l%, e = 1.79 pm/l%, f = 2.39 pm/l%). The enlargement of stomodeum semicircular length happens at 9.1 pm/l% over the stages studied. Scale bars = 50 pm in A-E,G, 100 pm in F,H.

whereas Z3 seems to be a major organizer of the hypocerebral ganglion, with some contribution of Zz. Nevertheless, a detailed lineage analysis is needed to precisely establish the contribution of each NZ.

At 33% of development the antibodies recognizing anti- gens present on neuronal processes (like sAb anti-HRP, mAbs 8B7, anti-Fasciclin I, anti-Lazarillo, and anti- Semaphorin I) revealed the beginning of axonogenesis in the developing hypocerebral ganglion. Neurons start send- ing axons anteriorly over the dorsal midline of the stomodeum. Some axons also emerge from cells located in the


lateral parts of the neuronal cluster and converge to a dorsomedial position where they fasciculate together. This results in the organization at 40% of development of an axon bundle, the future recurrent nerve (Fig. 4 0 , which runs anteriorly over the NZs and connects the frontal and hypocerebral ganglia. Later neuronal processes, as well as migrating neurons, seem to use these initial axons in their migration. A more detailed description of the formation of nerve tracts will be reported below.

The process of anterior migration finally organizes neurons into two ganglia. By 45% of development the dorsomedial cellular ridge becomes devoid of neurons, leaving a bare recurrent nerve, and the ganglia become fully separated with an ensheathing array of non-neuronal cells. However, some neurons emerging from Z2 can still be seen up to 53% of development joining the hypocerebral ganglion.

At 38%, while the anterior migration is still taking place, postmitotic neurons from the lateral part of the cellular cluster start to separate and migrate to the flanks of the stomodeum. This lateral migration occurs while axons from these neurons are being extended toward the dorsomedial recurrent nerve, which results in the neurons leaving behind their axons (Fig. 4D). All these axons join together and form a bundle (the future esophageal nerve) that serves as a pathway leading other migrating neurons to the incipient ingluvial ganglion (Fig. 4E).

At 40% of development the ingluvial ganglia are formed, and a new round of neurogenesis begins to generate precursor cells from Z3 (Fig. 4F). BrdU analysis at 45-70% of development shows the existence of mitotically active cells ingressed from 2 3 (Fig. 4G). The resulting daughter cells migrate bilaterally to the caudal part of the ingluvial ganglion (a process that continues until around 55%) and form a packet that enlarges and elongates posteriorly to the ingluvial ganglia (Fig. 4H, see also Fig. 6E). The laterally migrating cells do not incorporate BrdU along their way to the developing ingluvial ganglia, but do express the neuronal cell surface proteins Lachesin (not shown) and Fasciclin I (arrowheads in Fig. 4F). However, there is no labeling with the general neuronal marker anti-HRP (arrowheads in Fig. 4H). Unlike the first phase of lateral migration, cells involved in this second round do not extend axons while migrating. At 53% of development the ingluvial ganglia have acquired a structure similar to the other mature ganglia. Migrating cell bodies are no longer seen along the esophageal nerves.

We have described the formation of ganglia as a result of an active cell migration. However, our study does not include a real-time observation of neuron displacements that would unambiguously demonstrate the active migration process. A passive elongation and enlargement of the stomodeum could also be the cause of neurons being displaced and incorporated into the various ganglia. In order to clarify this issue we measured the stomodeum length (a), its half-diameter (b), and distances between the labral bend and the center of Z1 (c), from the center of Z1 to Z2 (d), from the center of Zz to Z3 (e), and between the stomodeal tip and the center of Z3 (f, at different stages of development (Fig. 41). It is apparent from these measurements that the developing foregut (23-50% of development) grows linearly both in the longitudinal and transver- sal axis at 14.2 and 9.1 pm/l% of development, respectively. Another characteristic of this growth is its heterogeneity. A faster growth is achieved by the proximal (anterior) part of the stomodeum that lies between Z1 and the labrum,

whereas the rest of the longitudinal rates of growth are rather slow. Taking into account these displacements, we measured the developmental time that it takes the neurons arising from Z3 to reach the next neurogenic zone Zz. Knowing the distance between these two NZs we estimated the rate of displacement for the neurons to be approximately 38 p,m/l% of development, 19 times faster than the stomodeal growth in this region (1.79 p,m/l% of development). This argues against a passive displacement of the stomodeum as the cause of ganglia formation and suggests an active process of migration. These results were con- firmed when estimating the rate of displacement of neurons originating in Z1 (50 km/l% of development) and that of the neurons separating from the dorsal neuronal cluster to form the ingluvial ganglia (38 pm/l% of development), which represents a 7 and 4 times, respectively, faster displacement than expected for a passive mechanism. These results support the idea that the formation of ganglia in the ENS occurs by an active migration of neurons from the NZs.

Formation of stomatogastric nerves The immunocytochemical results suggest that the first

developing axons that contribute to the recurrent and esophageal nerves grow in an anterior direction. We further investigated the process of nerve formation by labelling axons with the lipophilic dye DiI and looking for the location of their cell bodies. When labeling the recurrent nerve (Fig. 5, left), we found that in all cases under 40% of development (n = 5) the labeled cell bodies are in the hypocerebral ganglion, and their axons extend to the frontal ganglion where they branch profusely. After 40% of development, cell bodies appear labeled in the frontal ganglion as well (6 cases in n = 8), and, according to the injection site, the axons of these neurons might already have reached the hypocerebral ganglion.

A small number of axons comprise the esophageal nerve prior to 40% of development, which made it difficult to identify the bundle and localize the dye. However, ingluvial neurons can be labeled at this stage when applying the dye to the recurrent nerve (Fig. 5, left). This, along with the pattern of immunostained ingluvial neurons described above, suggests that the first axons running along the esophageal nerves originate from ingluvial neurons. After 40% of development the labelled axons of the esophageal nerve belong to cell bodies located both in the hypocerebral and ingluvial ganglia (n = 5) (Fig. 5, right).

Data from immunolabelling with the anti-Semaphorin I and 8B7 mAbs suggest that the frontal ganglion connectives form before 40% of development by axonal projections from the frontal ganglion. By 40% of development CNS neurons from the tritocerebrum send processes toward the frontal ganglion. At later developmental stages (45%) DiI- labeled axons originating in the hypocerebral ganglion extend along the entire recurrent nerve and the frontal ganglion connectives.

These results indicate that the initial consolidation of enteric nerves is polarized in an anterior direction. Once the pathways connecting the ganglia are formed, a bidirectional connection is established. Although the first extending axons are always seen along the dorsomedial aspect of the stomodeum we were unable to detect the existence of identified pioneer neurons. A similar direction of nerve outgrowth has been observed in Drosophila (Hartenstein et


Recurrent nerve 3 8 -4 0 Yo

Esophageal nerve

u. ‘9

Fig. 5. Formation of the nerve tracts assessed by iontophoretic application of the dye DiI at different developmental stages. Representa- tive examples are shown as camera lucida drawings. Anterior is left in all drawings and photomicrographs. The injection sites of the dye are marked by arrows. On the left, we show the results obtained when the recurrent nerve was labeled. At 40% of development the axons running along the recurrent nerve belong to cells in the hypocerebral and ingluvial ganglia, whereas frontal ganglion cells contribute to the recurrent nerve after that stage. As shown on the right, the esophageal

nerve injections identify cell bodies in both the hypocerebral and ingluvial ganglia after 40% of development. Examples of dye injections are shown at the bottom: an injection on the recurrent nerve at 40% (left) and one on the esophageal nerve at 42% (right). These images were observed with a Zeiss-Optronics fluorescence microscope equipped with a Cooled CCD camera. Images were digitally acquired and lightly enhanced by Adobe Photoshop software. fg, frontal ganglion; hg, hypocerebral ganglion; ig, ingluvial ganglion. Scale bars = 50 Fm.

al., 1994) andManduca (P.F. Copenhaver, personal commu- nication).

along the caudal part of the foregut (see Figs. lB, 7A). These caudal branches arise at 62% and extend initiallv

Generation of plexuses innervating the foregut and midgut

Although at halfway through embryogenesis the ENS ganglia have attained a mature organization, the foregut and midgut muscles still need to be innervated, and sensory neurons need to be located on the gut surface. At 60% of development, an assembly of neurons starts to migrate from the hypocerebral and ingluvial ganglia to form a plexus on the foregut (Fig. 6A). Neurons are arranged primarily in two bilaterally located dorsal and ventral trunks (Fig. 6B), a commissural band at the ventral surface (large arrow in Fig. 6A), and several branches running

between, and later over, the growing caeca buds. An arrangement of cells and processes (Fig. 6C) branch off from the main trunks covering the surface of the foregut. At later developmental stages, processes are also seen to connect the plexus branches with the esophageal and recurrent nerves (see Fig. 7A, small arrows). At 70% of development the arrangement of the foregut plexus is essentially complete. The migrating cells likely represent differentiated neurons, as they show no incorporation of BrdU (not shown) and bear long and branched processes that are labeled by specific neuronal markers such as mAb 8B7, sAb anti-HRP, or mAb anti-Fasciclin I (Fig. 6B,C). According to the plexus configuration in the adult Locusta


Fig. 6. A fourth phase of migration give rise to the neuronal plexuses innervating the foregut and midgut. All preparations shown are labeled with mAb anti-Fasciclin I except E. A: At 58% of development, neurons start migrating from the ingluvial ganglia (ig). Some are directed ventrally to form a commissure-like connection between both ingluvial ganglia (large arrow). Others migrate anteriorly and dorsally in a more dispersed way to contribute to the foregut plexus. The hypocerebral ganglion also contributes to some foregut plexus cells (arrowhead). The most posterior edge of the ingluvial ganglion is disorganized at this age, and cells acquire a “migrating” morphology with short processes extending posteriorly (small arrow). B: Ventral view of the foregut plexus at 70% of development. Neurons in the plexus do not have stereotyped positions but are organized in two main longitudinal arrays in the ventral side (arrowheads) and two on the dorsal side (not shown). The ventral commissure is out of focus on the right. C: Detail of foregut plexus cells (small arrows) and processes. D: At 60-62% of development, neurons from the ingluvial ganglia (ig) start spreading posteriorly as two compact tongues (arrowheads) that advance and cross the foregut-midgut border (dashed line). E: Cells migrating in the tongues (arrows) do not exhibit mitotic activity when assessed by BrdU incorporation in a 65% embryo. These cells start

(Plotnikova, 19671, the migrating neurons presumably are peripheral sensory neurons, whereas the processes are both afferent fibers from sensory neurons and efferent motor fibers from cells located in the ganglia.

We have found that the midgut plexus originates entirely from the developing ingluvial ganglia. At 58% of development the ingluvial ganglia are fully structured, but the posterior border starts to disorganize, and thin processes are directed to the midgut (Fig. 6A). Shortly afterward, a massive cell migration occurs from the cells located caudal to each ingluvial ganglion (arrowhead in Fig. 6E). Groups of cells organized into two “tongues” (arrowheads in Fig. 6D) migrate and cross the foregut-midgut boundary. The lead-

migrating from a compact group of cells (arrowhead) organized caudally to each ingluvial ganglion (ig) after the second round of lateral migration from Z3. Labelled nuclei that appear on the surface of the ingluvial ganglion could belong to glial cells. F After a rapid dispersal on the midgut surface, the cells of the tongues advance leaving behind axonal processes that form nerves. Newly migrating cells (arrowhead) use those nerves as a pathway, although some cells branch off them (small arrow). G The dispersal of neurons from the tongues gives rise to the midgut plexus (70% of development) composed of a ring of neurons at the foregut-midgut border (open arrow) and four longitudinal migratory pathways, two of which are indicated by arrows. H: Enlarged view of neurons migrating along one of the midgut nerves (arrow). Neuronal processes branch from the four main midgut nerves and innervate the muscles between them (arrowhead). I: View of midgut-hindgut border (dashed line) at 70% of development. Cells migrating along the longitudinal midgut pathways have reached the posterior part of the midgut and project axons posteriorly to connect with the hindgut nerves (arrow). The hindgut nerves appear to be formed only by axons (and not cell bodies) originated in the CNS. The malpighian tubules (arrowhead) are seen at the midgut-hindgut border. Scale bars = 100 K r n in A,B,D,G,I, 50 *rn in C,E,F,H.

ing cells composing the tongues bear neurites that are connected to the ganglion. Later migrating cells seem to fasciculate with previous processes in their movement toward the midgut. No further mitosis occurs in these cells as deduced from the lack of BrdU incorporation along the entire migratory pathway (arrows in Fig. 6E). Although some mitotic activity is detected within the elongated ingluvial ganglion, we cannot determine if it represents divisions engendering neurons or a late proliferation phase giving rise to glia as has been reported in Manduca (Copenhaver, 1993). These observations and the positive labeling obtained with neuronal markers (Fig. 6D) suggest that these migrating cells are postmitotic neurons and that


Fig. 7. ENS organization in the foregut at 80% of development. A: Side view of a foregut labelled with mAb anti-Fasciclin I showing the final arrangement of enteric ganglia and plexuses. Small arrows indicate branches connecting the recurrent and esophageal nerves with the foregut plexus. Dashed line indicates the foregut-midgut boundary. B,C: General organization of the frontal and hypocerebral ganglia (B), and ingluvial ganglion (C) as revealed by labeling with mAb anti- Fasciclin I: Cell bodies lie at the outer ring, whereas the neuropil is at the inner core of each ganglion. B: The frontal ganglion is linked to the CNS by two connectives (large arrows) and with the hypocerebral ganglion by the recurrent nerve that is composed of two main fascicles

the midgut plexus origin is primarily Z3. Neurons arise in Z3 as a result of the second round of neurogenesis, then migrate laterally to accumulate at the posterior edge of the ingluvial ganglia, and finally migrate posteriorly as described. At 62% of development the tongues begin to disperse. Small groups of cells detach from the tongues and move parallel to the longitudinal muscle bands on the surface of the midgut (Fig. 6F). The cells have a bipolar shape with short processes running posteriorly. The processes that are left behind establish four main pathways, the midgut nerves, two derived from each ingluvial ganglion (Fig. 6G). Some cells branch off these main pathways (small arrow in Fin. 6F) leavine: behind their axons as well.

(small arrows). These two fascicles remain essentially separated within the hypocerebral ganglion and are contiguous with the paired esophageal nerves that connect with the bilateral ingluvial ganglia. A posterior commissure within the hypocerebral ganglion (arrowhead) connects the two longitudinal pathways. C: Each ingluvial ganglion is connected with the frontal ganglion by an esophageal nerve. Two other nerves connect with the midgut plexus (arrowheads) and the rest with the foregut plexus and caeca. fg, frontal ganglion; hg, hypocerebral ganglion; rn, recurrent nerve; ig, ingluvial ganglia; en, esophageal nerves; fp, foregut plexus; c, caeca. Scale bars = 200 pm in A, 75 pm in B,C.

boundary (open arrow in Fig. 6G). Another nerve ring without cell bodies will form at 70% of development at the midgut-hindgut boundary around the base of the malpighian tubules (Fig. 61). Whereas the anti-Fasciclin I mAb labels all the foregut and midgut plexus cells, the neurons migrating along the midgut are not labeled by the anti-HRP sAb. The anti-Semaphorin I mAb only recognizes the processes and not the migratory cell bodies in both plexuses.

Morphology of the ganglia in late embryogenesis

" ,

Migrating cells can be seen al&g the entire length of the midgut nerves (Fig. 6H), and at 75% of development thin processes (arrowhead in Fig. 6H) are seen covering the space between the nerves.

Some cells will follow a circumferential migration pathway and form a nerve ring along the foregut-midgut

At 80% of development the ENS shows a mature organization. The frontal, hypocerebral, and ingluvial ganglia have reached their final location, and there is a stereotyped array of nerves and plexuses (Fig. 7A). The ganglia show a similar size and pear-like shape, with the apex pointing anteriorly (ingluvial and hypocerebral ganglia) or posteri-


33% A

Brain

50%

( Brz Foregut-midgut

boundary 4

co

Midgut plexus

Fig. 8. Schematic drawing summarizing the development of the enteric ganglia and plexuses in the grasshopper embryo. Postmitotic neurons are colored according to the neurogenic zone where they originate. Proliferating precursor cells are depicted in red. Cells in dark

orly (frontal ganglion) (Fig. 7B,C). The ENS ganglia are enclosed by a sheath of glial cells and show an outer domain of neuronal cell bodies and an inner neuropil region. The labeling with the anti-Fasciclin I mAb at 80% of development shows that the neuropil in the frontal (fg in Fig. 7B) and ingluvial ganglia (Fig. 7C) appears compact, corresponding to a region with abundant neuronal processes and hence synaptic connections. However, the hypocerebral ganglion neuropil (hg in Fig. 7B) displays a less structured central region, an external rind with a commissural tract in the caudal part of the ganglion (arrowhead in Fig. 7B), and two bilateral longitudinal fiber tracts (small arrows in Fig. 7B) that converge to the recurrent nerve. Each of these longitudinal tracts remains split along the recurrent nerve (rn in Fig. 7B) and is contiguous with each esophageal nerve.

ganglion

green represent the second round of lateral migration that contributes to the ingluvial ganglia and midgut plexus. Arrows indicate the direction of outgrowing neurites. The cells organizing the plexuses are shown in light brown. See text for details.

A summary of the ENS ontogenesis is schematically illustrated in Figure 8, and a timetable for some important cellular and molecular events during ENS development is shown in Figure 9.

DISCUSSION Morphogenesis and differentiation in the ENS

of the grasshopper Neuronal migration is not a common theme in insect

neural development, with only a few cases of migration documented in the CNS and PNS (Heathcote, 1981; Good- man et al., 1984; Bentley and Toroian-Raymond, 1989). However, this complex cellular behavior is fundamental in


I caeca formation

1-1 formation of foregut and midgut plexuses I midgut formation

1-1 2nd round of lateral migration

1-1 formation of IG;1 st round of lateral migration

H formation of axon tracts

1-1 formation of FG and HG

I starts proliferation of ENS precursors

I NZs arise; proctodeum invagination

I placodal transformation

I stomodeum invagination I I I I I I I I 1 I I I I I I I I

0 10 20 30 40 50 60 70 80 90 100 Developmental stage (O/.) Hatching

Fig. 9. Developmental timetable of the ENS ontogenesis. Grasshoppers embryogenesis occurs in 20 days at 31°C and 60% humidity. Staging is by percentage, with 5% corresponding to the development occurring in 1 day.

shaping the vertebrate nervous system in general, and the enteric nervous system in particular. The vertebrate enteric neurons and glia derive from the vagal and sacral neural crest cells, which undergo an extended period of migration from the dorsal aspect of the neural tube to the gut (reviewed by Gershon et al., 1993). Interestingly, an extensive cellular migration is also present during the development of the ENS in insects (Copenhaver and Tag- hert, 198913; Copenhaver, 1993; Hartenstein et al., 1994). In grasshoppers, an anteriorly directed migration is accomplished by neurons delaminating from the three NZs. The migration route for these neurons is along the dorsomedial aspect of the stomodeum and gives rise to the formation of the unpaired frontal and hypocerebral ganglia. A later round of cell migration is accomplished by Z3-derived cells that migrate laterally and results in a package of cells caudal to the ingluvial ganglia. The cells involved in these two phases of migration have not begun axonogenesis. A different type of lateral migration is seen during the initial formation of the ingluvial ganglia that involves the displacement of neuronal cell bodies from the developing hypocere- bra1 ganglion. This process does not start with an initial identified pioneer cell, but is carried out by several leading cells simultaneously. These neurons lack substantial processes extended in the direction of migration, but instead they leave behind trailing neurites that fasciculate together in a tight bundle that serves in turn as a pathway for later migrating cells. This pattern of migration with trailing processes resembles the migration of granule neurons in the vertebrate cerebellum.

In general, the lateral migration pattern in grasshopper, although organized in two rounds with different characteristics, resembles the slow circumferential migration of the

enteric plexus cells that gives rise to the foregut and midgut plexus in Munduca (Copenhaver and Taghert, 1989b). Not surprisingly, the lateral migrations in grasshopper also result in the organization of the ingluvial ganglia and the midgut plexus. The absence of labeling with the anti-HRP sAb in the postmitotic cells involved in the second round of lateral migration, as well as in the neurons of the midgut plexus, suggests that the cells that initially migrate from Z3 to the posterior part of the ingluvial ganglia are the ones that later migrate posteriorly to form the midgut plexus. Alternatively, as the migrating cells express other markers (Fasciclin I and Lachesin) that are present in neuronal precursors as well as in neurons, it is possible that they are not fully differentiated before reaching the ingluvial ganglia. The absence of anti-HRP labeling in midgut plexus cells suggests an interesting difference in the carbohydrate composition between the midgut plexus cells and the rest of the enteric neurons.

The midgut cells migrate first in the form of two compact bulges or tongues that detach from the posterior part of each ingluvial ganglion. Each tongue disperses and estab- lishes a midgut nerve, thus forming four nerves, two on each side of the midgut. InManduca the enteric plexus cells remain initially restricted to the posterior region of the foregut until the six major longitudinal muscle bands appear on the midgut surface, and then the cells disperse specifically along the muscle bands during a fast migratory phase (Copenhaver and Taghert, 198913). The establish- ment of midgut nerves in grasshopper seems to follow different cues as no major muscle bands are morphologi- cally recognized. The cells of the tongues cross the foregut- midgut boundary between the developing caeca buds, but we cannot detect physical or chemical (according to our


molecular markers) features that could serve as guidance cues to the migrating cells. By the end of migration Manduca enteric plexus cells have only traveled approximately one-fifth of the total length of the midgut (Copen- haver and Taghert, 1989b), in contrast with grasshoppers in which we have observed migrating cells over the entire midgut nerve pathways. Whether these cells have traveled through the entire length of the midgut or they represent cells incorporated from the midgut epithelium cannot be resolved with the methods used in this work, although the latter hypothesis seems unlikely, as our markers fail in identifying cell clusters originating in the midgut. Further- more, no cells have been observed migrating anteriorly from the hindgut.

The plexus that innervates the foregut is composed of cells that migrate from the ingluvial ganglia and a small number of cells from the hypocerebral ganglion. This contrasts with the foregut innervation in Manduca that is accomplished by processes branching from the recurrent nerve, with only a small number of neuronal cell bodies that migrate from the epithelial placode (Copenhaver and Tag- hert, 1989b).

The plexuses in the grasshopper ENS originate from cells that were initially part of developing ganglia, whereas the Manduca enteric plexus originates from a package of cells that invaginates from the stomodeum epithelium. There is a parallel between these two apparently different mecha- nisms to generate a plexus: Both seem to require plexus cells packed together prior to their dispersal. Since no en masse invagination of cells occurs in grasshopper embryos, the cells individually delaminating from the NZs migrate first to the developing hypocerebral or ingluvial ganglia; only afterward do these cells disperse to form plexuses. It would be of great interest to study how these cells become committed to a ganglionic or a plexus fate.

The detailed analysis carried out in Manduca (Copen- haver and Taghert, 1989a, 1991) reveals distinct differentiation events occurring between the enteric ganglia and the enteric plexus. The cells composing the enteric ganglia delay their differentiation until the ganglia are formed, and can then be recognized by anatomical or biochemical crite- ria. Enteric plexus cells also acquire a differentiated state and exhibit specific peptidergic phenotypes after they have concluded their migration across the gut surface, but they cannot be uniquely identified by their position, morphology, or transmitter phenotype. Interestingly, the vertebrate enteric neurons show the same differentiation pattern, as they adopt their terminal phenotype once they have reached the gut and stop migration (Gershon et al., 1993). Although we have not attempted a characterization of the morphological and transmitter phenotypes of the grasshopper enteric neurons, the differential expression of some cell surface molecules like Lazarillo (see Fig. 3D) suggests an early differentiation prior to their migration.

The consequence of cellular migration is the morphogenetic rearrangement of cells arising from the neurogenic zones into several ganglia and plexuses. The origin of cells contributing to each particular ganglion has been studied in a variety of insects. However, accurate results coming from the use of specific cellular markers or lineage tracers have only recently been reported. The coincidence of the number of neurogenic zones and ganglia supported the idea of a one-to-one correspondence, as previously reported in grasshoppers (Lal-Roonwal, 1937). However, doubts about this view have arisen by the observation that cells from a single

neurogenic zone can contribute to more than one ganglia. The frontal ganglion in Drosophila mainly derives from three clusters of delaminated rostral precursors, but neurons from the rostral and intermediate vesicles also contribute (Hartenstein et al., 1994). Cells from Manduca NZs contribute both to the frontal and hypocerebral ganglia, whereas cells derived from an epithelial placode later formed at Z3 give rise to the enteric plexus (Copenhaver and Taghert, 1990, 1991). The markers used in the present study also point to a mixed origin for the grasshopper enteric ganglia. The neurogenic zones Z1 and Z2 provide cells to the frontal ganglion, cells from 23 and Z2 contribute to the hypocerebral ganglion, and the ingluvial ganglia have their main origin in Z3. The foregut plexus is formed with Zz and Z3 neurons, whereas as in Manduca, the origin of the midgut plexus is the caudal neurogenic zone.

Neurogenesis in the grasshopper ENS The neurogenic program is perhaps the area of the ENS

ontogenesis most intensely studied. Two different programs of neurogenesis have been described in Manduca (Copenhaver and Taghert, 1990,1991). The enteric ganglia of this lepidopteran, namely the frontal and hypocerebral ganglia, develop from three NZs located on the dorsal surface of the stomodeum. The zone precursor cells delaminate and migrate to organize these ganglia. Some scattered epithelial cells also delaminate at the anterior part of the stomodeum and contribute to the foregut plexus. However, the enteric plexus is derived from an epithelial placode that differentiates at the same location as the caudal NZ, but at a different developmental time. This placode invaginates en masse, and then the cells dissociate and migrate to the midgut. The Drosophila ENS also displays two different developmental programs. Three clusters of epithelial cells invaginate and form transient vesicles. Once the vesicles are separated from the epithelium the cells dissociate and migrate to reach their final destination in the four enteric ganglia. In addition, cells from three small groups located rostral to the three vesicles delaminate individually and sequentially, and contribute to the frontal ganglion.

As is general in the insect embryo, the developing stomodeum of the grasshopper appears clearly polarized along the dorsoventral axis. The dorsomedial aspect of this initially single-layered epithelium thickens and undergoes a placodal transformation (Fristrom, 1988). A similar epithelial modification generates the Manduca enteric plexus (Copen- haver and Taghert, 1990) and is a general finding in other insect tissues (Fristrom, 1988) as well as in vertebrate sensory ganglia (Le Douarin and Smith, 1988). The transformation affects the entire length of the grasshopper stomodeum, but at equidistant locations along the dorsal midline three prospective NZs start expressing the surface protein Fasciclin I. A differential expression of molecules is also observed in Drosophila, where the ENS anlage ex- presses proneural genes of the achaete-schute complex, the gap gene Kruppel, and the nuclear protein fork head (Gonzalez-Gaitan and Jackle, 1995). A discrete protein expression reflects a state of competence in the grasshopper NZs to undergo a critical morphogenetic change: a slight invagination or infolding that makes the zones protrude onto the surface of the foregut. The boundaries of these zones appear demarcated by the absence of the Ig- superfamily protein REGA- 1 or the intracellular antigen 8B7. There is a central point in each zone where the constricted apical ends of the zone cells appear to be


anchored. At that location there is a differential expression of the cell adhesion molecules Fasciclin I, Fasciclin 11, and a putative Neuroglian-like protein. Their presence on these processes might be related to the detachment and posterior ingression of the zone cells. Whereas the rest of stomodeal epithelial cells are seen incorporating BrdU, the competent zone cells do not undergo mitotic divisions during the time they remain in the NZs. Only after their ingression do they start incorporating BrdU. Some mitotic profiles are seen in the packet of cells that is formed dorsally to each NZ. These mitoses are restricted to the zone domain and are not observed once the cells start their migration. The properties of the proliferation pattern of grasshopper ENS precursors differ significantly from the cell divisions observed in the neurogenic placode giving rise to the enteric plexus in Manduca (Copenhaver and Taghert, 1990) and the invaginating vesicles of Drosophila (Hartenstein et al., 1994) that undergo mitosis only before the segregation from the epithelium. In contrast, the proliferative program in grasshopper resembles that of the delaminating rostra1 ENS precursors in Drosophila (Hartenstein et al., 1994) and the zone neurogenesis in Manduca (Copenhaver and Taghert, 1991). The overall process of growth in the zone and the fact that no more replicating cells are seen in the anterior stream of migrating cells suggest a small number of divisions of the ENS precursors similar to reports in Manduca and Drosophila. The few progeny generated and the sym- metrical appearance of these divisions are features analo- gous to those of the insect CNS midline precursors (Bate and Grunewald, 1981; Goodman et al., 1981). The postmitotic migratory cells soon start expressing neuronal molecular markers, suggesting a neuronal identity. Some of these cells might also differentiate into glia, as demonstrated in Manduca (Copenhaver, 1993), but the markers used in this work would not identify ENS glial cells.

Despite the differences in the final arrangement of ganglia and nerves between orders of insects, most of the characteristics of the ENS ontogeny show a common pattern. Cellular migration, a rare event in the CNS and PNS, is a general finding in the ENS. The pattern of migration is also similar in several orders of insects. The embryonic origin of the ganglia is similar among the insects that have been studied with specific markers, and the formation of axon bundles occurs in the same sequence and with similar directionality. However, two different strategies can be seen in the proliferation pattern and some morphogenetic aspects of the neurogenic program: 1) a strategy based on delamination or ingression of individual cells, and 2) a program based on epithelial invagination followed by extrusion and dissociation of cells. The first strategy involves a proliferation pattern that is effective after the cells have delaminated from the epithelium. The cells involved in the second strategy divide while they share an epithelial character and stop after they dissociate. The notable variation in the gross morphology of the mature ENS suggests high degrees of freedom for the underlying developmental programs to accommodate to the functional idiosyncrasy of the digestive tract in each insect. Whether this freedom in design is also involved in the selection of a particular neurogenic program, or whether there exists an evolutionary trend to evolve more invagination-extrusion centers in the phyletic lines of insects, perhaps only in holometabo- lans, is unknown and worth studying.

Nevertheless, the differences make us wonder what might be the selective benefits, if any, represented by a

given neurogenic program. Hartenstein et al. (1994) propose an advantageous caudal transportation of invaginated structures based on the esophagus growth and head involu- tion in Drosophila. Despite the plausibility of this hypothesis, other solutions not requiring invagination, like the directed lateral migration that form the grasshopper ingluvial ganglia, have been exploited to overcome the passive rearrangement caused by stomodeal growth. A critical difference between both programs of neurogenesis is that cells arising from invagination-extrusion centers are closer to their mature phenotype as no further proliferation occurs in the extruded packet or vesicle, whereas cells undergoing delamination still have to divide and define their identity. Moreover, the invagination-extrusion strategy correlates with a poorly structured, although still stereotyped, terminal disposition of the resulting cells like the enteric plexus cells in Manduca or the three caudal ganglia in Drosophila. The Drosophila frontal ganglion and the Manduca and grasshopper enteric ganglia show a highly structured architecture with definite cellular com- partments, a complex neuropil with commissural and longitudinal tracts, and an elaborate array of output nerves. We believe that building such a complex organization is more appropriately regulated by the sequential addition of cells to the scaffold of the ganglia, rather than by the sudden appearance of many randomly positioned cells. Therefore, we propose an alternative hypothesis in which the invagination-xtrusion program would speed up building simple networks, and insects have evolved the ability to select between these two programs of neurogenesis depending on the complexity of the neuronal configuration to be as- sembled.

The analysis of Drosophila mutations that affect ENS development (Hartenstein et al., 1994; Gonzalez-Gaitan and Jackle, 1995) is beginning to uncover the molecular basis for the enteric neurogenic program in this insect. The present work describes the cellular events that organize the ENS in grasshoppers by studying the proliferation pattern with BrdU and the expression pattern of a number of nervous system-specific cell surface molecules. This information will be the basis for an analysis of the roles these molecules play in ENS development.

ACKNOWLEDGMENTS We are grateful to P. Copenhaver for support and critical

reading of the manuscript. We also thank C. Goodman, A. Kolodkin, A. Harrelson, and A. Bieber for their gift of mAbs 3Bl1, 6F8, 8C6, and 5A12. This work was supported by NIH grant NS25387 to M.J.B.

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Embryonic development of the enteric nervous system of the grasshopperSchistocerca americana