View - Development - The Company of Biologists

Development 128, 233-242 (2001) 

Printed in Great Britain © The Company of Biologists Limited 2001 

DEV7838 

Hindgut visceral mesoderm requires an ectodermal template for normal 

development in Drosophila 

Beatriz San Martin and Michael Bate* 

Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 2EJ, UK 

*Author for correspondence (e-mail: cmb16@cus.cam.ac.uk) 

Accepted 7 November; published on WWW 21 December 2000 

SUMMARY 

During Drosophila embryogenesis, the development of the 

midgut endoderm depends on interactions with the 

overlying visceral mesoderm. Here we show that the 

development of the hindgut also depends on cellular 

interactions, in this case between the inner ectoderm and 

outer visceral mesoderm. In this section of the gut, the 

ectoderm is essential for the proper specification and 

differentiation of the mesoderm, whereas the mesoderm is 

not required for the normal development of the ectoderm. 

Wingless and the fibroblast growth factor receptor 

INTRODUCTION 

In the Drosophila embryo, the mesoderm becomes subdivided 

soon after gastrulation into populations of cells that will give 

rise to the various mesodermal derivatives (Bate, 1993). 

Although we now have a good understanding of the 

mechanisms by which mesodermal cell fates are allocated in 

the trunk (or segmented region) of the embryo (Baylies et al., 

1998; Frasch, 1999), little is known about how the terminal 

mesoderm is organised, or the mechanisms by which it is 

patterned. 

Visceral muscle forms the outer layer of the gut. It arises 

from clusters of cells in both the trunk and terminal mesoderm 

and develops in concert with the inner layer of the gut, which 

is composed both of endodermal cells (midgut) and ectodermal 

cells (foregut and hindgut). Two subtypes of visceral muscle 

envelop the midgut endoderm, the circular visceral muscle, 

which arises in the trunk of the embryo (Tremml and Bienz, 

1989; Azpiazu and Frasch, 1993), and the longitudinal visceral 

muscle, which arises from the caudal mesoderm (Georgias et 

al., 1997; Broihier et al., 1998; Kusch and Reuter, 1999). To 

emphasise their distinct origins, the subtypes of visceral 

mesoderm have been referred to as the trunk visceral 

mesoderm and caudal visceral mesoderm (Broihier et al., 1998; 

Kusch and Reuter, 1999). Since the hindgut visceral muscle 

also arises from the caudal mesoderm and the longitudinal 

visceral muscle eventually ends up in the trunk of the embryo, 

we feel that this terminology could lead to confusion. For these 

reasons, we would like to propose a simple and logical 

nomenclature for the different subtypes of visceral mesoderm 

in the Drosophila embryo. We have designated the circular 

233 

Heartless act over sequential but interdependent phases of 

hindgut visceral mesoderm development. Wingless is 

required to establish the primordium and to enhance 

Heartless expression. Later, Heartless is required to 

promote the proper differentiation of the hindgut visceral 

mesoderm itself. 

Key words: Drosophila, Embryo, Visceral mesoderm, Hindgut 

ectoderm, Cell signalling 

visceral mesoderm (which gives rise to the circular visceral 

muscle) as the CVM, the longitudinal visceral mesoderm 

(which gives rise to the longitudinal visceral muscle) as the 

LVM and the hindgut visceral mesoderm (which gives rise to 

hindgut visceral muscle) as the HVM. Finally, we suggest the 

abbreviation of FVM for the foregut visceral mesoderm, 

though consideration of this subtype of visceral mesoderm is 

beyond the scope of this paper. 

Most of our understanding of visceral mesoderm 

development has centred on the CVM. Several observations 

indicate that distinct mechanisms govern the development of 

the HVM. First, whereas the CVM is derived from 11 

metamerically repeated clusters of cells on either side of the 

embryo (Tremml and Bienz, 1989; Azpiazu and Frasch, 1993), 

the HVM arises from a single cluster of cells that migrates en 

masse over the hindgut ectoderm. Second, unlike the CVM, 

cells of the HVM primordium express relatively high levels of 

Twist (Dunin Borkowski et al., 1995; Baylies and Bate, 1996). 

One feature of the CVM is its role in promoting the 

development of the midgut endoderm. The endoderm is 

derived from anterior and posterior embryonic cells that 

invaginate during gastrulation (Skaer, 1993; Campos-Ortega 

and Hartenstein, 1997). To form the midgut endoderm, these 

cells migrate over a considerable distance before they meet in 

the centre of the embryo. The CVM serves as a substrate on 

which the endodermal cells migrate and later undergo a 

mesenchymal-epithelial transition (Reuter et al., 1993; Tepass 

and Hartenstein, 1994). In addition, the CVM provides the 

endoderm with anteroposterior (A/P) cues. Thus, signals from 

the visceral mesoderm are required for the expression of genes 

in discrete domains within the endoderm (Bienz, 1994; Bienz,

234 

B. San Martin and M. Bate 

1996; Szüts et al., 1998). The best known example of this is 

the regulation of labial expression in the endoderm by signals 

(Decapentaplegic (Dpp), Wingless (Wg) and Vein (Vn)) 

derived from the visceral mesoderm. 

Unlike the midgut endoderm, the hindgut ectoderm 

maintains its tubular structure throughout embryogenesis and 

is intrinsically patterned, as seen by the restricted expression 

of a number of segmentation genes (Skaer, 1993; Bienz, 1994; 

B. S. M. and M. B., unpublished observations). This leads us 

to suggest that one of the features distinguishing the 

development of the CVM from the HVM might be the direction 

of instructional interactions between the visceral mesoderm 

and inner epithelial layer of the gut. 

Here we show that, unlike midgut endoderm, the hindgut 

epithelium is essential for the proper specification and 

differentiation of the HVM. In addition, hindgut ectoderm can 

develop largely wild-type characteristics in the absence of 

visceral mesoderm. Of the three signalling molecules known 

to be expressed in the hindgut ectoderm, (Wg, Hedgehog (Hh) 

and Dpp; Hoch and Pankratz, 1996), only Wg is indispensable 

for the formation of differentiated HVM. Wg is required to 

enhance Twist expression in the HVM, mirroring its role in 

promoting the development of the somatic mesoderm in the 

trunk by maintaining enhanced levels of Twist expression 

(Baylies and Bate, 1996; Riechmann et al., 1997). Further, we 

show that a fibroblast growth factor (FGF) receptor, Heartless 

(Htl), is also essential in the development of the HVM. 

Wg and Htl are required during different but overlapping 

phases of HVM development. Wg acts early in both the hindgut 

ectoderm and mesoderm to establish the HVM primordium and 

promote its development. Wg also regulates the expression of 

htl, which is required later to promote the specification and 

differentiation of the HVM by activating the mitogen-activated 

protein kinase (MAPK) cascade. Our work indicates that 

during the development of the HVM, the hindgut ectoderm is 

required as a template and acts as a source of signalling by Wg 

and a presently unknown FGF signal. 

MATERIALS AND METHODS 

Fly stocks 

The following flies were used: Oregon R, a null allele of twist, twi ID96 

(Simpson, 1983), a null allele of wingless, wg CX4 (Baker, 1987), a 

temperature sensitive allele of wg, wg IL114 (Gonzalez et al., 1991), a 

hypomorphic allele of heartless, htl S1-28 (Shishido et al., 1997), a lossof-function 

allele of string, stg 9M53 (Hoch et al., 1994) and an 

amorphic allele of fork head, fkh 1 (Weigel et al., 1989). We also used 

the following GAL4 and UAS lines: 455.2GAL4 (Hinz et al., 1994), 

twistGAL4 (Baylies and Bate, 1996), 24BGAL4 (Brand and 

Perrimon, 1993), UASCD2 (Dunin Borkowski and Brown, 1995), 

UASreaper (John and Brand, 1997, from W. Gehring), UASarm S10C 

(an activated form of arm, Pai et al., 1997), UASDNHtl (lacks the 

tyrosine kinase domain, Beiman et al., 1996), UASKMRaf 3.1 (a 

dominant negative form of Raf; Roch et al., 1998), UASRas1 Act (an 

activated from of Ras; Gisselbrecht et al., 1996, from X. Lu and N. 

Perrimon) and UASwg E1 (full-length wg from K. Gieseler and J. 

Pradel). The wg CX4 , UASRas1 Act /CyO, ftzlacZ stock was a gift from 

A. Michelson (Carmena et al., 1998) and the wg CX4 , twistGAL4/CyO, 

ftzlacZ stock had previously been made in our laboratory by M. 

Baylies (Baylies et al., 1995). In most cases, balancers carrying lacZ 

inserts were used to discriminate homozygous mutant embryos by 

double staining with anti-β-galactosidase. For all GAL4/UAS 

experiments we took GAL4 females and mated them to the UAS lines. 

Unless otherwise stated, these crosses were kept at 29°C. 

Temperature shift experiments 

wg IL114 embryos produce viable larvae at 18ºC whereas at 25ºC they 

exhibit a wg null phenotype (Baker, 1988; Bejsovec and Martinez 

Arias, 1991). The null phenotype at 25ºC is due to the retention of 

Wg in the secretory apparatus (Gonzalez et al., 1991). Thus loss of 

functional Wg at the restrictive temperature depends on the half-life 

of the protein, which has been estimated to be approximately 20 

minutes (Skaer and Martinez-Arias, 1992). 

We kept the wg IL114 stock in large cages at 18ºC. Prior to collection 

of embryos, the apple juice agar plates were changed several times to 

ensure that the female flies did not retain fertilised embryos. wg IL114 

embryos from hourly collections at 18ºC were transferred to small 

Eppendorf tubes and placed in a PCR machine where they were kept 

at 18ºC until the desired stage, before shifting to 29ºC. The embryos 

were then left to develop to stage 14 before being fixed. As a control 

for timing embryos at different temperatures, Oregon R embryos from 

1 hour lays at 18ºC or 29ºC were transferred to a PCR machine and 

left to develop for a number of hours at 18ºC or 29ºC, respectively. 

The embryos were fixed and stained with anti-Wg antibody before 

calculating the stage of the earliest embryo. 

Histochemistry 

Whole-mount in situ hybridisation of a digoxigenin-labelled bagpipe 

probe (bap; Azpiazu and Frasch, 1993; Boehringer Mannheim) was 

performed according to the method of Tautz and Pfeifle (Tautz and 

Pfeifle, 1989) as modified by Ruiz-Gómez and Ghysen (Ruiz-Gómez 

and Ghysen, 1993). Immunological staining of whole-mount embryos 

was performed essentially as in Rushton et al. (Rushton et al., 1995) 

using the Vectastain Elite ABC kit (Vector Laboratories) with the 

slight modification that we simultaneously incubated both primary 

antibodies in double-labelling experiments. To double label for anti- 

Wg and bap we first immunologically stained the embryos, as 

described above, developing the stain without salts. This was followed 

by in situ hybridisation (see above), changing from Triton-X to Tween 

as the detergent in PBT. 

The following primary antibodies were used: anti-muscle Myosin 

(Kiehart and Feghali, 1986), anti-Wg (available from Developmental 

Studies Hybridoma Bank, from S. Cohen), anti-β-galactosidase 

(Cappel and Promega), anti-CD2, (Dunin Borkowski and Brown, 

1995), anti-Connectin (Meadows et al., 1994), anti-Twist (Roth et al., 

1989), anti-Heartless (Shishido et al., 1997), anti-Dichaete (Sanchez- 

Soriano and Russell, 1998) and anti-diphospho-ERK (Gabay et al., 

1997). 

Embryos were analysed using a Zeiss Axioplan microscope. Any 

negatives taken were scanned with a Nikon Coolscan. Other images 

were captured using a JVC analogue camera linked to a PC. Images 

and figures were processed in Adobe Photoshop 5.0. 

RESULTS 

The origin and development of the hindgut ectoderm 

and visceral mesoderm 

The caudal mesoderm gives rise to two populations of visceral 

mesoderm, the HVM and the LVM (Georgias et al., 1997; 

Broihier et al., 1998; Kusch and Reuter, 1999; B. San Martin, 

PhD thesis, University of Cambridge, 1999). The HVM 

primordium is distinguishable at stage 10 both by the expression 

of bagpipe (bap, Fig. 1A) and by virtue of its relatively higher 

levels of Twist (Fig. 1B). It includes all mesodermal cells caudal 

to the 15th Wg stripe (Fig. 1C), which, unlike mesoderm cells 

in the trunk (Dunin Borkowski et al., 1995), are organised in

multiple layers soon after gastrulation (not shown). In contrast, 

the LVM primordium arises from cells adjacent and eggposterior 

to the HVM (Broihier et al., 1998; B. San Martin, PhD 

thesis, University of Cambridge, 1999). 

The hindgut ectoderm is derived from a ring of cells that lies 

just anterior to the posterior midgut primordium at the cellular 

blastoderm stage (Hartenstein et al., 1985, see reviews by 

Skaer, 1993; Campos-Ortega and Hartenstein, 1997). At stage 

7, these cells invaginate into the embryo and, by stage 10, form 

a hollow tube that extends by cell division and rearrangement. 

HVM cells become closely associated with the invaginated 

hindgut ectoderm at stage 11 (Fig. 1D and see Fig. 6). Later in 

this stage, all HVM cells begin to express Connectin (Fig. 2G, 

Gould and White, 1992; Nose et al., 1992) and this together 

with Twist expression, can be used to follow the cells as they 

move over the hindgut ectoderm tube. 

As the germband retracts, the hindgut tube undergoes 

considerable morphological rearrangements. By stage 13, it 

lies longitudinally (arrow in Fig. 2F) from the anus and bends 

at right angles to join the posterior midgut. Over time, the 

bend flattens laterally and the hindgut lengthens, revealing 

morphological subdivisions (Hoch and Pankratz, 1996; B. S. 

M. and M. B., unpublished observations). HVM cells continue 

to cover the ectodermal tube during these stages and as they 

mature, they begin to express Myosin (Fig. 2M). 

The hindgut ectoderm develops independently of 

mesoderm 

To determine whether hindgut ectoderm requires interactions 

with mesoderm to form normally, we followed its development 

in twist mutant embryos that lack mesoderm (Simpson, 1983; 

Leptin and Grunewald, 1990; Leptin, 1991). In these embryos, 

cells of the hindgut ectoderm can form a tube that bends 

anteriorly, lengthens partially at stage 13 and expresses Wg and 

Dichaete in restricted domains, similar to wild-type embryos 

(Fig. 2B,D, Hoch and Pankratz, 1996; Sanchez-Soriano and 

Russell, 2000). Thus, characteristic features of ectodermal gut 

development occur in the complete absence of surrounding 

visceral mesoderm. 

Hindgut ectoderm is required for the development of 

the surrounding visceral mesoderm 

To test whether hindgut ectoderm is needed for the proper 

development of the HVM, hindgut ectoderm cells were 

selectively killed early in their embryogenesis using the 

GAL4/UAS system (Brand and Perrimon, 1993). We used 

455.2GAL4 (Hinz et al., 1994), which is expressed in the 

primordium of the hindgut ectoderm from stage 9 onwards but 

not in the caudal mesoderm (Fig. 2E,F), to drive expression of 

reaper, a gene whose protein product promotes death of those 

cells in which it is expressed (White et al., 1994; Nordstrom et 

al., 1996; White et al., 1996). 

As in wild-type embryos, Twist is expressed strongly in the 

prospective HVM at early stage 11 in embryos carrying 

455.2GAL4;UASreaper constructs (compare Fig. 1D with 2I). 

However, even though the morphology of the hindgut ectoderm 

appears normal at this stage (not shown), the bulk of the HVM 

is not closely associated with the hindgut ectoderm (Fig. 2I). 

By late stage 11, the number of HVM cells expressing 

Connectin (Fig. 2H) or Twist (not shown) is clearly reduced. 

This is concomitant with the severe disruption in the 

Cell interactions in gut development 

235 

Fig. 1. Origin of the HVM. (A-C) Stage 10 embryos. (A) bap 

expression is initiated in the primordium of the HVM at stage 10 

(arrow). (B) The primordium also expresses relatively high levels of 

Twist (arrow). (C) Double labelling for bap (blue) and Wingless 

(brown) shows that the primordium arises from mesoderm cells 

caudal to the 15th Wg stripe (arrow). The numbering refers to the wg 

stripes. (D) By stage 11, the high Twist-expressing HVM cells have 

begun to envelop the hindgut ectoderm (arrow). Anterior is towards 

the left. A,B,D show ventral-caudal views; C shows a lateral-caudal 

view. 

morphology of the hindgut ectoderm. During stage 13, many 

HVM cells die (Fig. 2K). The surviving cells are those that 

attach to any remaining hindgut ectoderm (Fig. 2L); these cells 

persist, differentiating to form a variable amount of visceral 

muscle (Fig. 2N,O). Thus, we conclude that the hindgut 

ectoderm acts as a template to promote the development and 

differentiation of the HVM. 

Wg signalling is essential for HVM development 

The hindgut ectoderm might be required simply because it is 

an essential substrate for the development of the HVM. To test 

whether it is also the source of essential signals, we analysed 

the formation of the HVM in embryos with mutations in dpp, 

hh and wg, which are known to be expressed in the hindgut 

ectoderm during embryogenesis (Hoch and Pankratz, 1996). 

Of these, only Wg is essential for the differentiation of the 

HVM (Fig. 3B and Fig. 6, not shown for Dpp and Hh). In 

embryos mutant for wg, bap expression is reduced (Fig. 3A) 

and Twist expression fails to be enhanced in the HVM at stage 

10 (not shown). By stage 11, even fewer cells express 

Connectin and Twist (not shown). By mid stage 12, some 

embryos completely lack Connectin and Twist expression in 

the caudal mesoderm (not shown), while in other embryos only 

a few cells maintain their expression and these cells are unable 

to cover the whole of the hindgut ectoderm (shown for 

Connectin in Fig. 3D). The few remaining cells may eventually 

express Myosin (Fig. 3B), but this is difficult to assess because 

some syncitial somatic muscle cells appear to attach 

ectopically to the hindgut ectoderm in these embryos. 

In wg embryos, proctodeal cells fail to divide after gastrulation 

(Skaer and Martinez-Arias, 1992). Thus, the hindgut ectoderm 

is very small and this reduction in the size could indirectly cause 

the defects observed in the development of surrounding visceral 

mesoderm. To test this, we compared the development of the 

HVM in string (stg) embryos, where all cells fail to divide after 

the cellular blastoderm stage (cycle 13, Edgar and O’Farrell, 

1989). As in wg embryos, a very small ectodermal hindgut 

develops, but many more visceral mesoderm cells maintain

236 


Fig. 2. (A-D) Hindgut ectoderm 

development in the absence of visceral 

mesoderm. (A,B) Wg expression in the 

hindgut ectoderm of wild-type and 

twi ID96 embryos, respectively (stage 13, 

lateral views). Asterisks mark the 

lumen of the gut. Arrows and 

arrowheads point to the posterior and 

anterior hindgut Wg domains, 

respectively. (C,D) Dichaete expression 

in the hindgut ectoderm of wild-type 

and Df(2R)S60/twi ID96 embryos, 

respectively (stage 15, dorsal view). 

Though the hindgut ectoderm fails to 

elongate properly in Df(2R)S60/twi ID96 

embryos, Dichaete expression is still 

restricted to the central portion of the 

hindgut (arrows mark the extent of this 

expression and asterisks mark the 

lumen of the gut). Df(2R)S60/twi ID96 

embryos fail to retract their germband 

fully and this may lead indirectly to 

defects in the morphology of the 

hindgut ectoderm. (E,F) 455.2GAL4 

drives expression in the hindgut 

ectoderm. CD2 expression in 

455.2GAL4; UASCD2 embryos at 

stage 10 (E) and stage 13 (F) (lateral 

views). The CD2 expression that is 

initiated in the primordium of the 

hindgut ectoderm (arrow in E) is 

maintained throughout embryogenesis 

(arrow in F). By stage 13 (F), CD2 

expression has spread to the posterior 

midgut (arrowhead) and some cells of 

the ventral midline (asterisk). 

455.2GAL4 does not drive expression 

in the caudal mesoderm (asterisk in E). 

(G-O) The hindgut ectoderm is needed 

for the proper specification and 

differentiation of the visceral 

mesoderm. (G,H) Connectin expression in wild-type and 455.2GAL4;UASreaper embryos at late stage 11 (lateral-caudal views). Fewer HVM 

cells express Connectin in 455.2GAL4;UASreaper embryos (compare arrows in G and H). (I) Twist expression in 455.2GAL4;UASreaper 

embryos at early stage 11 (ventral-caudal view). Twist is expressed strongly in the prospective HVM (arrow in I), but the majority of cells are 

not in close association with the hindgut ectoderm. (J-L) Connectin expression in wild type (J) and 455.2GAL4;UASreaper (K,L) embryos at 

stage 13. (J,K) Lateral views; (L) dorsal view. Arrow in K indicates a Connectin-expressing cell that is no longer attached to the hindgut 

ectoderm and, by its condensed morphology, appears to be dying (compare with wild-type cell, arrow in J). (L) Visceral mesoderm cells 

maintain Connectin expression when attached to hindgut ectoderm (arrow) even if only a small remnant remains (arrowhead). Asterisk marks 

the lumen of the remaining tube. (M-O) Myosin expression in the HVM at stage 15 (dorsal views, asterisks mark the hindgut lumen). (M) Wildtype 

embryo, arrow indicates the visceral muscle. (N,O) 455.2GAL4; UASreaper embryos raised at 25ºC, arrows point to the visceral muscle 

that surrounds the remnant of hindgut ectoderm. Anterior is towards the left. 

Connectin expression (compare Fig. 3D with 3E). These cells 

eventually envelop the whole of the ectodermal tube and 

differentiate to express Myosin (not shown). Thus, the defects 

in wg embryos cannot solely be explained by the reduced size 

of the hindgut ectoderm, suggesting that Wg signalling is 

required directly for the normal development of the HVM. 

Wg signalling activity is required within the 

mesoderm 

If Wg signals to the surrounding mesoderm and promotes the 

development of visceral mesoderm, then the Wg signalling 

pathway should be activated in the caudal mesoderm. 

Consequently, in wg embryos, the development of the HVM 

might be rescued if the Wg signalling pathway was activated 

autonomously throughout the mesoderm. One of the 

components needed to transduce the Wg/Wnt signalling 

pathway is Armadillo (Arm; McGrea et al., 1990; Peifer and 

Wieschaus, 1990). We used a constitutively active form of arm 

(arm S10C ) under the control of the UAS promoter (Pai et al., 

1997) and two mesodermal GAL4 drivers, twistGAL4 and 

24BGAL4 (Brand and Perrimon, 1993; Baylies and Bate, 

1996), to activate the Wg pathway in the mesoderm. 

twistGAL4 activates expression throughout the mesoderm 

from stage 6 onwards and is maintained in the HVM 

throughout embryogenesis (B. S. M. and M. B., unpublished 

observations). In contrast, 24BGAL4 drives weak and patchy


Fig. 3. Wg signalling is necessary for 

the development of the HVM. (A) In 

wg − embryos, bap expression is 

reduced in the primordium of the HVM 

at stage 10 (arrow, ventral-caudal 

view). (B) Myosin expression in a wg − 

embryo. Though it is likely that some 

HVM fibres differentiate over the 

reduced hindgut ectoderm, this is 

generally obscured by syncitial somatic 

muscle cells attaching to the hindgut 

ectoderm; the arrow indicates one such 

syncitial muscle (stage 15, dorsal view, 

asterisk marks the lumen of the gut). 

(C-I) Lateral views of Connectin 

expression at mid-stage 12, arrows 

indicate the HVM. (C) Wild-type 

embryo; visceral mesoderm envelops 

the hindgut ectoderm (arrow). Whereas 

no, or very few (arrow), caudal 

mesoderm cells express Connectin in 

wg − mutant embryos (D), more cells 

express Connectin in stg − (E) and fkh 1 

(F) embryos. There is some rescue of 

Connectin expression in the HVM of 

wg − embryos that express arm S10C 

under the control of twistGAL4 (G) or 

455.2GAL4 (I), but not 24BGAL4 (H). 

(J-L) Lateral views of Connectin 

expression at stage 14 in wild-type (J) 

and wg IL114 (K,L) embryos; arrows 

indicate the HVM and arrowheads 

mark the extent of the hindgut 

ectoderm. (K) Only a few mesoderm 

cells maintain Connectin expression around the hindgut when wg IL114 embryos are raised at the restrictive temperature throughout 

embryogenesis. (L) When Wg function is removed at mid stage 11 by shifting wg IL114 embryos to 29ºC early in stage 11, the morphology of the 

hindgut ectoderm is improved and visceral mesoderm cells envelop the hindgut tube. Anterior is towards the left. 

expression at stage 9 (B. S. M. and M. B., unpublished 

observations), which is reinforced by late stage 10, and spreads 

to most mesoderm cells, including those of the HVM. 

Expression of arm S10C under the control of twistGAL4, but 

not 24BGAL4, partly rescues the development of the HVM in 

wg embryos as seen by an increase in the expression of 

Connectin at stage 12 (Fig. 3G,H). One explanation for the 

difference between the two drivers is that expression of 

arm S10C driven by 24BGAL4 is too late to rescue HVM 

development. However, twistGAL4 also drives transient and 

weak expression in the hindgut ectoderm (B. S. M. and M. B., 

unpublished observations), and it could be this expression that 

provides the rescuing effect. 

To clarify the results of the rescue experiments, we activated 

the Wg signalling pathway solely in the hindgut ectoderm of 

wg embryos using 455.2GAL4 (Fig. 2E,F). In these embryos, 

more HVM cells express Connectin at stage 12 than in wg 

embryos (compare Fig. 3D with 3I) and these cells eventually 

cover the whole of the hindgut ectodermal template (not 

shown). As the size of the hindgut ectoderm does not appear 

to be significantly rescued in these embryos, we suggest that 

it acquires the necessary properties to support visceral 

mesodermal development throughout its length. Importantly 

however, despite the continuous expression of arm S10C 

throughout the hindgut ectoderm, the rescue observed is not 

as strong as when using the twistGAL4 driver (Fig. 3G). Thus, 

237 

in wg embryos, both hindgut ectoderm and mesoderm respond 

to the activation of the Wg signalling pathway and promote 

the development of the HVM. 

Non-hindgut ectodermal sources of Wg can promote 

the development of the HVM 

Although wg is expressed in the primordium of the hindgut 

ectoderm, it is also expressed in epidermal stripes and transiently 

in the mesoderm (Baker, 1987; Baker, 1988; van den Heuvel et 

al., 1989; Gonzalez et al., 1991; Ingham and Hidalgo, 1993; 

Lawrence et al., 1994). Any of these sources of Wg might 

promote the development of the HVM. Indeed, in fork head (fkh) 

embryos, which lack Wg expression in the hindgut ectoderm 

(Hoch and Pankratz, 1996; B. S. M. and M. B., unpublished 

observations) many more caudal mesoderm cells begin to 

express Connectin (Fig. 3F) than in wg embryos (Fig.3D). By 

stage 12 however, Connectin expression begins to diminish quite 

rapidly in fkh embryos, but this is most likely due to the loss of 

hindgut ectoderm at later stages (Wu and Lengyel, 1998; B. S. 

M. and M. B., unpublished observations). This suggests that at 

least for the early stages of HVM development, the source of 

Wg need not be the hindgut ectoderm alone. 

Loss of Wg signalling after stage 11 does not affect 

HVM development 

Ablation of the hindgut ectoderm through directed expression

238 


Fig. 4. Htl is necessary for the 

development of the HVM. Although in 

htl − embryos, bap expression is initiated 

normally in the primordium of the HVM 

at stage 10 (arrow in A, ventral-caudal 

view), Connectin expression is reduced 

from its outset (arrow in B, lateral views 

of a mid-stage 11 embryo). (C) Dorsal 

view of Myosin expression in htl − 

embryos at stage 15. The hindgut lacks 

differentiated visceral muscle (asterisk 

marks the lumen of the hindgut). (D) Htl 

expression in wild-type embryos at 

stage 10; arrow indicates prospective 

HVM cells which express Htl strongly 

(ventral-caudal view). (E,F) Expression 

of diphospho-ERK in wild-type and htl − 

embryos respectively (stage 11, lateralcaudal 

views). Diphospho-ERK 

expression is reduced in the HVM of 

htl − embryos (compare arrows in E and 

F). (G-I) Lateral views of Connectin 

expression at mid-stage 12, arrows point 

to the HVM. (G) The htl mutant 

phenotype can be rescued by driving 

expression of Ras* in the mesoderm. (H,I) Fewer caudal mesoderm cells express Connectin when either KMRaf3.1 (H) or DNhtl (I) expression 

is driven in the mesoderm of wild-type embryos (cf. Fig. 3C). Anterior is towards the left. 

of reaper reveals that the HVM needs to be in contact with the 

hindgut ectoderm to differentiate (Fig. 2). Wg signalling might 

mediate this requirement for the hindgut ectoderm. To test this, 

we used the temperature-sensitive wg IL114 allele (Lindsley and 

Zimm, 1992) to remove Wg function after the HVM has begun 

to migrate over the hindgut ectoderm (mid stage 11, see 

Materials and Methods). 

As expected, more cells develop as hindgut ectoderm when 

Wg function is first removed at mid stage 11 (Fig. 3L), 

compared with wg IL114 embryos raised at the restrictive 

temperature throughout (Fig. 3K). In addition, Connectin 

expressing visceral mesoderm cells envelop the hindgut 

ectoderm (Fig. 3L). Thus, loss of Wg after the initial HVM 

migration over the hindgut ectoderm does not prevent HVM 

cells from developing further. 

Htl is essential for the development of the HVM 

Since Wg is not required after mid stage 11, the requirement 

for the hindgut ectoderm could reflect the need for a second 

signalling pathway in the interactive process that underlies 

HVM differentiation. Indeed we find that htl, which encodes 

the FGF receptor tyrosine kinase DFR1 (Shishido et al., 

1993; Beiman et al., 1996; Gisselbrecht et al., 1996; Shishido 

et al., 1997), is essential for HVM development (Fig. 4 and 

see Fig. 6). Although the development of the hindgut 

ectoderm appears relatively normal in htl embryos, hindgut 

visceral muscle fails to differentiate altogether (Fig. 4C). The 

early stages of HVM development appear relatively normal, 

thus at stage 10, bap expression is established normally in 

the caudal visceral mesoderm (Fig. 4A). Defects in HVM 

development first appear at stage 11, once the HVM has 

begun to move over the hindgut ectoderm. Initial Connectin 

expression is reduced both in intensity and in cell number 

(Fig. 4B), while Twist expression is rapidly lost from HVM 

cells late in stage 11 (not shown). Expression of both markers 

is lost completely between stages 12 and 13 (not shown). 

Thus, Htl is necessary to promote the development of the 

HVM as it migrates over the hindgut ectoderm and to allow 

its further differentiation. 

Htl functions through the activation of the MAPK 

signalling cascade 

Htl is initially expressed throughout the mesoderm, though its 

expression is particularly strong in the primordium of the HVM 

(Fig. 4D) where it is maintained throughout embryogenesis 

(not shown). This is consistent with the possibility that the 

visceral mesoderm responds to an FGF signal from the hindgut 

ectoderm. If so, we would also expect activation of signalling 

cascades downstream of Htl in HVM cells. 

Receptor tyrosine kinase activation can trigger a number of 

signalling cascades (Ullrich and Schlessinger, 1990), though 

these kinases generally activate the MAPK signalling 

pathway that uses the MAPK kinase (MEK) and extracellular 

signal-regulated kinase (ERK) isoforms (see reviews by 

Cobbs and Goldsmith, 1995; Seger and Krebs, 1995). 

Activation of this cascade can be followed by staining 

embryos against the double phosphorylated form of ERK 

(diphospho-ERK, Gabay et al., 1997). We find that 

diphospho-ERK is expressed weakly in HVM cells between 

stage 10-12 (Fig. 4E) and that this expression is reduced in 

htl embryos (Fig. 4F). 

That Htl functions through the activation of the MAPK 

pathway in the development of the HVM is suggested by two 

additional observations. First, the loss of Connectin and 

Myosin expression in the HVM of htl embryos can be 

partially rescued when an activated form of Ras (a 

component of the MAPK signalling pathway) is expressed 

throughout the mesoderm (Fig. 4G, not shown for Myosin). 

Secondly, targetted mesodermal expression of a dominant 

negative form of Htl (DNHtl, Beiman et al., 1996) or of a

Fig. 5. Wg and Htl are needed over sequential and overlapping 

phases of HVM development. (A) Htl expression at stage 10 in wg − 

embryos (ventral-caudal view). The caudal-most Htl expression in 

the prospective HVM is not enhanced and all mesoderm cells appear 

to express similar levels (arrow, compare with Fig. 4D). 

(B-D) Lateral and caudal views of Connectin expression at mid-stage 

12, arrows in C,D indicate the HVM. (B) All wg − ;htl − embryos lack 

Connectin expression in the caudal mesoderm. (C) There is weak 

expression of Connectin in htl − embryos. (D) 455.2GAL4-driven 

expression of wg E1 in htl − embryos can rescue some of the caudal 

mesodermal Connectin expression. Anterior is towards the left. 

dominant negative form of Raf (also a component of the 

MAPK signalling cascade), both lead to a reduction in the 

number of Connectin-expressing HVM cells (Fig. 4H,I, cf. 

Fig. 3C). 

Wg and Htl act over sequential and overlapping 

phases of HVM development 

Our analysis shows that the establishment of the HVM 

primordium at stage 10 relies on Wg but not on Htl. 

Consequently, there is an early Htl-independent role of Wg. 

However, in this first phase of HVM development, Wg is also 

required for the normal development of Htl expression in the 

HVM and in wg embryos, expression of Htl in the cells that 

normally give rise to the HVM does not become enhanced at 

stage 9-10 (Fig. 5A). 

At stage 11 and 12, Wg and Htl partly function 

independently of each other in controlling Connectin 

expression in the HVM. Thus, the loss of Connectin 

expression in the HVM is more severe in embryos mutant for 

both wg and htl than in either wg or htl mutant embryos. All 

wg;htl double mutant embryos lose expression of Connectin 

in the HVM by stage 12 (Fig. 5B), whereas at the same stage, 

some wg embryos maintain Connectin expression in a few 

cells (Fig. 3D) and there is weak expression of Connectin in 

htl embryos (Fig. 5C). If Wg can act in parallel with Htl to 

promote Connectin expression in the HVM, this explains why 

misexpression of Wg throughout the hindgut ectoderm of a 

htl mutant embryo directs strong expression of Connectin in 

HVM cells at stage 12 (Fig. 5D). 

Differentiation of the HVM requires Htl (Fig. 4C). Wg is not 

required past stage 11 for the continued development of the 

HVM (Fig. 3L), and the HVM fails to differentiate and express 

Myosin when Wg is misexpressed in the hindgut ectoderm of 

a htl mutant embryo (not shown). 

DISCUSSION 


239 

Fig. 6. Phases of HVM development. The HVM primordium can be 

distinguished at the onset of bap expression. Wg function (green 

arrow) is required from this first stage until after the HVM cells have 

begun to contact the hindgut ectoderm. Htl function (purple arrow) is 

required once the HVM cells become closely associated with the 

hindgut ectoderm at the onset of Connectin expression. There is a 

time window during which Wg and Htl function in parallel. Later Htl 

is also needed for the differentiation of the HVM. 

Distinct mechanisms govern the development of the 

visceral mesoderm in the midgut and hindgut 

The gut in Drosophila consists of two cell layers, an inner 

epithelial layer and an external muscle layer. Our work shows 

that interactions between the inner ectodermal layer of the 

hindgut and the surrounding mesoderm are essential for the 

normal specification and differentiation of the HVM. This is 

very different from the development of the CVM, which, in the 

absence of midgut endoderm (e.g. in serpent mutant embryos; 

Reuter, 1994), can develop normally until at least late stage 12 

(B. San Martin, PhD thesis, University of Cambridge, 1999). 

The differences between the development of the CVM and 

the HVM reflect the way in which they form. The CVM arises 

from metamerically repeated clusters of cells, from 

parasegment 2-12 (Tremml and Bienz, 1989; Azpiazu and 

Frasch, 1993), which on allocation may already have acquired 

information as to their relative position in the A/P axis. These 

cells form a continuous template in the A/P axis over which 

the midgut endoderm spreads before coalescing as a tube. The 

HVM, by contrast, develops as a single cluster of cells that 

employs the ectoderm of the hindgut as a template over which 

to spread and differentiate. 

The development of the midgut endoderm and 

hindgut ectoderm 

Whereas the hindgut ectoderm can develop fairly normally in 

the absence of visceral mesoderm, the development of the 

midgut endoderm requires the presence of the CVM. This may 

reflect distinct morphogenetic events in the development of the 

hindgut and midgut. Anterior and posterior midgut endoderm

240 


originate as clusters of cells and migrate over a considerable 

distance before they meet in the centre of the embryo (Skaer, 

1993; Campos-Ortega and Hartenstein, 1997). Thus the 

endoderm is probably not intrinsically patterned, at least in the 

A/P axis (Bienz, 1994). The circular midgut visceral 

mesoderm, which arises locally in a metamerically repeated 

fashion, not only acts as a scaffold for the migration of the 

endoderm but also provides A/P patterning cues (Reuter et al., 

1993; Bienz, 1994; Bienz, 1996; Tepass and Hartenstein, 

1994). In contrast to the midgut endoderm, the hindgut 

ectoderm maintains its tubular structure throughout 

embryogenesis and is intrinsically patterned, as seen by the 

restricted expression of a number of segmentation genes 

(Skaer, 1993; Bienz, 1994, B. S. M. and M. B., unpublished 

observations). Consequently, the hindgut ectoderm can develop 

independently of surrounding visceral mesoderm. 

The control of HVM development 

The control of HVM development by Wg and Htl is sequential 

and interdependent (Fig. 6). At stage 10, Wg, secreted from 

multiple sources, promotes the normal formation of the HVM 

primordium and enhances Htl expression in this tissue. 

However Htl itself is not needed until stage 11 for the normal 

development of the HVM. 

As in the embryonic somatic mesoderm, Wg may regulate 

Htl expression indirectly by first enhancing Twist expression. 

At stage 10, Twist is expressed at high and low levels in 

adjacent metamerically repeated domains (Dunin Borkowski et 

al., 1995). In the trunk mesoderm, modulation of this pattern 

of Twist expression depends on wg function (Bate and 

Rushton, 1993; Riechmann et al., 1997) and is thought to lead 

to the differential expression of Htl (Shishido et al., 1997). 

Twist expression is also modulated in the caudal mesoderm 

with the HVM cells expressing Twist most strongly (B. San 

Martin, PhD thesis, University of Cambridge, 1999). This 

strong Twist expression is lost in wg mutants as is the strong 

expression of Htl (B. San Martin, PhD thesis, University of 

Cambridge, 1999). 

Although Htl expression in the HVM depends on Wg, both 

Wg and Htl also function independently of each other in 

promoting the development of the HVM. Thus wg;htl double 

mutants exhibit a more severe phenotype than wg or htl 

mutants and loss of htl can be partly rescued by misexpression 

of Wg throughout the hindgut ectoderm. Later, Htl promotes 

the differentiation of the HVM whereas Wg is not required in 

this last phase of HVM development and is unable to substitute 

for Htl. 

The subdivision of the caudal mesoderm 

Although the primordia of the HVM and LVM arise next to 

each other, Twist is expressed at relatively high levels in the 

HVM but at relatively low levels in the LVM. As in the trunk 

of the embryo, this modulation of Twist expression is necessary 

for the correct allocation of mesodermal cell fates (Baylies and 

Bate, 1996; B. San Martin, PhD thesis, University of 

Cambridge, 1999). Thus, reduced Twist function appears to 

lead to a reduction in the HVM and overexpression of twist 

leads to a reduced LVM primordium (B. San Martin, PhD 

thesis, University of Cambridge, 1999). 

wg is required for the correct allocation of alternative cell 

fates in the trunk mesoderm. If wg were to play a similar role 

in the caudal mesoderm we would expect an expansion in the 

primordium of the LVM at the expense of the HVM in wg 

mutant embryos. This is not the case however, and the LVM 

primordium is specified normally in wg mutant embryos (B. 

San Martin, PhD thesis, University of Cambridge, 1999). 

Instead, it seems that the allocation of these two cell fates 

depends on the terminal class genes and their downstream 

targets (B. San Martin, PhD thesis, University of Cambridge, 

1999). 

htl also appears to play distinct roles in the trunk and caudal 

mesoderm. In htl embryos, the uncoordinated migration of the 

trunk mesoderm at stage 10 leads to a reduction in dorsal 

mesodermal cell fates (Beiman et al., 1996; Gisselbrecht et al., 

1996; Shishido et al., 1997). In contrast however, the primordia 

of the HVM and LVM are specified normally in htl mutant 

embryos (B. San Martin, PhD thesis, University of Cambridge, 

1999). 

Extrinsic factors in the development of the 

mesoderm 

The patterning of the trunk mesoderm depends on the 

integration of a combination of factors that are intrinsic and 

extrinsic to the mesoderm (Baylies et al., 1998; Frasch, 1999). 

Cells that are ectodermal in origin are major sources of 

inductive signals. First, the ectoderm is an essential source of 

inductive signals that reinforce A/P patterning and subdivide 

the mesoderm along the dorsoventral axis (Bate and Rushton, 

1993; Staehling-Hampton et al., 1994; Baylies et al., 1995; 

Frasch, 1995; Bate and Baylies, 1996; Riechmann et al., 1998). 

Second, the specification of the ventral mesodermal glial cell 

fate requires inducing activity from midline cells of the central 

nervous system, and these cells are also ectodermal in origin 

(Lüer et al., 1997). 

Our work indicates that the initial development of the 

HVM depends on the ectoderm of the developing hindgut. It 

is probably the case that the entire gut ectoderm functions as 

a source of signals necessary for the development of 

overlying visceral mesoderm. Indeed, the FVM fails to form 

in the absence of foregut ectoderm (B. San Martin, PhD 

thesis, University of Cambridge, 1999). Furthermore, since 

both wg and htl are required for the normal specification of 

FVM and HVM, it may well be that similar mechanisms 

operate in the development of both these tissues. Later in 

development, we find that the HVM is subdivided by 

localised patterns of gene expression that may represent 

different functional domains of the gut (B. S. M., unpublished 

observations). It will be important to show whether, like the 

early development of the HVM, these later refinements in the 

patterning process depend on signals derived from the 

ectoderm of the gut. 

We thank Helen Skaer and Matthias Landgraf for their helpful 

comments on the manuscript, as well as Mar Ruiz Gomez, Alfonso 

Martinez Arias, Fernando Roch and Keith Brennan for useful 

discussions. We would also like to thank the following people who 

generously provided us with reagents and fly stocks: Alfonso 

Martinez Arias, Keith Brennan, Mary Baylies, Jordi Casanova, Kaoru 

Saigo, Helen Skaer, Talila Volk, Andrea Brand, Enrique Martin 

Blanco, Thomas Klein, Claire Ainsworth, Rob White, Dan Keihart, 

Siegfried Roth, Uwe Hinz and Steve Russell. This work was supported 

by a Wellcome Trust Prize studentship and fellowship to Beatriz San 

Martin.

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