Anat Embryol (2005) 210: 187–197 DOI 10.1007/s00429-005-0014-8
O R I GI N A L A R T IC L E
Yegor Malashichev Æ Valentin Borkhvardt Bodo Christ Æ Martin Scaal
Differential regulation of avian pelvic girdle development by the limb field ectoderm
Accepted: 10 June 2005 / Published online: 17 September 2005 Ó Springer-Verlag 2005
Abstract Although limb development has been a subject of intense research over the last decades, development of the girdles has been poorly investigated. Particularly, a detailed analysis of pelvic girdle development including functional data is not available to date. Here, we describe the early steps of the formation of mesenchymal and cartilaginous anlagen of the pelvic elements using alcian blue staining in whole mount embryos and serial histological sections, and the expression pattern of several marker genes to provide an operative basis for further research in pelvis development. Moreover, we describe pelvis development after unilateral hindlimb bud amputation and somatopleural ectoderm extirpation. We show for the first time, that ectodermal signals at pre-limb bud stages are required for pelvis formation. We present evidence suggesting that the regulation of ilium development is different from the development of ischium and pubis. Keywords Ilium Æ Pubis Æ Ectoderm Æ Chick Æ Pelvic girdle
Introduction Pattern formation and morphogenesis during vertebrate limb development has been a focus of intense research over the last decades, making the vertebrate limb one of
Y. Malashichev Æ V. Borkhvardt Department of Vertebrate Zoology, St. Petersburg State University, Universitetskaya nab. 7/9, 199034 St. Petersburg, Russia E-mail:
[email protected] B. Christ (&) Æ M. Scaal Æ Y. Malashichev Anatomisches Institut der Universita¨t Freiburg, Albertstrasse 17, 79104 Freiburg, Germany E-mail:
[email protected] Tel.: +49-761-2035089 Fax: +49-761-2035091
the best studied developmental systems to date (reviewed e.g. in Tickle 2004; Niswander 2002; Capdevila and Izpisua Belmonte 2001). However, the most proximal elements of the vertebrate appendages that connect the skeleton of the free limbs to the axial skeleton, the shoulder and pelvic girdle, have to large extent escaped attention of modern developmental biology. In spite of the enormous clinical interest in human pelvic malformations, like congenital hip dysplasia (reviewed in e.g. Vitale and Skaggs 2001; Weinstein et al. 2004), or scapuloiliac dysostosis (reviewed in e.g. Elliot et al. 2000; Hauser et al. 1998; Williams 2003), morphogenesis and molecular regulation of pelvic girdle development is largely unknown. A classical description of the early development of the pelvic girdle in the chick, which was based on the study of hematoxylin stained histological sections, was given by Johnson (1893), later followed by Mehnert (1887) and Lebedinsky (1913) and reviewed by Romanoff (1960). They observed that differentiation of the pelvic girdle in chick starts between days 4 and 5 of development, corresponding to HH-stages 25–26, from a common mesenchymal condensation in the region between the axial skeleton and the free limb. Soon thereafter the first chondrification centers appear at about days 5 and 6. At day 8, the anlagen of ilium, ischium and pubis separate within the common presumptive mesenchyme. At around day 10 the final morphology of the avian pelvis is established (Lebedinsky 1913; Chevallier 1977). These studies are the primary basis for our current knowledge of pelvis morphogenesis in this species. As to the embryological origin of the pelvis, Bardeen and Lewis (1901) postulated from morphological observations that the human pelvic girdle originates from the lateral plate mesoderm, which was later experimentally confirmed in the chick by Chevallier (1977). More precisely, Chevallier (1977) found that the somatopleure at the level of somites 26 through 32 gives rise to the whole pelvic girdle. The first experimental approach to study pelvis development came from Spurling (1923), who
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amputated early leg buds, presumably with a portion of surrounding somatopleure, from HH-stage 17 embryos and observed defects in the pelvis on the operated side of the embryos. Specifically, the proximalmost pelvic element, the ilium, was always present though truncated, whereas the more distal elements, the ischium and the pubis, were severely malformed or lacking completely. Rogulska (1965), who removed limb buds without adjacent somatopleure in older embryos at HH-stages 20–23, did not observe apparent defects in the pelvis, indicating that a putative influence of limb structures on pelvis development is limited to very early stages of appendage development. In these ablation experiments, the pelvis inducing tissue has remained obscure. The dependence of pelvis development on limb formation is furthermore illustrated by the progressive absence of pelvic structures in squamate reptiles with vestigial or absent limb buds, where the extent of pelvic girdle development correlates to the size of the hindlimb bud. The smaller the hindlimb bud, the smaller the early mesenchymal gap in sacral muscle segments which gives rise to the sacro-iliac joint (Borkhvardt and Malashichev 2000), and the less developed the adult pelvic girdle (Raynaud 1985; Raynaud et al. 1975; Sewertzoff 1931). Saunders (1948) identified a distal ectodermal thickening, the apical ectodermal ridge (AER), as the prerequisite for growth of the limb bud. However, AER ablation experiments (Summerbell 1974; Rowe and Fallon 1982) and the phenotype of the limbless mutant chick (Prahlad et al. 1979), which lacks the AER demonstrated that the AER is not required for girdle formation. Moreover, after inactivation of FGF4 and FGF8 in mouse AER cells, pelvis development is not affected (Sun et al. 2002). Interestingly, similar to the limbless mutants in chick (Fallon et al. 1983; Carrington and Fallon 1988), in limbless reptiles the hindlimb buds form, but regress prior to AER formation and show no further outgrowth (Raynaud 1962, 1985). Together, these experimental and comparative observations indicate a very early influence of limb bud structures on pelvis development. Recently, mutant mice devoid of the homeobox transcription factor Emx2 (Pellegrini et al. 2001) were found to lack the ilium, but not the ischium and pubis, indicating a differential regulation of proximal and distal pelvic elements. This hypothesis is supported by the recent finding that aristaless-related genes have an impact on the development of the pubis, and in a less extent the ischium, but not the ilium (Kuijper et al. 2005). In Alx4 mutants mostly the proximal part of the pubis is absent; while Alx3 / /Alx4 / and Alx4 / /Cart1 / double mutant mice show the complete absence of the pubic cartilage and severe truncation of the ischium. To our knowledge, no further functional data on pelvis development are available to date. To shed more light on the development of the avian pelvic girdle, we provide here a detailed description of chick pelvis morphogenesis based on the study of serial sections and whole-mount skeletal preparations, and give an overview of the expression of various marker genes in
the developing pelvic region. Moreover, we show for the first time that avian pelvis development depends on ectodermal signals prior to overt limb development. Our results indicate that the regulation of ilium development differs from the regulation of ischium and pubis.
Materials and methods Chick embryos Fertilized eggs of White Leghorn domestic hen (Gallus gallus domesticus) were incubated at 37.8°C and 80% humidity. The embryos were staged according to Hamburger and Hamilton (1951). A series of embryos (n=59) at HH-stages 25–41 for whole-mount skeleton visualization and serial histological sections, and another series of embryos (n=28) at HH-stages 25–31 for in situ hybridization in whole-mounts and serial sections were used. For microsurgical experiments 145 embryos were operated at HH-stages 12–19. Microsurgery The general approach for microsurgery was performed as described in the literature (Ordahl and Christ 1998). To investigate the effect of the hindlimb bud and the somatopleure ectoderm on the development of the pelvic girdle, we carried out two series of experiments. In the first series we cut off the right hindlimb bud with small scissors or two electrolytically sharpened tungsten needles (n=65) at HH-stages 17 to 19, taking care to remove all the limb bud mesenchyme and the overlying ectoderm including the AER. In the second series we replaced the limb field ectoderm covering the somatopleure opposite the somites or presomitic paraxial mesoderm at approximate levels of somites 25–33 with gold foil at HH-stages 12–16 (n=80). Experimental embryos were re-incubated until HH-stages 30–36 when the pelvic muscle pattern and the cartilaginous pelvic girdle were fully developed. Of the surviving embryos we investigated histologically and as whole-mounts those operated embryos (n=15 for the first series and n=18 for the second series) which did not show any signs of limb development or severe nonspecific malformations (e.g. caused by excessive somatopleure cutting). Unoperated sides of operated embryos and non-operated embryos of comparable age served as controls. Both successfully operated and control embryos were fixed in alcohol and acetic acid solution for whole-mount skeleton visualization, Serra fixative (Serra 1946) for immunostaining, and DEPC–treated 4% paraformaldehyde in PBT for in situ hybridization. Whole-mount cartilage visualization To visualize the chondrifying skeleton, whole embryos ranging from day 7 to 11 were stained with Alcian Blue
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8GS (Serva) as described by Kant and Goldstein (1999) with some modifications. Briefly, embryos were fixed in 100% ethanol for 24 hours and stained in 0.015% solution of Alcian Blue dissolved in 80 ml of absolute ethanol and 20 ml of glacial acetic acid. After staining for four to 6 days, the embryos were cleared either in 100% methyl salicylate for 24–48 h or 1% KOH for one to two weeks. Embryos cleared with methyl salicylate were then examined under the microscope and processed further for sectioning. Older embryos from HH-stage 34 and up to pre-hatching were also stained with a mixture of Alcian blue and Alizarin red (Sigma) as described (Kessel and Gruss 1991) to visualize centers of ossification.
placed on three parallel rows of object slides. These three pools of sections were labeled in accordance to the position of the sections, but processed separately. Two of the pools were hybridized with each of the probes, the third one was stained with Alcian blue and anti-a-desmin antibodies as described above. This technique allowed us to visualize expression of two regulatory genes, as well as muscle desmin and cartilage mucopolysaccharides in the same embryo on adjacent sections offset by approximately 30–40 lm of each other and alternating at a regular interval. This approach allowed us to examine the co-expression of the marker genes in tissues of interest.
Results Immunostaining on sections Morphogenesis of the chicken pelvic girdle To visualize skeletal muscles in histological sections we performed immunostaining with anti-a-desmin antibodies. Serial sections of 7–10 lm were washed in 0.3% solution of H2O2 in methanol to block the endogenous peroxydases, preincubated in 1% solution of bovine serum albumin in phosphatase buffer and hybridized with primary monoclonal anti-a-desmin antibodies (Dako). After incubation with secondary goat antimouse antibodies, conjugated with horse-radish peroxidase (Sigma), antibodies were visualized with the help of diaminobenzidine (DAB-reaction). This staining was combined with alcian blue staining done either before (see above) or after immunostaining and nuclear red contour coloring. After this staining procedure, in sections muscles were brown to black, cartilage was blue, and other tissues were pink with red nuclei. Whole-mount in situ hybridization Normal and experimental embryos were fixed overnight at 4°C in 4% paraformaldehyde. Embryos were then washed twice in PBT, dehydrated in methanol and stored at 20°C. In situ hybridization was performed as previously described (Nieto et al. 1996). The following riboprobes were used: Emx2 (gift of Andrew Lumsden; Bell et al. 2001); Pax1 (kindly provided by Dr. Martin Goulding), MyoD (kindly provided by Dr. Bruce Paterson), and Sox9 (gift of Felicitas Pro¨ls, Pro¨ls et al. 2004). The sense and antisense riboprobes were labelled with digoxigenin RNA labelling kit as recommended by the manufacturer (Boehringer Mannheim, Germany). In situ hybridization on tissue sections In situ hybridization on tissue sections was made with the same riboprobes as used for whole-mount in situ hybridization (not shown). In situ hybridization with Emx2 and Sox9 riboprobes were performed in the same embryos. Serial sagittal or transverse sections were
The triradiate pelvic girdle of birds is morphologically well described in the comparative anatomical literature (Lebedinsky 1913; Boas 1933; Baumel et al. 1979; Wassif et al. 1980). The major and most proximal pelvic element, the ilium, consists of two large wings, Alae preand postacetabulares . The junction between the two iliac wings is situated at the level of vertebra 29 and over the acetabulum (Figs. 1a, 2). The whole ilium in chick stretches along approximately 14 synsacral vertebrae from vertebra 21 through 35 and contributes to the dorsal half of the acetabulum. The ischium is a mediolaterally flattened, almost triangular element, underlying the Ala postacetabularis ilii and contributing to the ventrocaudal sector of the acetabulum. The pubis is a long and thin bone, stretching along the ventral edge of the ischium, and contributing with its proximal end to the ventrocranial sector of the acetabulum, reaching with its distal end beyond the caudal part of the ilium and ischium (Fig. 1a). The first morphological evidence of the onset of pelvis development is the formation of an extended mesenchymal gap between the epaxial (back) and hypaxial (leg) muscles at HH-stages 25–29 opposite the prospective synsacral vertebrae and the hindlimb bud. This mesenchyme, which is of somatopleural origin (Chevallier 1977), fills the space left by the cells of the hypaxial dermomyotomes which have emigrated to the limb to form appendicular muscle (Figs. 1b, 2a). The following description of pelvis morphogenesis is summarized in Table 1 and schematically illustrated in Fig. 2. The ilium is the first pelvic element visible at HHstage 26–27 as a separate, densely packed mesenchymal anlage at the level of somite 27. It is located craniodorsal to the proximal extremity of the already well established femoral cartilage, spanning approximately the diameter of the latter (Fig. 1c). At HH-stage 28, the ilium encircles the femoral head in a cap-like fashion (Fig. 1d), and subsequently, with the onset of chondrification, elongates rapidly in caudal direction to form the Ala postacetabularis ilii (Fig. 1e, f). After having reached the
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caudal end of the synsacrum, the ilium grows craniad from the prospective acetabulum until, at HH-stage 32– 33, it reaches the caudalmost thoracic rib (Fig. 1h, j). The definite extension spanning the length of 12 synsacral vertebrae is reached at HH-stage 36. Only after HH stage 38, ossification in the ilium starts (Table 1). Pubis and ischium first appear as mesenchymal condensations at HH-stages 27 and 28, respectively. The
early pubic anlage is initially (until HH-stage 29) directed cranioventrally towards the caudal wall of the visceral cavity and localized in front of the prospective acetabulum. Subsequently, it grows caudad along the body wall and points more and more caudally in the course of development. The ischium anlage is first visible ventral to the acetabulum. At HH-stages 28–29 it grows caudad along the lateral wall of the cloacal part of the
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Fig. 1 Overview on the normal development of the chick pelvic girdle. Cranial is to the left, dorsal is to the top. Abbreviations: Ac Acetabulum, caJl: Caudal portion of the ilium (Ala postacetabularis ilii), crJl Cranial portion of the ilium (Ala praeacetabularis ilii), Fe Femur, Is Ischium, Pu Pubis, Sa (prospective) Synsacrum. a Completely ossified pelvis of an adult hen. Only the structures referred to in the text are labelled, for further anatomical details please refer to appropriate textbooks on avian anatomy (e.g. Starck 1979; Nickel et al. 1992). b Transverse section through a HH-stage 27 embryo at sacral level. Note the pre-pelvic mesenchyme (black arrow) between the epaxial and hypaxial muscle anlagen (white arrows). c–j Whole mount chick embryos stained with Alcian Blue to visualize the cartilaginous components of the pelvic girdle. c HHstage 27. The anlage of the ilum forms a small mesenchymal condensation next to the proximal extremity of the femur. Note that femur, tibia and fibula are already well developed whereas chondrification of the ilium is just about to start. d, e HH-stage 28 and 29. Chondrification of the ilium has started in the acetabular region. f HH-stage 30. The ilium has grown caudad to form the Ala postacetabularis ilii. Growth of the pubis has started ventromedial to the acetabular region of the ilium (poorly visible here). The mesenchymal anlage of the ischium is not yet chondrified. g HHstage 31. The ilium has reached the caudal level of the synsacrum and elongates craniad to form the Ala praeacetabularis ilii. Chondrification of the ischium has started ventrocaudal to the acetabulum (not visible here). h HH-stage 32. Pubis and ischium are well distinguishable ventrocranial and ventrocaudal to the acetabulum, respectively. The pubis is changing its orientation from dorsoventral to craniocaudal by bending caudad. j HH-stage 33. The pubis has extended caudal to the acetabulum and approaches the ilium and ischium from ventral in craniocaudal orientation Fig. 2 Schematic illustration of pelvis development as displayed in Alcian blue stainings in Fig. 1. a–g correspond to c–j in Fig. 1, respectively. Dotted lines: Skeletal elements of the free limbs. Red: Ilium. Blue: Pubis. Yellow: Ischium. Abbreviations see Fig. 1
Table 1 Timetable of skeletal development in the chick pelvic girdle
Mesenchymal condensation Chondrification Perichondral ossification Endochondral ossification
Ilium
Pubis
Ischium
26–27 27–28 38 38–39
27 29 38–39 40
28 31 40 41
Developmental stages (Hamburger and Hamilton 1951) are indicated in cells
visceral cavity, flattening more and more medio-laterally. At stage 29 the small pubis anlage is completely chondrified, while the larger primordium of the ischium chondrifies last at HH-stage 31. Until the onset of osteogenesis (Table 1), development of the pubis precedes the ischium considerably. Thus, the temporal sequence of development of the chick pelvic elements is ilium, pubis, and lastly ischium. Marker gene expression during pelvic girdle development To provide a basis for future research on the molecular regulation of pelvis development, we describe the expression patterns of four marker genes illustrating
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early steps of pelvic differentiation, MyoD Pax1, Sox9, and Emx2 (Fig. 3). MyoD, which is a marker of myogenic differentiation, is expressed in the axial and limb girdle musculature. Starting at HH-stage 26, the lack of MyoD expression proximal to the base of the hindlimb illustrates the prospective pelvic mesenchyme (Fig. 3a–d). Pax1 has been shown to be expressed in the developing avian shoulder girdle, where it precedes chondrification in the chondrogenic mesenchyme (Huang et al. 2000). Similarly, in the pelvic girdle, Pax1 is expressed in the mesenchyme ventral and cranial to the proximal
Fig. 3 Whole-mount in situ hybridizations of chick embryos from HH-stage 26 to HH-stage 29 to illustrate the expression of MyoD Sox9, Emx2, and Pax1 during early stages of pelvis development. Cranial is to the right, dorsal is to the top. a–d MyoD expression pattern. Note that the pre-pelvic mesenchyme is forming between the epaxial (back-) and hypaxial (limb-) muscle anlagen (arrows). e–h Sox9 expression pattern. As a marker of chondrogenesis, expression is first seen in the ilium anlage (arrows) from HH-stage 27. j–m Emx2 expression pattern. Expression precedes Sox9 expression in the postacetabular ilium (white arrow) and a small domain dorsal and cranial to the acetabulum (black arrow). n–q Pax1 expression. Prior to chondrogenesis, Pax1 is expressed in a mesenchymal region ventrocranial to the acetabulum (arrow)
portion of the hindlimb bud (Fig. 3n–q), but not in cartilage. At HH-stage 28, Pax1 expression demarcates the position of the mesenchymal anlage of the pubis and the cranial part of the acetabulum. At HH-stage 29–30, it is expressed in the mesenchyme surrounding the pubic cartilage, as well as weakly in the mesenchymal ilium adjacent and cranial to the prospective acetabulum. However, Pax1 expression seemed to exclude the ischium anlage at stages studied. Thus, except this last observation, the expression pattern of Pax1 in chick is similar to that found in the pelvic region of the mouse (Timmons et al. 1994). Emx2 is required for the formation of the scapula blade and the ilium (Pellegrini et al. 2001). It is expressed in the presumptive scapular blade, but not in the dermomyotomes (Pro¨ls et al. 2004) where the precursors of the scapular blade originate (Huang et al. 2000). In the pelvic region, Emx2 is only expressed in the mesenchyme at the base of the hindlimb bud corresponding to the prospective Ala postacetabularis ilii, and in a narrow craniocaudal strip demarcating the dorsal limb base (Fig. 3j–m). Expression ceases with the onset of chondrification as monitored by Sox9 expression. Thus it precedes the expression of Sox9 in a way similar to that found in the shoulder region (Pro¨ls et al. 2004). Sox9 is
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expressed in all pelvic anlagen starting at HH-stage 27, most intensively in the ilium (Fig. 3e–h, and data not shown), and marks the progress of chondrification as described in the above section. The presence of the early hindlimb bud is required for the formation of pelvic elements Classical studies on limb bud development have suggested that total removal of very early limb buds in 65-h old embryos (HH-stage 17–18), deleting both ectoderm and mesoderm, lead to the absence of distal pelvic elements (pubis and ischium), and to a truncated ilium (Spurling 1923). These pelvic defects were not seen after limb ablation in 3.5 day old embryos (HH-stage 20–23) (Rogulska 1965). To reinvestigate in more detail the timing of a putative influence of limb bud structures on pelvis development, we ablated limb buds including mesenchyme and ectoderm at the critical HH-stages 17 to 19. After limb bud extirpation at HH-stage 17 (n=6), in all cases pelvis development was disturbed. Specifically, the ilium was shortened due to a truncation of its preacetabular part, and abnormally inclined towards the sacrum at a greater angle (Fig. 4a; see also figures in Laing 1982). The ischium was present, but smaller in size and abnormally shaped. The pubis was entirely absent (n=2, Table 2) or displayed a lack of the proximal part, while, interestingly, the distal part was present without proximal connection to the acetabulum (n=2, Fig. 4b). In contrast, after limb bud extirpation at HH-stage 18 or older (n=9), all embryos showed a complete set of pelvic elements, including in some cases a dislocated rudiment of the proximal part of the femur (n=3). However, they always displayed defective morphogenesis of the acetabulum and at least a slight shortening of the preacetabular wing of the ilium (Fig. 4a, c). Since such defects have also been reported in paralyzed chick embryos (Laing 1982), they are likely to be due to the lack of femoral articulation and missing physical stress of the tissue. Thus, our results suggest that an influence of the limb bud tissues on pelvis formation is required only until HH-stage 17, and is not required any more from HH-stage 18 onwards. Early limb field ectoderm is required for pelvis formation Limb bud ablation experiments in the literature (Spurling 1923; Rogulska 1965), and those reported above, did not discriminate between pelvis inducing signals from the limb bud ectoderm or mesenchyme. Classical experiments have suggested that signals from the limb bud ectoderm are required for hindlimb outgrowth, but not for pelvis formation. After removal of the AER at HH-stage 18 (Saunders 1948; Summerbell 1974) and in limbless mutant embryos devoid of a functional AER (Prahlad et al. 1979), the limbs were lacking all skeletal
Fig. 4 Defective pelvis development after hindlimb bud excision. a and b dorsal views of Alcian blue stained whole mount specimens, cranial is to the top. c transverse section of an operated embyo at acetabular level. Sections stained with Alcian blue to visualize cartilage (blue color), and with an antibody against Desmin to visualize muscle (brown color). Abbreviations see Fig. 1. a After excision of the hindlimb bud at HH-stage 17, the ilium is truncated cranially Arrows demarcate the lost portion of the ilium as compared to the contralateral side of the embryo. The ischium has formed aberrantly, the pubis is lacking. b After excision of the hindlimb bud at HH-stage 18, the proximal portion of the pubis is missing whereas the distal part is formed. The dotted line indicates the plane of section in c. c Transverse section through the specimen in b at acetabular level. The acetabulum (arrow on the control side) is missing on the operated side
elements distal to the girdle, but the girdles developed normally. Here, to investigate whether pelvis formation depends on ectodermal signals prior to AER formation, we removed the ectoderm over the somatopleure, corresponding to somite levels 25–33, in embryos of HHstages 12 through 16. In all stages examined, after ectoderm removal, we observed severe defects in the pelvic skeleton. The earlier the manipulation was done, the more proximal structures were affected, and the more severe the overall defect was found (Table 2, Fig. 5). Ectoderm removal at HH-stages 13 through 16 lead to a complete loss of pubis only. Ectoderm removal at HH-stage 12 always resulted in a total loss of ischium
194 Table 2 The resultant pelvic phenotypes after removal of the hindlimb bud (at HH-stage 17) or the lateral ectoderm over the somatopleure opposite the level of somites 25–33 (at HH-stages 12–16) The numbers of successfully operated embryos with corresponding phenotypes after each experiment are shown
Operation stage 12 13 14 15 16 17
Complete pelvis (minor defects)
Pubis ischium, ilium +
Pubis, ischium + (defective)
ilium
2 1 3 1 2 2
2 2 4
Fig. 5 Defective pelvis development after ectoderm ablation. a dorsal view of Alcian blue stained whole mount specimen, cranial is to the top. b transverse section of operated embyos at acetabular level. Sections stained with Alcian Blue to visualize cartilage (blue color), and with an antibody against Desmin to visualize muscle (black color). a After ectoderm ablation in the hindlimb field at HH-stage 15, the ilium is severely truncated, pubis and ischium are entirely missing. Note that on the contralateral side, the ischium is hidden underneath the ilium and therefore not visible in this view. The arrow indicates the pubis. b Transverse section through the acetabular region after limb field ectoderm ablation at HH-stage 16. The acetabulum (black arrow) is missing on the operated side (white arrow pointing at non-chondrified mesenchyme in the acetabular region)
and pubis. The ilium, and in the latter case also the ischium, were severely truncated. The ilium never disappeared completely, its caudal part (Ala postacetabularis ilii) being always present after operation at HH-stage 12, while the cranial portion was shortened or lacking in all cases. As ectoderm ablations in the hindlimb field are not possible earlier than HH-stage 12, we cannot yet determine whether the ilium depends on earlier ectodermal signals, or whether the ilium is regulated differently from ischium and pubis. Our results insinuate, however, that the cranial portion of the ilium might be differently regulated, or of different origin, than the posterior portion. The sacral ribs and diapophyses, which form the connection of the ilium to the sacrum, were never affected in these experiments. In summary, we show for the first time that avian pelvis development depends on ectodermal signals emanating from the limb field prior to limb bud and
3 1 1
AER formation, and that the individual pelvic elements seem to depend on these signals to a different extent.
Discussion Following the early descriptions of avian pelvis development based on histological sections (Johnson 1893; Mehnert 1887; Lebedinsky 1913), no descriptive study of pelvis morphogenesis based on whole-mount specimens has been available. Here, we have analyzed chick pelvis development in serial paraffin sections and in Alcian blue and Alcian blue/Alizarin red stained whole-mount preparations of chicken embryos from HH-stage 27 to 41 (Table 1). We have shown that the pelvic elements arise from HH-stage 26 as local condensations in the mesenchyme between the epaxial trunk muscles and the limb muscles at the rostro-caudal level of somites 25–34. This mesenchyme fills the space left by hypaxial dermomyotomal cells after their emigration as muscle and endothelial precursor cells into the limb bud (Scaal and Christ 2004). As it interrupts the continuity of muscle anlagen between epaxial and hypaxial locations at the level of the synsacrum, the pre-pelvic mesenchyme has been described in reptile embryos as sacral gap (Borkhvardt and Malashichev 2000). This mesenchyme gives rise to the pelvic anlage (see Borkhvardt 1995; Borkhvardt and Malashichev 2000; Malashichev 2001 for more details). The diverse pelvic elements appear in the temporal sequence ilium, pubis, ischium, and maintain this succession during further differentiation. Thus,
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the onset of chondrification is as early as at HH-stage 27 in the ilium and at HH-stage 31 in the ischium, and the onset of perichondral ossification is at HH-stage 38 in the ilium and HH-stage 40 in the ischium (Table 1). We have monitored the expression patterns of a number of marker genes to illustrate the earliest steps of pelvis formation at molecular level. Starting at HH-stage 25, the pre-pelvic mesenchyme is devoid of MyoD expression and expresses high levels of Emx2. Emx2 is expressed temporarily prior to chondrogenesis in the upper part of the pelvic girdle (ilium anlage). With the onset of chondrification, as revealed by Alcian blue staining and Sox9 expression, Emx2 expression is downregulated. Thus, development of the ilium follows the general regulatory cascade leading to ossification as described in the scapula (Pellegrini et al. 2001; Akiyama et al. 2002; Pro¨ls et al. 2004). In the shoulder girdle, Pax1 is also expressed in the pre-scapula mesenchyme prior to chondrification (Huang et al. 2000). Interestingly, our data indicate that Pax1 is not expressed in all pelvic precursor cells but seems to be restricted to mesenchymal regions which will give rise to the pubis, raising the possibility that the pubis is regulated differently than the other pelvic bones. Data in the literature suggest a differential regulation also of the ilium. In mice Emx2 expresses at the base of hindlimb bud in the region fated to prospective ilium. In Emx2 mutant mice, both the scapular blade and the major part of the ilium, which normally connects to the vertebral column, are absent (Pellegrini et al. 2001). In chick we observed the expression of Emx2 in the mesenchymal ilium anlage and mostly in its caudal portion. This argues for an Emx2 dependent developmental program in the postacetabular ilium, but not in the other pelvic elements. Similarly to the dual origin of the scapula (Huang et al. 2000), this might reflect a different embryonic origin of the ilium. However, Chevallier (1977) concluded from quail-to-chick transplantations that the entire pelvis originates from a single source, the somatopleure. Cell lineage experiments to re-examine this issue are under way in our laboratory. The notion of developmental heterogeneity of the pelvic girdle is supported by our finding that a part of the caudal ilium persists after ectoderm ablations even at HH-stage 12, while other parts of the ilium and the rest of the girdle are lacking or severely truncated after ectoderm ablations at various stages, and also after early limb bud amputation. Moreover, comparative morphology indicates that proximal (ilium) and distal (pubis and ischium) parts of the pelvic girdle might undergo different developmental programs. In terrestrial squamate reptiles, which show distal to proximal limb and girdle reduction, the most evolutionarily stable pelvic element is the ilium, which may even retain the connection to the axial skeleton (Sewertzoff 1931; Raynaud et al. 1975). Extinct marine ichthyosaurs have a reduced proximal part of the ilium and the ilio-sacral joint, whereas the rest of the girdle and limb skeleton is formed (McGowan and Motani
2003). Similarly, whales with preserved hindlimb rudiments usually lack the ilium and girdle connection to the vertebral column, while the ischium–pubis cartilage is maintained (Hosokawa 1951). The regulation of avian pelvis formation is still largely unknown. In classical limb bud ablation experiments, Spurling (1923) found that development of the pelvic bones depends on the presence of the early limb bud. Later Rogulska (1965) showed that older limb buds are dispensable for pelvis formation. To determine the exact timing of limb bud influence on pelvis development, we repeated these limb ablation experiments in the critical stages from HH-stage 17 to 19 and specified that limb bud signals are necessary until HH-stage 17, but are dispensable already at HH-stage 18. This coincides well with the onset of AER formation at HH-stage 18 (Todt and Fallon 1986), illustrating that the influence from the limb bud to the pre-girdle mesenchyme is AER independent. In the limb bud ablation experiments reported above, the authors did not distinguish between the effects of ectodermal and mesenchymal components on pelvis development. Even though it is known that the AER and AER-derived FGF signals are not necessary for pelvis formation (Summerbell 1974; Prahlad et al. 1979; Sun et al. 2002), we hypothesized that the limb field ectoderm prior to AER formation might be involved in pelvis development. Indeed, after ectoderm ablations from HH-stages 16 to 12, pelvic elements were truncated or missing to a progressive extent the earlier the ectoderm was ablated (Table 2). Interestingly, the sequence of disappearance of pelvic elements after progressively earlier ectoderm ablations did not simply correspond to the reverse order of their formation in the course of normal embryogenesis (Table 1). The element most prone to defects was the pubis, which showed severe truncation of its proximal portion or was entirely absent after ectoderm removal as late as HH-stages 16 and injured after later limb bud amputation. Defects in development of the ischium, which forms last in normal development, were first observed after ectoderm ablation at HH-stage 16, but complete absence of the ischium occurred only after ectoderm ablation at HHstage 12. The ilium never disappeared completely, its caudal portion (Ala postacetabularis ilii) being present even after operation at HH-stage 12. Earlier operations were not possible because the prospective hindlimb bud and pelvic region have not yet formed in the embryo at stages 11 or earlier. These findings substantiate the hypothesis that the influence of the ectoderm on the development of the various pelvic elements is differential. Moreover, these results suggest that pelvic girdle development is not following the growth zone model, which has been proposed for the limb skeletal elements by Summerbell et al. (1973) and which would predict a proximal to distal succession of pelvic element formation. An alternative model proposed by Dudley et al. (2002) postulates that cell fates are established very early in the limb field by intercellular signaling events, and
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that AER-mediated limb outgrowth is expanding these preformed populations rather than laying them down. A similar mode of early specification of skeletal elements prior to differentiation seems to be active during pelvis development. In summary, we present an overview of chick pelvis development based on whole-mount skeletal preparations, sections, and expression patterns of various marker genes. We demonstrate that the limb field ectoderm is necessary for pelvis formation, and that the ilium, ischium and pubis depend on ectodermal signals at different time points and to a different extent. We establish that the influence of limb bud tissues on pelvis development is required at stages prior to HH-stage 18 and that proximal (ilium) and distal (pubis and ischium) parts of the pelvic girdle might undergo different developmental programs. Acknowledgements We thank Ulrike Pein, Ellen Gimbel, Lydia Koschny, Susanna Konradi for excellent technical assistance, and Florian Ehehalt and Ruijin Huang for discussion of experiments. This work was supported by EMBO and Alexander von Humboldt Foundation (Y.M.), and by the Medical Faculty of University of Freiburg and DFG (SFB592, A1) to B.C. and M.S.
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