The zygotic mutant tailless affects the anterior and posterior ectodermal regions of the Drosophila embryo

DEVELOPMENTAL

113,64-76

BIOLOGY

(1986)

The Zygotic Mutant Tailless Affects the Anterior and Posterior Ectodermal Regions of the Drosophila Embryo TERESA

R.

STRECKER,*

KRITAYA

Department

of Biology,

Received

November

KONGSUWAN, University

JUDITH of California,

8, 19X4; accepted

A.

LENGYEL,

Los Angeles,

in re?iiaed form

AND Califmia

July

JOHN

R.

MERRIAM

90024

19, 1985

The recessive zygotic lethal mutation tailless maps to region lOOA5,6-B1,2 at the tip of the right arm of chromosome 3, and results in shortened pharyngeal ridges in the head skeleton of the mature embryo and the elimination of the eighth abdominal segment and telson. Although they have a normal body length, tailless embryos have a smaller number of abdominal segments, some of which are larger than normal. The mutant phenotype is seen as early as 8 hr postfertilization, when tailless embryos are observed to have fewer tracheal pits than wildtype. At 9 hr, tailless embryos appear to be missing segments A8, AS, and A10 and have an abnormal clypeolabrum, optic lobes, and procephalic lobe. Segments A4, A5, A6, and A7 appear larger in tailless embryos than wildtype at this stage. The tailless mutation, although affecting anterior and posterior ectodermal structures in the mature embryo, does not affect the formation of pole cells, the posterior midgut, or the proctodeum, which arise from the most posterior region of the embryo. The mutation does result, however, in the failure of Malpighian tubule formation. Consistent with its effect on ectodermal segments, tailless leads to a reduction in the number of segmented, paired ganglia in the ventral nerve cord as well as to an abrupt alteration in the posterior region of the tracheal system. The role the taillessgene may play in the formation of the most anterior and posterior regions of the embryo’s ectodermal body plan is discussed. o 1986 Academic press, I~C. INTRODUCTION

each thoracic and abdominal segment (Ntisslein-Volhard and Wieschaus, 1980). A number of zygotic mutations affecting the embryonic segment pattern, however, do not fall within the gap, pair-rule, and segment polarity categories. One of these mutations, tailless, is unusual in its phenotype since it affects only the most anterior and posterior regions of the mature embryo (Jiirgens et al., 1984). Mature tailless embryos have an abnormal cephalopharyngeal skeleton and are missing the eighth abdominal segment and telson. The tailless mutant phenotype thus defines an additional category of pattern mutants representing genes involved in the development of the anterior and posterior ends of the segmented ectoderm in the embryo. We report here a detailed analysis of the tailless mutant phenotype. In addition to further mapping the tailless gene, we have studied the mutant phenotype at several stages during embryogenesis using optical and scanning electron microscopy. Examination of both whole and sectioned embryos was used to further clarify the internal structures affected by the tailless mutation. The results of this analysis point to an early role of the normal tailless gene in establishing the most anterior and posterior ectodermal regions of the embryonic body plan.

The study of zygotic lethal mutations which affect the segmentation pattern of the Drosophila embryo is an effective means of identifying the role certain genes play in embryonic pattern formation. An alteration in the number and/or polarity of segments in the mature Drosophila embryo has been the basis for selecting mutations in genes involved in segment number and pattern formation (Ntisslein-Volhard et al., 1984; Jtirgens et al., 1984; Wieschaus et al., 198413). The mature Drosophila embryo (1st instar larva) consists of a pseudocephalon, three thoracic, eight abdominal segments, and a terminal telson. The cuticularized segments of the hypoderm arise from embryonic segments which are determined by the cellular blastoderm stage (Sander, 1960; Chan and Gehring, 1971; Herth and Sander, 1973; Schubiger, 1976; Simcox and Sang, 1983). Mutations which alter the normal segment pattern identify genes required for segment formation. Furthermore, the phenotypes of such mutations shed light upon the steps involved in establishing the body plan of the embryo. Zygotic mutations which alter the number and pattern of cuticularized segments have been placed into three groups according to their mutant phenotype: gap mutants which affect a large region of the thorax and abdomen, pair-rule mutants which affect pairs of segments, and segment polarity mutants which affect a portion of * To whom 0012-1606/86 Copyright All rights

correspondence

should

$3.00

0 1986 by Academic Press, Inc. of reproduction in any form reserved.

MATERIALS

AND

METHODS

Tailless mutant alleles. The original tailless (tll’) allele was isolated and kindly donated to us by C. Niisslein-

be addressed. 64

STRECKER

ET AL.

Volhard (Jiirgens et al., 1984). This particular allele was isolated in an EMS mutagenesis screen on a 3rd chromosome marked with ebony (e) and scarlet (st) and is kept in a stock balanced by TM3, which is marked with Stubble (Sb) and ebony (e). The chromosome also carries another lethal mutation which we have mapped to 99F9,10-lOOA by deletion mapping (data not shown). The tll’ allele was subsequently used in an X-ray mutagenesis screen for additional tll alleles (Strecker and Merriam, in preparation). In the first 4500 chromosomes screened, three new tailless alleles (tlla, tll”, and tile) were recovered (see Table 1). Each X-ray induced allele was isolated on a 3rd chromosome marked with claret (ca) and is kept in a stock balanced by In(3R)C which is marked with Stubble (Sb), Tubby (Tb), cardinal (cd), and claret (ca). During our genetic study of tailless we recovered what appears to be a spontaneous tll allele, tll’ (Table l), which was also isolated on a 3rd chromosome marked with claret (co) and is kept in a stock balanced by In(3R)C (Strecker and Merriam, in preparation). During our characterization of the tll mutant phenotype the (Y;3) translocation, A113 (Lindsley et al., 1972), was used to uncover the tailless mutation. Segregants of the All3 translocation yield larvae bearing a terminal deficiency from IOOA. Gene and chromosome symbols are described by Lindsley and Grell (1968). Preparation and examination of mature embryonic cuticle. Eggs were collected for l-2 hr at 25°C from each of the tailless stocks using specially prepared agar plates (Niisslein-Volhard, 1977) dotted with a moist yeast paste. Eggs were aged 24 hr at 25°C at which time the ratio of hatched to unhatched embryos was recorded. All unhatched embryos were prepared for examination of the larval cuticle. The chorion of these embryos was removed

THE

Origin

Tailless

TABLE 1 ALLELES DESCRIBED

Allele

Cytology

EMS

tll’

Normal

X Ray

fll”

Norma1

X Ray

tr

Norma1

X Ray

tll”

Df(3R)

Spontaneous

tlP

h(3R)C

IWA~,%CI

85FlO-86Al;

lOOA5,6-B1,2

IN THIS

PAPER

Lethal embryonic phenotype (tll”/tZl’) A8 and telson absent; shortened pharyngeal ridges (tP/tll’) A8 and telson absent; shortened pharyngeal ridges (tll”/tll’) A8 and telson absent; shortened pharyngeal ridges (tZl”/tll’) A8 and telson absent; shortened pharyngeal ridges (tlle/tll’; tllz/tl12) T&on missing; normal A8 denticle belt and pharyngeal ridges

Tu illess

embryos

65

by rolling them on double stick tape. The dechorionated embryos were transferred to water, freed of the vitelline membrane with tungsten needles, and then placed in fixative (acetic acid:glycerol, 4:l) at 60°C overnight. Embryos were then mounted on slides using Hoyer’s medium (van der Meer, 1977) and left overnight at 45°C. Cuticle preparations of unhatched mature embryos were examined using light microscopy and phase contrast optics. The head skeleton and associated head structures as well as the thoracic and abdominal regions of each embryo were examined in detail. While examining the head region, attention was focused on the following head structures: cirri, antenno-maxillary complex of sense organs, mouth hooks, median (or hatching) tooth, labial sense organs, H-piece, pharyngeal ridges, and the floor of the hypopharynx (Fig. 8A; Lohs-Schardin et al., 1979; Turner and Mahowald, 1979; Struhl, 1983). The length of certain elements of the head skeleton was measured using an ocular micrometer. Tailless embryos were also examined for any evidence of structures derived from segments A8, A9, and AlO, namely, the eighth abdominal denticle belt, anal pads and anus, anal tuft, dorsal posterior spiracles, filzkiirper, and the anal sense organs (Lohs-Schardin et al., 1979; Turner and Mahowald, 1979; Struhl, 1983). Measurement qf total bodlg and segment length in mature embryos. In addition to examining the cuticle of mature tailless embryos, we measured the total body length of each embryo using an ocular micrometer. Only embryos which did not possess any folds or other artifacts of the mounting process were measured. Total body length was defined as beginning at the level of the mouth hooks and ending at the posterior end of the eighth abdominal segment and telson. The dorsal posterior spiracles were not included in the total body measurement since they project more dorsally than posteriorly and their position is variable in each whole mount preparation. The average fraction that each body segment contributes to the total body length of tailless and wildtype embryos was determined as follows. The length of each body segment was measured using an ocular micrometer. The pseudocephalon was interpreted as beginning at the anterior tip of the mouth hooks and terminating at the most anterior end of the first thoracic setal belt. The thoracic and abdominal segments were each measured as the distance from the most anterior row of denticles of a particular segment to the most anterior row of dentitles of the next segment. For each embryo measured, individual segment lengths were converted to a percentage of the total body length of the mature embryo. Results from large numbers of embryos were then averaged to determine the average fraction each body segment contributes to the embryo’s total body length.

66

DEVELOPMENTAL BIOLOGY

Preparation of embryos for scanning electron microscopy (SEM). Eggs were collected from fly stocks as described above and were aged at 25°C. When embryos had reached the desired developmental stage, their chorions were removed by rolling on the surface of doublestick Scotch tape. The vitelline membrane was then made permeable to fixative by immersing the embryos in icecold glutaraldehyde-saturated heptane (Mahowald and Turner, 1978). After 1 min, the heptane solution was removed and evaporated, and the embryos were immediately transferred to a slide well lined with electrician’s tape (Zalokar, 1977) containing ice-cold trialdehyde fixative (Kalt and Tandler, 1971). Embryos were stuck to the surface of the tape and were maintained at 4°C from 30 min to as long as overnight. Embryos were then rolled out of their vitelline membrane which remained stuck to the tape. Fixation of embryos was allowed to reach completion overnight, after which the embryos were post-fixed and dehydrated as described by Mahowald and Turner (1978). Embryos were dried by the critical point technique using a Polaron (E3000 series II) critical point dryer, in CO2 with absolute ethanol as the intermediate fluid. Following the drying procedure, embryos were placed on stubs which had been covered by double-stick tape. They were then sputter-coated with a 400 angstrom layer of gold (Polaron ISI-5400) and were viewed with an IS1 DS-130 SEM operated at 20 kV. Images were photographed using Polaroid Type 52 Land film. Acetylcholinesterase staining of ventrd nerve cord. To visualize the ventral nervous system, ll- to 12-hr-old embryos from the tlP/k(3R)C stock were dechorionated and briefly treated with ice-cold heptane solution. The vitelline membrane was removed as described above and embryos were fixed and stained using the technique of Wakimoto et al. (1984). Stained embryos were mounted in halocarbon oil, and photographed using bright field optics. Serial sectioning of embryos. Embryos were collected from the tll’/TM3 stock as described above and aged to 13-15 hr post-fertilization at 25°C. The embryos were prepared for fixation in a trialdehyde fixative (Kalt and Tandler, 1971) according to the protocol used for SEM described above. Following overnight fixation, embryos were dehydrated through a graded series of alcohols ranging from 30% through absolute and then transferred to propylene oxide. This was followed by infiltration in a 50:50 mixture of propylene oxide and Medcast epoxy (Ted Pella, Inc.) for 2 hr, followed by overnight incubation in Medcast epoxy. Embryos were embedded and sectioned at 4 pm using a LKB Ultrotome V Microtome. Sections were stained with 1% toluidine blue in water, overnight followed by destaining in 70% ethanol, and

VOLUME 113,1986

were photographed optics.

using bright field and phase contrast

RESULTS

The tailless locus was originally mapped to region lOOA-B (Jtirgens et al., 1984) based on the observation that this locus is uncovered by DfsA113 and J55. We have confirmed this localization by isolating an X-rayinduced tll allele, tll”, which is a deficiency for region lOOA1,2-100Cl (Strecker and Merriam, in preparation). We have also identified a spontaneous, hypomorphic tailless allele, tll’ (In(3R)C 85FlO-86Al; lOOA5,6-B1,2), with a distal breakpoint allowing the more precise localization of tailless to lOOA5,6-B1,2 (Strecker and Merriam, in preparation). The tailless mutant phenotype in the cuticle of the mature embryo has been described as an abnormal head skeleton and the absence of the eighth abdominal segment and the telson (Jtirgens et al., 1984). As described below, our observations of tll’/Df(3R)tll” embryos confirm that the absence of the eighth abdominal denticle belt and telson structures is a result of the tailless mutation (Fig. 1B). Furthermore, we show that the abnormal head skeleton is due specifically to the shortened length of the pharyngeal ridges in the cephalopharyngeal skeleton (Fig. lB, arrow; Figs. 8B, C). All other head skeletal structures are normal in tailless embryos. The amorphic tailless mutant phenotype is defined as that observed in tll”/Df(3R)tll’ embryos, which is the absence of the eighth abdominal denticle belt and telson, and shortened pharyngeal ridges (Fig. 1B). This phenotype is also consistently observed in tll’/tll” and tll’/ tll” embryos. Furthermore, we are able to observe a less extreme, hypomorphic tailless phenotype due to the spontaneous tailless allele, tll’. This hypomorphic tll phenotype is not fully penetrant in homozygotes or in the transheterozygote, tl11/tl12. Embryos with either of these genotypes consistently lack any telson structures, but have normal pharyngeal ridges and the eighth abdominal denticle belt (Fig. 1C). Eflects of Tailless on th>ePosterior Segmentation

Ectodermal

Cuticular structures. Effects of the tailless mutation on the thoracic and abdominal segmentation pattern in mature embryos are confined to the absence of the most posterior abdominal segments. Tailless embryos have the normal thoracic and abdominal ventral setal belts and dorsal hairs characteristic of segments Tl through A7. These embryos are missing, however, the structures of

PC Tl T2 T3 Al A2 A3 A4 A5 A6 A7 A8

FIG 1. (Comparison of tailless and wildtype cuticular phenotypes in mature embryos, 24 hr post-fertilization, using darkfield microscopy. (A) Wildt yw, ventral view (25X). Note the elongated pharyngeal ridges (arrow). (B) Amorphic fll phenotype (tll’/tll”), ventral view (25X). Note the at xen ce of the eighth abdominal denticle belt and the telson as well as the shortened pharyngeal ridges (arrow). (C) Hypomorphic t/l pheno 'tYP( e (tll’/tlP), lateral view (25X). Note the presence of the eighth abdominal denticle belt and the absence of the telson. At the most poster .ior tip of these embryos is a cluster of dorsal hairs. PC = pseudocephalon; Tl-T3 = thoracic segments; Al-A8 = abdominal segments; Te = telson

the larval cuticle derived from the embryonic segments A8, A9, and AlO. These structures are the denticle belt and dorsal hairs of the eighth abdominal segment, and the structures which comprise the telson: the anus, anal tuft, anal pads, anal sense organs, filzkorper, and spiracles. Although lacking a telson, tailless embryos retain a remnant of the proctodeum; this is evident as a slit in the cuticle on the dorsal side of A7. In wildtype embryos, the proctodeal opening becomes the anal opening which lies on the ventral side of the telson just posterior to the eighth abdominal denticle belt. Se.qmcwt .formation. Consistent with the mutant cutitular phenotype observed in mature tailless embryos at 24 hr, there is a characteristic difference between

and wildtype embryos at 9 hr post-fertilization as observed by SEM. While wildtype embryos possess thirteen (three thoracic and ten abdominal) body segments (Figs. 2C, E), tailless embryos were consistently observed to have only 10 body segments (Figs. 2D, F). Due to the absence of these segments and their structural derivatives in the mature tailless embryo, the segments absent in tailless embryos are interpreted as representing A8, A9, and AlO. Despite the absence of the presumed terminal segments, tailless embryos have a clearly formed proctodeal opening at the dorsal posterior tip of the most caudal body segment (Fig. 2F, arrow). This is consistent with the normal formation of the terminal portion of the fate

tailless

FIG. 2. A comparison of the segmentation pattern of tuille.~ and wildtype embryos. Scanning electron micrographs of embryos, 8 hr postfertilization (lateral view): (A) wildtype (274X); (B) tuikw, tll’/tll’ (275X). Note the fewer, more widely spaced trachea1 pits (arrow heads) on the dorsal side of the tailless embryo. Scanning electron micrographs of embryos, 9.5 hr post-fertilization (lateral view): (C) wildtype (274X); 68

STRECKER

ET AL.

T2

A7

FIG. 3. Acetylcholinesterase-stained preparations of (A) wildtype (40X) and (B) tailless tll”/tll” (40X) embryos, approximately llL12 hr post-fertilization showing the segmented ventral nerve cord. Eleven segmented ganglia, three thoracic (Tl-T3) and eight abdominal (AlA8) are observed in wildtype. A reduced segmented ganglion corresponding to A9 is observed at the most posterior end of the ventral nerve cord. In both wt and t/l embryos the first segmented ganglion corresponding to Tl is obscured in these photographs by the brain. Tailless embryos were identified by the absence of posterior filzkorper. These embryos consistently exhibited a reduction in the number of segmented ganglia. Only seven abdominal ganglia are observed in failless embryos, with the seventh appearing abnormal in morphology.

plan that gives rise to the posterior midgut and the proctodeum as well as the observation of a small dorsal opening at the posterior of mature tailless embryos. Ventral yzerve co&. The absence of the hypodermal segments AS, A9, and A10 in the 9-hr tailless embryo suggests that tailless may affect other ectodermal derivatives in the embryo, namely the segmented ventral nerve cord. Acetylcholinesterase staining of whole embryos reveals that there are also fewer segmented ganglia in the ventral nerve cord of tailless embryos at ll(tailless embryos were distin12 hr post-fertilization guished from wildtype on the basis that the former do not have a telson which can be easily recognized in wildtype embryos in the light microscope) (Fig. 3). The segmented nature of the nerve cord can be observed at this stage in embryogenesis, prior to the condensation of the central nervous system. Eleven segmented ganglia, three

(D) tailless, tZl’/tll’ (274X). Note the fewer, but larger heads) at posterior of 9 hr embryos in (E) wildtype embryonic abdominal segments; D, dorsal; V, ventral.

abdominal (450X) and

Toilless

69

wdryos

thoracic and eight abdominal, are observed in wildtype embryos (Fig. 3A). A reduced segmented ganglion attributed to A9 is observed at the most posterior end of the ventral nerve cord. Tailless embryos consistently exhibited a reduction in the number of segmented ganglia (Fig. 3B). Only seven abdominal ganglia are observed in tailless embryos, and the seventh abdominal ganglion appears abnormal in morphology. Formation of tracheal pits. An alteration in posterior segmentation resulting from the tll mutation is first observed during tracheal pit formation at 8 hr post-fertilization (Figs. 2A, B). Seven out of 39 embryos were observed by SEM to have a consistent reduction in the number of tracheal pits distributed along the extended germ band. While wildtype embryos have 10 clearly visible tracheal pits (Fig. 2A; Fullilove and Jacobson, 1978), these embryos had only nine (Fig. 2B) and were assumed to be tailless. The tracheal pits on these embryos were evenly distributed over the extended germ band and appeared more widely spaced apart on the dorsal side of the embryo than are the pits in normal embryos. Since the 10 tracheal pits in wildtype embryos are located at segments T2, T3, and Al-S, it is reasonable to conclude that the missing tracheal pit in tailless embryos is associated with the missing segment AS. Tracheal system. The absence of the eighth abdominal tracheal pit in S-hr tailless embryos suggests that the terminal branch of the tracheal system should be missing in mature tailless embryos. This hypothesis was tested in a comparison between the tracheal system of and wildtype embryos. Embryos mature tailless (tP/tlP) mounted on slides in water were examined using bright field microscopy. The tailless embryos clearly differ from wildtype in the posterior region of the tracheal system (Fig. 4). The main tracheal trunk ends abruptly in mature tailless embryos at the level of A6, and thus does not open to the exterior at the posterior tip of the embryo. Every segment present in tailless embryos is associated with a branch from the main trunk. The branch which is associated with A7 in tailless embryos originates from the posterior end of the shortened tracheal trunk (Fig. 4B, arrow). Thus the elements of the tracheal system normally associated with A8 and the telson, namely the eighth abdominal branch from the tracheal trunk and the extension of this trunk into the filzkorper, are absent in tailless embryos. These results are consistent with the observation of seven abdominal tracheal pits in tailless embryos at 8 hr.

segments in the tailless embryo. Dorsal view (F) tailless (449X). H, head; Tl-T3, embryonic

of proctodeal opening (arrow thoracic segments; Al-AlO,

70

DEVELOPMENTAL

BIOLOGY

113, 1986

= 30) for wildtype. Mature tailless embryos are of normal overall body length apparently due to the increase in the length of their abdominal segments (Fig. 5). This increase in segment length is noticed as anteriorly as A2, and increases posteriorly, being most pronounced in segments A4, A5, A6, and A7 of tailless embryos. Consistent with these observations on the cuticle, SEM observations of 9-hr tailless embryos show that they are also of normal body length when compared to wildtype embryos. These tll embryos exhibit a measurable increase in abdominal segment length beginning at the level of A2 (Figs. 2C, D). This increase in the segment length seen in SEM preparations parallels that found in the cuticle of the mature mutant embryos. The increase in segment length is just barely detectable in the more anterior affected abdominal segments (AZ and A3), but becomes quite significant in the more posterior segments (A4 < A5 < A6 < A7) (Fig. 2D). The normal body length observed in tailless embryos, despite the absence of segments A8-10, may arise as a result of a change in cell fate. In other words, cells which normally form the A8-10 posterior segments, rather than dying, may be reprogrammed to contribute to fewer, but larger abdominal segments in tailless em-

A

FIG. 4. The tracheal system in tailless and wildtype embryos was compared using mature embryos mounted in water on a slide and viewed with bright field optics. (A) Frontal view of wildtype embryo at hatching stage (41X). Note the termination of the tracheal trunk in the form of the posterior filzkiirper, the segmented tracheal branches associated with each thoracic and abdominal segment, and Malpighian tubules (arrow). (B) Lateral view of tailless (tll’/tll’) embryo, 24 hr post-fertilization (41X). Note the abrupt shortening of the tracheal trunk (arrow), the seven abdominal denticle belts and the seven abdominal branches of the tracheal system. The seventh branch arises from the very posterior tip of the shortened tracheal trunk (arrow), and descends into the seventh abdominal segment.

Tailless Results in Lengthening Segmentation Pattern

VOLUME

of the Abdominal

Since tailless embryos lack the structures derived from the three most posterior abdominal segments, they might be expected to have a shortened body length. We found, however, that the total body length of mature tailless embryos does not differ significantly from that of wildtype embryos. The average body length of a sample of tailless embryos was 1.08 -+ 0.16 mm (n = 58), compared to an average length of 1.07 + 0.06 mm (n

f F

14_

s <

12_

t

lo-

"0 I-

a-

PC Tl

T2

T3

Al Body

A2

A3

A4

A5

A6

A7

A8

Segment

FIG. 5. The average proportion each body segment contributes to the total body length of tailless (open circles) versus wildtype (closed circles) embryos. The lengths of the pseudocephalon, and the thoracic and abdominal segments of thirty tZl’/DfAU3 embryos which exhibit the amorphic tll phenotype and thirty wildtype (Oregon-R) were measured. The raw measurements were divided by the total body length for each embryo to yield the percent each segment contributes to the embryo’s total body length. The average proportion of each segment’s contribution to the total body length is graphically illustrated with the standard deviation represented as a vertical bar.

STRECKER

ET AL.

FIG. 6. Scanning electron micrograph comparison of the fifth abdominal segment at 9.5 hr post-fertilization in (A) wildtype versus (B) toilless (tlZ’/tll’) embryo (both 1720x). The line drawn across the abdominal segment was used as an aid in counting the number of cells which span the length (anterior to posterior) of each segment. In this particular comparison, A5 in the tailless embryo was observed to have 17% more cells along the white line when compared to the wildtype embryo.

bryos. To investigate this possibility, we counted the number of cells in each thoracic and abdominal segment from scanning electron micrographs (Fig. 6 and Table 2). The number of cells that intersect a line drawn from anterior to posterior, across each body segment, is compared in Table 2 for tailless and wildtype embryos. As illustrated in a comparison of scanning electron micrographs of A5 from a wildtype and a tailless embryo (Fig. 6), segments A5, A6, and A7 display a marked increase in cell number in tailless versus wildtype embryos.

Tailless

Endodermal

Derivatives

71

observed with the light microscope from the blastoderm to the hatching stage. By 24 hr, eight of these embryos (out of a total of 28) developed a tailless phenotype. All of these tailless embryos were recorded as having had clearly distinguishable pole cells. No differences in gastrulation, posterior midgut invagination or germband extension could be distinguished between these eight mutant embryos and their normal sibs. In addition, we did not observe a difference in the extent of germband elongation between wt and tll embryos at 8 hr. To confirm this observation, the dorsal extension of the germband in eight developing embryos from the tl11/TM3 stock was measured using an ocular micrometer. Two of these eight embryos were later identified as tailless when they were examined in cuticular preparations at 24 hrs. The extent of germband extension in these embryos was identical to that of their wildtype sibs. To analyze the same stages at higher resolution, 3.5to 12-hr embryos from the tll/TM3 stock were prepared and examined by SEM (Turner and Mahowald, 1976, 1977). From the earliest stages through the completion of germband extension at 7.5 hr, tailless and wildtype embryos are indistinguishable. We observed clearly distinguishable pole cells in 65 embryos examined at the cellular blastoderm stage. Gastrulation was normal among 47 embryos ages 3.5-5.5 hr.

TABLE

Wildtype Average cell number

in Tailless Embryos

Formation of pole cells and gastrulation. The posterior segments missing from the hypoderm, ventral nerve cord, and tracheal system of tailless embryos are derived from a region located on the blastoderm fate plan at about 20% of the egg length from the posterior tip (LohsSchardin et al., 1979; Underwood et al., 1980; Anderson and Niisslein-Volhard, 1984). We wished to determine whether the tailless mutation selectively eliminates only the posterior ectoderm or whether it also affects cells derived from the entire posterior portion of the blastoderm fate plan, such as the pole cells, the posterior midgut, and the proctodeum. A series of living embryos from the balanced W/TM3 stock was placed under halocarbon oil and continually

2

CELLNUMBERALONGTHORACICAND ABDOMINAL SEGMENTSINS-~~EMBRYO

Segment

Posterior

ewhryoa

Tl T2 T3 Al A2 A3 A4 A5 A6 A7

11 10

10 8 9

10 12 12 11 12

Tailless

Std. error (2)

1.4 2.1 2.1 1.4 1.4 0.3 0.7 0.3 0.0 1.2

Average cell number 12 9

10 10 12 12 14 16 17

Std. error (*I 2.5 0.7 0.0 0.7 0.7

1.1 1.1 0.4 1.5 1.7

Notes. The number of cells observed along a line drawn from anterior to posterior in each thoracic and abdominal segment in tailless (tU’/ tU’) (n = 2) versus wildtype (n = 2) embryos was determined by a blind counting done separately by two observers; the averages and standard deviations of these numbers are shown. Note that the difference in cell number between tailless and wildtype embryos becomes significant in the posterior abdominal segments.

DEVELOPMENTAL

BIOLOGY

VOLUME

113. 1986

Internal organs. To determine if the tailless mutation affects endodermal organs, i.e., those not derived from the ectoderm, embryos from the tll’/TM3 Sb stock were serially sectioned after fixation at 13-15 hr post-fertilization (Figs. 7A-D). Tailless embryos are distinguished from wildtype based upon the absence of filzkorper, and the presence of only seven abdominal segments. Embryos which clearly exhibited these characteristics were presumed to be tailless and were compared to wildtype for the presence and relative position of the following internal structures: cephalopharyngeal apparatus, brain and ventral nerve cord, proventriculus, midgut, posterior midgut, hindgut, Malpighian tubules and the proctodeal opening or anus (Figs. 7A, B). Tailless embryos possess all the structures listed above, with the exception of the Malpighian tubules, which are not found in serial sections or whole mount preparations of these embryos. They have, however, a continuous gut which terminates in a posterior, proctodeal opening (Fig. 7C). Although the midgut, posterior midgut, and hindgut are present in tailless embryos, the posterior midgut and hindgut are not in the same orientation as they are found in the wildtype embryo (Figs. 7C, D). (The cephalopharyngeal apparatus extends outside the tll’/tll’ embryo through the stomodeum due to an arrest in head involution (Figs. 7C, D). This is due, however, to the second mutation, which disrupts head involution, at a locus other than tailless on the original till chromosome.)

Zflect of Tailless on the Anterior

Ectodermal

Pattern

Mature cuticular phenotype. The cephalopharyngeal skeleton of mature tailless embryos was compared to wildtype using phase contrast microscopy of cuticular preparations (Figs. 8A-C). The only skeletal defect consistently observed in tailless embryos is the shortened length of the pharyngeal ridges (Figs. SB, C, arrow). All other head structures are normal in these embryos. Head phenotype at 9 hr. The presumptive head structures in tailless and wildtype embryos were examined at 9 hrs. At this time in embryogenesis, the presumptive head structures have not involuted through the stomodeum, thus it is possible to compare embryonic head

FIG. 7. Comparison of serial sections from 13 to 15-hr wildtype and tailless embryos (42X). (A) Dorsal frontal section of wildtype embryos; note filzkiirper, brain, and proventriculus, Malpighian tubules, and orientation of hindgut and posterior midgut. (B) Midfrontal section of wildtype embryo; note involuted cephalopharyngeal musculature and posterior midgut. (C) Frontal section of tailless (tll’/tlZ’) embryo;

note presence of the proventriculus, midgut and hindgut which ends in a posterior, proctodeal opening. (D) Frontal section of tailless (tll’/ tU’) embryo; note partially involuted cephalopharyngeal skeleton, brain, posterior end of ventral nerve cord, midgut, and enlarged posterior midgut. Cp, cephalopharyngeal skeleton; B, brain; Vnc, ventral nerve cord; Pv, proventriculus; Mg, midgut; Pg, posterior midgut; Hg, hindgut; P, proctodeum; F, filzkorper; Mt, Malpighian tubules.

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ET AL.

structures between embryos with the * characteristic posterior tailless phenotype. Embryos were compared with respect to the presence and form of the following head structures: clypeolabrum, procephalic lobe, hypopharyngeal lobes, mandibular lobes, maxillary lobes, labial lobes, dorsal ridge, and optic lobes (Figs. 8D, F). Ventrally, the only deviation from wildtype that was observed consistently in tailless (n = 40) embryos was a significant decrease in the size of the clypeolabrum (Fig. 8E). Laterally, the mutant clypeolabrum in these embryos appeared to be deviant in overall shape from wildtype, appearing most aberrant on the dorsal side (Fig. 8G). The dorsally oriented cleft (Fig. 8F, arrow) which separates the clypeolabrum from the procephalic lobe in wildtype was not present in tailless embryos and the procephalic lobe appeared to be smaller in size in these mutants. The optic lobe was present and in its normal position (just anterior to the dorsal ridge), but was now round rather than rectangular in shape (Fig. 8G). Thus, tailless mutants are defective in three regions of the head: the clypeolabrum, the procephalic lobe, and the optic lobe. DISCUSSION

The existence of zygotic lethal mutations and their effect upon embryonic segmentation is evidence that zygotic gene activity plays an important role in embryonic pattern formation. Information about ectodermal pattern formation acquired from the study of the gap, pair-rule and segment polarity mutants can be coupled with what we have gained from our study of tailless This leads to a more complete description of the classes of genes involved in the formation of the embryonic segmentation pattern. The zygotic lethal mutation tailless, mapped to the region lOOA5,6-B1,2, results in a specific and consistent alteration of the segment pattern in the mature embryo. The amorphic tailless phenotype is observed as shortened pharyngeal ridges and the absence of A8 and the telson. Tailless embryos exhibit altered segmentation during the earliest stage in embryogenesis when the segment pattern is evident (8 hr). At this time, tailless embryos consistently have one fewer tracheal pit compared to wildtype. Also, from the earliest stage when the segments are themselves present (9 hr), the tailless phenotype lacks the three posterior-most segments. Consistent with its effect on the external embryonic segmentation pattern at 9 and 24 hr, the tll mutation affects other segmented structures derived from the presumptive ectoderm of the cellular blastoderm. Specifically, the tll mutation affects not only the larval hypoderm, but other ectodermal derivatives, such as the

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segmented ganglia of the ventral nerve cord and the tracheal system of the mature embryo. While the tll mutation affects segmented derivatives of the posterior ectoderm, it does not affect all posterior structures. Derivatives of the non-ectodermal regions at the posterior tip of the embryonic fate plan, such as the proctodeum, pole cells, and posterior midgut are present in tailless embryos. Thus, the tailless mutation appears to specifically affect the structures derived from the ectoderm in the early embryo. We did observe one endodermal structure, however, which is affected by the tailless mutation. The Malpighian tubules, which arise from the gut at the interface of the posterior midgut and hindgut (Poulson, 1965), are missing in tailless embryos. The absence of these structures in tll embryos could be due to a primary effect of tailless upon their determination and subsequent development, or to the fact that the gut in ta,illess embryos does not have a normal spatial orientation relative to wildtype. Specifically, the hindgut in 15-hr tailless embryos has not undergone elongation which normally occurs concomitant with the development of the Malpighian tubules (Poulson, 1965). It may be significant that the gap mutation, Kriippel, also results in the absence of Malpighian tubules (Gloor, 1950). We consider it likely that the elimination of the three posterior-most segments A8, A9, and A10 in tailless embryos is a result of a reprogramming of the blastoderm fate map rather than from cell death. We have not observed any evidence of a decrease in cell number or necrosis in either living embryos or those observed using SEM. Furthermore, tailless embryos are of normal body length at both 9 and 24 hr. Since the posterior segments, A8, A9, and AlO, span more than 10% of the egg length of the cellular blastoderm (Underwood et al., 1980), a loss of these cells by cell death would be expected to result in a shortened embryo. The absence of segments A8, A9, and AlO, coupled with the larger size of and increased number of cells in segments A4 through A7 at both 9 and 24 hr, suggests that cell death is not the mechanism by which tailless embryos lack A8, A9, and AlO. Rather, these results suggest that in tailless embryos, cells which normally give rise to the posterior abdominal ectoderm are now contributing to the formation of larger, more anterior abdominal segments. At the anterior end of the embryo, the tailless mutation affects the formation and size of the clypeolabrum, procephalic and optic lobes, while appearing not to affect the development of the hypopharyngeal, mandibular, maxillary, and labial lobes and the dorsal ridge. The three affected embryonic head structures (clypeolabrum, procephalic and optic lobes) are thought to share a common origin in the early embryo as derivatives of the

A A

t-l

PIG. 8. Comparison of wildtype and tailless head phenotypes using phase SEM of embryonic head structures at 9.5 hr (D-G). (A) Wildtype, tha t the head skeleton appears normal with the exception of the shortened

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74

contrast microscopy of cuticle preparations of mature embryos ( Am ventral view, (45X). (B) T&less (tZl”/tZZy), ventral view (45X). N ote pharyngeal ridges (arrow). (C) Tailless (tZla/tZla), lateral view (45 N.

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dominal segments. This suggests that in addition to the gap mutant genes, which affect a central trunk region of the embryo, there is another class of genes that affects only the anterior and posterior ectodermal regions of the embryo. The division of the body plan of the embryo into anterior and posterior ends separate from a large central region has in fact been observed in less highly evolved arthropods. During the early development of the Coleopteran, Teuebrio molitor (flour beetle; Ullman, 1964, 1967; Anderson, 1972) the most anterior and most posterior ends of the embryonic primordium are enlarged into the head lobes and a rounded posterior end, respectively. Between these enlarged opposing ends of the embryo, the remainder of the body is formed: the gnathal segments which are delineated posterior to the head lobes, the thoracic segments, and finally, a growth zone, which proliferates the rudiments of the abdominal segments as the germ band elongates. Although Drosophilu lacks a formal growth zone, some significant comparisons can be drawn. The anterior head lobes of Tenebrio later form the labrum, protocerebral lobes and optic region of the head of the embryo. These structures appear to share homology to those Drosophila head derivatives (clypeolabrum, procephalic, and optic lobes) affected by the tailless mutation. Furthermore, the posterior end of the embryonic primordium in Tenebrio molitor later gives rise, in the feeding larva, to the most posterior body region which contains the anus. This may be analogous to the eighth abdominal segment and telson in the mature Drosophila embryo, which is also the region affected by the tailless mutation. The similarity between steps in ectodermal pattern determination postulated from the study of zygotic lethal mutants and those steps in pattern formation clearly visible in lower insects is intriguing. It suggests the possibility that the specialization of the embryonic ectodermal primordium into anterior and posterior ends separate from a central region is a function which has been preserved during evolution and that tailless may be a gene normally involved in the execution of this function in Drosophila. The significance of the tuilless gene is that its mutant phenotype suggests there is a step early in Drosophila embryogenesis when both anterior and posterior ectodermal regions are determined as separate from the

most anterior presumptive head segments. The segmental origin of head structures in both lower and higher insects has been described (Jura, 1972; Anderson, 1972; Rempel, 1975), and most recently summarized by Struhl (1981) as follows. Immediately anterior to the thoracic segments are, in order (from posterior to anterior), the labial, maxillary, and mandibular segments which comprise the gnathocephalon. Continuing anteriorly are the premandibular and antennal, and in some insects, the preantennal (labral) regions, which together comprise the procephalon and optic region of the head (Rempel, 1975). Additional evidence supports the view that the clypeolabrum, procephalic, and optic lobes in the Drosophila embryo arise from the anterior end of the presumptive ectoderm. First, a blastoderm fate map in Drosophila has been made of the regions which give rise to the adult head (Struhl, 1981) placing the clypeolabrum at the most anterior head segment (i.e., the labral or pre-antenna1 segment). Second, the area of the presumptive optic lobe in the hemimetabolous polyneopteran embryo (Anderson, 1972) is adjacent to the clypeolabrum at the very anterior of the embryonic head. Third, the anterior origin of the clypeolabrum, procephalic and optic regions distinct from mandibular, maxillary and labial segments is well illustrated in the Coleopteran, Tenebrio molitor (flour beetle; Ullman, 1964, 1967; Anderson, 1972) (see below). Thus, failless appears to affect the development of the most anterior ectodermal region of the embryonic head as well as result in the absence of the most posterior abdominal segments. Our observations of the tailless mutant phenotype suggests that the most anterior and most posterior ectodermal regions of the embryo are established as areas separate from the central trunk region affected by “gap” segmentation mutants (Ntisslein-Volhard and Wieschaus, 1980). The total domain of the gap mutants appears to be phenotypically complementary to that of the tailless mutation: Kruppel embryos lack the thoracic and anterior abdominal segments, kxirps embryos lack segments Al through A7, and finally, hunchback embryos lack the gnathal and thoracic segments (Wieschaus et al., 1984a; Ntisslein-Volhard and Wieschaus, 1980). These mutations, however, do not appear to alter the anteriorly derived structures of the head or the most posterior ab-

Note the shortened length and stunted processes of the pharyngeal ridges. Head structures are labeled as follows: Mh, mouth hooks; Amc, antenno-maxillary complex of sense organs; C, cirri; L, labial sense organs; Hp, H-piece; Pr, pharyngeal ridges; Hph, hypopharynx; Mt, median tooth. (D) Wildtype embryonic head phenotype, ventral view (545~); (E) Tailless (tll’/DfA~U), same orientation as (D) (544~); note reduced size of clypeolabrum; (F) wildtype, lateral view (560X); the dorsal cleft which lies between the clypeolabrum and the procephalic lobe is indicated by the arrow. (G) TuilZess (tll’/DfAll8’), lateral view (560X); note reduced clypeolabrum and procephalic lobe and the rounded optic lobe. Embryonic head structures are labeled as follows: C, clypeolabrum; H, hypopharyngeal lobe; P, procephalic lobe; Mx, maxillary lobe; Md, mandibular lobe; L, labial lobe; 0, optic lobe; D, dorsal ride; S, stomodeum.

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central trunk region. Molecular cloning of the tailless gene will permit us to directly test our hypothesis that this gene has regional specificity in expression at the anterior and posterior ends of the presumptive ectoderma1 regions of the early embryo. We thank E. Underwood, F. R. Turner, F. Eiserling, and B. Mueller for technical advice on SEM, T. Kaufman and G. Struhl for discussion on embryonic head development, and Margrit Lohs-Schardin for instruction in cuticular preparations. We also thank Dr. C. NiissleinVolhard and Dr. G. Jiirgens for sending us fly strains and for sharing their results with us prior to publication. Research was supported by NSF Grant PCM 21830 to J.A.L. and J.R.M., NIH Grant HD 09948 to J.A.L., and a University of California research award to J.R.M.; the SEM facility was supported in part by USPHS5SO?-RR07009; T.R.S. was supported by a Genetics Training Grant, USPHS National Research Service award GM 7104. REFERENCES ANDERSON, D. T. (1972). The development of holometabolous insects. In “Developmental Systems: Insects” (S. J. Counce and C. H. Waddington, eds.), Vol. 1, pp. 165-242. Academic Press, London/New York. ANDERSON, K. V., and NUSSLEIN-VOLHARD, C. (1984). Genetic analysis of dorsal-ventral pattern in Drosoph,ila. In “Pattern Formation” (G. M. Malacinski and S. Bryant, eds.), pp. 269-289. Macmillan, New York. CHAN, L. H., and GEHRING, W. (1971). Determination of blastoderm cells in Drosophila melancgaster. Proc. NutL Acad Sci. USA 68,22172221. FULLILOVE, S. L., and JACOBSON, A. G. (1978). Embryonic developmentDescriptive. In “The Genetics and Biology of Drosophila” (M. Ashburner and T. R. F. Wright, eds.), Vol. 2c, pp. 106-227. Academic Press, New York. GLOOR, H. (1950). Schadigungsmuster eines Letalfaktors (Kr) von Drosophila melanogaster. Arch. Jul. Klaus Stifiung 25, 38-44. HERTH, W., and SANDER, K. (1973). Mode and timing of body pattern formation (Regionalization) in the early embryonic development of cyclorrhaphic dipterans (Protophormia, Drosophila). Wilhelm Roux Arch. 172, l-27. JORGENS, G., KLUDING, H., NUSSLEIN-VOLHARD, C., and WIESCHAUS, E. (1984). Mutations in Drosophila melanogaster. II. Zygotic loci on the third chromosome. Wilhelm Roux Arch. 193,183-195. JURA, C. (1972). Development of apterygote insects. In “Developmental Systems: Insects” (S. J. Counce and C. H. Waddington, eds.), Vol. 1, pp. 49-95. Academic Press, London/New York. KALT, M. R., and TANDLER, B. (1971). A study of fixation of early amphibian embryos for electron microscopy. J. Ultrustruct. Res. 36, 633645. LINDSLEY, D. L., and GRELL, E. H. (1968). Genetic Variations of Drosophila melanogaster. Carnegie Institution of Washington, Washington D. C. LINDSLEY, D. L., SANDLER, L., BAKER, B. S., CARPENTER, A. T. C., DENNELL, R. E., HALL, J. C., JACOBS, P. A., MIKLOS, G. L. G., DAVIS, B. K., GETHMANN, R. C., HARDY, R. W., HESSLER, A., MILLER, S. M., NOZAWA, H., PARRY, D. M., and GOULD-SOMERO, M. (1972). Segmental aneupoloidy and the genetic gross structure of the Drosophila genome. Genetics 71, 157-184. LOHS-SCHARDIN, M., CREMER, C., and NUSSLEIN-VOLHARD, C. (1979). A fate map for the larval epidermis of D. melanogaster: Localized cuticle defects following irradiation of the blastoderm with an ultraviolet laser microbeam. Dev. BioL 73, 239-255. MAHOWALD,

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