Recent Advances in Insect Embryology in Japan and Poland
Edited by H. Ando and Cz. Jura
Arthropod. EmbryoL. Soc. Jpn.
(ISEBU Co. Ltd., Tsukuba) 1987

237

Formation of Brain of the Normal and the Embryonic Lethal,"Star-spots Duplication" Embryos in the Silkworm,

Bombyx mori (Bombycidae, Lepidoptera)

Susumu HASHIMOTO
andKeiichiro MIYA
Synopsis
Formation of the brain is described in the normal and Dus/Dus embryos of Bombyx. In bothtypes of embryos the brain originates similarly from the neuroblasts in the neurogenic region of
the protocl!phalon and there is no remarkable difference between them. From the appendage
formation stage, each neuroblast divides unequally to produce a larger and a smaller daughter
cell, and these are organized into three groups of cells, anlagen of the proto-, deuto- and tritocerebrum. The larger cell repeats unequal divisions as the neuroblast and the smaller cell divides equally as the ganglion mother cell, and the number of cells increases remarkably. With
formation of the head, these three cerebra migrate and unite to form a brain. In Dus/Dus embryothese divisions occur more than in the normal ones, resulting in hypertrophy of the brain and failure of the head capsule formation. This process is discussed comparatively in both types ofembryos.

Introduction
The embryonic development of the central nervous system has been studied bymany authors in various insect species, but formation of the brain has been reported
usually as a part of these studies. The insect brain originates from the neuroblasts
which differentiate in the ectoderm of the protocephalon during the early development
similarly as the ganglia of the ventral nerve cord. Different from the ganglia of the ventral nerve cord, however, the brain is not only a complex structure consisting of several

238

S. Hashimoto and K. Miyagroups of nerve cells (see Johannsen and Butt, 1941; Anderson, 1972a, b), but also integrates various informations includi!1g those from the sensory organs of the head tocontrol the behavior of the individual (see Chapman, 1969). And the fate map of thecentral nervous system is made in several species, for example, in Drosophila (Poulson,1950) and in Tenebrio (see Anderson, 1972b; Sander, 1976).
Recently, segregation of the neuroblasts in Drosophila was studied in detail by Hartenstein and Campos-Ortega (1984) and interesting results were reported. Further,
they discussed the function of the genes relating to segregation of the neuroblasts incomparison with mutants affecting the early neurogenesis.
On the other hand, the embryonic development of the central nervous system in
Bombyx was described already by Toyama (1909) and Ikeda (1913), but there was no
detailed description on formation of the brain. Afterwards, the fate map of theneurogenic region was made clear also in Bombyx by Takami (1946) with the cauterization experiment and by Katsuki et al. (1980) through the analysis of mosaic larvae induced by super-cooling treatment. Furthermore, we found a new embryonic lethal
mutant, "star-spots duplication", which affected on development of various organs andtissues, especially on the neurogenesis (Miya and Hashimoto, 1977). The phenotype
shows hypertrophy of the central nervous system similarly as in Drosophila. Hence wefelt that the re-examination of Bombyx neurogenesis might elucidate a better understanding of this mutant, but the present paper deals only with formation of the brain incomparison with the normal and the mutant embryos.

Material and Method"Star-spots duplication" (symbol Dus) is a dominant gene and shows additional
star-spots on the sixth abdominal segment, and it is a recessive embryonic lethal andthe homozygotes represent various abnormalities and die at the setae formation stage.
Non-hibernating eggs of Dus/ + X Dus/ + were used as material.
For light microscopic observation, the eggs were fixed with Carnoy's fluid for 24
hr, stained with carbol thionin, dehydrated with ethanol, cleared with benzene, andmounted in ceder oil for total preparations. The fixed and dehydrated eggs wereembedded in paraffin and cut 8 p m thick. The sections were stained with Delafield'shematoxylin and eosin.
For electron micros~opy, the eggs were fixed with 2.5% glutaraldehyde in 0.1 M
cacodyrate buffer (pH 7.4) at 4·C for 1.5 hr, rinsed with the same buffer and postfixedwith 1 % osmium tetroxide in O. 1 M cacodyrate buffer (pH 7.4) at 4·C for 2 hr. Afterdehydration with ethanol series, the fixed eggs were embedded in Epon 812 throughQY-I. Ultrathin section were stained with uranyl acetate and lead nitrate.

Results
1. Formation of the brain in the normal embryoa. Differentiation of the neuroblasts in the protocephalon

Brain formation in normal and embryonic lethal Bombyx embryos

239

am

Fig. 1.

Cross section of the protocephalon in 36-hour old embryo. am, amnion; ecd,
ectoderm; msd, mesoderm; nb, neuroblast.

stom

Fig. 2.

Cross section of the protocephalon in 48-hour old embryo. am, amnion; dc, ganglion mother cell; deut, anlage of deutocerebrum; ecd, ectoderm; msd, mesoderm;prot, anlage of protocerebrum; stom, stomodaeum.

At 36 hour after oviposition the embryo becomes elongated and the boundary between the protocephalon and the trunk is clearly distinguised, but metamerism of theectoderm does not occur yet. The mesoderm is arranged segmentally, but in the posterior part of the embryo it does not spread laterally along the dorsal side of theectoderm. In the middle region of the protocephalon the mesoderm exists as a large

240

s. Hashimoto and K. Miyacell group, an oral cell mass, and several cells are being isolated from the oral cell massinto the yolk.
The neuroblasts, anlagen of the future brain, are recognized at first at this stage,earlier than their differentiation in the trunk. The neuroblast has a round larger nucleus than that of the other ectoderm cells composing the protocephalon, and in the
nucleus a distinct nucleolus is recognized. The neuroblasts are rich in light-stained
cytoplasm and are distributed separately and wide-spreadly in the ectoderm of the protocephalon (Fig. 1). The nucleus of the ectoderm cells is elliptical and the cells arearranged in a layer.
The ultrastructures of the neuroblasts are hardly different from those of theepidermis precursor cells, except for their size. The cytoplasm of the neuroblasts contains rod-like mitochondria, poor-developed rough-surfaced endoplasmic reticulum(rER) , free ribosomes, lipid droplets, and lacunae containing glycogen granules (Fig.
3). In the nucleus chromatin granules are dispersed and look less dense than that of
the epidermis precursor cells.
b. Proliferation of the neuroblasts

In the 42-hour old embryo, the metamerism of the ectoderm becomes clear, andthe embryo consists of the protocephalon, three gnathal, three thoracic, and elevenabdominal segments. Further, the appendages begin to be formed in the gnathal andthoracic segments. From this stage the neuroblasts divide unequally to form a larger
and a smaller daughter cells. The larger cell repeats unequal divisions as the neuroblast to produce the smaller cells, and the smaller cell is so-called "ganglion mothercell" and its nucleus is dark-stained.
c. Division of the neurogenic region in the protocephalon

In the 48-hour old embryo, the labral appendages project slightly at the anterior
end of the protocephalon. The antennal, gnathal, and thoracic appendages develop
more and more, and the appendages also appear in the abdominal segments. At thisstage many neuroblasts and smaller ganglion mother cells are distributed widely along
the dorsal (inner) side of the ectoderm of the protocephalon. Many neuroblasts, which
do not divide yet, also are recognized, and the fact suggests that the segregation of theneuroblasts in the neurogenic region does not occur at the same time, but extends forseveral hours (Fig. 2).
Then the protocephalon is divided into three areas by occurrence of the slight
lobes; that is, a wider area occupying from the anterior end of the protocephalon to theanterior end of the antennal appendages, an area from both sides of the stomodaeum tothe base of the antennal appendages, and an area behind the stomodaeum. With theprogress of development, the neuroblasts and their daughter cells are aggregated as one
pair of groups at the dorsal side of each area. These are anlagen of the proto-, deutoand tritocerebrum.
In 60 - 72-hour old embryo, all appendages, and the stomodaeum and the procto-

Brain formation in normal and embryonic lethal Bombyx embryos

241

Figs. 3, 4. Electron micrographs representing neuroblasts in 36- and 48-hour old embryo,
respectively. as, autosome; Ec, ectoderm cell; gl, glycogen granule; lip, lipid droplet; mit, mitochondria; Nb, neuroblast. Scales: I f-L m.

242

s. Hashimoto and K. Miyadaeum develop remarkably. The neuroblasts repeat unequal divisions actively to produce abundant daughter cells, especially in the area of the protocerebrum. The daughter cells produced by divisions of each neuroblast are not arranged radially, but scattered irregularly within the group. The equal divisions of daughter cells also are
observed, but it is unclear whether all cells derived from each neuroblast divide or not.
The electron micrographs at this stage indicate several new features. The nerve
cells, produced by the unequal division of the neuroblasts and/or the equal divisions of
the ganglion mother cells, extend several cytoplasmic projections. In the ectoderm cells
autosomes appear, which seem to correspond to differentiation of the ectoderm cells as
the anlagen of epidermis forming the head capsule later (Fig. 4).

d. Morphological changes with advance of the head formation and differentiation of
constituents of the brain
At about 72 hours after oviposition, the gnathal segments move forward and begin
to form the head together with the protocephalon. The deutocerebrum and the tritocerebrum migrate forward with the ectoderm of the protocephalon. The protocerebrum also moves dorsad with ectoderm forming the dorsal side of the future
head. Projections of the nerve cells begin to form the distinct nerve fibers (Fig. 5).
In the 78-hour old embryo, the boundary between the head and the thorax becomes distinct. The deutocerebrum and the tritocerebrum go ahead and the former occupies its position under the ventral side of the protocerebrum and contacts with it. The
latter connects with the posterior end of the deutocerebrum and lies at both sides of thestomodaeum. In the peripheral region of the brain many neuroblasts are arranged andwithin the brain several mitotic figures are observed. In the central region the nerve fibers assemble to form the neuropile.
At 84 hours after oviposition, the embryo shortens its length and the mouth parts
begin to be formed. The protocerebrum migrates posteriorly along the dorsal side of
the head and the deutocerebrum unites with the lower part of the protocerebrum infront of the opening of the stomodaeum. The tritocerebrum occupies its position behindthe deutocerebrum. Then each cerebrum continues migration to form the brain on theupper part of the oesophagus. The distinct space appears between the brain and alayer of the ectoderm cells, which secrete later cuticle composing the future head capsule (Fig. 6). Figure 7 represents diagrammatically the process of migration of eachcerebrum with progress of the head formation.
At 96 hours after oviposition, the labial appendages unite to form a single labium
and the abdominal segments separate slightly from the egg surface to curve into theyolk.
At 120 hours after oviposition, the embryo completes blastokinesis and the appendages of the gnathal segments assemble around the opening of the stomodaeum. The
brain is covered by a layer of cells with a flat nucleus, the perineurium, and the glial
cells with dark-stained nucleus appear at the boundary between the perineurium andthe outer layer of nerve cells and among the nerve cells themselves.
Figures 8 and 9 are electron micrographs representing the periphery of the brain

Brain formation in normal and embryonic lethal Bombyx embryos

243

Fig. 5. Cross section of the head in 72-hour old embryo, showing appearance of nerve fibers. Ir, labrum; nb, neuroblast; nf, nerve fiber; prot, protocerebrum.

ehFig. 6. Cross section of the head in 84-hour old embryo. br, brain; eh, epidermis of head;np, neuropile; stom, stomodaeum.
and the neuropile, respectively. The surface of the brain is covered with a layer of cells,
the perineurium, the cell of which is flat and contains autosomes. The periphery of thebrain is occupied with the abundant nerve cells, among which the glial cells intervene.
The neuropile is constituted with the abundant nerve fibers intermingled complicatedly.

244

s. Hashimoto and K. Miya

B

thl

Fig. 7.

oDiagrammatic figures representing the process of migration of proto-, deuto- andtritocerebrum with progress of head formation. A. 48 hours, B. 72 hours, C. 84
hours, D. 90 hours after oviposition. ant, antenna; deut, deutocerebrum; Ib,
labium; lr, labrum; md, mandible; mx, maxilla; prot, protocerebrum; stom, stomodaeum; thl, thoracic leg; trit, tritocerebrum.

e. Ultrastructure of the completed brain
Figures 10 and 11 are electron micrographs representing the peripheral and central part of the brain, respectively. As mentioned above, the brain of Bombyx also consists of the right and left hemispheres, each of which is complex body of the proto-,
deuto- and tritocerebrum. The perineurium secretes a non-cellular membrane, the neurallamella. The neural lamella is a felt-like membrane similarly to the basement membrane of other tissues. The cell of the perineurium is flat and its cytoplasm is filledwith many small lipid droplets, mitochondria, and rER parallel to the surface of thebrain.

Brain formation in normal and embryonic lethal Bombyx embryos

245

The peripheral region of the brain is occupied with abundant nerve cells, the cytoplasm of which is filled with tubular rER. Some nerve cells represent several different
features from the ordinary ones, that is, they contain well-developed vesicular rER and
Golgi bodies, larger mitochondria, and groups of small secretion granules (Fig. 12).
These cells seem to be neurosecretory cells. Among the nerve cells numerous glial cells
intervene and their cytoplasmic processes enclose the nerve cells. In the cytoplasmic
processes there are abundant pigment granules. The cytoplasm of the glial cells are denser than that of the nerve cells, because of rich free ribosomes, and contains many
vesicles.
The central region, the neuropile, is filled with numerous axons, which containmitochondria and longitudinal neurotubules. The cytoplasmic processes enclose thebundles ofaxons and also tracheoles intervene among them.

2. Formation of the brain in Dusl Dus embryoa. Characteristics of "star-spots duplication"
As reported already, "star-spots duplication" gene (symbol Dus) is located at 3.9
on the tenth linkage group (Doira et al., 1984) and Dusl+ larvae show additional starspots on the sixth abdominal segment (Fig. 13; Miya and Hashimoto, 1977). Furthermore, this mutant is a recessive embryonic lethal and Dusl Dus embryos indicate various conspicuous features: failure of formation of the head capsule and the midgut
epithelium, hypertrophy of the central nervous system including brain, supernumerarylegs in the seventh abdominal segment, supernumerary spiracles in the ninth abdominal segment, and so on (Fig. 14). The homozygous embryos develop until the setae
formation stage, but they cannot carry out blastokinesis because of incompleteness ofthe dorsal closure.
Among the above characteristics, the most conspicuous one is hypertrophy of thecentral nervous system. Figures 15 and 16 represent the frontal section of the head andthe longitudinal section of the abdomen, respectively. The brain is exposed completely
without enclosing by the head capsule, and the ventral nerve cord is connected witheach other to form a tube-like organ.
b. Segregation of the neuroblasts in the protocephalon
When the Dusl + female is mated with the Dusl + male, it is expected in the laid
egg batch that Dusl Dus embryos will occupy about one forth of all eggs. Until the early
appendage formation stage, however, any distinct difference could not be observed between the normal and Dusl Dus embryos even by the histological examination. Therefore, the first segregation of the neuroblasts in the protocephalon and division into
three groups seem to occur normally.

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S. Hashimoto and K. Miya

Figs. 8, 9. Electron micrographs representing periphery of the brain (Fig. 8) and theneuropile (Fig. 9) in 120-hour old embryo. as, autosome; Gc, glial cell; Nc, nerve
cell; Np, neuropile; Pn, perineurium. Scales: 1 f1 m.

Brain formation in normal and embryonic lethal Bombyx embryos

247

Figs. 10, 1l. Electron micrographs representing peripheral (Fig. 10) and central region(Fig. 11) of the brain at body pigmentation stage. Gc, glial cell; Nc, nerve cell;
Np, neuropile; Pn, perineurium. Scales: 1 fL m.

248

S. Hashimoto and K. Miyac. Hypertrophy of the brain
As mentioned above, segregation of the neuroblasts in the neurogenic region of the
protocephalon is not carried out at the same time at the definite developmental stage,
but continues for several stages. Hence during this period the new neuroblasts not only
segregate in the neurogenic region and produce the daughter cells by unequal divisions,but also these daughter cells continue to divide, resulting in increase of the number.
In Dus/Dus embryos, increase of the neuroblasts might be produced by normal processof segregation, though the exact number could not be calculated (Fig. 17), while theequal divisions of the ganglion mother cells occur more than in the normal embryos,and consequently the size of the brain becomes larger than in the normal (Fig. 18).
These results are summarized in Table 1; in the 72-hour old Dus/ Dus embryo the sizeof the brain is twice as large as the normal embryo and the mitotic figures are observed
three times as many as in the normal brain. Because of hypertrophy of the brain, thelateral epidermis could not spread over the brain, resulting in failure of the head capsule in spite of normal formation of the labral and antennal appendages in the head.

Discussion
The embryonic development of the central nervous system in insects begins at thestage of differentiation of the neuroblasts. The neuroblasts, anlagen of the ganglia, aresegregated from the ectoderm on each side of the ventral midline of the embryo behindthe stomodaeum, diverging into broader area over the protocephalon. The neurogenic
region, from which the neuroblasts are segregated, is traced back the position to a longitudinal strip of blastoderm cells located between the anlagen of the mesoderm and of
the ventral epidermis, for example, in Drosophila (Poulson, 1950) and in Tenebrio (seeAnderson, 1972b; Sander, 1976).
Also in Bombyx a fate map concerning to the neurogenic region was made by
Takami (1946) with cauterization experiment. According to him, the presumptive
neurogenic region existed as a pair of slender strips in both sides of the presumptive
mesoderm region. A more detailed similar fate map was given by Katsuki et al. (1980)
through the analysis of mosaic larvae induced by super-cooling method.
As to the embryonic development of the brain, the similar pattern of the differentiation has been described in Holometabola as well as in Hemimetabola, namely
the neuroblasts are segregated from the ectoderm of the protocephalon and then areorganized into three pairs of groups to form the anlagen of the proto-, deuto- and tritocerebrum, which unite later to produce a compound body, the brain (see Johannsen
and Butt, 1941; Ando, 1970, 1978; Anderson, 1972a, b).
Formation of the brain in Bombyx embryo proceeds almost similarly as in the otherlepidopterans, but several differences are known in given species. In Pieris (Eastham,1930) the areas of the proto-, deuto- and tritocerebrum were distinguishable at the early stage of the neuroblast segregation the same as in Bombyx, while in Antheraea (Saito,1937) the neuroblasts distributed evenly in the wide-spread area of the protocephalon,and so distinction of each cerebrum was impossible until the later stage.

Brain formation in normal and embryonic lethal Bombyx embryos

Fig. 12.

249

Electron micrograph representing a neurosecretory cell at body pigmentation
stage. mit, mitochondria; nu , nucleus; rer, rough-surfaced endoplasmic reticulum;sg, secretion granule. Scale: 1 J.L m.

14
Fig. 13. Dorsal view of fifth ins tar Dus/ + larva (from Miya and Hashimoto, 1977).
Fig. 14. Light micrograph representing Dus/ Dus embryo at setae formation stage (fromMiya and Hashimoto, 1977). Br, brain.

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S. Hashimoto and K. Miya

Recently Hartenstein and Campos-Ortega (1984) studied in detail on spatial andtemporal pattern of the segregation of the neuroblasts in Drosophila. According to them,
the neurogenic ectoderm contains neural precursors intermingled with epidermal precursors, extending from the midline up to the primordia of tracheal tree along the germ
band and latero-dorsally in the procephalic lobe and these two regions are separated
from each other by the cephalic furrow, differently from the fate map made by Pou1son(1950). Germ band neuroblasts segregate from the neurogenic ectoderm during aperiod of several hours according to characteristic spatial and temporal patterns, on theother hand protocephalic neuroblasts are segregated continuously during a definite
period.
Furthermore, in Drosophila several mutants affecting the early neurogenesis havebeen known and their function are being analysed vigorously (Pou1son, 1937, 1940;
Lehmann et al., 1981; Jimenez and Campos-Ortega, 1982; Lehmann et al., 1983; Campos-Ortega, 1983; Hartenstein and Campos-Ortega, 1984). Summarizing their assumption, all cells of the neurogenic ectoderm develop into neuroblasts initially, then suppressed in order to allow to become epidermal cells. Therefore, these mutants misroutethe ectoderm cells in wild type that would have developed as epidermal cell precursors
into the neural developmental pathway, resulting in leading to a conspicuous hypertrophy of the central nervous system and to a lack of several epidermal derivatives.
In Bombyx spatial and temporal segregation of the neuroblasts in the neurogenic
region could not be observed in detail, but in formation of the brain the following phenomena were confirmed: (1) segregation of the neuroblasts did not occur at the sametime, but continuously during a definite period, (2) the newly formed neuroblast soon
begins to divide unequally to produce a smaller daughter cell, the ganglion mother cell,(3) these neuroblasts and the daughter cells are organized to three pairs of groups, theanlagen of the proto-, deuto- and tritocerebrum, and (4) during migration and union ofeach cerebrum, the neuroblasts and the ganglion mother cells divide actively to produce abundant nerve cells, which elongate cytoplasmic processes to form axons.
The embryonic lethal mutant Dus affects the early neurogenesis to induce hypertrophy of the central nervous system similarly as the mutants in Drosophila. As to
hypertrophy of the brain and the related failure of the head capsule formation, the following causes are assumed: (1) abnormal segregation of the neuroblasts in the protocephalon, accompanying with decrease of the epidermal precursors the same as in
Drosophila, (2) abnormal divisions of the ganglion mother cells, resulting in productionof more numerous nerve cells than in the normal brain, and (3) abnormality by both
two causes. As shown in Figs. 5 and 17, there is no remarkable difference in number ofthe neuroblasts in the brain between the normal and the Dus embryos, and the factsuggests impossibility of the first cause the same as in Drosophila, while Table 1 indicates that abnormal proliferation of the ganglion mother cells might be the principal
cause. The tube-like ventral nerve cord in Dus embryo is not exposed usually, as shownin Fig. 16. Therefore, from these results abnormality of the head including the brain in
Dus embryo might be induced chiefly by abnormal proliferation of the ganglion mothercells.

Brain formation in normal and embryonic lethal Bombyx embryos

251

,
15
Figs. 15, 16. Light micrographs representing frontal section of the head (Fig. 15) andlongitudinal section of the abdomen (Fig. 16) of Dus/ Dus embryos. Ag6-8, sixth
to eighth abdominal ganglion; Br, brain; Stom, stomodaeum. X 160.
Table 1.

Size of the brain and number of mitotic figures in the normal and Dus/ Dusembryos.

Hours
after Oviposition
72

84

96

Phenotype

Relative
Size

Number of
Mitotic Figures*

+

1

16.5

Dus

1 .8

52.0

+

1.1

24.8

Dus

2.1

62.7

+

1.2

12.5

Dus

2.2

46.5

(* Number in hemisphere of the brain)

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S. Hashimoto and K. Miya

Ir

stom
Fig. 17. Cross section of the head in 72-hours old Dus/Dus embryo. am, amnion; lr, labrum; nb, neuroblast; nf, nerve fiber; stom, stomodaeum.

Ir

stom
Fig. 18.

Cross section of the head in 84-hours old Dus/Dus embryo, showing lack of
epidermis. br, brain; lr, labrum; np, neuropile; stom, stomodaeum.

Brain formation in normal and embryonic lethal Bombyx embryos

253

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S. Hashimoto and K. Miya

Authors' addresses: MAg. S. Hashimoto
Agricultural Administration Department, I wate Prefectural Government,
Uchimaru 10-1, Morioka, Iwate 020,
Japan
Dr. K. Miya, Prof. Emeritus (Iwate
U niversi ty)
Yamagishi 3-11-10, Mrioka, I wate 020,
Japan