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Fate map and cell lineage relationships of thoracic and abdominalmesodermal anlagen inDrosophila melanogaster

Robert Klapper, Anne Holz, Wilfried Janning*

Institut fur Allgemeine Zoologie und Genetik der Westfa¨ lischen Wilhelms-Universita¨t, Schlossplatz 5, D-48149 Mu¨nster, Germany

Received 29 August 1997; revised version received 9 December 1997; accepted 10 December 1997

Abstract

We have examined the cell lineage of larval and imaginal precursors of the mesodermal anlage between 10% and 60% egg length (EL) byhomotopic single-cell transplantations at the blastoderm stage. Clones in the larval somatic muscles and in the fat body were derived fromtransplantations everywhere between 10% and 60% EL along the ventral side of the embryo. Clones frequently overlap these tissues andcan extend over a maximum of four segments in the larval somatic muscles or over two morphologically-distinct parts in the fat body.Clones in the gonadal mesoderm overlap with other mesodermal derivatives and exhibit different mitotic behaviour in the two sexes. Wepresent a blastoderm fate map for the fat body, the larval somatic muscles and the gonadal mesoderm. Clones in the imaginal muscleprecursors of the abdomen, as well as of the thorax, always show a common cell lineage with larval somatic muscles and partly with othermesodermal tissues. These clones of imaginal derivatives are always found within a single segment, while the overlapping clone parts in thelarval somatic muscles can label up to three segments. 1998 Elsevier Science Ireland Ltd.

Keywords:Blastoderm stage; Single-cell transplantation; Fat body; Larval somatic muscles; Imaginal muscle precursor

1. Introduction

The mesoderm gives rise to different larval as well asimaginal tissues, including the fat body, somatic and visc-eral muscles, the heart and the blood (Poulson, 1950; forreview, see Bate, 1993). While the blastoderm fate map forthe ectodermal anlagen ofDrosophila melanogasterhasbeen continuously refined with various experimental meth-ods (for example, see Poulson, 1950; Lohs-Schardin et al.,1979; Hartenstein et al., 1985; Technau, 1987; Meise andJanning, 1994), a detailed fate map of the mesoderm is stilllacking. Only the expansion of the mesoderm anlage (Thisseet al., 1987; Leptin and Grunewald, 1990; Leptin, 1991;Reuter and Leptin, 1994) is represented in the blastodermfate map. At this stage the mesoderm anlage extends fromabout 10% EL to 90% EL (EL: egg length; 0% EL: posteriorpole) on the ventral side of the embryo. The dorsoventralwidth comprises 0% VD to 15 or 20% VD (VD: ventrodor-sal; 0% VD: ventral) (Poulson, 1950; Campos-Ortega and

Hartenstein, 1985) on each half of the embryo. Currentmesoderm fate maps refer to embryos from late stage 9 on(Hartenstein and Jan, 1992; Hoshizaki et al., 1994), whenthe parasegmental organisation of the mesodermal anlagenbecomes visible (Dunin-Borkowski et al., 1995; Azpiazu etal., 1996; Riechmann et al., 1997). Here we present a fatemap of the larval somatic muscles, the fat body, the gonadalmesoderm, the visceral muscles of the hindgut and the ade-pithelial cells, referring to the blastodermal mesodermanlage between 10% and 60% EL.

While the main portion of the larval somatic muscles ishistolysed during metamorphosis (Robertson, 1936; Boden-stein, 1950; Crossley, 1978), the imaginal muscles developfrom groups of cells that remain in a proliferative, undiffer-entiated state during the larval stages (Crossley, 1978; Bateet al., 1991; Currie and Bate, 1991; Fernandes et al., 1991).In the larval thorax these imaginal muscle precursors arearranged as adepithelial cells in a characteristic pattern onthe epithelium of the thoracic imaginal discs (Poodry andSchneiderman, 1970; Bate et al., 1991; Holz et al., 1997). Inthe abdomen the imaginal muscle precursors form tiny rowsof cells at three different positions in each hemisegment

Mechanisms of Development 71 (1998) 77–87

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* Corresponding author. Tel.: +49 251 8323847; fax +49 251 8324723;e-mail: [email protected]

(Bate et al., 1991). Investigation of the imaginal muscleprimordia at the blastoderm stage by mitotic recombinationhas given evidence that the thoracic muscles of each seg-ment are of separate origin (Lawrence, 1982), indicating anearly segmental restriction of their primordial cells. How-ever, the results from single-cell transplantation reveal thatthe progeny of cells giving rise to larval tissues are notrestricted to a single segment: the clones contributing tolarval somatic muscles extended over as many as threesegments (Beer et al., 1987; Holz et al., 1997; and thework described here). Analyses of expression patterns ofthe proneural genelethal of scute(l’sc) andS59by Carmenaet al. (1995) prove a common cell lineage of imaginalmuscle precursors and larval somatic muscles in the abdo-men. Transplantation data obtained by Holz et al. (1997)show a common cell lineage between larval somatic mus-cles and adepithelial cells. Without exception, our trans-plantation results demonstrate a common cell lineagebetween imaginal muscle precursors and larval somaticmuscles of the thorax as well as of the abdomen at theblastoderm stage. Furthermore, clones in larval somaticmuscles can cross segmental borders, whereas the imaginalfraction of the same clone is always restricted to one singlesegment.

2. Results

To examine the cell lineages of larval as well as imaginalmesodermal derivatives at the blastoderm stage we per-formed single-cell transplantations between 10% and 60%EL and between 0% and 30% VD at the blastoderm stage sothat mesodermal cells of the thoracic and abdominal seg-ments could be followed. As donor strains, we used enhan-

cer trap lines with strong nuclearb-galactosidase expressionin most tissues of third instar larvae. Due to the weak stain-ing in the visceral musculature of the midgut, it is possiblethat an unknown fraction of clones in this derivative was notdetected (see Section 4). All clones were analysed in thirdinstar larvae, because this stage allows larval and imaginaltissues to be examined at the same time. We carried out atotal of 2236 homotopic transplantations. A total of 1468(66%) hosts survived until they were dissected and stainedas third instar larvae. In 706 of these larvae (48% of surviv-ing larvae) a clone was detected. Of the clones,622 marked mesodermal derivatives (Table 1), in 83 casesectodermal, and in one case endodermal tissues werelabelled. Ectodermal clones derived from transplantationsof donor cells from neighbouring germ layers (Beer et al.,1987) e.g. cells originating from the lateral border ofthe mesoderm anlage between 10% VD and 30% VD(Table 2). Clones overlapping different germ layers werenot observed. Comparing the clone frequencies betweentransplantations in which the sexes of the donor and thehost were identical or not, we found no differences (datanot shown).

2.1. Fate map of the fat body

The entire transplantation region was divided into tensubregions of 5% EL each between 10% and 60% ELalong the embryo. Clones in the fat body were obtained atall subregions (Fig. 1). Of the 232 clones with labelled cellsin the fat body (Table 1), 123 could be classified as belong-ing to morphologically-distinguishable fat body parts. Sinceclones can overlap two fat body parts (e.g. parts 1 and 2a orparts 2a and 2b; modified after Rizki, 1978), it is not possi-ble to attribute a distinct fat body part to a certain transplan-tation site. Nevertheless, there is a correlation betweentransplantation sites and fat body parts: clones in the ante-riormost fat body parts 1, 2a and 2b, were obtained onlyafter transplantations between 50% and 60% EL with acomplete overlap of these primordia. Also, the subregionsgiving rise to the posterior fat body parts frequently overlap,but the labellings appeared in a consecutive array dependingon their transplantation site. We also examined whether therelative clone frequencies in the fat body differ dependingon the dorsoventral integration site of the transplanted cell.No such differences were found (Table 3).

Table 1

Distribution of mesodermal clones in different tissues after transplantationbetween 10% and 60% EL

Combinations of marked tissues Frequency

Larvalsomaticmuscles

Fatbody

Visceral muscles of Gonadalmesoderm

Imaginalmuscles

n %

Midgut Hindgut

X 367 59X 94 15

X 2 ,1X 4 1

X X 126 20X X X 1 ,1X X X X 1 ,1X X 3 ,1X X X 7 1X X 14 2

X X 1 ,1X X 2 ,1

Total 622 100

Table 2

Relationship between VD position of the donor and the germ layer of theresulting clone

VD position of the donor

0–10% 10–30%

Mesodermal clones 478 (92%) 42 (60%)Ectodermal clones 44 (8%) 28 (40%)Total 522 (100%) 70 (100%)

78 R. Klapper et al. / Mechanisms of Development 71 (1998) 77–87

2.2. The size of the fat body primordium

The clone size in 94 pure fat body clones (Table 1) variesfrom 2–16 cells, four and eight cells being most frequentlylabelled. All blastodermal precursor cells for this tissueundergo at least one mitosis, and in some cases up to fourmitoses are possible. The average clone size is 6.7 cells,corresponding to an average number of two to three mitoses.Most of the clones (73%) can be explained by regularmitoses of the daughter cells, i.e. clones with two, four,eight or 16 labelled cells. Besides pure fat body labellingswe found 135 clones that participate in the formation oflarval somatic muscles and fat body (9 of these cloneswith additional overlaps, see Table 1). The fat body fraction

of these clones varies from one to 12 cells. Clones with twoor four labelled fat body cells are most frequent. The aver-age clone size of the fat body part of these mixed clones is4.4 cells, more than two cells smaller than the average clonesize in pure fat body clones. Assuming in principle equalmitotic behaviour in pure and mixed fat body clones, thismeans that on average two to three cells were set aside forthe partial clone in other mesodermal derivatives. The aver-age size of pure and overlapping fat body clones is 5.5 cells.

The fat body of the donor strain consists of about 1970cells. Dividing this number by the average clone size of 5.5cells, we obtain about 360 cells for the size of the blasto-derm anlage of the fat body. The fat body develops from tensegmental clusters, which become visible at embryonicstage 11 (Hoshizaki et al., 1994; Azpiazu et al., 1996;Rehorn et al., 1996; Sam et al., 1996). According to thiscalculation, then, each of the ten future mesodermal seg-ments will contain about 36 cells, the descendants ofwhich will give rise to the fat body.

2.3. Fate map of larval somatic muscles

All 467 clones in larval somatic muscles whose segmentidentities could be determined are shown, in order of theirtransplantation sites, in Fig. 2. For each transplantation-siterange, the number of clones with a particular segmentaldistribution is indicated. Evidently, there is no precise cor-relation between a specific transplantation region and a sin-gle segment; indeed, a single clone can overlap up to foursegments. However, there is a clear trend: the further ante-rior the transplantation site is, the more likely it is that athoracic or anterior abdominal segment will be labelled.From these data we constructed a blastoderm fate map forthe somatic muscles of segments T2 to A8/9 (Fig. 3). Incorrelation with the expression pattern offushi tarazu,depicted here as an example from which to deduce the posi-tions of the prospective ectodermal segments, the centre ofthe muscle primordium of a given segment matches theposition of the same segment in the ectoderm. This can beseen in the exemplary graph of the two abdominal segmentsA4 and A7 (Fig. 3A). Furthermore, this graph shows the

Fig. 1. Blastoderm fate map for the fat body parts 1 to 5. The right side ofthe figure is a schematic drawing of one half of the bilaterally-symmetricalfat body, showing parts 1–5 (modified after Rizki, 1978). Because someclones could not unambiguously be assigned to a particular fat body part,the transitional parts 3a/b, 3b/4 and 4/5 are also shown. Part 6 could not beevaluated because it was often damaged or absent, hence it is not includedhere. The left side shows the relationship between transplantation regionsand labelled fat body parts. For each transplantation region, the distribu-tion among fat body parts is shown for all the clones from that site. Thebackground colours correspond to those associated with the fat body partsin the drawing. Sg, salivary gland; go, gonad primordium.

Fig. 2. Segmental distribution of the muscle clones, depending on transplantation region. All 467 muscle clones whose segment identities could bedetermined were assigned to ten transplantation regions along the A/P-axis. The numbers below the shaded part in each column represent the number ofclones in that category. (The two columns on the left in each region show the most frequent clone types in the whole region (dark grey). The other columnsshow the remaining clone types in an anterior-posterior sequence (light grey).) For example, in the transplantation region between 55% and 60% EL, 16clones were found in T3 and 14 clones in T2, while one clone overlapped T1 and T2.

79R. Klapper et al. / Mechanisms of Development 71 (1998) 77–87

diffuse extension of the blastodermal muscle primordia,which results in partial overlap of two primordia that areseparated in the ectoderm by two segments. The relativeclone frequency in the larval somatic muscles is indepen-dent of the dorsoventral transplantation site of the hostembryos within the region examined (Table 3).

2.4. Estimation of muscle clone sizes

The clones we observed in larval somatic muscles appearto cover a large range of clone sizes. There are cases inwhich very few nuclei are labelled, as well as clones with30 or more labelled nuclei in several muscle groups span-ning up to four segments. The clone sizes in the musculaturecannot be determined simply by counting the labelled

nuclei, because we detected clones in which only one, orfew, nuclei are stained intensely, while the surroundingnuclei exhibit a gradually weaker expression (Fig. 4A,B).An explanation for the variability in nuclear staining might

Fig. 3. Blastoderm fate map for the thoracic and abdominal mesoderm. (A) Example of the localisation of mesodermal primordia for the abdominal segmentsA4 and A7. An embryo in the blastoderm stage is shown in ventrolateral view, and the region studied here is indicated by the white background (0–10% VD,10–60% EL). The expression pattern of the pair rule genefushi tarazu(grey stripes) is used to deduce the prospective ectodermal segments from T1 to A8. Asexamples, the abdominal segments A4 and A7 are marked in colour. The musculature of A4 and A7 was localised on the basis of our transplantation data; thecolour intensity reflects the relative clone frequency. (B) Schematic blastoderm fate map for the larval somatic musculature of T2 to A8/9, the fat body parts 1to 5, the gonadal mesoderm (go), the visceral musculature of the hindgut (vm) and the adepithelial cells (ac). From the clone frequencies in the larvalmusculature, polygonal distribution curves (third order) were calculated; each of these shows both the centre of the associated primordium and its extentalong the A/P-axis. The areas representing abdominal segments A4 and A7 are coloured to correspond with (A). The primordia for the fat body parts 1 to 5,aswell as those for the adepithelial cells and the gonadal mesoderm, are indicated by rectangles.

Table 3

Relationship between clone frequencies in larval somatic muscles and fatbody along the VD-axis

VD position of the host

0–10% 10–30%

Pure larval muscle clones 78% (278) 80% (51)Pure fat body clones 22% (79) 20% (13)Total 100% (357) 100% (64)


Fig. 4. Labelled tissues in third instar larvae after single-cell transplantation. (A) Bilateral clone overlapping two abdominal segments (A2 and A3) in theventral larval somatic musculature. White dots mark the segment boundaries. (B) Clone in muscle 4 (according to the nomenclature of Crossley, 1978,modified according to Hooper, 1986) in the lateral somatic musculature of A2. Note that some nuclei strongly expressb-galactosidase (the nucleus that seemsto exhibit the strongest expression is marked with an asterisk), while its expression gradually declines in adjacent nuclei (arrowheads). (C,D) A clone aftersingle-cell transplantation overlapping larval muscle 8 and the lateral imaginal muscle precursors in A3 (C) and additional larval muscles in comparison withcytoplasmictwist-lacZ expression in the lateral imaginal muscle precursors of the abdomen (D). (E) Clone in the visceral muscles of the hindgut (furtherstained nuclei out of focus). (F) Clone overlapping the male gonadal mesoderm and four nuclei in the fat body (also overlapping larval somatic muscles). (G)Clone overlapping four nuclei in the fat body and the female gonadal mesoderm (also overlapping larval somatic muscles).


be that after translation of the mRNA forb-galactosidase inthe syncytial cytoplasm, the enzyme is imported into adja-cent nuclei of different clonal origin, leading to a distance-dependent gradient of staining intensity. In many cases wefound stained and unstained nuclei together in one muscle(Fig. 4A,B), and therefore the fusing cells obviously do nothave a common cell lineage.

In order to obtain an approximate minimum clone size wecalculated the number of labelled muscles from a sample of100 clones. Each muscle contributing to the clone shouldconsist of at least one descendant of the transplanted cell. Inour sample we observed clones ranging from a singlelabelled muscle to clones that were composed of morethan ten labelled muscles. The average clone comprises

four labelled muscles and therefore a minimum of four des-cendants of the transplanted cell. Due to the multi-layeredstructure of the musculature, the actual clone size was prob-ably larger than estimated in many cases. Differences in theaverage number of stained nuclei between pure and over-lapping muscle clones could not be determined with thisapproach.

2.5. Imaginal myoblasts always share common precursorswith larval muscles

Most of the imaginal thoracic muscles develop from ade-pithelial cells of imaginal discs (Reed et al., 1975; Fer-nandes et al., 1991; Fernandes and Keshishian, 1996). Allclones comprising adepithelial cells share a common celllineage with larval somatic muscles at the blastoderm stage(Holz et al., 1997). The imaginal abdominal muscles aredifferentiated from myoblasts, which – like the adepithelialcells – are characterised by a persistenttwist-expression inthe larva (Bate et al., 1991; Currie and Bate, 1991; Fer-nandes et al., 1991).

After transplantation between 50% and 60% EL we found18 clones of adepithelial cells, each overlapping with larvalsomatic muscles in the same hemisegment (Holz et al.,1997). In four cases we observed clones overlappingabdominal imaginal muscle precursor cells and larvalabdominal somatic muscles. Three of these clones werefound in the lateral abdominal muscle precursors (Fig.4C) and one in a ventral abdominal muscle precursor. Forcomparison, Fig. 4D shows the array of the lateral abdom-inal muscle precursors in atwist/lacZ-line with cytoplasmicb-galactosidase expression in imaginal muscle precursorcells. The imaginal clone fraction is always – as observedfor clones of adepithelial cells – restricted to one segment,while the larval clone fraction can cross the segmentalboundary. In three additional cases we found clones over-lapping larval muscles and cells which, to judge by theirposition, are likely to have been precursors of the lateralimaginal abdominal muscles.

2.6. Clones in the visceral muscles and in the gonadalmesoderm

In four cases we found clones in the visceral muscles ofthe hindgut (Table 1; Fig. 4E) after transplantation at≤13%EL. These clones, as well as six additional clones resultingfrom a series of transplantations at,10% EL (data notshown), never overlap with other mesodermal derivatives.We suppose that the primordium of the visceral musculatureof the hindgut represents a separate anlage at the time oftransplantation. We therefore included the correspondingregion as a presumptive primordium in the blastodermfate map (Fig. 3B).

In three cases of a total of 622 we observed clones in thevisceral muscles of the midgut, one of them overlappingwith the fat body (Table 1). The non-overlapping clones

Fig. 5. Model explaining segmental restriction of imaginal muscle precur-sors. In this model, the potential developmental capacities of three blas-todermal cells (1,2 and 3) belonging to two different segments (a in yellowand b in green) are presented. For better comprehension, only two mitosesare depicted in each case. Arrows represent mitoses and solid lines connectidentical cells in different stages of development. Transplantation of cell 1results in a clone labelling one muscle founder cell and one fusion-com-petent cell in a muscle of segment a and two nuclei in the fat body.Transplantation of cell 2 gives rise to a clone labelling two fat bodycells and larval somatic muscles of segments a and b, consisting only offusion-competent cells. Transplantation of cell 3 results in a clone label-ling an imaginal muscle precursor solely in segment b and a larval musclefounder cell in the same segment, while the two fusion-competent cellsmark muscles overlapping both segments. Note that not every larval mus-cle founder cell is the sibling of an imaginal muscle precursor cell, but thismodel provides that each imaginal muscle precursor is the sibling of afounder cell of the same segment.


resulted from transplantations between 35% and 40% EL,while the overlapping clone originated from a transplanta-tion at 53% EL. The musculature of the midgut consists oftwo layers of fibres: an inner circular set and an outer long-itudinal set (Elder, 1975; Tepass and Hartenstein, 1994). Inthose cases in which we were able to determine the orienta-tion of the marked fibres, we found stained nuclei only in thecircular muscles. Heterotopic transplantations with cellstaken from 40% EL and transplanted to 30% EL (data notshown) gave rise to two further clones in the longitudinalvisceral muscles of the midgut.

After transplantation into the region between 22% and32% EL (see also fate map Fig. 3B) ten clones (includingthree clones from other experimental series, data not shown)were found in the gonadal mesoderm (Fig. 4F,G). All clonesoverlap with additional mesodermal derivatives. If theseclone parts label muscles, they are always located in seg-ment A5 and overlap with A4 or A6. Overlapping parts ofthe larval fat body are located in the region of fat body part 4to 5. While the seven clones in the male gonadal mesoderm(Fig. 4F) are smaller than ten labelled cells, the three clonesin the female gonadal mesoderm label more than 20 cells(Fig. 4G). Furthermore, the labelled cells are differentlydistributed in the two sexes: the cells in the male gonadalmesoderm are more dispersed, while in the female gonadalmesoderm the cells are clustered tightly.

3. Discussion

3.1. Fate mapping in the mesoderm

From our transplantation data we constructed a blasto-derm fate map (Fig. 3B) for the larval somatic muscles,the fat body, the gonadal mesoderm, the visceral musclesof the hindgut and the thoracic adepithelial cells. This fatemap illustrates the considerable overlap of the primordia forsomatic muscles in neighbouring segments and of the dif-ferent fat body parts. The primordium of the gonadal meso-derm is restricted to an area between 22% and 32% EL andoverlaps with other mesodermal tissues. The primordium ofthe visceral musculature of the hindgut presumably repre-sents a separate anlage posterior to 13% EL. Clones label-ling the adepithelial cells of the thoracic imaginal discsappear from 51% EL to the anterior border of the transplan-tation region, corresponding to the ectodermal thoracicanlagen (Technau and Campos-Ortega, 1985; Meise andJanning, 1993). Additionally, the fate map shows the corre-lation between the position of the prospective ectodermalsegments and the origin of the primordia of the larvalsomatic muscles.

Clones overlapping up to four segments in the larvalsomatic musculature (Fig. 2) clearly show that it is notpossible to define their blastodermal primordia more pre-cisely, in contrast to the ectoderm, in which segmentaldetermination has already occurred at this stage (Garcia-

Bellido et al., 1973; Garcia-Bellido et al., 1976; Morataand Ripoll, 1975; Wieschaus and Gehring, 1976; Meiseand Janning, 1993). On the other hand, the future cell fateis shown to depend on the position along the A/P-axis,which therefore roughly reflects the anatomy of the prospec-tive mesodermal derivatives.

From our transplantation results we conclude that themesodermal cells between 50% and 60% EL differentiatethe larval anterior fat body (parts 1, 2a and 2b). The frequentoverlap of clones between these anterior fat body parts indi-cates that their precursors either originate from a singlecommon primordium or are mixed after the first postblasto-dermal mitosis.

In a comparative analysis of the fat body in wild-typelarvae and in homeotically-transformed larvae, which, inthe ectoderm, differentiate a second mesothorax instead ofa metathoracic segment, Rizki and Rizki (1978) observed aduplication of anterior fat body structures in the homeoticmutant. Based on this finding, they postulate a phylogene-tically-metameric organisation of the larval fat body. Morerecent cell-lineage analyses with enhancer trap lines andgene expression patterns (Hartenstein and Jan, 1992; Abelet al., 1993; Hoshizaki et al., 1994; Azpiazu et al., 1996;Riechmann et al., 1997) also indicate a metameric organisa-tion of the embryonic fat body precursor cells at stage 11.The furthest anterior cluster of fat body precursor cells issituated in segment T2 (Hoshizaki et al., 1994). Therefore,at stage 11 only two fat body anlagen exist for the corre-sponding primordia of the three thoracic segments. Theanalysis of Rizki und Rizki (1978) shows that a large portionof the anterior fat body structures originates from themesothorax. This could conceivably explain why the ante-rior fat body clones occupy spatially-separated fat bodyparts (e.g. parts 1 and 2b).

The transplantation area giving rise to clones in the gona-dal mesoderm corresponds to the primordia of segments A5to A7 in the mesoderm. Whenever clones in the gonadalmesoderm overlap somatic muscles, labellings in segmentA5 are always found. This is in agreement with results ofgynandromorph analyses (Szabad and No¨thiger, 1992),which correlate the primordium for gonadal mesodermwith the segments A4 and A5. Following back markersspecific for the gonadal mesoderm to earlier developmentalstages indicates that the primordia are situated in paraseg-ments 10 to 12 (Brookman et al., 1992; Boyle et al., 1997),corresponding to a region between the posterior compart-ment of A4 and the anterior compartment of A7. Analysis ofthe effects of segmentation gene defects on gonad formationshowed that only the posterior compartments of A5 and A6are likely to take part in the specification of the gonadalmesoderm (Warrior, 1994).

Beer et al. (1987) obtained high frequencies of clones inthe visceral muscles of the midgut after heterotopic trans-plantations from the mesodermal anlage into the dorsolat-eral ectodermal anlage. Our transplantation results give noevidence as to the localisation of the primordium of the


visceral musculature of the midgut, although the whole lat-eral extent of the mesodermal anlage was examined.

3.2. Clone sizes in the mesoderm

The labelling of up to 16 cells in the fat body shows thatthe larval mesodermal tissues can undergo as many as fourpostblastodermal mitoses. Beer et al. (1987) also observedmesodermal clones that can only be explained by more thanthree mitoses. Bate (1993) describes a fourth mitosis for themesoderm in late stage 11. Most pure fat body clones sug-gest two to four regular mitoses of all cells making up theclone and show that most cells apparently have an indivi-dual proliferation behaviour. We cannot exclude the possi-bility that clones in the visceral muscles of the midgut wereoften not detected due to the weak staining intensity in thistissue (see Section 4). Therefore, it might be possible thatthe small clones in the fat body share clonally-related cellsin the visceral muscles of the midgut which were notdetected.

Determination of clone size by counting labelled nuclei isnot feasible in the larval somatic muscles because of vari-able staining intensities in the nuclei (Holz et al., 1997),presumably due to the syncytial character of muscles. Wecan only give a minimum estimate of clone sizes. From anaverage of four labelled muscles per clone we infer thatthe transplanted cell must have undergone at least twomitoses.

3.3. Cell lineage in the mesoderm: determination after thesecond postblastodermal mitosis

That clones can overlap more than two mesodermal tis-sues (Table 1) – one clone overlapping four different tissues(somatic muscles, fat body, gonadal mesoderm and imagi-nal muscle precursor cells) – indicates that determination ofthe ultimate primordia and also the determinative decisionof larval versus imaginal fate conceivably does not takeplace until after the second postblastodermal mitosis. Wefurther infer that determination occurs after the second post-blastodermal mitosis from the difference in clone distribu-tion in pure and overlapping fat body clones, as follows. Thelargest pure fat body clones, consisting of 16 labelled cells,correspond to four regular postblastodermal mitoses. Pre-suming the same mitotic behaviour for overlapping fat bodyclones, a maximum of four cells are allotted to anothertissue in the largest overlapping clones, with 12 fat bodycells. If determination were to occur after the first postblas-todermal mitosis, only eight labelled cells could be presentin the fat body part. Under these assumptions, a separationinto distinct tissues would not take place until after thesecond mitosis. Lu¨er et al. (1997) also presented evidencefor the splitting of a common cell lineage within the meso-derm after the second postblastodermal mitosis. Similarresults were obtained for the ectoderm by Meise and Jan-ning (1993). In the mesoderm, the second postblastodermal

mitotic wave begins at 4:15 h after egg deposition (stage 9,Campos-Ortega and Hartenstein, 1985). At this stage ofdevelopment, an initial patterning of the mesoderm estab-lishes the parasegmental primordia of the somatic and visc-eral muscles, the heart and the fat body mediated by geneslike tinman, bagpipe and serpent (Azpiazu and Frasch,1993; Bodmer, 1993; Staehling-Hampton et al., 1994;Azpiazu et al., 1996; Riechmann et al., 1997).

Clones can overlap different segments in the larvalsomatic muscles (Beer et al., 1987; Holz et al., 1997; andthis work). This seems to indicate that the mesodermal germlayer is not yet segmentally-restricted at the blastodermstage. In contrast, clones in imaginal muscles induced bymitotic recombination in the blastoderm show a segmentalorigin of their precursor cells (Lawrence, 1982), indicatingan early segmental restriction of the anlagen for the imagi-nal muscles. Also, clones of adepithelial cells and imaginalabdominal muscle precursor cells are always restricted toone segment. Nevertheless, partial clones in the larval mus-cles, which always appear together with imaginal muscleclones, can extend across segmental boundaries.

How can a precursor cell be segmentally-restricted forthose daughter cells that produce imaginal tissue but notfor descendants that take part in larval muscle formation?

It could be that the descendants of the transplanted cell donot spread up to the stage when the imaginal muscle pre-cursors are separated from cells giving rise to larval mus-cles. Since imaginal muscle precursors arise at tightlyrestricted locations (Bate et al., 1991; Currie and Bate,1991) that are separated from one another by the fullwidth of a segment, the apparent segmental restriction ofimaginal precursors could be a result of statistical events.Migrations of muscle founder cells across segmentalboundaries (Dohrmann et al., 1990) could be an additionalreason for the observed spreading of larval somatic muscleclones across different segments. However, clones overlap-ping adepithelial cells of dorsal and ventral imaginaldiscs within one segment (Lawrence, 1982; Holz et al.,1997) reveal that the descendants of transplanted cellsare capable of expanding, at least in the dorsoventral direc-tion.

We therefore favour a different explanation of the seg-mental restriction of the imaginal muscle clones (Fig. 5). Itmight be that segmental restriction of all mesodermal cellshas already taken place at the blastoderm stage, as in theectoderm. While only a few descendants of these cells uti-lise the information for segmental identity, namely the pre-cursors of the imaginal muscles as well as the larval musclefounder cells, the fusion-competent cells do not maintainthis segmental identity after the larval muscle foundercells and the precursors of the imaginal muscles havebeen determined.

The formation of larval somatic muscles is started by thefusion of single muscle founder cells with additional fusion-competent cells to form muscle precursors. These syncytialmuscle precursors fuse with further fusion-competent cells,


producing one larval muscle each (Bate, 1990; Dohrmann etal., 1990; Rushton et al., 1995). Several muscle foundercells express specific genes, e.g.nautilus (Michelson etal., 1990; Paterson et al., 1991; Abmayr et al., 1992),S59(Dohrmann et al., 1990) orapterous (Bourgouin et al.,1992). These cells, a priori, appear in an organised segmen-tal pattern. A first expression of theS59gene product can bedetected 6 h to 7 h after egg deposition (stage 11) in a singlecell of the somatic mesoderm of each abdominal hemiseg-ment (Dohrmann et al., 1990), indicating that these cellsmust have precise positional information. This informationmight either be ensured by the cells’ competence to main-tain a state of segmental identity, possibly experienced inthe blastoderm or be restored through exogenous processes,for instance, certain kinds of positional information can beprovided by the ectoderm.

Supposing that the fusion-competent cells do not retainany information about segmental identity, they can berecruited by founder cells of adjacent segments to formsyncytial muscles with cells of different segmental origin.Since clones of imaginal muscle precursors of the thorax aswell as of the abdomen always overlap larval somatic mus-cles, we postulate that these cells obligatorily share a com-mon cell lineage. Carmena et al. (1995) support thispostulate by finding a common cell lineage between theventral imaginal muscle precursor of the abdomen and aspecific larval founder cell.

4. Experimental procedures

4.1. Stocks

As donor for transplantation, we used the strain SW005with the genotypeP[lArB]; ry 506 (2;3) (Sachs, Wedekindand Janning, unpublished data), which exhibits uniformlyintenseb-galactosidase expression in almost all larval andimaginal tissues of the third instar larva. In the gonads andthe visceral muscles of the midgut this enzyme is weaklyexpressed. To map the gonadal mesoderm, a modified donorstrain was used in which an additionalP[lArB] -element onthe third chromosome (Rellecke and Janning, unpublisheddata), with strong expression in the gonads, was crossed intothe original donor strain. The strain CG9 is a null mutationfor the b-galactosidase-1 gene (2L, 26A-B) (Knipple andMacIntyre, 1984) and served as the recipient in transplanta-tion experiments. In larvae of this strain, there is back-ground staining of pericardial and garland cells. Forhistological observations of abdominal imaginal myoblastswe used the strain CPS9 with the genotypeP[w+, twi/lacZ](3) (Thisse et al., 1988). Histochemical demonstration ofb-galactosidase expression was carried out as described byMeise and Janning (1993). All stages are according to Cam-pos-Ortega and Hartenstein (1985). Muscle nomenclature isaccording to Crossley (1978), modified according to Hooper(1986).

4.2. Single-cell transplantation

Cells were transplanted from the mesodermal anlagebetween 10% and 60% EL and between 0% and 30% VDof donor embryos into homotopic positions of host embryos.The VD-positions of the donor- as well as of the host-embryos could not be measured but were estimated by fol-lowing the gastrulation movements. Most transplantationswere carried out at 0–10% VD. To avoid possible hetero-topic effects only those transplantations were considered forfurther analysis in which the sites of cell removal and ofintegration did not deviate by more than 5% EL. The trans-plantations followed the technique of Meise and Janning(1993). Single cells were transplanted at the cellular blas-toderm (late stage 5(2) or 5(3) of Lawrence and Johnston(1989)), before invagination of the mesoderm begins.Further treatment of the embryos and preparation of larvaewere carried out according to Holz et al. (1997).

Acknowledgements

We would like to thank Elke Naffin and Rita Hassenru¨ckfor excellent technical assistance, and Renate Renkawitz-Pohl, Volker Hartenstein, Ruth Harbecke and Martin Meisefor valuable comments on the manuscript. Suggestions ofboth anonymous reviewers of the previous version of themanuscript are gratefully acknowledged. We are grateful toRolf Reuter for sending us theP[twi/lacZ] line. This workwas supported by the Deutsche Forschungsgemeinschaft (Ja199/12).

References

Abel, T., Michelson, A.M., Maniatis, T., 1993. ADrosophila GATAfamily member that binds toAdh regulatory sequences is expressed inthe developing fat body. Development 119, 623–633.

Abmayr, S.M., Michelson, A.M., Corbin, V., Young, M.W., Maniatis, T.,1992.nautilus, a Drosophilamember of the myogenic regulatory genefamily. In: Blau, H.M., Kelly, A.M. (Eds.), Neuromuscular Develop-ment and Disease. Raven Press, New York, pp. pp. 1–16.

Azpiazu, N., Frasch, M., 1993.tinmanandbagpipe: two homeobox genesthat determine cell fates in the dorsal mesoderm ofDrosophila. GenesDev. 7, 1325–1340.

Azpiazu, N., Lawrence, P.A., Vincent, J.P., Frasch, M., 1996. Segmenta-tion and specification of theDrosophila mesoderm. Genes Dev. 10,3183–3194.

Bate, M., 1990. The embryonic development of larval muscles inDrosophila. Development 110, 791–804.

Bate, M., 1993. The mesoderm and its derivatives. In: Bate, M., MartinezArias, A. (Eds.), The Development ofDrosophila melanogaster, Vol. II.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp.1013–1090.

Bate, M., Rushton, E., Currie, D.A., 1991. Cells with persistenttwistexpression are the embryonic precursors of adult muscles inDrosophila. Development 113, 79–89.

Beer, J., Technau, G.M., Campos-Ortega, J.A., 1987. Lineage analysis oftransplanted individual cells in embryos ofDrosophila melanogaster.IV. Commitment and proliferative capabilities of mesodermal cells.Roux’s Arch. Dev. Biol. 196, 222–230.


Bodenstein, D., 1950. The postembryonic development ofDrosophila. In:Demerec, M. (Ed.), Biology ofDrosophila. Hafner, New York, pp.275–364 (reprinted 1965).

Bodmer, R., 1993. The genetinman is required for specification ofthe heart and visceral muscles ofDrosophila. Development 118, 719–729.

Bourgouin, C., Lundgren, S.E., Thomas, J.B., 1992.apterousis a Droso-phila LIM domain gene required for the development of a subset ofembryonic muscles. Neuron 9, 549–561.

Boyle, M., Bonini, N., Dinardo, S., 1997. Expression and function ofcliftin the development of somatic gonadal precursors within theDrosophilamesoderm. Development 124, 971–982.

Brookman, J.J., Toosy, A.T., Shashidhara, L.S., White, R.A.H., 1992. The412 retrotransposon and the development of gonadal mesoderm inDrosophila. Development 116, 1185–1192.

Campos-Ortega, J.A., Hartenstein, V., 1985. The Embryonic Developmentof Drosophila melanogaster. Springer-Verlag, Berlin.

Carmena, A., Bate, M., Jimenez, F., 1995.Lethal of scute, a proneuralgene, participates in the specification of muscle progenitors duringDro-sophilaembryogenesis. Genes Dev. 9, 2373–2383.

Crossley, C., 1978. The morphology and development of theDrosophilamuscular system. In: Ashburner, M., Wright, T.R.F. (Eds.), The Genet-ics and Biology ofDrosophila, Vol. 2b. Academic Press, New York, pp.499–560.

Currie, D.A., Bate, M., 1991. The development of adult abdominal mus-cles in Drosophila: myoblasts expresstwist and are associated withnerves. Development 113, 91–102.

Dohrmann, C., Azpiazu, N., Frasch, M., 1990. A newDrosophilahomeo-box gene is expressed in mesodermal precursor cells of distinct musclesduring embryogenesis. Genes Dev. 4, 2098–2111.

Dunin-Borkowski, O.M., Brown, N.H., Bate, M., 1995. Anterior-posteriorsubdivision and the diversification of the mesoderm inDrosophila.Development 121, 4183–4193.

Elder, H.Y., 1975. Muscle structure. In: Usherwood, P.N.R. (Ed.), InsectMuscle. Academic Press, New York, pp. 1–74.

Fernandes, J., Bate, M., Vijayraghavan, K., 1991. Development of theindirect flight muscles ofDrosophila. Development 113, 67–77.

Fernandes, J.J., Keshishian, H., 1996. Patterning the dorsal longitudinalflight muscles (DLM) ofDrosophila: insights from the ablation of larvalscaffolds. Development 122, 3755–3763.

Garcia-Bellido, A., Ripoll, P., Morata, G., 1973. Developmental compart-mentalisation of the wing disk ofDrosophila. Nature 245, 251–253.

Garcia-Bellido, A., Ripoll, P., Morata, G., 1976. Developmental compart-mentalization in the dorsal mesothoracic disc ofDrosophila. Dev. Biol.48, 400–410.

Hartenstein, V., Technau, G.M., Campos-Ortega, J.A., 1985. Fate-map-ping in wild-type Drosophila melanogaster. III. A fate map of theblastoderm. Roux’s Arch. Dev. Biol. 194, 213–216.

Hartenstein, V., Jan, Y.N., 1992. StudyingDrosophilaembryogenesis withP-lacZ enhancer trap lines. Roux’s Arch. Dev. Biol. 201, 194–220.

Holz, A., Meise, M., Janning, W., 1997. Adepithelial cells inDrosophilamelanogaster: origin and cell lineage. Mech. Dev. 62, 93–101.

Hooper, J.E., 1986. Homeotic gene function in the muscles ofDrosophilalarvae. EMBO J. 5, 2321–2329.

Hoshizaki, D.K., Blackburn, T., Price, C., Ghosh, M., Miles, K., Ragucci,M., Sweis, R., 1994. Embryonic fat-cell lineage inDrosophilamelanogaster. Development 120, 2489–2499.

Knipple, D.C., MacIntyre, R.J., 1984. Cytogenetic mapping and isolationof mutations of theb-Gal-1 locus ofDrosophila melanogaster. Mol.Gen. Genet. 198, 75–83.

Lawrence, P.A., 1982. Cell lineage of the thoracic muscles ofDrosophila.Cell 29, 493–503.

Lawrence, P.A., Johnston, P., 1989. Pattern formation in theDrosophilaembryo: allocation of cells to parasegments byeven-skippedand fushitarazu. Development 105, 761–767.

Leptin, M., Grunewald, B., 1990. Cell shape changes during gastrulation inDrosophila. Development 110, 73–84.

Leptin, M., 1991.twist andsnailas positive and negative regulators duringDrosophilamesoderm development. Genes Dev. 5, 1568–1576.

Lohs-Schardin, M., Sander, K., Cremer, C., Cremer, T., Zorn, C., 1979.Localized ultraviolet laser microbeam irradiation of earlyDrosophilaembryos: fate maps based on location and frequency of adult defects.Dev. Biol. 68, 533–545.

Luer, K., Urban, J., Kla¨mbt, C., Technau, G.M., 1997. Induction of iden-tified mesodermal cells by CNS midline progenitors inDrosophila.Development 124, 2681–2690.

Meise, M., Janning, W., 1993. Cell lineage of larval and imaginal thoracicanlagen cells ofDrosophila melanogaster, as revealed by single-celltransplantations. Development 118, 1107–1121.

Meise, M., Janning, W., 1994. Localization of thoracic imaginal-disc pre-cursor cells in the early embryo ofDrosophila melanogaster. Mech.Dev. 48, 109–117.

Michelson, A.M., Abmayr, S.M., Bate, M., Martinez Arias, A., Maniatis,T., 1990. Expression of aMyoD family member prefigures musclepattern inDrosophilaembryos. Genes Dev. 4, 2086–2097.

Morata, G., Ripoll, P., 1975. Minutes: mutants ofDrosophila autono-mously affecting cell division rate. Dev. Biol. 42, 211–221.

Paterson, B.M., Walldorf, U., Eldridge, J., Du¨bendorfer, A., Frasch, M.,Gehring, W.J., 1991. TheDrosophila homologue of vertebrate myo-genic-determination genes. Natl. Acad. Sci. 88, 3782–3786.

Poodry, C.A., Schneiderman, H.A., 1970. The ultrastructure of the devel-oping leg of Drosophila melanogaster. Wilhelm Roux’s Arch. Dev.Biol. 166, 1–44.

Poulson, D.F., 1950. Histogenesis, organogenesis, and differentiation inthe embryo ofDrosophila melanogasterMeigen. In: Demerec, M. (Ed.),Biology of Drosophila. Hafner, New York, pp. 168–274 (reprinted1965).

Reed, C.T., Murphy, C., Fristrom, D., 1975. The ultrastructure of thedifferentiating pupal legs ofDrosophila melanogaster. Roux’s Arch.Dev. Biol. 178, 285–302.

Rehorn, K.P., Thelen, H., Michelson, A.M., Reuter, R., 1996. A molecularaspect of hematopoiesis and endoderm development common to verte-brates andDrosophila. Development 122, 4023–4031.

Reuter, R., Leptin, M., 1994. Interacting functions ofsnail, twist andhuckebeinduring the early development of germ layers inDrosophila.Development 120, 1137–1150.

Riechmann, V., Irion, U., Wilson, R., Grosskortenhaus, R., Leptin, M.,1997. Control of cell fates and segmentation in theDrosophilamesoderm. Development 124, 2915–2922.

Rizki, T.M., Rizki, R.M., 1978. Larval adipose tissue of homoeoticbithorax mutants ofDrosophila. Dev. Biol. 65, 476–482.

Rizki, T.M., 1978. The fat body. In: Ashburner, M., Wright, T.R.F. (Eds.),The Genetics and Biology ofDrosophila, Vol. 2b. Academic Press, NewYork, pp. 561–601.

Robertson, C.W., 1936. The metamorphosis ofDrosophila melanogaster,including an accurately timed account of the principal morphologicalchanges. J. Morphol. 59, 351–399.

Rushton, E., Drysdale, R., Abmayr, S.M., Michelson, A.M., Bate, M.,1995. Mutations in a novel gene,myoblast city, provide evidence insupport of the founder cell hypothesis forDrosophila muscledevelopment. Development 121, 1979–1988.

Sam, S., Leise, W., Hoshizaki, D.K., 1996. Theserpentgene is necessaryfor progression through the early stages of fat-body development. Mech.Dev. 60, 197–205.

Staehling-Hampton, K., Hoffmann, F.M., Baylies, M.K., Rushton, E.,Bate, M., 1994. dpp induces mesodermal gene expression inDrosophila. Nature 372, 783–786.

Szabad, J., No¨thiger, R., 1992. Gynandromorphs ofDrosophila suggestone common primordium for the somatic cells of the female and malegonads in the region of abdominal segments 4 and 5. Development 115,527–533.

Technau, G.M., 1987. A single cell approach to problems of cell lineageand commitment during embryogenesis ofDrosophila melanogaster.Dev. Biol. 100, 1–12.


Technau, G.M., Campos-Ortega, J.A., 1985. Fate-mapping in wild-typeDrosophila melanogaster. Roux’s Arch. Dev. Biol. 194, 196–212.

Tepass, U., Hartenstein, V., 1994. The development of cellular junctions inthe Drosophilaembryo. Dev. Biol. 161, 563–596.

Thisse, B., Stoetzel, C., El Messal, M., Perrin-Schmitt, F., 1987. Genes ofthe Drosophilamaternal dorsal group control the specific expression ofthe zygotic genetwist in presumptive mesodermal cells. Genes Dev. 1,709–715.

Thisse, B., Stoetzel, C., Gorostiza-Thisse, C., Perrin-Schmitt, F., 1988.

Sequence of thetwist gene and nuclear localization of its protein inendomesodermal cells of earlyDrosophila embryos. EMBO J. 7,2175–2183.

Warrior, R., 1994. Primordial germ cell migration and the assembly of theDrosophilaembryonic gonad. Dev. Biol. 166, 180–194.

Wieschaus, E., Gehring, W., 1976. Clonal analysis of primordial disc cellsin the early embryo ofDrosophila melanogaster. Dev. Biol. 50, 249–263.


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