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doi: 10.1242/10.1242/dev.00395

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A hedgehog homolog regulates gut formation in leech (Helobdella)

Dongmin Kang^1,*, Françoise Huang¹, Dongling Li^2,, Marty Shankland², William Gaffield³ and David A. Weisblat^1,

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

View larger version (45K): [in a new window]   Fig. 1. Schematic depiction of relevant stages of Helobdella development. (A-E) Lateral views with animal pole at top and anterior to left; (F) dorsal view. (A) Stage 4b (10 hours AZD); cells DNOPQ and DM arise from macromere D' at third cleavage and give rise to the teloblasts that generate segmental mesoderm and ectoderm. Macromeres A''', B''' and C''' are endodermal precursor cells. Micromeres are evident at the animal pole. (B) Early stage 8 (61 hours AZD); DM and DNOPQ have cleaved to generate the full complement of 10 teloblasts (circles, only 8 are shown in the drawing) plus additional micromeres. Each teloblast produces a column of segmental founder cells (blast cells); ipsilateral bandlets merge, forming left and right germinal bands (gb; shaded; only left gb is visible in this lateral view), which are covered by micromere-derived epithelium stretching over the animal pole (net pattern). As blast cells are added to their posterior (pos) ends, the germinal bands elongate and move ventrovegetally (arrows) and coalesce from anterior (ant) to posterior (pos) along the ventral midline, forming the germinal plate (gp), accompanied by the expansion of the micromere-derived epithelium. (C) Late stage 8 (94 hours AZD); the completed germinal plate (shaded) extends from anterior to posterior, defining the ventral territory of the embryo. (D) Mid stage 10 (155 hours AZD); during stage 10, segmental tissues arise from the proliferation and differentiation of cells within the germinal plate (shaded), the edges of which gradually expand dorsally and meet at the dorsal midline, closing the definitive body tube. Macromeres, teloblasts and supernumerary blast cells have fused into a syncytial yolk cell (hatched) that constitutes the midgut; foregut arises largely from micromere progeny (the micromere-derived epithelium is omitted for clarity). (E) Early stage 11 (172 hours AZD). By this stage, the foregut has generated distinct proboscis and esophagus, while the midgut has given rise to crop, intestine and rectum; segmental tissues are well-differentiated, including ganglia within the ventral nerve cord (vnc). (F) Late stage 11 (195 hours AZD); crop caeca are well-differentiated [adapted from Kang et al. (Kang et al., 2002)].

View larger version (63K): [in a new window]   Fig. 2. Hro-hh is an hh homolog. (A) Domain alignment of HH-homologs in leech (Hro-hh; accession number AF517943), mouse (Mus-shh, Q62226; Mus-ihh, P97812; Mus-dhh, Q61488), human (Hom-shh, Q15465), zebrafish (Dan-shh, A49426), Amphioxus (Amp-hh, CAA74169) and fruitfly (Dro-hh, L02793): secreted HH amino terminus (HH-N, gray box); within the less well conserved carboxy-terminal fragments (HH-C), putative autoprocessing sequence motifs (PASM, black box) and the cell division sequence motif (CDSM, hatched box) in Hro-hh are indicated. (B) Alignment of HH-Ns, including the available sequence from a mollusc, Patella vulgata (Pat-hh, AF435840). (C) Alignment of PASMs in HH-Cs. Gray shading in B and C indicates conserved residues. shh, sonic hedgehog; ihh, Indian hedgehog; dhh, desert hedgehog.

View larger version (11K): [in a new window]   Fig. 3. Unrooted phylogram for hh-class genes recreates the accepted evolutionary relationships among protostomes (Dro-hh; Hro-hh and Pat-hh) and deuterostomes (all others). Well-supported branches are indicated by numbers (percentage of 100-replicate bootstrap trials). Abbreviations and accession numbers as in Fig. 1, plus chick (Gal-ihh, Q98938 and Gal-shh, Q91035), Xenopus (Xen-chh, Q91610 and Xen-hh4, Q91611) and another zebrafish gene (Dan-ehh, Q98862). See text for details.

View larger version (27K): [in a new window]   Fig. 4. Semiquantitative RT-PCR demonstrates late zygotic transcription of Hro-hh. (A) Digital images of ethidium bromide-stained agarose gels. Fragments of Hro-hh and 18S rRNA were amplified in separate reactions carried out in parallel. (B) Amount of Hro-hh mRNA during development, relative to stage 10 (100%). At each stage, the intensity of the Hro-nos band was normalized against the 18S rRNA fragment (see Materials and Methods for details). Each reaction sample contained template cDNA equivalent to 4 embryos at the stage indicated [stage 4b 10 hours AZD, stage 6a 19 hours AZD, stage 7 40 hours AZD, early/mid stage 8 (E/M8) 65 hours AZD, late stage 8 (L8) 88 hours AZD, early stage 9 (E9) 112 hours AZD, late stage 9 (L9) 140 hours AZD, stage 10 150 hours AZD, late stage 10/stage 11(L10/11) 170185 AZD, late stage 11(L11) 195 AZD]. Squares indicate the data obtained from the gels shown in (A); circles and triangles represent data obtained starting with independent sets of embryos; black circles show the data from independent PCR.

View larger version (89K): [in a new window]   Fig. 5. Early expression of Hro-hh in gut tissues, but not in germinal plate, prior to the establishment of segmental boundaries. Photomicrographs of embryos processed by in situ hybridization for Hro-hh. In these and all subsequent illustrations, embryos are shown in lateral views, with anterior to left and ventral down, unless otherwise stated. (A) Anteroventral view of a stage 8 embryo (78 hours AZD) showing the partially formed germinal plate (dotted outline); transcripts (arrow) occur at the anterior, micromere-derived end of the germinal plate, from which prostomial tissues and proboscis arise, but not in the more posterior, teloblast-derived region that will form segmental ectoderm and mesoderm. (B) Lateral view of the same embryo, at higher magnification. (C,D) An embryo at early stage 9 (100 hours AZD), showing the presence of two distinct groups of cells expressing Hro-hh at the anterior end of the germinal plate; there are still no transcripts visible within the segmental portion of the germinal plate. (E) By early stage 10 (135 hours AZD), the proboscis is starting to differentiate in the everted position. Hro-hh transcripts are present in the central core of the proboscis (extent indicated by double-headed arrow), in a ring of cells defining the future oral opening (white arrow) and in transverse bands of cells at the posterior end of the SYC, corresponding to posterior midgut (black arrows). Relatively weak expression is also observed in the prospective esophagus, between the circumoral ring and the yolk cell. (F) A higher magnification view of the embryo shown in E, looking down along the longitudinal axis of the foregut and focussed at the level of the circumoral ring. In this view, Hro-hh-expressing longitudinally oriented fibers (arrows) appear as dots surrounding the core of the proboscis. (G) At mid stage 10 (145 hours AZD), transcripts are clearly present throughout the extent of esophagus and proboscis (doubleheaded arrow), in the circumoral ring (white arrow), and in the rectum (black arrows), which is becoming morphologically distinct from the crop. Patches of Hro-hh expression are also visible at the surface of the anterior portion of the crop. (H) An embryo at the same age as that in E, in which the color reaction was allowed to proceed longer. This image is focused on the lateral edge of the germinal plate. Hro-hh transcripts are visible along the edge of the germinal plate (black arrowhead) and in transverse segmentally iterated bands (white arrowheads). (I) Parasagittal section (ventral is down, posterior to right) through the posterior segments of an embryo at a similar stage to that shown in H (paired arrows in H). In three segments, precursors of the rings of stained rectal muscle (arrows) lie at the dorsal edge of the intersegmental septa. Staining is also present in the body wall, with a clear boundary at the boundary between the prospective midbody and caudal sucker (black arrowhead). Scale bar: A, 150 µm; B 75 µm; C,E,G,H, 100 µm; D, 50 µm; F, 25 µm; I, 20 µm.

View larger version (101K): [in a new window]   Fig. 6. Later expression of Hro-hh in gut, body wall, reproductive tissue and nervous system. (A) Late stage 10 and (B) early stage 11 embryos (160 and 180 hours AZD) from two separate hybridization experiments. The embryo shown in B was later sectioned in a roughly transverse orientation. Selected sections, corresponding to the planes indicated by the paired arrows and arrowheads, are shown in C-G. In each embryo, Hro-hh transcripts are visible within the oral opening (black arrowhead in A,B), along the inner portion of the proboscis (inverted by this stage; extent indicated by double-headed white arrow in A) and esophagus (bracket in A), in reproductive organs (box in A), around the crop (vertical arrows in B), at the crop-intestine and intestine-rectum boundaries (white arrowheads in B) and rectum (white arrows in B). (A) The staining reaction was carried out for somewhat longer for this embryo, revealing low transcript levels in the ventral nerve cord (small arrows) and in the epidermis except for the future anterior and posterior suckers (black horizontal arrows indicate boundaries). (C) Section through the anterior portion of the proboscis (leftmost paired arrows in B) shows Hro-hh transcripts in cells near the tri-radiate lumen of the proboscis, and in a pair of neurons (arrows) in the ventral ganglion. (D) Section through a more posterior portion of the proboscis (left paired arrowheads in B) shows transcripts in a ring of longitudinally oriented fibers. (E) Section through the anterior end of the crop (middle paired arrows in B) reveals transcripts in a U-shaped pattern corresponding to the reproductive organs. (F) Section through the middle of the crop (right paired arrowheads in B) shows transcripts in visceral mesoderm and/or endoderm. (G) Section through the posterior portion of the embryo (rightmost paired arrows in B) shows transcripts in visceral mesoderm and/or endoderm of the posterior crop caeca, in muscles associated with the rectum (white arrowhead and arrows), and in the body wall bordering the posterior sucker (black arrow). (H) In later stage 11 (190 hours AZD), transcripts are largely confined to four rings of presumptive muscle surrounding the rectum (box), shown at higher magnification in I. Scale bar: 100 µm in A, B,H; 90 µm in C-G; 70 µm in I.

View larger version (112K): [in a new window]   Fig. 7. Cells expressing Hro-hh in proboscis arise from specific micromere lineages. (A) Transverse section through the proboscis of an adult Helobdella; the triangular lumen (*) is surrounded by an inner ring (i) comprising mainly the thick ends and nuclei of radial muscles, a middle ring (m) comprising circumferential muscles and an outer ring (o) comprising longitudinal muscles and salivary gland ductules. (B) Magnified view of the boxed area in A highlighting two radial muscles (green), a circumferential muscle fiber (m, pink), plus several longitudinal muscle fibers (pink) and ductules (circles). (Labels i and o have been omitted from B for clarity.) (C) Combined bright-field and fluorescence micrographs showing anterior portion of an embryo in which micromeres a', b' and d' had been injected with RDA (red), FDA (green) and both (yellow), respectively, at stage 4 (8 hours AZD); the embryo was processed for in situ hybridization at early stage 10 (135 hours AZD), and then sectioned in obliquely transverse orientation through the long axis of the embryo (dorsal is up in all sections). (D) A section through the anterior proboscis (left paired arrows in C). Micromeres a', b' and d' contribute cells to the left dorsal, right dorsal and left ventral quadrants of the outer ring (o) of the proboscis, respectively (black arrows). Other experiments demonstrated that the unlabeled right ventral quadrant contains progeny of micromere c' (data not shown), consistent with the established symmetry of the clones of these four cells (Nardelli-Haefliger and Shankland, 1993; Smith and Weisblat, 1994; Huang et al., 2002). Hro-hh transcripts (dark grey) are localized to the inner ring (i) of presumptive radial muscles surrounding the lumen (white *); some of these cells are co-labeled with FDA (green, partially masked by the in situ signal), indicating their descent from micromere b'. Progeny of a' are also present in the proboscis sheath (white arrow). Note also the presence of a middle ring (m, black arrowhead) containing neither lineage tracer nor Hro-hh transcripts. (E) A section through the mid-portion of the proboscis (middle arrows in C) shows Hro-hh transcripts in presumptive longitudinal muscles at the inner edge of the outer ring (white arrowheads); some of these cells appear to co-label with lineage tracer. At this level, all three of the labeled micromeres contribute to the proboscis sheath (white arrow). (F) A section near the posterior end of the proboscis (right arrows in C) includes part of the left side of the supraesophageal ganglion (sg), containing progeny of a' and d'; at this level, the outer portions of the section pass through definitive epidermis ventrally and a temporary embryonic covering [provisional integument (Weisblat et al., 1984)] dorsally. Progeny of all three micromeres are seen in the epithelium of the provisional integument (black arrow) and in the inner (i) and outer (o) rings of the proboscis, including cells that express Hro-hh. (G,H) Obliquely transverse sections (at roughly the level and orientation indicated by the paired arrowheads in C) through an embryo in which micromere dm' (G) or a'' (H) had been injected with RDA (red) at stage 4 (10 hours AZD). (G) Progeny of dm' occupy the middle ring (black arrowhead) of the proboscis, between the inner and outer rings of cells expressing Hro-hh. [The seemingly double-labeled cell (white arrow) is an artifact resulting from the thickness and obliquity of the section.] Other dm' progeny occupy the outer ring (black arrow) and external surface (white arrowhead) of the proboscis. (H) Progeny of micromere a'' contribute to the proboscis sheath (white arrow), and to both the outer (o) and inner (i, white arrowhead) rings of the proboscis. Scale bar: (A,C-H) 50 µm, (B) 30 µm.

View larger version (87K): [in a new window]   Fig. 8. Cyclopamine treatment disrupts formation of the gut and coelomic mesenchyme (see Materials and Methods for details of treatment). (A,B) Dorsal views of the anterior and posterior portions, respectively, of a control embryo at stage 11 (200 hours AZD) showing the extent of the proboscis (double headed white arrow in A) and crop (double headed white arrow in B); note the well-differentiated crop caeca (black arrowheads). (C-E) Combined bright-field and fluorescence views of transverse sections (ventral is down; counterstained with Hoechst 33258, blue) through the proboscis (at level of black arrows in A), esophagus (left black arrows in B) and crop (right black arrows in B), of a comparable embryo, at roughly the levels indicated in A and B. Note that the proboscis (arrow in C) has well-defined inner and outer layers separated by a middle layer containing relatively few nuclei and that the visceral mesoderm has spread to form a thin layer of nuclei surrounding the crop [arrowhead in E; E' is an enlarged view of the box in E showing the ventral blood vessel (arrowhead)]. (F,G) Lateral views of the anterior and posterior portions of a sibling embryo treated with 5 µM cyclopamine. Note the shortened proboscis (double headed white arrow in F) and crop (double headed white arrow in G) and the incomplete differentiation of the crop caeca (white arrowheads in G) relative to those in the control. (H,I) Dorsal views of the anterior and posterior portion of another embryo treated with 5 µM cyclopamine. The proboscis (double headed arrow in H) and crop (double headed arrow in I) are similarly affected, whereas the intestine (box in I) appears normal. (J,K) Lateral views of the anterior and posterior portions of a sibling embryo treated with 10 µM cyclopamine. The proboscis (extent indicated by white double headed arrow in J) is shortened and has failed to invert. The esophagus (extent indicated by black double headed arrow in J) is thin and elongated. The crop lacks even the large posterior caeca, but intestine and rectum are still present (arrowhead in K). (L-O) Views of transverse sections at successively more posterior levels (indicated by black arrows and arrowheads in J) through the proboscis (white arrow in L), esophagus (white arrows in M and N) and crop (white arrow in O), of a comparable cyclopamine-treated embryo. In such embryos, the tri-radiate geometry of the proboscis lumen is less well defined (arrowhead in L) and the middle ring of circumferential muscles is missing (compare L with C). In addition, the coelomic lacunae remain largely devoid of cells (compare N with D) and visceral mesoderm (arrowhead in O) has failed to expand around the crop, but the ventral blood vessel is still present (arrowhead in O'). Scale bar, 100 µm in A, B, F-K; 50 µm in C-E, L-O; 12 µm in E' and O'.

View larger version (73K): [in a new window]   Fig. 9. Cyclopamine treatment disrupts formation of circumferential muscles in proboscis. Fluorescence micrographs of control embryos at early stage 11 (180 hours AZD; top row) and siblings treated with cyclopamine at 5 µM (middle row) or 10 µM (bottom row). In each embryo, micromere dm' had been injected with FDA (green) at stage 4 (10 hours AZD) and an N or OPQ cell with RDA (red) at stage 6a (20 hours AZD). The left column (A,D,G) shows intact embryos; in each row, the center and right columns are higher magnification views of the anterior (B,E,H) and posterior (C,F,I) of the same embryo, after dissecting the germinal plate from the yolk. In the control embryo, the segmentally iterated pattern of neurons arising from the O, P and Q lineages is visible along the ventral nerve cord (arrowheads in A); dm'-derived circumferential muscles are present throughout the inverted proboscis (bracket in A and B) and a network of dm'-derived fibers (arrows in C) is present in the caudal sucker. In the embryo treated with 5 µM cyclopamine, the segmental pattern of N-derived neurons (arrowheads in D) is not affected; circumferential fibers have formed in the proboscis, though it has failed to invert (bracket D and E) and the posterior fibers (arrows in F) appear as in controls. In the embryo treated with 10 µM cyclopamine, the N-derived neurons (arrowheads in G) and dm'-derived posterior fibers (arrows in I) are still comparable to those in controls, but the dm'-derived circumferential muscles are absent (G,H) and the proboscis is reduced in length. Scale bar: (A,D,G) 100 µm; (B,C,E,F,H,I) 40 µm.