1 Department of Molecular and Cell Biology, 385 LSA, University of California,
Berkeley, CA 94720-3200, USA
2 Section of Molecular Cell and Developmental Biology, Institute for Cellular
and Molecular Biology, University of Texas, Austin, Texas 78712, USA
3 Western Regional Research Center, ARS, USDA, Albany, CA 94710, USA
* Present address: Department of Biological Sciences, Stanford University, CA
94305, USA
Present address: Cancer Research Laboratory, University of California,
Berkeley 94720,USA
Author for correspondence (e-mail:
weisblat{at}uclink4.berkeley.edu)
Accepted 17 January 2003
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SUMMARY |
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Key words: hedgehog, Helobdella robusta, Gut formation, Cell signaling
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INTRODUCTION |
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The consensus HH protein contains a secreted domain
(NH2-terminal fragment; HH-N) that functions in extracellular
signaling pathways and a C-terminal domain (HH-C) that is involved in
intracellular post-translational autoprocessing and covalent attachment of
cholesterol (Chuang and Kornberg,
2000; Lee et al.,
1994
). Sequence alignments among known hh homologs reveal
that the HH-N domains are usually more conserved than HH-C domains.
Current molecular phylogenies support the organization of all or most
extant bilaterian animals into 3 ancient clades, Deuterostomia, Ecdysozoa and
Lophotrochozoa (Adoutte et al.,
2000; Aguinaldo et al.,
1997
). Until recently, all of the hh-class genes reported
in the literature were from members of the former two clades. The recent
discovery that a hh-class gene from the limpet Patella
vulgata (Lophotrochoazoa) is expressed along the ventral midline has been
interpreted as supporting the dorsoventral axis inversion theory and as
supporting a role for hh-class genes in neural patterning
(Nederbragt et al., 2002
), but
functional tests of these ideas are yet to be reported. Thus, the question of
which aspect(s) of hh-class gene function are conserved between
Lophotrochozoans and the other two groups remain of interest, both for
deducing the nature of early bilaterian ancestors and for understanding the
evolution of body plans by modification of developmental mechanisms.
For example, in Drosophila embryos hh plays a pivotal
role in segmentation. The hh gene is co-expressed with
engrailed (en) in the posterior compartment of each nascent
segment, and the secreted HH protein functions to specify the fates of cells
in the adjoining anterior compartment in a concentration-dependent manner
(Heemskerk and DiNardo, 1994;
Lee et al., 1992
). Later in
Drosophila development, the hh and en genes are
also expressed in identical compartment-specific patterns in the imaginal
discs (Tabata and Kornberg,
1994
). An en-class gene has been described in
glossiphoniid leeches, a group of segmented lophotrochozoans (phylum Annelida,
class Hirudinea). But while leech-en is expressed during segmentation
and neurogenesis (Lans et al.,
1993
; Wedeen and Weisblat,
1991
), more recent work suggests that it does not play a direct
role in segmentation (Shain et al.,
1998
) and that the cells that express leech en are not
required to specify the fates of cells in the remainder of the segment
(Seaver and Shankland, 2001
).
These observations raise the question as to whether hh-class genes
are expressed coincident with en-class genes in the leech embryo,
and, if so, how that expression might relate to segmentation.
Here, we report the identification and characterization of a hh
homolog (Hro-hh) from the glossiphoniid leech, Helobdella
robusta. Hro-hh is expressed zygotically in gut and some other tissues,
but Hro-hh RNA was not detected in the cells that express
Hro-en during segmentation. In addition, we found that
bath-application of cyclopamine, a known blocker of hh signaling in
vertebrates (Helms et al.,
1997; Incardona et al.,
1998
; Incardona et al.,
2000
) induced malformation of foregut, anterior midgut and
coelomic mesenchyme in Helobdella, but had no apparent effect on the
segmental patterning of mesoderm and ectoderm. Along with data on the function
of hh-class genes in vertebrates and insects, our results support a
scenario in which the ancestral role of hedgehog family genes in
bilaterian animals was associated with gut formation and/or neural
differentiation, rather than segmentation.
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MATERIALS AND METHODS |
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The relevant aspects of glossiphoniid leech development are illustrated in
Fig. 1. During cleavage (stages
1-7), 5 bilateral pairs of stem cells (M, N, O/P, O/P and Q teloblasts), 3
macromeres (A''', B''' and
C''') and 25 micromeres are formed. Teloblasts undergo
repeated, highly unequal divisions to generate columns (bandlets) of founder
cells (blast cells) for segmental mesoderm and ectoderm in an
anterior-to-posterior progression (stages 5-8). The five bandlets on each side
of the embryo join together in parallel to form the germinal bands, which then
coalesce from anterior to posterior along the ventral midline into the
germinal plate (stage 8); the segmented nervous system, nephridia, epidermis,
and musculature differentiate from the germinal plate during stages 9-11.
Micromeres contribute definitive progeny to non-segmented tissues such as the
supraesophageal ganglion and the foregut of the adult leech, and also a
squamous epithelium that covers the germinal bands and the intervening
prospective dorsal territory during gastrulation
(Fig. 1)
(Bissen and Weisblat, 1989;
Weisblat et al., 1984
). The
midgut epithelium forms by cellularization of a syncytial yolk cell (SYC),
that arises by the stepwise fusion of the
A'''-C''' macromeres, teloblasts (after
they have completed blast cell production), and supernumerary blast cells
(Fig. 1) (Desjeux and Price, 1999
;
Isaksen et al., 1999
;
Liu et al., 1998
;
Nardelli-Haefliger and Shankland,
1993
).
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Isolation of Hro-hh
An initial fragment of Hro-hh was amplified from an H.
robusta cDNA library (embryonic stages 7-10; Stratagene) by the
polymerase chain reaction (PCR), using the following degenerate primers:
The PCR reaction mixture contained 50 mM KCl, 10 mM Tris, pH 9.0, 2.5 mM
MgCl2, 0.1% Triton X-100, 0.2 mM each dNTP, 0.8 mM each primer, 1
µl H. robusta cDNA library (1010 pfu/ml;
Stratagene), and 1 unit Taq polymerase (Perkin-Elmer) in a 50 µl volume.
For the amplification, we employed a 3-step `touchdown' protocol: 5 cycles of
94°C for 1 minute, 53°C for 1 minute, 72°C for 1 minute; 10 cycles
of 94°C for 1 minute, 50°C for 1 minute, 72°C for 1 minute; then
20 cycles of 94°C for 1 minute, 48°C for 1 minute, 72°C for 1
minute. The amplified DNA was purified from an agarose gel using Gel
Extraction Kit (Qiagen) and cloned into pGEM T easy vector (Promega).
The 150 bp Hro-hh sequence was amplified and labeled with [32P]dCTP from cloned pGEM T easy plasmid by PCR. We used this probe to screen an H. robusta cDNA library (stages 7-10; Stratagene). Phage plating and hybridization were carried out according to manufacturer's instructions with minor modifications. Hybridization was performed at 45°C in 6x SSC, 5x Denhardt's solution, 0.5% SDS, 500 µg/ml herring sperm DNA and 50% deionized formamide. The filters were washed in 2x SSC, 0.5% SDS at 65°C for 1 hour and in 0.1x SSC, 0.1% SDS for 3 hours. pBlueScript SK() phagemids were excised in vivo according to the manufacturer's instructions. A plasmid (pHrohh) containing 3.49 kb Hro-hh fragment was sequenced. This sequence (GenBank accession number AF517943) was confirmed by RT-PCR from cDNA prepared from total RNA as described below.
Independently, another hh-class gene fragment was isolated by degenerate PCR from the Helobdella population collected in Austin, Texas, using primers corresponding to nucleotides 934-952 of Hro-hh in the forward direction and nucleotides 1060-1077 in the reverse direction, as follows:
From the 107 nucleotide sequence obtained, there was only a single synonymous change (position 1053). In keeping with this high degree of sequence identity, the same probe was used for in situ hybridization experiments on both types of embryos and the results obtained were indistinguishable.
Developmental RT-PCR
Total RNA samples were prepared from H. robusta embryos at
selected stages with RNA Wiz (Ambion) according to the manufacturer's
instructions, using 40 embryos for each sample. The RNA samples were added to
solutions containing 1x reverse transcription buffer (Gibco), 3.33 mM
DTT, 0.33 mM dNTP, 3.33 µM random decamer (Ambion), and 200 Units reverse
transcriptase (Gibco). The mixture was incubated at 42°C for 50 minutes
and the resultant first strand cDNA (30 µl) was stored at 20°C.
PCR conditions were as described by the manufacturer of ampliTaq Polymerase
(Perkin-Elmer Cetus, Norwalk, CT) except that 3 µl of cDNA template were
used in 50 µl of reaction mixture.
To amplify an Hro-hh-specific fragment (96 bp in length), a pair of PCR primers was designed as follows:
PCR conditions for amplifying the Hro-hh fragment were: 1 minute at 94°C, 1 minute at 60°C, and 1 minute at 72°C for 5 cycles, followed by 1 minute at 94°C, 1 minute at 58°C, and 1 minute at 72°C for 17 cycles. A 15 µl aliquot of each sample was removed after 22 cycles and the remaining material underwent 5 more cycles of amplification.
As an internal standard to adjust for differences in efficiency of RNA extraction between samples, a 488 bp fragment of 18S rRNA was amplified in parallel to each Hro-hh sample. For this purpose, and to attenuate the signal obtained from the abundant rRNAs, bona fide and non-extending 18S primers (competimers; Ambion) were used in a 3:7 ratio, respectively. PCR conditions for amplifying the 18S rRNA fragment were: 1 minute at 94°C, 1 minute at 58°C and 1 minute at 72°C for 1 minute for 5 cycles, followed by 1 minute at 94°C, 1 minute at 56°C, and 1 minute at 72°C for 10 cycles. A 15 µl aliquot of each sample was removed after 15 cycles and the remaining material underwent 5 more cycles of amplification.
To quantitate PCR products, each sample was run out in a 2% agarose gel and stained with ethidium bromide. Band intensity was measured with an Alphaimager (Alpha Innotech Corp.) using Alphaease (v3.3b) program.
In situ hybridization
In situ hybridization was performed using a digoxigenin-labeled RNA probe
with some modifications of methods described previously
(Harland, 1991;
Nardelli-Haefliger and Shankland,
1992
). Sense and antisense probes (each of 3.6 kb length) were
obtained by T7 and T3 in vitro transcription (MEGAscript kit, Ambion) using
linearized pHrohh (cut with BamHI to generate the sense probe, and
with SalI to generate the antisense probe) and subsequently
hydrolyzed into shorter fragments (
300 bp) in an alkaline solution
(Cox et al., 1984
).
Early embryos (stage 1-8) were fixed in 4% formaldehyde in PBS (130 mM NaCl, 10 mM phosphate buffer, pH 7.4) for 1 hour, then permeabilized by a 5-minute incubation with 50 µg/ml proteinase K (Gibco); late embryos (stages 9-11) were fixed as described above for 30 minutes, then treated for 25 minutes in 0.5 mg/ml Pronase E (Sigma).
Hybridization was carried out overnight at 59°C in a 1:1 mixture of deionized formamide and 5x SSC, 0.2 mg/ml tRNA, 0.1 mg/ml heparin, 1x Denhardt's solution, 0.1% Tween 20 and 0.1% Chaps. Alkaline phosphatase (AP)-conjugated anti-digoxigenin Fab fragments (Roche) were added to a dilution of 1:5000 in 1x PBS, 0.1% Tween 20 for late embryos. To remove unhydrolyzed probe, embryos were treated with RNase A (Sigma; 50 µg/ml for 1 hour for early embryos and 5 µg/ml for 30 minutes for late embryos) at 37°C. Alkaline phosphatase (AP)-conjugated anti-digoxigenin Fab fragments (Boehringer-Mannheim) were added (1:2000 in 1x PBS, 10% normal goat serum, 0.1% Tween 20, 0.2% Triton X-100 for early embryos; 1:5000 in 1x PBS, 0.1% Tween 20 for late embryos) and the color reaction was carried out using nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indoyl-phosphate (NBT/BCIP; Roche) by standard procedures. Intact embryos were dehydrated in ethanol and propylene oxide, followed by infiltration with plastic embedding medium (Poly/Bed 812; Polysciences).
Cyclopamine treatment
Cyclopamine was obtained from Veratrum californicum as described
previously (Gaffield et al.,
1986) and was diluted to a final concentration of 10 µM, 5
µM and 1 µM in HL saline (from a stock solution of 10 mM in ethanol).
Experimental embryos (early or mid stage 8; 60-68 AZD) were cultured in
cyclopamine for 3-6 days (sibling controls were cultured in HL saline with
0.1% ethanol). Treated embryos were then examined morphologically.
Histology and microscopy
For closer examination than was possible in whole mount, some embryos were
dehydrated through a series of graded alcohols to 95% and then embedded in
glycolmethyacrylate resin (JB-4; Polysciences, Inc) according to the
manufacturer's instructions. Embedded specimens were sectioned with glass
knives on a Sorvall MT2-B microtome. Sections were mounted on glass slides and
stained with Hoechst 33258 (1 µg/ml).
In our hands, the AP reaction product obtained by in situ hybridization is
not stable in JB-4 embedding resin. So to examine such embryos in section, the
selected specimens were dehydrated and embedded in epoxide embedding resin
(Poly/Bed 812; Polysciences) following the manufacturer's instructions, and
sectioned at 10 µm thickness with glass knives using a Sorvall MT2-B
microtome or at
100 µm thickness using hand-held razor blades.
Sections were mounted on glass slides under coverslips in a non-fluorescing
medium (Fluoromount; BDH Laboratory Supplies, Ltd.), and photographed with
Nomarski optics (Zeiss Axiophot) or observed with confocal microscope (MRC
600; Bio-Rad) in 0.1 µm optical sections.
Whole embryos or sections were viewed with DIC optics (Zeiss Axiophot or Nikon E800 microscope) and photographed using Ektachrome 160 film (Kodak) or with a `Spot' CCD camera (Diagnostics, Inc.). Live embryos were paralyzed in relaxant HL saline prior to photography.
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RESULTS |
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In other organisms, HH-C is important for autoproteolysis of the HH
propeptide and for covalently attaching cholesterol to the carboxyl terminus
of HH-N. HRO-HH-C is less well conserved than HRO-HH-N, based on amino acid
sequence comparison with other HH-class proteins
(Fig. 2) and is longer than any
other known HH-C peptides, but a region resembling the proposed active site
motif (PASM) of HH-C peptides [including the invariant T and H residues
required for autoproteolysis (Lee et al.,
1994)] was also found in HRO-HH-C at residues 561-567
(Fig. 2A,C).
We also note the presence in HRO-HH-C of a putative cell division sequence
motif (CDSM) not found in other HH proteins. The consensus motif
([ND]-C-[TES]X[DE][EDTS][DE], where X is a spacer of 1-8 amino acids) exists
in the v-myc and c-myc oncoproteins, CDC25, and other proteins thought to
carry out one of the required steps in the cell division competency cascade of
deactivating a cell division repressor
(Figge and Smith, 1988). The
presence of this motif in Hro-hh
(DKTCDSIDSE, residues 954-963;
Fig. 2A), suggests that
HRO-HH-C may also be involved in intracellular signaling related to the
control of cell division.
To investigate the relationships between Hro-hh and previously
described hh-class genes, we used ClustalX 1.81 to construct an
unrooted phylogram, comparing that portion of the conserved HH-N domain for
which sequences were available for all species of interest
(Fig. 3). The best supported
tree grouped Hro-hh with hh from Drosophila and
Patella, separate from the deuterostome hh-class genes, in
accord with the accepted metazoan phylogenetic tree based on rRNA sequence
(Adoutte et al., 2000;
Aguinaldo et al., 1997
).
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The proboscis of glossiphoniid leeches is a tri-radiate, muscular tube (see Fig. 7A) that is everted during feeding to pierce host or prey tissues and suck out blood or soft body parts. This process is aided by secretions from ductules arising in the salivary glands and running the length of the proboscis. As illustrated in Fig. 1, the proboscis occupies the everted position during the early stages of its morphogenesis (stages 9 and early 10), and then assumes the inverted position (late stage 10 and beyond).
Hro-hh expression was largely confined to the inner layer of the developing proboscis, proximal to the lumen (Fig. 5E,G). Beginning in stage 9, staining was also seen in a ring of external tissue surrounding the base of the proboscis (Fig. 5E-G). This ring is apparently transformed into the epithelial lining of the oral opening once the proboscis has inverted (Fig. 6A,B). Hro-hh expression continued within the proboscis through early stage 11 (Fig. 6A,B), and transverse sections of such embryos revealed that the lumen proximal staining was in presumptive radial muscle cells throughout the length of the proboscis (Fig. 6C,D). Within the posterior portion of the proboscis, staining was also seen in presumptive longitudinal muscle cells, more distal to the lumen (Fig. 5F, Fig. 6D).
In addition to the expression in the foregut described above, Hro-hh expression was evident in cells associated with the midgut. Beginning in late stage 8 and continuing into stage 9, Hro-hh was seen in faint, segmentally iterated, transverse stripes at the interface of the germinal plate and the SYC (prospective midgut; Fig. 5H). These stripes appeared only after segmental morphology was established, and were associated with the developing gut wall at the dorsal edge of the intersegmental septa (Fig. 5I). During stage 9, most of these stripes fade, but five persist and become stronger during stage 10, so that by stage 11 Hro-hh staining was seen in a series of five rings around the posterior midgut derivatives: one at the boundary between crop and intestine; one near the boundary of the intestine and rectum; and three around the rectum (Fig. 6B,G). Also by stage 11, a speckled pattern of staining was evident at the surface of the crop (Fig. 6B,F,G). By mid-stage 11, much of the expression appears to have disappeared, except for the rings of presumptive muscle associated with the intestine and rectum (Fig. 6H,I).
Staining was also seen in cells at the lateral edges of the germinal plate during stage 9 (Fig. 5H), and during stage 10, in the male and female reproductive tissues (Fig. 6A,E) and throughout most of the definitive epidermis of the body wall. The epidermal staining was more intense in the posteriormost midbody segments, and absent at the ends of the embryo, corresponding to the presumptive anterior and posterior suckers (Fig. 5I, Fig. 6A,G). Finally, by stage 11, Hro-hh expression was also seen in a small number of segmentally iterated cell bodies in the ganglia of the ventral nerve cord (Fig. 6A,C).
Identity and embryonic origins of cells expressing Hro-hh in
proboscis
A prominent structural feature of the proboscis is an extensive array of
radial muscles (Fig. 7A,B),
whose contractions open the lumen of the proboscis to draw in food; their
actions are opposed by a thick, interwoven band of circumferential muscles
situated roughly midway along the radius of the proboscis
(Fig. 7A,B). Nuclei of the
radial muscles are situated within the ring of circumferential muscle. Outside
this ring, the space between the radial muscles is occupied by relatively
sparse sets of longitudinal muscles and a large number of ductules that carry
salivary gland secretions to the tip of the proboscis
(Fig. 7B).
Thus, given the observed staining patterns, the prime candidates for cells
expressing Hro-hh near the lumen of the developing proboscis are the
radial muscles and/or circumferential muscles, while the longitudinally
oriented fibers near the outer surface in the posterior portion of the
proboscis correspond to longitudinal muscles. We have previously established
that presumptive circumferential muscles of the proboscis arise from
micromeres c''' and dm'
(Huang et al., 2002). Careful
examination of sectioned, in situ-stained embryos in which cell dm' had
been injected with lineage tracer, revealed no doubly labeled cells
(Fig. 7G). All the cells
colored with the in situ reaction product lay within or outside the ring of
tracer-labeled circumferential muscles. From this, we conclude that it is the
radial muscles and longitudinal muscles, and not the circumferential muscles,
that express Hro-hh in the proboscis at this stage.
The embryonic origins of the radial and longitudinal muscles are less clear, but on the basis of our previous results, it seemed that the primary quartet micromeres (a'-d') and secondary trio micromeres a'' and c'' were good candidates for contributing at least some of these muscles. Consistent with this interpretation, Hro-hh-stained embryos in which micromere a'' had been injected with lineage tracer did contain some doubly labeled cells near the core of the proboscis that appear to be radial muscle precursors (Fig. 7H). Embryos in which primary quartet micromeres (a', b' and d') had been injected showed double labeling of some radial muscle precursors, and also of some longitudinal fibers in the outer layers of the proboscis that we assume are presumptive longitudinal muscles (Fig. 7D-F).
Identity and embryonic origins of cells expressing Hro-hh in
midgut (crop, intestine and rectum)
The wall of the midgut comprises two closely apposed layers of cells. One,
an outer layer of visceral mesoderm arises from the same m blast cells that
contribute segmentally repeating mesodermal structures such as body wall and
nephridia, is laid down in an anteroposterior progression and expands dorsally
with the rest of the germinal plate during stages 9-10. The other layer is
endoderm that arises by cellularization of the SYC
(Nardelli-Haefliger and Shankland,
1993), under mesodermal influence
(Wedeen and Shankland, 1997
).
The SYC arises by a stepwise series of cell-cell fusions, and thus contains
nuclei derived from the macromeres, teloblasts, and supernumerary blast cells
(Desjeux and Price, 1999
;
Liu et al., 1998
). Because the
two layers of cells are thin and tightly apposed, it is difficult to tell them
apart, even using lineage tracer injections. The difficulty is compounded by
the fact that the M teloblasts have already fused with the SYC by the time
visceral mesoderm forms, so it is not possible to label visceral mesoderm
without also labeling the endoderm. The rings of staining in the posterior
midgut clearly lay outside the endoderm, and presumably corresponded to
M-derived muscles ringing the intestine and rectum. But examination of
fortuitous sections through the anterior midgut (prospective crop) suggest
that some Hro-hh expression was also occurring within the endodermal
layer (not shown).
Hro-hh signaling is required for normal gut development
To obtain information regarding the functional significance of
Hro-hh expression in the developing leech, we examined intact and
sectioned stage 10-11 embryos that had been grown from early stage 8 in the
presence of cyclopamine, a steroidal alkaloid that inhibits HH signaling in
vertebrates (Cooper et al.,
1998; Incardona et al.,
1998
; Taipale et al.,
2000
). In these experiments, epiboly, germinal plate formation and
the differentiation of segmental tissues proceeded in parallel between
experimental and control embryos (not shown), but the differentiation of the
proboscis and crop were clearly disrupted by cyclopamine
(Fig. 8).
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To further test the specificity of the cyclopamine effects, we examined control and cyclopamine-treated embryos in which selected cells had been injected with cell lineage tracer. Ectodermal lineages in cyclopamine-treated embryos were indistinguishable from those in controls (Fig. 9A, D, G). In the M lineage, contributions to the provisional integument and ventral nerve cord appeared normal; mesodermal derivatives were also present in the body wall, but the definition and organization of muscle fibers was less clear than in controls. This latter observation correlated with the fact that the germinal plate was very fragile and difficult to dissect in cyclopamine-treated embryos and that the embryos failed to flatten in the DV axis (data not shown).
|
In addition to the foregoing defects, cyclopamine-treated embryos were more
transparent than controls. In normal development, most of the coelom of the
leech embryo becomes filled with mesenchyme and other mesodermal derivatives,
so that the remaining space is reduced to a relatively narrow system of
interconnecting channels (reviewed by
Sawyer, 1986)
(Fig. 8C-E); this process
failed to occur in embryos treated with cyclopamine
(Fig. 8M-O). Moreover, in
sectioned, cyclopamine-treated embryos, we were unable to recognize any
morphogically defined gonads, which appear at this stage in normal embryos as
U-shaped structures connected to the ventral ectoderm (see
Fig. 6E). In contrast, the
ventral blood vessel was still present
(Fig. 8E',O'),
which is further evidence for the specificity of the cyclopamine effects.
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DISCUSSION |
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Based on these results, we can try to interpret which elements of the Hro-hh expression pattern are responsible for the developmental defects produced by cyclopamine. For example, it seems likely that the expression of Hro-hh by the radial and/or longitudinal muscle fibers of the proboscis is required for the normal development of the circular muscle fibers derived from micromere dm'. The source of the Hro-hh signals that regulate crop morphogenesis and mesenchyme formation is less obvious, but they may originate in the faint stripes of germinal plate expression seen at stage 9 (Fig. 5H).
Though expression of hh and en are tightly coupled during
segmentation in insects, we found no evidence for patterned expression of a
leech hh gene in the germinal bands or early germinal plate at the
time and place where the segmental pattern of engrailed expression is
first manifest (Wedeen and Weisblat,
1991; Lans et al.,
1993
), nor is there a clear correlation between the patterns of
hh- and en-class gene expression in the ventral ganglia.
Subject to the caveats necessary for any negative result, the apparent
dissociation of en and hh gene expression during leech
segmentation is consistent with other studies suggesting that the segmentation
processes in Helobdella and Drosophila are not homologous at
the molecular level (Iwasa et al.,
2000
; Pilon and Weisblat,
1997
; Savage and Shankland,
1996
; Seaver and Shankland,
2000
; Seaver and Shankland,
2001
; Shain et al.,
1998
; Song et al.,
2002
).
Speculation as to the ancestral role of hh-family genes
Given the diverse functions of hh-class genes in various
organisms, what can we conclude regarding the function of the
hedgehog gene in the common ancestor of the three main bilaterian
clades? The eponymous hh was identified as a segment polarity gene in
Drosophila, and vertebrate hh-class genes are critical for
patterning limbs and somites. The question is still open
(De Robertis, 1997;
Dewel, 2000
;
Collins and Valentine, 2001
;
Valentine and Collins, 2000
),
but it seems unlikely to us that the ancestral bilaterian had these particular
features, in which case the ancestral hh gene could not have
functioned in any of those roles.
The first description of a hh homolog in Lophotrochozoa has
recently been published for the limpet Patella vulgata, a gastropod
mollusc; its expression in anterior and ventral midline ectoderm of the
trochophore larva of that species was interpreted as supporting the
dorsoventral axis inversion theory
(Nederbragt et al., 2002). Our
results, that Hro-hes is expressed at low levels throughout the
epidermis of the germinal plate of the leech, but with no particular
relationship to the developing nerve cord, does not particularly support this
interpretation. Of course, Helobdella is a derived annelid, so the
expression and function of Hro-hh may bear little resemblance to
those of the ancestral hh gene. But the same argument holds for all
extant species, including those whose external morphology resembles ancestral
forms. Thus, whether the pattern of hh-class gene expression seen in
the Patella trochophore reflects its expression in the urbilateria or
an adaptation associated with torsion of the molluscan body plan during
gastropod evolution, for example, remains to be seen. In any event, the
expression of hh-class genes in the nervous system of vertebrates,
insects, annelids and (presumably) mollusks suggests that the ancestral
hh gene(s) may have been involved in some aspect of neural
differentiation.
Another candidate function of the ancestral hh gene is in gut
formation (Pankratz and Hoch,
1995). That the gut is a plesiomorphic trait of bilaterian animals
seems beyond dispute, and it has been suggested that signaling between
invaginating gut and ectoderm, leading to the formation of stomodeum/foregut
and/or proctodeum/hindgut, was among the first inductive interactions to have
evolved (Wolpert, 1994
). In
chick, sonic hedgehog (shh, one of 3 hh-class genes
known in vertebrates) is expressed in the endodermal epithelium throughout
much of the gut (Echelard et al.,
1993
; Roberts et al.,
1995
; Roberts et al.,
1998
; Sukegawa et al.,
2000
). In Amphioxus, which arose from a basal branch of
the chordate lineage, the one known hh-class gene is also expressed
in endoderm, among other tissues (Shimeld,
1999
). In Drosophila, epithelial expression of
hh is an essential aspect of foregut and midgut formation, but in
contrast to chick and Amphioxus, the source of the signal is
ectodermal rather than endodermal (Hoch
and Pankratz, 1996
; Pankratz
and Hoch, 1995
). And now in leech, we find hh-class gene
expression primarily in mesoderm, the third germ layer, during gut
formation.
Similarities between hedgehog signaling pathways may also extend to the
level of target genes. The inductive Shh signal regulates the concentric
patterning of the surrounding mesenchyme in chick, negatively regulating the
differentiation of smooth muscle, in part by activation of BMP4, a
member of the TGFß superfamily, in those cells
(Roberts et al., 1995;
Roberts et al., 1998
;
Sukegawa et al., 2000
).
Members of the multi-gene NK-2 family of homeobox genes are also
activated by Shh signaling in vertebrates
(Barth and Wilson, 1995
;
Briscoe et al., 1999
). In
Amphioxus, AmphiBMP2/4 is expressed in hypoblast and endodermal
derivatives (Panopoulou et al.,
1998
), and AmphiNk2-2 is expressed in anterior nervous
system and endoderm (Holland et al.,
1998
). In Drosophila, as in vertebrates, hh
activates the expression of TGFß-related genes (dpp and
60A) in adjacent visceral mesoderm. Moreover, an NK2-class
gene (tinman) is also a downstream target of hh signaling in
Drosophila heart differentiation
(Yin and Frasch, 1998
). In
leech, Lox10, an NK2-class gene, is known to be expressed in
the crop (Nardelli-Haefliger and
Shankland, 1993
); a TGFß-related gene has been
identified, but its expression remains unknown (Isaksen, 1992). Thus,
comparison of hh-class gene expression and function in a vertebrate
(chick), fly and leech reveals some parallels, supporting the hypothesis that
signaling in gut formation was an ancestral role for hh-class gene in
ancient bilaterians, but there are also significant differences. Further work
should reveal whether this situation reflects divergence or convergence.
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ACKNOWLEDGMENTS |
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