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Developmental Biology 220, 62–75 (2000)doi:10.1006/dbio.2000.9623, available online at http://www.idealibrary.com on

Signals from Trunk Paraxial Mesoderm InducePronephros Formation in ChickIntermediate Mesoderm

Teri Jo Mauch,*,†,1 Guizhi Yang,* Mindi Wright,† Danielle Smith,†nd 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 expressedcaudal to the fifth somite by stage 9. To determine whether the pronephros was induced by adjacent tissues and, if so, to identifythe inducing tissues and the timing of induction, we microsurgically dissected one side of chick embryos developing in cultureand then incubated them for up to 3 days. The undisturbed contralateral side served as a control. Most embryos cut parallel tothe rostrocaudal axis between the trunk paraxial mesoderm and IM before stage 8 developed a pronephros on the control sideonly. Embryos manipulated after stage 9 developed pronephric structures on both sides, but the caudal pronephric extension wasattenuated on the cut side. These results suggest that a medial signal is required for pronephric development and show that thesignal is propagated in a rostral to caudal sequence. In manipulated embryos cultured for 3 days in ovo, the mesonephros as wellas the pronephros failed to develop on the experimental side. In contrast, embryos cut between the notochord and the trunkparaxial 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 controlfor cuts between the PM and the IM and showed that signaling itself was blocked in the former experiments, not the migrationof pronephric or mesonephric precursor cells from the primitive streak. Additional control experiments ruled out the need forsignals from lateral plate mesoderm, ectoderm, or endoderm. To determine whether the trunk paraxial mesoderm caudal to thefifth somite maintains its inductive capacity in the absence of contact with more rostral tissue, embryos were transected. Thosetransected below the prospective level of the fifth somite expressed Pax-2 in both the rostral and the caudal isolates, whereasembryos transected rostral to this level expressed Pax-2 in the caudal isolate only. Thus, a rostral signal is not required toestablish the normal pattern of Pax-2 expression and pronephros formation. To determine whether paraxial mesoderm issufficient for pronephros induction, stage 7 or earlier chick lateral plate mesoderm was cocultured with caudal stage 8 or 9 quailsomites in collagen gels. Pax-2 was expressed in chick tissues in 21 of 25 embryos. Isochronic transplantation of stage 4 or 5 quailnode into caudal chick primitive streak resulted in the generation of ectopic somites. These somites induced ectopic pronephroiin lateral plate mesoderm, and the IM that received signals from both native and ectopic somites formed enlarged pronephroiwith increased Pax-2 expression. We conclude that signals from a localized region of the trunk paraxial mesoderm are bothrequired and sufficient for the induction of the pronephros from the chick IM. Studies to identify the molecular nature of theinduction are in progress. © 2000 Academic Press

Key Words: Pax-2; Lim 1/2; Lmx-1; kidney development; pronephros; in situ hybridization; immunohistochemistry;paraxis; somites.

and dysplasia, or abnormal kidney development, accountsf1mSk1

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INTRODUCTION

Congenital anomalies of the genitourinary tract are de-tected in about 1 in 500 fetal ultrasounds (Colodny, 1987),

1 To whom correspondence should be addressed at the Univer-sity of Utah School of Medicine 2R063, 50 N. Medical Drive, SaltLake City, UT 84132. Fax: (801) 581-8043. E-mail: [email protected].

62

or one-third of end-stage renal disease in children (USRDS,997). Embryonic kidney development has been describedorphologically (Waddington, 1938; Abdel-Malek, 1950;

axen and Sariola, 1987), and some of the genes involved inidney development have now been identified (Eccles,998; Lipschutz, 1998).Most studies of kidney induction have focused on the

ondensation of the metanephric mesenchyme in response

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All rights of reproduction in any form reserved.

to the invading ureteric bud (Rothenpieler and Dressler,

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63Induction of Chick Pronephros

1993; Dressler, 1995). Although this system is appropriatefor the investigation of conversion of mesenchyme toepithelium, it is not particularly useful for studying howthe kidney primordia are initially specified, because thenephrogenic mesenchyme is already determined by thisdevelopmental stage and lacks only the terminal condensa-tion signals provided by the ureteric bud (Saxen and Sariola,1987). The vertebrate kidney develops in three major stepsvia specific tissue interactions (Saxen and Sariola, 1987).The first kidney, the pronephros, is required for furtherdevelopment of the mesonephric and metanephric kidneys(Vise et al., 1997; Carroll et al., 1999a). The pronephros canbe analyzed throughout the period of mesodermal pattern-ing and it has the advantage of less complex anatomy,making it an ideal system in which to explore the earlystages of intermediate mesodermal patterning (Vise et al.,1997).

Conversion of undifferentiated tissue to committed celllineages requires the transcriptional activation of structuralgenes. The Pax genes are paired-box transcription factorsexpressed in distinct spatiotemporal patterns during em-bryogenesis (Gruss and Walther, 1992; Eccles, 1998). Pax-2,important in the differentiation of the definitive or meta-nephric kidney, is expressed during early embryonic devel-opment in the pronephroi, eyes, otic vesicles, and neuraltube (Dressler et al., 1990; Lechner and Dressler, 1997).Pax-2 expression is tightly regulated during normal humandevelopment; overexpression has been observed in Wilms’tumors and renal cell carcinomas (Dressler, 1996). Haplo-insufficiency of Pax-2 results in kidney hypoplasia, vesi-coureteral reflux, and optic nerve dysplasia in the autoso-mal dominant renal coloboma syndrome (Sanyanusin et al.,1995) and renal agenesis or hypoplasia in Senior–Lokensyndrome (Warady et al., 1994). Haploinsufficiency of Pax-2n three mouse mutants is associated with ear, eye, andrain anomalies as well as renal cystic changes and hypo-lasia of varying degrees (Keller et al., 1994; Torres et al.,

1995; Favor et al., 1996). In the studies described here, wesed Pax-2 expression as a molecular marker for pronephrosnduction.

Like Pax-2, Lim 1 is expressed in both the tubules and theuct of the early pronephros and persists in the adultenopus (Carroll et al., 1999a,b), zebrafish (Carroll et al.,999a), and murine (Barnes et al., 1994) kidney. Miceacking functional Lim 1 do not have pro-, meso-, or

etanephroi (Shawlot and Behringer, 1995). Staining withnti-Lim 1/2 antibody served as a confirmatory marker forronephros induction.Another LIM homeobox gene, Lmx-1, is first expressed in

he mesonephros (Fernandez et al., 1997). Mutations inmx-1B have been described in the nail–patella syndrome,hich includes proteinuria and renal dysplasia (Chen et al.,998; Dreyer et al., 1998).All three kidney forms arise from embryonic intermedi-

te mesoderm, which lies between the trunk paraxial me-oderm and lateral plate mesoderm in the early embryo.

Copyright © 2000 by Academic Press. All right

ntermediate mesoderm (Vise et al., 1997). Mesoderm cul-ured in isolation does not differentiate into specializedissue, making self-determination unlikely (Slack, 1994).ather, inducing signals presumably control when andhere structures form. A midline signal is required for thepregulation of Pax-2 expression in the developing eye

Macdonald et al., 1995). In Xenopus, somitic tissue willorm pronephric tubules if explanted and grown away fromhe influence of the notochord (Yamada, 1940), and anterioromites have recently been identified as the inducing tissuen frog pronephric development (Seufert et al., 1999).

We hypothesized that signals from adjacent tissues arenvolved in embryonic kidney induction. Using whole-

ount in situ hybridization and immunohistochemistry,e monitored Pax-2 and Lmx-1 expression and Lim 1/2

ntibody staining during the conversion of intermediateesoderm into pronephric tissue. We sought to identify the

issue interactions responsible for pronephros induction inhe early chick kidney.

MATERIALS AND METHODS

Embryos

Fertile White Leghorn chicken or Japanese quail (Coturnixcoturnix japonica) eggs were incubated at 38°C in forced-draft,humidified incubators until embryos had reached the appropriatestage (Hamburger and Hamilton, 1951).

Microsurgery and Culture

For most experiments, embryos were placed into Spratt culture(Spratt, 1947), subjected to microsurgery, and incubated overnight.Operations were done aseptically using the tips of cactus needles.Longitudinal cuts were made between the trunk paraxial meso-derm (PM)2 and the intermediate mesoderm (PM/IM), between thenotochord and the paraxial mesoderm (N/PM), or between theintermediate and the lateral plate mesoderm (IM/LP), and thetissues were teased apart. Rostral/caudal (R/C) transections weremade at several somitic levels. Following manipulation, embryoswere either fixed immediately for in situ hybridization or culturedin vitro for up to 24 h (Spratt, 1947).

For longer incubations, embryos were cultured in ovo (Criley,1966). A window was made in the shell and the embryo was lightlystained with 0.1% neutral red. Longitudinal incisions were madeusing cactus needle tips between the PM and the IM, and thetissues were teased apart. Windows were sealed with a glasscoverslip and paraffin, and eggs were incubated for up to 3 days at38°C in forced-draft, humidified incubators, then fixed and sub-jected to in situ hybridization.

Putative inducing and responding tissues were isolated andrecombined in collagen gels. Quail somites caudal to the fifthsomite were excised, along with their overlying ectoderm and

2 Abbreviations used: IM, intermediate mesoderm; LP, lateralplate mesoderm; N, notochord; PM, paraxial mesoderm; R/C,rostral caudal transection.

s of reproduction in any form reserved.

64 Mauch et al.

underlying endoderm. One-half of the somite explants also con-tained notochord. Chick lateral mesoderm (i.e., prospective inter-mediate and lateral plate mesoderm) was isolated from HH4 or

FIG. 1. Whole-mount in situ hybridization shows Pax-2 expressioax-2 expression in the paraxial mesoderm (fifth somitic level) ofection. The Hamburger and Hamilton stage is indicated in the lowube; S, somite; LP, lateral plate; MB/HB, midbrain/hindbrain level00 mm; section, 100 mm.

FIG. 2. Whole-mount in situ hybridization shows Pax-2 and Lmx-the lower row shows Lmx-1. At the right, cross sections show exarrowheads), and mesonephric ducts (large arrowheads). The Hambphoto. Abbreviations: N, notochord; NT, neural tube; A, aorta; V,IG. 3. Microsurgical disruption of embryos developing for 24 h intage 8 prevented Pax-2 expression. Embryos cut at stage 9 alreadyut did not extend caudally with further incubation. (Row 2) Tissueith the morphologic pronephric anlage. The Hamburger and Habbreviations: N, notochord; NT, neural tube; S, somite; LP, later


HH7 embryos. The endoderm was removed to expose the meso-derm, and the mesoderm was wrapped around the quail somites.These tissues were embedded in a collagen gel, overlaid with

IM destined to become pronephros (arrows). Arrowheads indicatestage 81 embryo. The bottom shows Pax-2 expression in cross

eft corner of each photo. Abbreviations: N, notochord; NT, neurale neural tube; PS, primitive streak; TB, tailbud. Bars: whole mount,

ression in mesonephros. The top row shows Pax-2 expression, andion in mesonephric tubules (arrows), connecting segments (smallr and Hamilton stage is indicated in the lower left corner of eachcardinal vein. Bars: whole mount, 300 mm; section, 100 mm.att culture. (Row 1) Longitudinal cuts between PM and IM prior tossed Pax-2 at the time of manipulation; such expression persisted

ons of an embryo cut at HH7 show that Pax-2 expression correlatedon stage at the time of manipulation is indicated in each photo.ate. Bars: whole mount, 300 mm; section, 100 mm.

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FIG. 4. Lim 1/2 antibody staining shows that pronephros formation requires a medial signal. A longitudinal cut between PM and IM atHH7 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 morphologicpronephric 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 pronephrosformation in whole mount and serial sections at time 5 4 or 8 h after IM was separated from PM. By t 5 8 h, the pronephros was detectablemorphologically, 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 andcultured 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 mm; section, 100 mm.

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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University of Michigan, Ann Arbor). For Lim 1/2 detection (Figs. 4

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TABLE 1

66 Mauch et al.

Dulbecco’s minimal essential medium supplemented withL-glutamine, penicillin, streptomycin, and 10% fetal bovine serumArtinger and Bronner-Fraser, 1993), and cultured for up to 28 h.ultured chick explants that included presumptive chick PM

erved as positive controls; negative control cultures comprisednly chick lateral mesoderm (absent the endoderm and any pro-pective PM).

In some experiments Hamburger and Hamilton stage 4 or 5HH4, HH5) (Hamburger and Hamilton, 1951) quail node (theostral 125 mm of the primitive streak) was transplanted isochroni-ally and heterotopically to the chick caudal primitive streak250–500 or 500–750 mm caudal to the chick node). Chimericembryos were cultured in vitro for up to 28 h (New, 1955) prior tofixation and analysis.

In Situ Hybridization

When experiments were terminated, embryos were fixed in 4%buffered paraformaldehyde, dehydrated in graded methanol, andfrozen at 220°C. Rehydrated embryos were subjected to whole-mount in situ hybridization with digoxigenin-labeled antisenseRNA probes for Pax-2 or Lmx-1 (Nieto et al., 1996). DomingosHenrique provided pDNA containing a 1-kb fragment of chickPax-2 cDNA obtained by PCR and inserted into the EcoRV site ofpKS by TA cloning. It was linearized with XbaI and transcribedwith T3 RNA polymerase in the presence of UTPdig. Sheep anti-igoxigenin antibody conjugated to alkaline phosphatase revealedhe probe in pro- and mesonephros (Figs. 1–3, 5, and 7–10). Randyohnson kindly provided a probe to Lmx-1 that labeled mesone-hros and limbs (Figs. 2 and 6). The antisense strand was cut fromBSK using T7 and EcoRI. A probe for paraxis (Fig. 10) was obtainedrom D. Sosic and E. Olson (Garcia-Martinez et al., 1997). Histo-ogic examination of cross sections was performed by sectioningaraffin-embedded embryos previously subjected to whole-mountn situ hybridization.

Immunohistochemistry

Whole embryos were fixed in 4% buffered paraformaldehyde andthen subjected to immunohistochemistry (Darnell and Schoen-wolf, 1995). An epitope in early embryonic quail cells (Figs. 9 and10) was detected using a monoclonal antibody (QCPN; B. Carlson,

Pronephros Formation Following Microsurgical Separationof Paraxial from Intermediate Mesoderm

HH stage Label

Kidney development on manipulated side

None Truncated Normal

7 Pax-2 30/32 (94%) 2/32 (6%) 0/32 (0%)Lim 1/2 13/13 (100%) 0/13 (0%) 0/13 (0%)

8 Pax-2 5/12 (42%) 7/12 (58%) 0/12 (0%)92 Pax-2 1/2 (50%) 1/2 (50%) 0/2 (0%)9 Pax-2 2/14 (14%) 12/14 (86%) 0/14 (0%).91 Pax-2 0/6 (0%) 6/6 (100%) 0/6 (0%)

Note. Data show the number of expressing embryos/total num-ber of cases, with the percentage either expressing Pax-2 or stainedwith anti-Lim 1/2 antibody on the manipulated side in parentheses.


and 10), embryos were blocked with normal goat serum, thenstained with a 1:2 dilution of mouse anti-Lim 1/2. The secondaryantibody was goat anti-mouse IgG conjugated to peroxidase (Jack-son No. 115-035-003), used at a dilution of 1:200. Hybridoma cellssecreting anti-Lim 1/2 or anti-quail were obtained from the Devel-opmental Studies Hybridoma Bank maintained by the Departmentof Pharmacology and Molecular Sciences, Johns Hopkins Univer-sity School of Medicine (Baltimore, MD), and the Department ofBiology, University of Iowa (Iowa City), under Contract 1-HD-6-2915 from the National Institute of Child Health and HumanDevelopment.

RESULTS

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 inthe developing kidney, brain, eye, and ear (Dressler et al.,1990). Figure 1 shows that Pax-2 is a molecular marker forronephric tissue. In some embryos, Pax-2 was weaklyxpressed in the intermediate mesoderm at HH7 (Ham-urger and Hamilton, 1951) (not shown); its level of expres-ion increased with maturation. By HH9, pronephric Pax-2xpression was limited to intermediate mesoderm caudal tohe fifth somite (Fig. 1). In the earlier embryo (HH7 andH8, Fig. 1), Pax-2 was also transiently expressed in trunkaraxial mesoderm at the level of the fifth somite. Pax-2xpression persisted in the mesonephros, which was alsoabeled with Lmx-1 (Fig. 2).

In contrast to Pax-2, Lmx-1 (Riddle et al., 1995; Fernan-ez et al., 1997) was not expressed in the pronephros; rather,ts expression was first seen in the mesonephros and limbuds after HH15. Thus, the pronephros was molecularlyharacterized by expression of Pax-2, but not Lmx-1 (Fig. 1).n contrast, the mesonephros expressed both markers, butheir expression patterns differed. Tissue sections of labeledmbryos showed Pax-2 expression in the nephric duct, itsonnecting segment, and the mesonephric tubules, whereasmx-1 was expressed only in the tubules (Fig. 2).

Trunk Paraxial Mesoderm Is Required forPronephros Formation from IntermediateMesoderm

To determine whether the intermediate mesoderm self-differentiates or whether inductive or suppressive signalsfrom adjacent tissues regulate the conversion of intermedi-ate mesoderm to pronephric tissue, we microsurgicallydissected embryos to separate putative inducing tissuesfrom intermediate mesoderm. Embryos at several develop-mental stages were incised along the rostrocaudal axis andthe tissues were teased apart. The contralateral uncut sideserved as the control. Dissected embryos were culturedovernight (Spratt, 1947), fixed, and subjected to in situhybridization (Nieto et al., 1996).

When embryos were cut between trunk PM and IM priorto HH8, the majority of embryos formed a pronephros from


intermediate mesoderm on the control side only (Fig. 3,

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Table 1). Pronephros formation was ascertained based onPax-2 expression and characteristic morphology and loca-tion in tissue sections. Embryos cut at HH9 or later alreadyhad bilateral Pax-2 expression at the time of manipulationnot shown) that persisted after culture, but the caudalxtent of Pax-2 expression was attenuated on the cut sideFig. 3, row 1). Embryos manipulated at HH8 gave interme-iate results; Pax-2 expression on the cut side was unde-ectable in some embryos, whereas others had attenuatedax-2 expression in truncated pronephroi.Some embryos were stained with an alternate marker for

ronephros formation. PM was separated from IM at HH7nd the embryo was cultured overnight as described aboveSpratt, 1947). Figure 4 shows a representative embryotained with a mouse antibody to Lim 1/2. In 100% of 13anipulated embryos, Lim 1/2 antibody staining and pro-

ephros development were seen on the control side only.hese results are in complete agreement with those ob-

ained with Pax-2 labeling and morphologic analysis ofronephric structure, providing additional support for ouronclusion that pronephros formation requires a medialignal.

Quantitative results are summarized in Table 1. In 43 of5 (96%) embryos manipulated prior to HH8, separation ofrunk paraxial mesoderm from intermediate mesodermrevented pronephros development on the experimentalide. Two (4%) embryos had truncated Pax-2 expression onhe experimental side, and none had normal expression. Inmbryos manipulated at HH8, 5 of 12 (42%) embryos hado pronephros formation on the experimental side; a trun-ated pronephros was seen in 7 (58%). The effect of surgicalanipulation on pronephros development decreased with

dvancing developmental stage, such that 50, 14, and 0% ofmbryos manipulated at HH92, 9, and $91, respectively,

had no Pax-2 expression or histologically evident prone-hros on the experimental side. However, the pronephrosas caudally truncated on the experimental side, even at

ater stages in which some development was evident at theime of microsurgery.

FIG. 7. Separation of IM from axial or lateral mesoderm did not altt t 5 0 and incubated for 24 h in Spratt culture. (Row 1) Cuts betwe

pronephroi formed lateral to somites on both the control (notoch(notochord and floor plate absent) sides. (Row 2) Separation of IMexpressed in intermediate mesoderm lateral to the somites, but watissue. The Hamburger and Hamilton stage at the time of manipulanotochord; NT, neural tube; S, somite; LP, lateral plate mesodermIG. 8. Removal of endoderm, ectoderm, or rostral tissues did nohown in cartoons at t 5 0 and incubated for 24 h in Spratt cultuissue sections showed that the pronephros developed and that the eemoval did not impair Pax-2 expression or morphologic pronephreparation of rostral from caudal tissues above the level of the fifthet segregated) resulted in Pax-2 expression only in the caudal segmegments in embryos transected below the level of the presumptiveP, lateral plate; MB/HB, Pax-2 expression in the midbrain/hindbra

ime of manipulation, we examined the time course ofronephros development in embryos in which the PM waseparated from the IM at HH7. Some embryos had weakax-2 expression in IM immediately after manipulation,ut most were negative, and pronephros formation was notet morphologically evident (not shown). After incubationor 4 h, weak Pax-2 expression was detected on the unma-ipulated control side, but had been lost from the cut side,nd there was no morphologic evidence for pronephrosormation on the cut side (Fig. 5, row 1). Embryos fixed ath had increased Pax-2 expression in the pronephric anlagef the control side only (Fig. 5, row 2), and by 24 h ofulture, Pax-2 was strongly expressed in the pronephroseveloping in the control, but not the cut, side of theanipulated embryo (Fig. 3, rows 1 and 2). These studies

onfirmed that proximity to the PM was required for bothax-2 expression and morphologic development of theronephros in embryos manipulated at HH7.

Pronephros Induction Is Required for MesonephrosDevelopment

Pronephros formation was required for further kidneydevelopment. We separated PM from IM in HH7 embryosand cultured the embryos in ovo for up to 3 days, until they

ad reached a developmental stage consistent with meso-ephros formation ($HH15). In 5 of 5 embryos, meso-ephros developed only on the unmanipulated side, asvidenced by both Lmx-1 expression and morphologic cri-eria (Fig. 6).

Neither Axial nor Lateral Signals Are Requiredfor Pronephros Induction, and Failure to FormPronephros after Microsurgery Is Not Dueto Blocking the Migration of ProspectiveIM Cells from the Primitive Streak

To determine whether the inducing signal arose directlyfrom the PM or more medially (e.g., from the notochord or

nephros development. Embryos were incised as shown in cartoonsand PM in early embryos did not impair Pax-2 expression. Normal

and floor plate of the neural tube present) and the experimentalLP in early embryos did not impair Pax-2 expression. Pax-2 was

t expressed in lateral plate mesoderm cultured apart from somiticis shown in the lower left corner of each photo. Abbreviations: N,s: whole mount, 300 mm; section, 100 mm.r pronephros development from the IM. Embryos were incised asow 1) Removal of endoderm did not impair Pax-2 expression anderm did not regrow during the incubation period. (Row 2) Ectodermvelopment. The endoderm did not regrow within 24 h. (Row 3A)ite (or above the level of the prospective fifth somite, if it had not

, whereas (Row 3B) Pax-2 was expressed in both rostral and caudalsomite. Abbreviations: N, notochord; NT, neural tube; S, somite;

rtion of the neural tube; OV, otic vesicle. Bars: whole mount, 300

er proen Nordfroms notion. Bart altere. (Rndodos desoment

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mm; section, 100 mm.Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

68 Mauch et al.


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70 Mauch et al.

the floor plate of the neural tube), embryos were dissectedparallel to the rostrocaudal axis between the notochord andthe trunk PM. Even embryos dissected at HH7 formednormal pronephroi on both the cut and the uncut sides afterovernight culture (Fig. 7, row 1). Importantly, these cutsalso showed that microsurgery did not block the migrationof prospective IM cells from the primitive streak. That is, ifthe more lateral cuts blocked migration, so should the moremedial cuts; these results show clearly that the latter donot. Rather, the pronephros failed to develop followingseparation of IM from the inducing signals produced by thePM. Separation of IM from LP did not impair or enhancepronephros formation in the IM on the experimental side(Fig. 7, row 2), showing that a more lateral signal from thelateral plate mesoderm, in contrast to the medial signalfrom the PM, is not required for pronephros induction.

uantitative results are summarized in Table 2.

Neither Endoderm, Ectoderm, nor Rostral TissuesAre Required for Pronephros Induction

To ascertain whether the inducing signal arises in the PMor in its underlying endoderm or overlying ectoderm, eachof these tissues was removed prior to culture. To avoid thepossibility of regrowth of ectoderm (Obara-Ishihara et al.,1999), the lateral portion of some embryos was also re-

FIG. 9. Pronephros induction following tissue explantation andecombined with HH7 chick lateral tissue (containing prospectiveollagen gel, and cultured in vitro for 28 h. Pax-2 expression (bluomites (S) are stained brown. Bars: whole mount, 300 mm; sectionIG. 10. (A) Fate map of the HH4 avian embryo. Hensen’s nodostrocaudal fashion to give rise to PM, IM, and LP mesoderm. (Bransplanted into the caudal primitive streak of chick embryos. Thomites (mostly composed of chick cells) in chick lateral tissues.uail-derived (brown); the remaining tissues arose from the chickblue) was expressed normally in the pronephros that arose from ixpressed in an ectopic pronephros arising from trans-fated chickeceived inducing signals from both native and ectopic somites (arrobrown) and somites were identified by in situ hybridization withM (arrows), an ectopic pronephros arose from trans-fated chick Lnducing signals from both native and ectopic somites (arrowheaeural folds; H, heart endoderm; NT, neural tube; LP, lateral plate

Normal Pax-2 Expression on the Manipulated Side Following Vari

Hamburger andHamilton stage

Cut betweenN and PM

Cut betwIM and

6 — —7 12/12 (100%) 7/7 (1008 10/10 (100%) 1/1 (100

Note. Data show the number of expressing embryos/total numberin parentheses.


oved prior to Spratt culture (Spratt, 1947). Examination oferial sections demonstrated that regrowth of endodermnd ectoderm did not occur (Fig. 8, rows 1 and 2). Removalf endoderm or ectoderm neither impaired nor enhancedronephros formation (Fig. 8, rows 1 and 2). Results ofmbryos dissected between HH6 and HH8 are summarizedn Table 2.

To establish the rostral boundaries of the pronephriceld, embryos were transected at various somitic levels. Inhese embryos, Pax-2 expression in the midbrain/hindbrain

region of the neural tube served as a positive control.Embryos transected above the level of the fifth somite hadbilateral Pax-2 expression only in the caudal isolate (Fig. 8,row 3, and Table 3), whereas embryos transected below thelevel of the sixth somite at HH8 or later expressed Pax-2 inoth the rostral and the caudal isolates (Fig. 8, row 3, andable 3). Five of ten embryos transected between the fifthnd the sixth somites expressed Pax-2 in both the rostralnd the caudal isolates. These studies showed that rostralissues such as head mesoderm are unlikely to be the sourcef the inducing signal and indicated that there was noostrocaudal shift over time in the character of eithernducing or responding tissue; tissue that had the charac-eristics found below the fifth somitic level at the time oficrosurgery retained those characteristics later and de-

pite microsurgical separation from more rostral tissues.

mbination in collagen gel. HH9 quail somites were isolated andmediate and lateral plate mesoderm and ectoderm), embedded in arved as a marker of pronephros induction (arrows). Quail-derived0 mm.es rise to the notochord, and primitive streak cells ingress in ansen’s node from HH4 or HH5 quail embryos was isochronicallynsplanted quail node formed quail notochord and induced ectopiche ectopic notochord (QN) and part of one ectopic somite were

ryo. On the control side (no ectopic notochord or somites), Pax-2ediate mesoderm (arrows). On the experimental side, Pax-2 was

sterisks), and an enlarged pronephros expressed Pax-2 in IM thatad). (D) The pronephros was characterized by anti-Lim 1/2 stainingbe for paraxis (blue). A normal pronephros was induced in nativesterisks) and an enlarged pronephros formed in IM that receivedbbreviations: S, somite; QN, quail notochord; N, notochord; NF,oderm. Bars: whole mount, 300 mm; tissue section, 100 mm.

icrosurgical Separations

Ectoderm removed Endoderm removed

2/2 (100%) —12/12 (100%) 11/11 (100%)

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Paraxial Mesoderm Is Sufficient for PronephrosInduction

Collectively, the experiments described above showthat signals from paraxial mesoderm are required forpronephros induction from intermediate mesoderm. Todetermine whether paraxial mesoderm is also sufficientfor pronephros induction, we cocultured the putativeinducing and responding tissues in collagen gels (Artingerand Bronner-Fraser, 1993). Caudal somites isolatedfrom HH8 to HH9 quail embryos were recombined withlateral mesoderm (containing prospective IM and LP)isolated from HH4 or HH7 chick embryos, embeddedin a collagen gel, and cultured in vitro for up to 28 h.Pax-2 expression served as a marker of pronephros induc-tion. Fixed tissues were also stained with anti-quailantibody to determine the origin of the pronephric tissue(Darnell and Schoenwolf, 1995). A representative speci-

Pax-2 Expression in the Rostral versus Caudal Isolate Following T

Hamburger andHamilton stage

1/2 2/3

C R C R C

7 9/9 0/9 3/3 0/38 1/181 1/192 1/191102

Note. The designation “1/2” indicates transection between thransection was made at the level at which somites were expected tor example, a HH81 embryo has only five somites, but some of thetween the sixth and the seventh somite was expected to appear., Pax-2 was expressed in the IM in the rostral isolate.

ABLE 4ronephros Induction in Chick Lateral Plate Following Coculture

Tissues cocultured

Negative control:HH7 chick LP

Positive controls:HH7 chick PM 1 LPHH7 chick N 1 PM 1 LP

Experimental:HH7 chick LP 1 HH9 quail PMHH7 chick LP 1 HH8 quail PMHH4 chick LP 1 HH8 quail PM

Note. Chick LP includes chick intermediate and lateral mesodernderlying endoderm. The Hamburger and Hamilton stage at the trospective PM was cultured with chick tissues. At stage 9, onlyxpressing embryos/total number of cases, with the percentage ex


men (Fig. 9) shows that the inducing PM was quailderived, whereas the responding (i.e., Pax-2-expressing)issue was of chick origin. Of 25 tissue recombinants, 21xpressed Pax-2 in chick tissue, indicating that the quailomites had induced pronephric anlage in the chickesoderm. This rate is comparable to that observed in

ositive controls in which caudal chick PM was left inpposition to IM and cultured (i.e., in isolation from thendoderm and the rostral and more medial tissues) inollagen gel (Table 4). It compares favorably with rateseported by Seufert and co-workers for experiments inhich they recombined anterior somites with two stage 9enopus animal caps. In their studies, 7 of 20 sampleseveloped pronephric tubules (Seufert et al., 1999). Inontrast, chick IM and LP cultured without PM (negativeontrols) did not express Pax-2. Quantitative results arehown in Table 4.

ction at Various Somitic Levels

4/5 5/6* 6/7*

R C R C R C R

4/4 0/40/1 3/3 0/3 6/6 5/60/1 1/1 0/1 1/1 0/1 9/9 9/90/1 1/1 0/1 1/1 0/1

1/1 0/11/1 0/1

st and the second pairs of somites. Asterisks indicate that theear, in embryos that had not yet developed that number of somites.s were made more caudally, at the level at which the demarcation

reviations: C, Pax-2 was expressed in the IM in the caudal isolate;

Quail Somites in Collagen Gels

-2 expressed No Pax-2 expression

/8 (0%) 8/8 (100%)

/11 (82%) 2/11 (18%)/7 (100%) 0/7 (0%)

/21 (81%) 4/21 (19%)/2 (100%) 0/2 (0%)/2 (100%) 0/2 (0%)

d associated ectoderm. Quail PM includes overlying ectoderm andf isolation is noted. At stage 8, the fourth somite and more caudalcaudal to the fifth somite was used. Data show the number of

ing Pax-2 in parentheses.

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The Generation of Ectopic Somites Leads to the

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Hamilton, 1952). Pax-2 expression persisted in the meso-n1cmwL

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72 Mauch et al.

Development of Ectopic and Enlarged PronephroiFigure 10A depicts a fate map of the avian embryo.

Hensen’s node gives rise to the notochord, and cells ingressfrom the more caudal primitive streak to give rise to PM,IM, and LP mesoderm in a rostrocaudal fashion (Schoenwolfet al., 1992). When Hensen’s node from HH4 or HH5 quailembryos was isochronically transplanted into the caudalprimitive streak of chick embryos (Fig. 10B), an ectopicnotochord self-differentiated, and the node induced ectopicsomites (Streit and Stern, 1999; Schoenwolf, unpublisheddata). Pronephric induction by paraxial mesoderm (somites)varied, depending upon both having adequate respondingtissue and the intensity of the inducing signal. In 10 of 13embryos, the native pronephros was elongated on the sidewith ectopic somites, but in 3, a supernumerary pronephroswas induced (Figs. 10C and 10D). In the embryo shown inFig. 10C, the pronephros was labeled with Pax-2, and quailtissues were identified by immunohistochemistry. To con-firm pronephric induction using another marker, and tobetter identify the somites, the pronephros in the embryoshown in Fig. 10D was labeled with antibody to Lim 1/2,and the somites were identified by paraxis expression. Asshown in Fig. 10C, the notochord and part of one somite arequail derived; the remaining tissues arose from the chickembryo. In both embryos, on the control side (lackingectopic notochord or somites), the pronephros developednormally in the intermediate mesoderm. On the experi-mental side, large ectopic somites formed between thenative intermediate mesoderm and the lateral plate meso-derm. An additional pronephros developed lateral to theectopic somite. In addition, between the native and theectopic somites, competent intermediate mesoderm ex-pressed pronephric markers. Moreover, presumably becauseit received signals from both the native and the largeectopic somite, the pronephros derived from this interme-diate mesoderm was greatly enlarged (Figs. 10C and 10D).This indicates that the inducing signal produced by theparaxial mesoderm acts in a dose-dependent manner.

DISCUSSION

The Pax genes are paired-box transcription factors ex-ressed in distinct spatiotemporal patterns during embryo-enesis (Walther et al., 1991). Pax-2, important in theevelopment of the metanephric kidney, is expressed dur-ng early embryonic development in a highly localizedattern in the pronephroi, eyes, otic vesicles, and midbrain/indbrain level of the neural tube (Dressler et al., 1990;orres et al., 1995). In our hands, weak Pax-2 expressionas detected in the intermediate mesoderm by HH7, andax-2 was strongly expressed caudal to the fifth somite byH9. Pax-2 expression correlated with morphologic evi-ence for pronephros development in tissue sections andas consistent with classical reports describing develop-ent of the pronephros in the intermediate mesoderm

audal to the level of the fifth somite (Abdel-Malek, 1950;


ephros, which was also labeled by Lmx-1 (Fernandez et al.,997). Therefore, we used Pax-2 as our primary marker ofonversion of pluripotent intermediate mesoderm to com-itted pronephric tissue. Antibody staining for Lim 1/2as used as a confirmatory pronephric marker, whereasmx-1 served as a marker for the mesonephros.The pronephros, mesonephros, and metanephros all arise

rom intermediate mesoderm, and similar gene expressionatterns in all three kidney forms argue for conservation ofevelopmental pathways and mechanisms of inductionVise et al., 1997). These similarities are useful; by studyingnduction and patterning in the pronephros, we also learnbout the development of the subsequent kidneys.The differences between pronephric determination and

evelopment of later phases can also be exploited to under-tand better the molecular mechanisms responsible foridney induction. Signals emanating from the nephric ductre required for meso- and metanephric kidney induction,hereas the pronephros arises directly from the intermedi-

te mesoderm and gives rise to the aforementioned nephricuct (Vise et al., 1997). If pronephros development involveshe same signaling molecules used at later kidney stages,hen these molecules must be produced by other embryonictructures (Yates and Pate, 1989; Vise et al., 1997).

The experiments described here ruled out the possibilityf self-determination. Lateral embryonic isolates contain-ng intermediate and lateral plate mesoderm failed to de-elop pronephroi in the absence of a medial signal. Theronephros was absent or truncated when the intermediateesoderm was separated from the adjacent medial tissues,

emonstrating that the inducing signal was produced by themmediately adjacent PM or perhaps by the notochord/eural tube. Normal pronephros formation in the IM left inpposition to the PM but separated from the notochord andoor plate of the neural tube ruled out an axial signal.eufert and co-workers similarly found that notochord wasot required for pronephros induction in Xenopus embryos

Seufert et al., 1999).We considered the possibility that the potential respond-

ng tissue had been ablated by surgical manipulation. Theidth of the IM labeled by either Pax-2 in situ hybridizationr Lim 1/2 antibody staining was 40–50 mm, whereas the

tip of the cactus needle used for separation of IM from PMwas significantly less than 10 mm in diameter, makingxtirpation of the entire width of responding tissue impos-ible. Each cut constituted an incision, not an ablation.oreover, medial-cutting errors would result in apposition

f the IM to the LP mesoderm, whereas lateral-cuttingrrors would result in apposition of IM to PM, with eacherving as a useful control. Because cuts were made frombout the first somitic level caudally through the tail end ofhe embryo, one might reason that ingression of prospectiveM from the primitive streak (Garcia-Martinez et al., 1993)as disrupted by cuts between PM and IM. However,

ngression of pronephric precursor cells has likely occurredrior to HH7, and if not, its occurrence would also be


prevented by cuts made between notochord/neural tube and

os

optrPiot(ec(

discrepancy between our results and those of Obara-


PM, which did not affect pronephros development. More-over, quail–chick chimeras have shown that adjacent me-soderm cells can migrate to new positions and form newintermediate mesoderm (Garcia-Martinez and Schoenwolf,1992), making ablation of all potential responding tissueunlikely.

Over 95% of the embryos cut between the PM and the IMbefore HH8 formed pronephros on the control side only,whereas embryos manipulated after HH9 had pronephrosdevelopment on both sides, but the length of the prone-phros on the cut side was truncated rostrocaudally com-pared to the control side. The persistence of the pronephrosat the rostral end of the cut side in HH9 embryos showedthat the inducing signal was only transiently required.However, the caudal extent of pronephros development wastruncated on the experimental side, even at later stages inwhich Pax-2 expression was evident at the time of micro-surgery, indicating that the inducing signal was producedby PM mesoderm in a rostral to caudal fashion.

Separation of intermediate mesoderm from LP mesodermneither impaired nor enhanced pronephros development,indicating that no lateral signal was required for IM induc-tion. We reasoned that the lateral tissues could emit inhibi-tory signals that restrict or otherwise determine the com-petence of the IM to respond to the medial-inducing signal,much as noggin inhibits BMP signaling in the medialsomite (Marcelle et al., 1997). However, based on the failuref the IM/LP isolates to show expansion of Pax-2 expres-ion, this possibility can be ruled out.

Removal of endoderm did not impair pronephros devel-pment, showing that a ventral signal was not required forronephros induction. Neither did we find evidence thathe ectoderm produced a dorsal signal required for proneph-ic induction, because both pronephric morphology andax-2 expression were normal on the manipulated side evenn embryos dissected at HH7. This contrasts with thebservation by Obara-Ishihara and co-workers that extirpa-ion of the ectoderm in chicken embryos cultured to HH16Hamburger and Hamilton, 1951) in ovo prevented Pax-2xpression in the IM and that nephric duct developmentould be rescued by the addition of exogenous BMP-4Obara-Ishihara et al., 1999). Our embryos were cultured invitro in Spratt culture, and they were considerably youngerat the time of manipulation; thus, it is possible that stagedifferences account for our differing observations. Indeed,these authors noted Pax-2 and cSim-1 labeling of a nephro-genic cord of cells derived from IM at HH10 in theirmanipulated embryos, but focused their observations onthe morphologic differentiation of the nephric duct. Thepronephric duct is not a tubular structure, but acquires alumen upon further development of the mesonephros,whereupon it is renamed the mesonephric or Wolffian duct(Hamilton, 1952). Thus, it is possible that endoderm orectoderm are important in the further differentiation of themesonephric duct, although they are not required for pro-nephros induction. However, another explanation for the


Ishihara and co-workers exists. BMP-4 has been demon-strated to be important in somitogenesis (Pourquie et al.,1996), and the effect of BMP-4 on pronephros induction inthe IM may be, therefore, indirectly mediated by trunkparaxial mesoderm.

Rostrocaudal transection experiments showed both thatPax-2 expression occurred only caudal to the level of theprospective fifth somite and that a rostral signal was notrequired for induction of Pax-2 expression in the IM. Thispattern was observed regardless of the developmental stageat the time of microsurgery, indicating that the character-istics of the inducing and responding tissues around level ofthe prospective fifth somite did not change with time ormanipulation.

Recombination experiments showed that quail somitesare sufficient for the induction of Pax-2 expression (prone-phros formation) in chick intermediate and lateral meso-derm. Our findings are consistent with those of Seufert etal., who reported that UV-ventralized Xenopus embryoslacked somites and subsequently failed to develop pro-nephroi. These investigators further reported that proneph-ric tubules, but not ducts, could be induced in embryonicisolates when lateral mesoderm was heterochronically cul-tured within an ectodermal sandwich that also containedanterior somitic tissue (Seufert et al., 1999). The embryonickidney of Xenopus is among the simplest of vertebratekidneys, consisting of a single large nephron with anexternal glomus (Vise et al., 1997). It is the functionalembryonic kidney in fish and amphibia, whereas the meso-nephros is the definitive kidney (Brandli, 1999). In contrast,avian kidney development more closely approximates thatof mammals, in that both proceed from pronephros throughmesonephros and have a metanephros as the definitivekidney. Our findings in the avian pronephros confirm thefrog studies, and the evolutionary conservation of signalingstresses the importance of the somite in the patterning ofthe early embryonic kidney.

When quail node was transplanted into chick caudalprimitive streak, ectopic somites were generated. Interme-diate mesoderm that received signals from two rows ofsomites produced an enlarged pronephros. One might arguethat an absence of suppressing signals from lateral platemesoderm explains the enhanced pronephric development,but microsurgical separation of IM from LP did not enhancePax-2 expression in the pronephros. Rather, these experi-ments provide the first evidence that the inducing signalproduced by the paraxial mesoderm acts in a dose-dependent manner.

In summary, we have shown that rostral tissue, axial orlateral plate mesoderm, endoderm, and ectoderm neitherinduce nor inhibit pronephric induction. In contrast, inter-action with trunk paraxial mesoderm is both required andsufficient for pronephros induction in the chick intermedi-ate mesoderm. Studies are in progress to define the molecu-lar nature of the inducing signals.


ACKNOWLEDGMENTS

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coloboma syndrome and results in developmental defects of the

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74 Mauch et al.

The Pax-2 and Lmx-1 probes were kindly provided by Drs.Domingos Henrique and Randy Johnson, respectively. A probe forparaxis was obtained from D. Sosic and E. Olson. We thankShipeng Yuan for technical advice during this study. Dr. Mauchgratefully acknowledges the support of the National Institutes ofHealth 1K08DK02490-01A1 and the Primary Children’s MedicalFoundation. Dr. Schoenwolf received support from the NationalInstitutes of Health NS18112.

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Received for publication January 7, 2000Accepted January 10, 2000


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