Signals from Trunk Paraxial Mesoderm Induce Pronephros Formation in Chick Intermediate Mesoderm

Home , Mesonephros, Paraxial mesoderm, Pronephric duct, Pronephros

Developmental Biology 220, 62–75 (2000) View metadata,doi:10.1006/dbio.2000.9623, citation and similar papers available at onlinecore.ac.uk at http://www.idealibrary.com on brought to you by CORE provided by Elsevier - Publisher Connector Signals from Trunk Paraxial Mesoderm Induce Pronephros Formation in Chick Intermediate Mesoderm

Teri Jo Mauch,*,†,1 Guizhi Yang,* Mindi Wright,† Danielle Smith,† and Gary C. Schoenwolf† *Department of Pediatrics and †Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, Utah 84132

We used Pax-2 mRNA expression and Lim 1/2 antibody staining as markers for the conversion of chick intermediate mesoderm (IM) to pronephric tissue and Lmx-1 mRNA expression as a marker for mesonephros. Pronephric markers were strongly expressed caudal to the fifth somite by stage 9. To determine whether the pronephros was induced by adjacent tissues and, if so, to identify the inducing tissues and the timing of induction, we microsurgically dissected one side of chick embryos developing in culture and then incubated them for up to 3 days. The undisturbed contralateral side served as a control. Most embryos cut parallel to the rostrocaudal axis between the trunk paraxial mesoderm and IM before stage 8 developed a pronephros on the control side only. Embryos manipulated after stage 9 developed pronephric structures on both sides, but the caudal pronephric extension was attenuated on the cut side. These results suggest that a medial signal is required for pronephric development and show that the signal is propagated in a rostral to caudal sequence. In manipulated embryos cultured for 3 days in ovo, the mesonephros as well as the pronephros failed to develop on the experimental side. In contrast, embryos cut between the notochord and the trunk paraxial mesoderm formed pronephric structures on both sides, regardless of the stage at which the operation was performed, indicating that the signal arises from the paraxial mesoderm (PM) and not from axial mesoderm. This cut also served as a control for cuts between the PM and the IM and showed that signaling itself was blocked in the former experiments, not the migration of pronephric or mesonephric precursor cells from the primitive streak. Additional control experiments ruled out the need for signals from lateral plate mesoderm, ectoderm, or endoderm. To determine whether the trunk paraxial mesoderm caudal to the fifth somite maintains its inductive capacity in the absence of contact with more rostral tissue, embryos were transected. Those transected below the prospective level of the fifth somite expressed Pax-2 in both the rostral and the caudal isolates, whereas embryos transected rostral to this level expressed Pax-2 in the caudal isolate only. Thus, a rostral signal is not required to establish the normal pattern of Pax-2 expression and pronephros formation. To determine whether paraxial mesoderm is sufficient for pronephros induction, stage 7 or earlier chick lateral plate mesoderm was cocultured with caudal stage 8 or 9 quail somites in collagen gels. Pax-2 was expressed in chick tissues in 21 of 25 embryos. Isochronic transplantation of stage 4 or 5 quail node into caudal chick primitive streak resulted in the generation of ectopic somites. These somites induced ectopic pronephroi in lateral plate mesoderm, and the IM that received signals from both native and ectopic somites formed enlarged pronephroi with increased Pax-2 expression. We conclude that signals from a localized region of the trunk paraxial mesoderm are both required and sufficient for the induction of the pronephros from the chick IM. Studies to identify the molecular nature of the induction are in progress. © 2000 Academic Press Key Words: Pax-2; Lim 1/2; Lmx-1; kidney development; pronephros; in situ hybridization; immunohistochemistry; paraxis; somites.

INTRODUCTION and dysplasia, or abnormal kidney development, accounts for one-third of end-stage renal disease in children (USRDS, Congenital anomalies of the genitourinary tract are de- 1997). Embryonic kidney development has been described tected in about 1 in 500 fetal ultrasounds (Colodny, 1987), morphologically (Waddington, 1938; Abdel-Malek, 1950; Saxe´n and Sariola, 1987), and some of the genes involved in kidney development have now been identiﬁed (Eccles, 1 To whom correspondence should be addressed at the Univer- 1998; Lipschutz, 1998). sity of Utah School of Medicine 2R063, 50 N. Medical Drive, Salt Lake City, UT 84132. Fax: (801) 581-8043. E-mail: tmauch@ Most studies of kidney induction have focused on the hsc.utah.edu. condensation of the metanephric mesenchyme in response

0012-1606/00 $35.00 Copyright © 2000 by Academic Press 62 All rights of reproduction in any form reserved. Induction of Chick Pronephros 63 to the invading ureteric bud (Rothenpieler and Dressler, Little is known about the regional specification of the 1993; Dressler, 1995). Although this system is appropriate intermediate mesoderm (Vise et al., 1997). Mesoderm cul- for the investigation of conversion of mesenchyme to tured in isolation does not differentiate into specialized epithelium, it is not particularly useful for studying how tissue, making self-determination unlikely (Slack, 1994). the kidney primordia are initially specified, because the Rather, inducing signals presumably control when and nephrogenic mesenchyme is already determined by this where structures form. A midline signal is required for the developmental stage and lacks only the terminal condensa- upregulation of Pax-2 expression in the developing eye tion signals provided by the ureteric bud (Saxeń and Sariola, (Macdonald et al., 1995). In Xenopus, somitic tissue will 1987). The vertebrate kidney develops in three major steps form pronephric tubules if explanted and grown away from via specific tissue interactions (Saxeń and Sariola, 1987). the influence of the notochord (Yamada, 1940), and anterior The first kidney, the pronephros, is required for further somites have recently been identified as the inducing tissue development of the mesonephric and metanephric kidneys in frog pronephric development (Seufert et al., 1999). (Vise et al., 1997; Carroll et al., 1999a). The pronephros can We hypothesized that signals from adjacent tissues are be analyzed throughout the period of mesodermal pattern- involved in embryonic kidney induction. Using whole- ing and it has the advantage of less complex anatomy, mount in situ hybridization and immunohistochemistry, making it an ideal system in which to explore the early we monitored Pax-2 and Lmx-1 expression and Lim 1/2 stages of intermediate mesodermal patterning (Vise et al., antibody staining during the conversion of intermediate 1997). mesoderm into pronephric tissue. We sought to identify the Conversion of undifferentiated tissue to committed cell tissue interactions responsible for pronephros induction in lineages requires the transcriptional activation of structural the early chick kidney. genes. The Pax genes are paired-box transcription factors expressed in distinct spatiotemporal patterns during em- bryogenesis (Gruss and Walther, 1992; Eccles, 1998). Pax-2, MATERIALS AND METHODS important in the differentiation of the definitive or metanephric kidney, is expressed during early embryonic devel- Embryos opment in the pronephroi, eyes, otic vesicles, and neural Fertile White Leghorn chicken or Japanese quail (Coturnix tube (Dressler et al., 1990; Lechner and Dressler, 1997). coturnix japonica) eggs were incubated at 38°C in forced-draft, Pax-2 expression is tightly regulated during normal human humidified incubators until embryos had reached the appropriate development; overexpression has been observed in Wilms’ stage (Hamburger and Hamilton, 1951). tumors and renal cell carcinomas (Dressler, 1996). Haplo- insufficiency of Pax-2 results in kidney hypoplasia, vesi- Microsurgery and Culture coureteral reflux, and optic nerve dysplasia in the autoso- mal dominant renal coloboma syndrome (Sanyanusin et al., For most experiments, embryos were placed into Spratt culture 1995) and renal agenesis or hypoplasia in Senior–Loken (Spratt, 1947), subjected to microsurgery, and incubated overnight. syndrome (Warady et al., 1994). Haploinsufficiency of Pax-2 Operations were done aseptically using the tips of cactus needles. Longitudinal cuts were made between the trunk paraxial meso- in three mouse mutants is associated with ear, eye, and derm (PM)2 and the intermediate mesoderm (PM/IM), between the brain anomalies as well as renal cystic changes and hypo- notochord and the paraxial mesoderm (N/PM), or between the plasia of varying degrees (Keller et al., 1994; Torres et al., intermediate and the lateral plate mesoderm (IM/LP), and the 1995; Favor et al., 1996). In the studies described here, we tissues were teased apart. Rostral/caudal (R/C) transections were used Pax-2 expression as a molecular marker for pronephros made at several somitic levels. Following manipulation, embryos induction. were either fixed immediately for in situ hybridization or cultured Like Pax-2, Lim 1 is expressed in both the tubules and the in vitro for up to 24 h (Spratt, 1947). duct of the early pronephros and persists in the adult For longer incubations, embryos were cultured in ovo (Criley, Xenopus (Carroll et al., 1999a,b), zebrafish (Carroll et al., 1966). A window was made in the shell and the embryo was lightly stained with 0.1% neutral red. Longitudinal incisions were made 1999a), and murine (Barnes et al., 1994) kidney. Mice using cactus needle tips between the PM and the IM, and the lacking functional Lim 1 do not have pro-, meso-, or tissues were teased apart. Windows were sealed with a glass metanephroi (Shawlot and Behringer, 1995). Staining with coverslip and paraffin, and eggs were incubated for up to 3 days at anti-Lim 1/2 antibody served as a confirmatory marker for 38°C in forced-draft, humidified incubators, then fixed and sub- pronephros induction. jected to in situ hybridization. Another LIM homeobox gene, Lmx-1, is first expressed in Putative inducing and responding tissues were isolated and the mesonephros (Fernandez et al., 1997). Mutations in recombined in collagen gels. Quail somites caudal to the fifth Lmx-1B have been described in the nail–patella syndrome, somite were excised, along with their overlying ectoderm and which includes proteinuria and renal dysplasia (Chen et al., 1998; Dreyer et al., 1998). All three kidney forms arise from embryonic intermedi- 2 Abbreviations used: IM, intermediate mesoderm; LP, lateral ate mesoderm, which lies between the trunk paraxial me- plate mesoderm; N, notochord; PM, paraxial mesoderm; R/C, soderm and lateral plate mesoderm in the early embryo. rostral caudal transection.

FIG. 1. Whole-mount in situ hybridization shows Pax-2 expression in IM destined to become pronephros (arrows). Arrowheads indicate Pax-2 expression in the paraxial mesoderm (ﬁfth somitic level) of a HH stage 8ϩ embryo. The bottom shows Pax-2 expression in cross section. The Hamburger and Hamilton stage is indicated in the lower left corner of each photo. Abbreviations: N, notochord; NT, neural tube; S, somite; LP, lateral plate; MB/HB, midbrain/hindbrain level of the neural tube; PS, primitive streak; TB, tailbud. Bars: whole mount, 300 ␮m; section, 100 ␮m. FIG. 2. Whole-mount in situ hybridization shows Pax-2 and Lmx-1 expression in mesonephros. The top row shows Pax-2 expression, and the lower row shows Lmx-1. At the right, cross sections show expression in mesonephric tubules (arrows), connecting segments (small arrowheads), and mesonephric ducts (large arrowheads). The Hamburger and Hamilton stage is indicated in the lower left corner of each photo. Abbreviations: N, notochord; NT, neural tube; A, aorta; V, postcardinal vein. Bars: whole mount, 300 ␮m; section, 100 ␮m. FIG. 3. Microsurgical disruption of embryos developing for 24 h in Spratt culture. (Row 1) Longitudinal cuts between PM and IM prior to stage 8 prevented Pax-2 expression. Embryos cut at stage 9 already expressed Pax-2 at the time of manipulation; such expression persisted but did not extend caudally with further incubation. (Row 2) Tissue sections of an embryo cut at HH7 show that Pax-2 expression correlated with the morphologic pronephric anlage. The Hamburger and Hamilton stage at the time of manipulation is indicated in each photo. Abbreviations: N, notochord; NT, neural tube; S, somite; LP, lateral plate. Bars: whole mount, 300 ␮m; section, 100 ␮m.

underlying endoderm. One-half of the somite explants also con- HH7 embryos. The endoderm was removed to expose the meso- tained notochord. Chick lateral mesoderm (i.e., prospective inter- derm, and the mesoderm was wrapped around the quail somites. mediate and lateral plate mesoderm) was isolated from HH4 or These tissues were embedded in a collagen gel, overlaid with

FIG. 4. Lim 1/2 antibody staining shows that pronephros formation requires a medial signal. A longitudinal cut between PM and IM at HH7 prevented pronephros formation. Left: A whole-mount embryo ﬁxed after incubation for 24 h following manipulation. The pronephros (arrows) was stained with Lim 1/2 antibody. Right: Tissue sections show that Lim 1/2 antibody staining correlated with the morphologic pronephric structure in the control, but not the cut side. FIG. 5. Time course of pronephros formation following manipulation. Pax-2 expression (arrows) was used as a marker for pronephros formation in whole mount and serial sections at time ϭ 4 or 8 h after IM was separated from PM. By t ϭ 8 h, the pronephros was detectable morphologically, as well as by Pax-2 expression, on the control, but not the cut, side. FIG. 6. Pronephros formation is required for mesonephros development. An embryo was incised longitudinally between PM and IM and cultured for 3 days in ovo. Lmx-1 was expressed in limbs (L) and mesonephric tubules (arrows), but not in mesonephric ducts (arrowhead). Abbreviations: N, notochord; NT, neural tube; H, head; A, aorta. Bars: whole mount, 300 ␮m; section, 100 ␮m.

TABLE 1 University of Michigan, Ann Arbor). For Lim 1/2 detection (Figs. 4 Pronephros Formation Following Microsurgical Separation and 10), embryos were blocked with normal goat serum, then of Paraxial from Intermediate Mesoderm stained with a 1:2 dilution of mouse anti-Lim 1/2. The secondary antibody was goat anti-mouse IgG conjugated to peroxidase (Jack- Kidney development on manipulated side son No. 115-035-003), used at a dilution of 1:200. Hybridoma cells secreting anti-Lim 1/2 or anti-quail were obtained from the Devel- HH stage Label None Truncated Normal opmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins Univer- Pax-2 7 30/32 (94%) 2/32 (6%) 0/32 (0%) sity School of Medicine (Baltimore, MD), and the Department of Lim 1/2 13/13 (100%) 0/13 (0%) 0/13 (0%) Biology, University of Iowa (Iowa City), under Contract 1-HD-6- Pax-2 8 5/12 (42%) 7/12 (58%) 0/12 (0%) 2915 from the National Institute of Child Health and Human Ϫ Pax-2 9 1/2 (50%) 1/2 (50%) 0/2 (0%) Development. 9 Pax-2 2/14 (14%) 12/14 (86%) 0/14 (0%) Ͼ9ϩ Pax-2 0/6 (0%) 6/6 (100%) 0/6 (0%)

Note. Data show the number of expressing embryos/total num- RESULTS ber of cases, with the percentage either expressing Pax-2 or stained with anti-Lim 1/2 antibody on the manipulated side in parentheses. Pax-2, but Not Lmx-1, Is Expressed in Pronephros, whereas Both Are Expressed in Mesonephros Previous studies have shown that Pax-2 is expressed in the developing kidney, brain, eye, and ear (Dressler et al., Dulbecco’s minimal essential medium supplemented with 1990). Figure 1 shows that Pax-2 is a molecular marker for L-glutamine, penicillin, streptomycin, and 10% fetal bovine serum pronephric tissue. In some embryos, Pax-2 was weakly (Artinger and Bronner-Fraser, 1993), and cultured for up to 28 h. expressed in the intermediate mesoderm at HH7 (Ham- Cultured chick explants that included presumptive chick PM burger and Hamilton, 1951) (not shown); its level of expres- served as positive controls; negative control cultures comprised only chick lateral mesoderm (absent the endoderm and any pro- sion increased with maturation. By HH9, pronephric Pax-2 spective PM). expression was limited to intermediate mesoderm caudal to In some experiments Hamburger and Hamilton stage 4 or 5 the fifth somite (Fig. 1). In the earlier embryo (HH7 and (HH4, HH5) (Hamburger and Hamilton, 1951) quail node (the HH8, Fig. 1), Pax-2 was also transiently expressed in trunk rostral 125 ␮m of the primitive streak) was transplanted isochroni- paraxial mesoderm at the level of the fifth somite. Pax-2 cally and heterotopically to the chick caudal primitive streak expression persisted in the mesonephros, which was also (250–500 or 500–750 ␮m caudal to the chick node). Chimeric labeled with Lmx-1 (Fig. 2). embryos were cultured in vitro for up to 28 h (New, 1955) prior to In contrast to Pax-2, Lmx-1 (Riddle et al., 1995; Fernan- fixation and analysis. dez et al., 1997) was not expressed in the pronephros; rather, its expression was first seen in the mesonephros and limb In Situ Hybridization buds after HH15. Thus, the pronephros was molecularly characterized by expression of Pax-2, but not Lmx-1 (Fig. 1). When experiments were terminated, embryos were fixed in 4% In contrast, the mesonephros expressed both markers, but buffered paraformaldehyde, dehydrated in graded methanol, and their expression patterns differed. Tissue sections of labeled frozen at Ϫ20°C. Rehydrated embryos were subjected to whole- mount in situ hybridization with digoxigenin-labeled antisense embryos showed Pax-2 expression in the nephric duct, its RNA probes for Pax-2 or Lmx-1 (Nieto et al., 1996). Domingos connecting segment, and the mesonephric tubules, whereas Henrique provided pDNA containing a 1-kb fragment of chick Lmx-1 was expressed only in the tubules (Fig. 2). Pax-2 cDNA obtained by PCR and inserted into the EcoRV site of pKS by TA cloning. It was linearized with XbaI and transcribed Trunk Paraxial Mesoderm Is Required for with T3 RNA polymerase in the presence of UTPdig. Sheep anti- Pronephros Formation from Intermediate digoxigenin antibody conjugated to alkaline phosphatase revealed the probe in pro- and mesonephros (Figs. 1–3, 5, and 7–10). Randy Mesoderm Johnson kindly provided a probe to Lmx-1 that labeled mesone- To determine whether the intermediate mesoderm self- phros and limbs (Figs. 2 and 6). The antisense strand was cut from differentiates or whether inductive or suppressive signals PBSK using T7 and EcoRI. A probe for paraxis (Fig. 10) was obtained from adjacent tissues regulate the conversion of intermedi- from D. Sosic and E. Olson (Garcia-Martinez et al., 1997). Histo- ate mesoderm to pronephric tissue, we microsurgically logic examination of cross sections was performed by sectioning paraffin-embedded embryos previously subjected to whole-mount dissected embryos to separate putative inducing tissues in situ hybridization. from intermediate mesoderm. Embryos at several developmental stages were incised along the rostrocaudal axis and the tissues were teased apart. The contralateral uncut side Immunohistochemistry served as the control. Dissected embryos were cultured Whole embryos were fixed in 4% buffered paraformaldehyde and overnight (Spratt, 1947), fixed, and subjected to in situ then subjected to immunohistochemistry (Darnell and Schoen- hybridization (Nieto et al., 1996). wolf, 1995). An epitope in early embryonic quail cells (Figs. 9 and When embryos were cut between trunk PM and IM prior 10) was detected using a monoclonal antibody (QCPN; B. Carlson, to HH8, the majority of embryos formed a pronephros from

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved. Induction of Chick Pronephros 67 intermediate mesoderm on the control side only (Fig. 3, Because some embryos already expressed Pax-2 at the Table 1). Pronephros formation was ascertained based on time of manipulation, we examined the time course of Pax-2 expression and characteristic morphology and loca- pronephros development in embryos in which the PM was tion in tissue sections. Embryos cut at HH9 or later already separated from the IM at HH7. Some embryos had weak had bilateral Pax-2 expression at the time of manipulation Pax-2 expression in IM immediately after manipulation, (not shown) that persisted after culture, but the caudal but most were negative, and pronephros formation was not extent of Pax-2 expression was attenuated on the cut side yet morphologically evident (not shown). After incubation (Fig. 3, row 1). Embryos manipulated at HH8 gave interme- for 4 h, weak Pax-2 expression was detected on the unma- diate results; Pax-2 expression on the cut side was unde- nipulated control side, but had been lost from the cut side, tectable in some embryos, whereas others had attenuated and there was no morphologic evidence for pronephros Pax-2 expression in truncated pronephroi. formation on the cut side (Fig. 5, row 1). Embryos ﬁxed at Some embryos were stained with an alternate marker for 8 h had increased Pax-2 expression in the pronephric anlage pronephros formation. PM was separated from IM at HH7 of the control side only (Fig. 5, row 2), and by 24 h of and the embryo was cultured overnight as described above culture, Pax-2 was strongly expressed in the pronephros (Spratt, 1947). Figure 4 shows a representative embryo developing in the control, but not the cut, side of the stained with a mouse antibody to Lim 1/2. In 100% of 13 manipulated embryo (Fig. 3, rows 1 and 2). These studies manipulated embryos, Lim 1/2 antibody staining and pro- conﬁrmed that proximity to the PM was required for both nephros development were seen on the control side only. Pax-2 expression and morphologic development of the These results are in complete agreement with those ob- pronephros in embryos manipulated at HH7. tained with Pax-2 labeling and morphologic analysis of pronephric structure, providing additional support for our Pronephros Induction Is Required for Mesonephros conclusion that pronephros formation requires a medial Development signal. Quantitative results are summarized in Table 1. In 43 of Pronephros formation was required for further kidney 45 (96%) embryos manipulated prior to HH8, separation of development. We separated PM from IM in HH7 embryos trunk paraxial mesoderm from intermediate mesoderm and cultured the embryos in ovo for up to 3 days, until they prevented pronephros development on the experimental had reached a developmental stage consistent with meso- side. Two (4%) embryos had truncated Pax-2 expression on nephros formation (ՆHH15). In 5 of 5 embryos, meso- the experimental side, and none had normal expression. In nephros developed only on the unmanipulated side, as embryos manipulated at HH8, 5 of 12 (42%) embryos had evidenced by both Lmx-1 expression and morphologic cri- no pronephros formation on the experimental side; a trun- teria (Fig. 6). cated pronephros was seen in 7 (58%). The effect of surgical manipulation on pronephros development decreased with Neither Axial nor Lateral Signals Are Required advancing developmental stage, such that 50, 14, and 0% of for Pronephros Induction, and Failure to Form embryos manipulated at HH9Ϫ,9,andՆ9ϩ, respectively, Pronephros after Microsurgery Is Not Due had no Pax-2 expression or histologically evident prone- to Blocking the Migration of Prospective phros on the experimental side. However, the pronephros IM Cells from the Primitive Streak was caudally truncated on the experimental side, even at later stages in which some development was evident at the To determine whether the inducing signal arose directly time of microsurgery. from the PM or more medially (e.g., from the notochord or

FIG. 7. Separation of IM from axial or lateral mesoderm did not alter pronephros development. Embryos were incised as shown in cartoons at t ϭ 0 and incubated for 24 h in Spratt culture. (Row 1) Cuts between N and PM in early embryos did not impair Pax-2 expression. Normal pronephroi formed lateral to somites on both the control (notochord and floor plate of the neural tube present) and the experimental (notochord and floor plate absent) sides. (Row 2) Separation of IM from LP in early embryos did not impair Pax-2 expression. Pax-2 was expressed in intermediate mesoderm lateral to the somites, but was not expressed in lateral plate mesoderm cultured apart from somitic tissue. The Hamburger and Hamilton stage at the time of manipulation is shown in the lower left corner of each photo. Abbreviations: N, notochord; NT, neural tube; S, somite; LP, lateral plate mesoderm. Bars: whole mount, 300 ␮m; section, 100 ␮m. FIG. 8. Removal of endoderm, ectoderm, or rostral tissues did not alter pronephros development from the IM. Embryos were incised as shown in cartoons at t ϭ 0 and incubated for 24 h in Spratt culture. (Row 1) Removal of endoderm did not impair Pax-2 expression and tissue sections showed that the pronephros developed and that the endoderm did not regrow during the incubation period. (Row 2) Ectoderm removal did not impair Pax-2 expression or morphologic pronephros development. The endoderm did not regrow within 24 h. (Row 3A) Separation of rostral from caudal tissues above the level of the fifth somite (or above the level of the prospective fifth somite, if it had not yet segregated) resulted in Pax-2 expression only in the caudal segment, whereas (Row 3B) Pax-2 was expressed in both rostral and caudal segments in embryos transected below the level of the presumptive sixth somite. Abbreviations: N, notochord; NT, neural tube; S, somite; LP, lateral plate; MB/HB, Pax-2 expression in the midbrain/hindbrain portion of the neural tube; OV, otic vesicle. Bars: whole mount, 300 ␮m; section, 100 ␮m. Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved. 68 Mauch et al.

TABLE 2 Normal Pax-2 Expression on the Manipulated Side Following Various Microsurgical Separations

Hamburger and Cut between Cut between Hamilton stage N and PM IM and LP Ectoderm removed Endoderm removed

6 — — 2/2 (100%) — 7 12/12 (100%) 7/7 (100%) 12/12 (100%) 11/11 (100%) 8 10/10 (100%) 1/1 (100%) — —

Note. Data show the number of expressing embryos/total number of cases, with the percentage expressing Pax-2 on the manipulated side in parentheses.

the floor plate of the neural tube), embryos were dissected moved prior to Spratt culture (Spratt, 1947). Examination of parallel to the rostrocaudal axis between the notochord and serial sections demonstrated that regrowth of endoderm the trunk PM. Even embryos dissected at HH7 formed and ectoderm did not occur (Fig. 8, rows 1 and 2). Removal normal pronephroi on both the cut and the uncut sides after of endoderm or ectoderm neither impaired nor enhanced overnight culture (Fig. 7, row 1). Importantly, these cuts pronephros formation (Fig. 8, rows 1 and 2). Results of also showed that microsurgery did not block the migration embryos dissected between HH6 and HH8 are summarized of prospective IM cells from the primitive streak. That is, if in Table 2. the more lateral cuts blocked migration, so should the more To establish the rostral boundaries of the pronephric medial cuts; these results show clearly that the latter do field, embryos were transected at various somitic levels. In not. Rather, the pronephros failed to develop following these embryos, Pax-2 expression in the midbrain/hindbrain separation of IM from the inducing signals produced by the region of the neural tube served as a positive control. PM. Separation of IM from LP did not impair or enhance Embryos transected above the level of the fifth somite had pronephros formation in the IM on the experimental side bilateral Pax-2 expression only in the caudal isolate (Fig. 8, (Fig. 7, row 2), showing that a more lateral signal from the row 3, and Table 3), whereas embryos transected below the lateral plate mesoderm, in contrast to the medial signal level of the sixth somite at HH8 or later expressed Pax-2 in from the PM, is not required for pronephros induction. both the rostral and the caudal isolates (Fig. 8, row 3, and Quantitative results are summarized in Table 2. Table 3). Five of ten embryos transected between the fifth and the sixth somites expressed Pax-2 in both the rostral and the caudal isolates. These studies showed that rostral Neither Endoderm, Ectoderm, nor Rostral Tissues tissues such as head mesoderm are unlikely to be the source Are Required for Pronephros Induction of the inducing signal and indicated that there was no To ascertain whether the inducing signal arises in the PM rostrocaudal shift over time in the character of either or in its underlying endoderm or overlying ectoderm, each inducing or responding tissue; tissue that had the charac- of these tissues was removed prior to culture. To avoid the teristics found below the fifth somitic level at the time of possibility of regrowth of ectoderm (Obara-Ishihara et al., microsurgery retained those characteristics later and de- 1999), the lateral portion of some embryos was also re- spite microsurgical separation from more rostral tissues.

FIG. 9. Pronephros induction following tissue explantation and recombination in collagen gel. HH9 quail somites were isolated and recombined with HH7 chick lateral tissue (containing prospective intermediate and lateral plate mesoderm and ectoderm), embedded in a collagen gel, and cultured in vitro for 28 h. Pax-2 expression (blue) served as a marker of pronephros induction (arrows). Quail-derived somites (S) are stained brown. Bars: whole mount, 300 ␮m; section, 100 ␮m. FIG. 10. (A) Fate map of the HH4 avian embryo. Hensen’s node gives rise to the notochord, and primitive streak cells ingress in a rostrocaudal fashion to give rise to PM, IM, and LP mesoderm. (B) Hensen’s node from HH4 or HH5 quail embryos was isochronically transplanted into the caudal primitive streak of chick embryos. The transplanted quail node formed quail notochord and induced ectopic somites (mostly composed of chick cells) in chick lateral tissues. (C) The ectopic notochord (QN) and part of one ectopic somite were quail-derived (brown); the remaining tissues arose from the chick embryo. On the control side (no ectopic notochord or somites), Pax-2 (blue) was expressed normally in the pronephros that arose from intermediate mesoderm (arrows). On the experimental side, Pax-2 was expressed in an ectopic pronephros arising from trans-fated chick LP (asterisks), and an enlarged pronephros expressed Pax-2 in IM that received inducing signals from both native and ectopic somites (arrowhead). (D) The pronephros was characterized by anti-Lim 1/2 staining (brown) and somites were identiﬁed by in situ hybridization with a probe for paraxis (blue). A normal pronephros was induced in native IM (arrows), an ectopic pronephros arose from trans-fated chick LP (asterisks) and an enlarged pronephros formed in IM that received inducing signals from both native and ectopic somites (arrowhead). Abbreviations: S, somite; QN, quail notochord; N, notochord; NF, neural folds; H, heart endoderm; NT, neural tube; LP, lateral plate mesoderm. Bars: whole mount, 300 ␮m; tissue section, 100 ␮m.

TABLE 3 Pax-2 Expression in the Rostral versus Caudal Isolate Following Transection at Various Somitic Levels

1/2 2/3 3/4 4/5 5/6* 6/7* Hamburger and Hamilton stage CRCRCRCRCRCR

7 9/9 0/9 3/3 0/3 4/4 0/4 8 1/1 0/1 3/3 0/3 6/6 5/6 8ϩ 1/1 0/1 1/1 0/1 1/1 0/1 9/9 9/9 9Ϫ 1/1 0/1 1/1 0/1 1/1 0/1 9ϩ 1/1 0/1 10Ϫ 1/1 0/1

Note. The designation “1/2” indicates transection between the ﬁrst and the second pairs of somites. Asterisks indicate that the transection was made at the level at which somites were expected to appear, in embryos that had not yet developed that number of somites. For example, a HH8ϩ embryo has only ﬁve somites, but some of the cuts were made more caudally, at the level at which the demarcation between the sixth and the seventh somite was expected to appear. Abbreviations: C, Pax-2 was expressed in the IM in the caudal isolate; R, Pax-2 was expressed in the IM in the rostral isolate.

Paraxial Mesoderm Is Sufﬁcient for Pronephros men (Fig. 9) shows that the inducing PM was quail Induction derived, whereas the responding (i.e., Pax-2-expressing) tissue was of chick origin. Of 25 tissue recombinants, 21 Collectively, the experiments described above show expressed Pax-2 in chick tissue, indicating that the quail that signals from paraxial mesoderm are required for somites had induced pronephric anlage in the chick pronephros induction from intermediate mesoderm. To determine whether paraxial mesoderm is also sufﬁcient mesoderm. This rate is comparable to that observed in for pronephros induction, we cocultured the putative positive controls in which caudal chick PM was left in inducing and responding tissues in collagen gels (Artinger apposition to IM and cultured (i.e., in isolation from the and Bronner-Fraser, 1993). Caudal somites isolated endoderm and the rostral and more medial tissues) in from HH8 to HH9 quail embryos were recombined with collagen gel (Table 4). It compares favorably with rates lateral mesoderm (containing prospective IM and LP) reported by Seufert and co-workers for experiments in isolated from HH4 or HH7 chick embryos, embedded which they recombined anterior somites with two stage 9 in a collagen gel, and cultured in vitro for up to 28 h. Xenopus animal caps. In their studies, 7 of 20 samples Pax-2 expression served as a marker of pronephros induc- developed pronephric tubules (Seufert et al., 1999). In tion. Fixed tissues were also stained with anti-quail contrast, chick IM and LP cultured without PM (negative antibody to determine the origin of the pronephric tissue controls) did not express Pax-2. Quantitative results are (Darnell and Schoenwolf, 1995). A representative speci- shown in Table 4.

TABLE 4 Pronephros Induction in Chick Lateral Plate Following Coculture with Quail Somites in Collagen Gels

Tissues cocultured Pax-2 expressed No Pax-2 expression

Negative control: 0/8 (0%) 8/8 (100%) HH7 chick LP Positive controls: HH7 chick PM ϩ LP 9/11 (82%) 2/11 (18%) HH7 chick N ϩ PM ϩ LP 7/7 (100%) 0/7 (0%) Experimental: HH7 chick LP ϩ HH9 quail PM 17/21 (81%) 4/21 (19%) HH7 chick LP ϩ HH8 quail PM 2/2 (100%) 0/2 (0%) HH4 chick LP ϩ HH8 quail PM 2/2 (100%) 0/2 (0%)

Note. Chick LP includes chick intermediate and lateral mesoderm and associated ectoderm. Quail PM includes overlying ectoderm and underlying endoderm. The Hamburger and Hamilton stage at the time of isolation is noted. At stage 8, the fourth somite and more caudal prospective PM was cultured with chick tissues. At stage 9, only PM caudal to the ﬁfth somite was used. Data show the number of expressing embryos/total number of cases, with the percentage expressing Pax-2 in parentheses.

The Generation of Ectopic Somites Leads to the Hamilton, 1952). Pax-2 expression persisted in the meso- Development of Ectopic and Enlarged Pronephroi nephros, which was also labeled by Lmx-1 (Fernandez et al., Pax-2 Figure 10A depicts a fate map of the avian embryo. 1997). Therefore, we used as our primary marker of Hensen’s node gives rise to the notochord, and cells ingress conversion of pluripotent intermediate mesoderm to com- from the more caudal primitive streak to give rise to PM, mitted pronephric tissue. Antibody staining for Lim 1/2 IM, and LP mesoderm in a rostrocaudal fashion (Schoenwolf was used as a confirmatory pronephric marker, whereas et al., 1992). When Hensen’s node from HH4 or HH5 quail Lmx-1 served as a marker for the mesonephros. embryos was isochronically transplanted into the caudal The pronephros, mesonephros, and metanephros all arise primitive streak of chick embryos (Fig. 10B), an ectopic from intermediate mesoderm, and similar gene expression notochord self-differentiated, and the node induced ectopic patterns in all three kidney forms argue for conservation of somites (Streit and Stern, 1999; Schoenwolf, unpublished developmental pathways and mechanisms of induction data). Pronephric induction by paraxial mesoderm (somites) (Vise et al., 1997). These similarities are useful; by studying varied, depending upon both having adequate responding induction and patterning in the pronephros, we also learn tissue and the intensity of the inducing signal. In 10 of 13 about the development of the subsequent kidneys. embryos, the native pronephros was elongated on the side The differences between pronephric determination and with ectopic somites, but in 3, a supernumerary pronephros development of later phases can also be exploited to under- was induced (Figs. 10C and 10D). In the embryo shown in stand better the molecular mechanisms responsible for Fig. 10C, the pronephros was labeled with Pax-2, and quail kidney induction. Signals emanating from the nephric duct tissues were identified by immunohistochemistry. To con- are required for meso- and metanephric kidney induction, firm pronephric induction using another marker, and to whereas the pronephros arises directly from the intermedi- better identify the somites, the pronephros in the embryo ate mesoderm and gives rise to the aforementioned nephric shown in Fig. 10D was labeled with antibody to Lim 1/2, duct (Vise et al., 1997). If pronephros development involves and the somites were identified by paraxis expression. As the same signaling molecules used at later kidney stages, shown in Fig. 10C, the notochord and part of one somite are then these molecules must be produced by other embryonic quail derived; the remaining tissues arose from the chick structures (Yates and Pate, 1989; Vise et al., 1997). embryo. In both embryos, on the control side (lacking The experiments described here ruled out the possibility ectopic notochord or somites), the pronephros developed of self-determination. Lateral embryonic isolates contain- normally in the intermediate mesoderm. On the experi- ing intermediate and lateral plate mesoderm failed to de- mental side, large ectopic somites formed between the velop pronephroi in the absence of a medial signal. The native intermediate mesoderm and the lateral plate meso- pronephros was absent or truncated when the intermediate derm. An additional pronephros developed lateral to the mesoderm was separated from the adjacent medial tissues, ectopic somite. In addition, between the native and the demonstrating that the inducing signal was produced by the ectopic somites, competent intermediate mesoderm ex- immediately adjacent PM or perhaps by the notochord/ pressed pronephric markers. Moreover, presumably because neural tube. Normal pronephros formation in the IM left in it received signals from both the native and the large apposition to the PM but separated from the notochord and ectopic somite, the pronephros derived from this interme- floor plate of the neural tube ruled out an axial signal. diate mesoderm was greatly enlarged (Figs. 10C and 10D). Seufert and co-workers similarly found that notochord was This indicates that the inducing signal produced by the not required for pronephros induction in Xenopus embryos paraxial mesoderm acts in a dose-dependent manner. (Seufert et al., 1999). We considered the possibility that the potential respond- DISCUSSION ing tissue had been ablated by surgical manipulation. The width of the IM labeled by either Pax-2 in situ hybridization The Pax genes are paired-box transcription factors ex- or Lim 1/2 antibody staining was 40–50 ␮m, whereas the pressed in distinct spatiotemporal patterns during embryo- tip of the cactus needle used for separation of IM from PM genesis (Walther et al., 1991). Pax-2, important in the was significantly less than 10 ␮m in diameter, making development of the metanephric kidney, is expressed dur- extirpation of the entire width of responding tissue impos- ing early embryonic development in a highly localized sible. Each cut constituted an incision, not an ablation. pattern in the pronephroi, eyes, otic vesicles, and midbrain/ Moreover, medial-cutting errors would result in apposition hindbrain level of the neural tube (Dressler et al., 1990; of the IM to the LP mesoderm, whereas lateral-cutting Torres et al., 1995). In our hands, weak Pax-2 expression errors would result in apposition of IM to PM, with each was detected in the intermediate mesoderm by HH7, and serving as a useful control. Because cuts were made from Pax-2 was strongly expressed caudal to the fifth somite by about the first somitic level caudally through the tail end of HH9. Pax-2 expression correlated with morphologic evi- the embryo, one might reason that ingression of prospective dence for pronephros development in tissue sections and IM from the primitive streak (Garcia-Martinez et al., 1993) was consistent with classical reports describing develop- was disrupted by cuts between PM and IM. However, ment of the pronephros in the intermediate mesoderm ingression of pronephric precursor cells has likely occurred caudal to the level of the fifth somite (Abdel-Malek, 1950; prior to HH7, and if not, its occurrence would also be

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved. Induction of Chick Pronephros 73 prevented by cuts made between notochord/neural tube and discrepancy between our results and those of Obara- PM, which did not affect pronephros development. More- Ishihara and co-workers exists. BMP-4 has been demon- over, quail–chick chimeras have shown that adjacent me- strated to be important in somitogenesis (Pourquie´ et al., soderm cells can migrate to new positions and form new 1996), and the effect of BMP-4 on pronephros induction in intermediate mesoderm (Garcia-Martinez and Schoenwolf, the IM may be, therefore, indirectly mediated by trunk 1992), making ablation of all potential responding tissue paraxial mesoderm. unlikely. Rostrocaudal transection experiments showed both that Over 95% of the embryos cut between the PM and the IM Pax-2 expression occurred only caudal to the level of the before HH8 formed pronephros on the control side only, prospective fifth somite and that a rostral signal was not whereas embryos manipulated after HH9 had pronephros required for induction of Pax-2 expression in the IM. This development on both sides, but the length of the prone- pattern was observed regardless of the developmental stage phros on the cut side was truncated rostrocaudally com- at the time of microsurgery, indicating that the character- pared to the control side. The persistence of the pronephros istics of the inducing and responding tissues around level of at the rostral end of the cut side in HH9 embryos showed the prospective fifth somite did not change with time or that the inducing signal was only transiently required. manipulation. However, the caudal extent of pronephros development was Recombination experiments showed that quail somites truncated on the experimental side, even at later stages in are sufficient for the induction of Pax-2 expression (prone- which Pax-2 expression was evident at the time of micro- phros formation) in chick intermediate and lateral meso- surgery, indicating that the inducing signal was produced derm. Our findings are consistent with those of Seufert et by PM mesoderm in a rostral to caudal fashion. al., who reported that UV-ventralized Xenopus embryos Separation of intermediate mesoderm from LP mesoderm lacked somites and subsequently failed to develop pro- neither impaired nor enhanced pronephros development, nephroi. These investigators further reported that proneph- indicating that no lateral signal was required for IM induc- ric tubules, but not ducts, could be induced in embryonic tion. We reasoned that the lateral tissues could emit inhibi- isolates when lateral mesoderm was heterochronically cul- tory signals that restrict or otherwise determine the com- tured within an ectodermal sandwich that also contained petence of the IM to respond to the medial-inducing signal, anterior somitic tissue (Seufert et al., 1999). The embryonic much as noggin inhibits BMP signaling in the medial kidney of Xenopus is among the simplest of vertebrate somite (Marcelle et al., 1997). However, based on the failure kidneys, consisting of a single large nephron with an of the IM/LP isolates to show expansion of Pax-2 expres- external glomus (Vise et al., 1997). It is the functional sion, this possibility can be ruled out. embryonic kidney in fish and amphibia, whereas the meso- Removal of endoderm did not impair pronephros devel- nephros is the definitive kidney (Bra¨ndli, 1999). In contrast, opment, showing that a ventral signal was not required for avian kidney development more closely approximates that pronephros induction. Neither did we find evidence that of mammals, in that both proceed from pronephros through the ectoderm produced a dorsal signal required for pronephric induction, because both pronephric morphology and mesonephros and have a metanephros as the definitive Pax-2 expression were normal on the manipulated side even kidney. Our findings in the avian pronephros confirm the in embryos dissected at HH7. This contrasts with the frog studies, and the evolutionary conservation of signaling observation by Obara-Ishihara and co-workers that extirpa- stresses the importance of the somite in the patterning of tion of the ectoderm in chicken embryos cultured to HH16 the early embryonic kidney. (Hamburger and Hamilton, 1951) in ovo prevented Pax-2 When quail node was transplanted into chick caudal expression in the IM and that nephric duct development primitive streak, ectopic somites were generated. Interme- could be rescued by the addition of exogenous BMP-4 diate mesoderm that received signals from two rows of (Obara-Ishihara et al., 1999). Our embryos were cultured in somites produced an enlarged pronephros. One might argue vitro in Spratt culture, and they were considerably younger that an absence of suppressing signals from lateral plate at the time of manipulation; thus, it is possible that stage mesoderm explains the enhanced pronephric development, differences account for our differing observations. Indeed, but microsurgical separation of IM from LP did not enhance these authors noted Pax-2 and cSim-1 labeling of a nephro- Pax-2 expression in the pronephros. Rather, these experi- genic cord of cells derived from IM at HH10 in their ments provide the first evidence that the inducing signal manipulated embryos, but focused their observations on produced by the paraxial mesoderm acts in a dose- the morphologic differentiation of the nephric duct. The dependent manner. pronephric duct is not a tubular structure, but acquires a In summary, we have shown that rostral tissue, axial or lumen upon further development of the mesonephros, lateral plate mesoderm, endoderm, and ectoderm neither whereupon it is renamed the mesonephric or Wolffian duct induce nor inhibit pronephric induction. In contrast, inter- (Hamilton, 1952). Thus, it is possible that endoderm or action with trunk paraxial mesoderm is both required and ectoderm are important in the further differentiation of the sufficient for pronephros induction in the chick intermedi- mesonephric duct, although they are not required for pro- ate mesoderm. Studies are in progress to define the molecu- nephros induction. However, another explanation for the lar nature of the inducing signals.

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