Osr1 expression demarcates a multi-potent population of intermediate mesoderm that undergoes progressive restriction to an Osr1-dependent nephron progenitor compartment within the mammalian kidney

doi:10.1016/j.ydbio.2008.09.010

Dev Biol. Author manuscript; available in PMC 2009 December 1.

Published in final edited form as:

Dev Biol. 2008 December 1; 324(1): 88–98.

Published online 2008 September 19. doi: 10.1016/j.ydbio.2008.09.010

PMCID: PMC2642884

NIHMSID: NIHMS81008

Osr1 expression demarcates a multi-potent population of intermediate mesoderm that undergoes progressive restriction to an Osr1-dependent nephron progenitor compartment within the mammalian kidney

Joshua W. Mugford,¹ Petra Sipilä,^1,² Jill A. McMahon,¹ and Andrew P. McMahon¹^*

¹Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA

^*Author for correspondence: Email: mcmahon/at/mcb.harvard.edu

²Current address: Department of Physiology, Institute of Biomedicine, University of Turku, FIN-20520, Turku, Finland

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Abstract

The mammalian metanephric kidney is derived from the intermediate mesoderm. In this report, we use molecular fate mapping to demonstrate that the majority of cell types within the metanephric kidney arise from an Osr1⁺ population of metanephric progenitor cells. These include the ureteric epithelium of the collecting duct network, the cap mesenchyme and its nephron epithelia derivatives, the interstitial mesenchyme, vasculature and smooth muscle. Temporal fate mapping shows a progressive restriction of Osr1⁺ cell fates such that at the onset of active nephrogenesis, Osr1 activity is restricted to the Six2⁺ cap mesenchyme nephron progenitors. However, low-level labeling of Osr1⁺ cells suggests that the specification of interstitial mesenchyme and cap mesenchyme progenitors occurs within the Osr1⁺ population prior to the onset of metanephric development. Furthermore, although Osr1⁺ progenitors give rise to much of the kidney, Osr1 function is only essential for the development of the nephron progenitor compartment. These studies provide new insights into the cellular origins of metanephric kidney structures and lend support to a model where Osr1 function is limited to establishing the nephron progenitor pool.

Keywords: Osr1, metanephros, kidney, progenitor

Introduction

The nephron is the basic functional unit of the metanephric kidney. Depending on the mammalian species, tens to hundreds of thousands of nephrons are formed over an extensive period of fetal and neonatal nephrogenesis (Cullen-McEwen et al., 2001; Nyengaard and Bendtsen, 1992). The main body of the nephron is a tubular epithelial network comprised of specialized segments along it proximal to distal axis (Yu et al., 2004). The interface of the vasculature and the nephron epithelium occurs at the proximal end of the nephron in the glomerulus where podocytes of the nephron epithelia lie opposed to a specialized vascular and supporting cell (mesangial cell) network (Spinelli, 1974). Blood filtrate travels along the nephron tubule where all essential small molecules in the filtrate are resorbed into a closely associated vasculature (Reeves et al., 2001). The distal end of the nephron fuses with the epithelial collecting system enabling the passage and final adjustment of the water/salt balance of the urine prior to its removal via the ureter into the bladder (Meneton et al., 2004). Smooth muscle surrounding the medullary collecting system and ureter facilitate the fluid movement in these final stages (Yu et al., 2002). In addition to the cell populations above, the nephrons are embedded within an interstitial mesenchyme whose function is poorly understood (Hatini et al., 1996; Levinson et al., 2005).

Recent studies have started to probe the cellular origins and cellular relationships among the component parts of the kidney to determine the developmental mechanisms that establish a functional upper urinary system (Boyle et al., 2008; Kobayashi et al., 2008; Yu et al., 2002). The metanephric kidney of the mouse arises from within the posterior intermediate mesoderm (IM) around embryonic day 10.5 (E10.5) (Saxén, 1987; Vize et al., 2002). At this time, Gdnf expression within pre-specified metanephric IM mesenchyme at the hindlimb level induces an outgrowth of the adjacent nephric duct, the ureteric bud (Grieshammer et al., 2004; Kume et al., 2000; Sanchez et al., 1996b). On entering the metanephric mesenchyme, cell interactions between the ureteric bud and adjacent mesenchyme drive assembly of the functional kidney. In this, the mesenchyme continues to stimulate branching growth of the epithelium, generating the arborized network of the urine transporting collecting system (Grieshammer et al., 2004; Pepicelli et al., 1997; Sanchez et al., 1996b; Yu et al., 2002).

With each branching event, a portion of the metanephric mesenchyme adjacent to the ureteric bud tips, the cap mesenchyme, is induced by the ureteric epithelium to undergo a mesenchymal to epithelial transition (MET). This induction establishes the renal vesicle, the epithelial precursor of the main body of the nephron (Carroll et al., 2005; Dressler, 2002; Park et al., 2007; Stark et al., 1994). The cap mesenchyme is a self-renewing population of renal vesicle progenitors and expresses the transcriptional regulator Six2, which is required for its self-renewal (Boyle et al., 2008; Kobayashi et al., 2008; Self et al., 2006). From E11.5 through post-natal day 3, this process reiterates in conjunction with branching growth of the ureteric epithelium to generate the full complement of nephrons (Hartman et al., 2007).

A second population of Foxd1⁺ mesenchyme surrounds Six2⁺ nephron progenitors (Hatini et al., 1996; Levinson et al., 2005). While the fate of this cell population has not been addressed, they are thought to give rise to the interstitial components of the metanephric kidney. Origins of the vascular, mesangial and smooth muscle compartments are not fully resolved, though lineage-tracing studies suggest that the vasculature may arise intrinsically within the early metanephros or extrinsically by migration into the developing kidney (Bernstein et al., 1981; Loughna et al., 1997; Loughna et al., 1998; Sariola et al., 1984).

The transcriptional regulator Osr1 is expressed broadly in the early IM mesenchyme beginning at E7.5 and its activity is essential for development of multiple IM derived structures (James et al., 2006; Wang et al., 2005). Notably, in the presumptive metanephric mesenchyme of E10.5 Osr1 mutant embryos, the expression of several key regulators of kidney development, including Pax2, Six2, Sall1, Eya1 and Gdnf, is absent and the metanephric mesenchyme undergoes apoptosis. Thus, Osr1 has an essential function at the outset of metanephric kidney development (James et al., 2006; Wang et al., 2005).

In this study, we have used a temporal fate mapping approach to address the relationship between Osr1⁺ cells and multiple cell lineages in the mouse kidney. We demonstrate that many distinct cellular compartments emerge from the Osr1⁺ population prior to the appearance of the metanephric anlagen. Furthermore, there is a separation of molecularly distinct Six2⁺ and Foxd1⁺ progenitor pools prior at the onset of metanephric development. Finally, Osr1 function is itself required for formation of the Six2⁺ renal vesicle progenitors, but not for the formation of interstitial precursors or ureteric epithelium components.

Results

Generation of an Osr1^eGFPCreERt2 driver line

To examine the relationship between Osr1⁺ descendants and distinct cell lineages in the kidney, we targeted a cDNA encoding an eGFPCreERt2 (GCE) cassette to the endogenous Osr1 locus, replacing the start codon of Osr1 with the start codon of the GCE (Fig. S1A) (Mugford et al., 2008). The use of GCE allows for both a spatial and temporal analysis of Osr1⁺ cell fate. Though this strategy creates a null mutation in one Osr1 allele, Osr1^{eGFPCreERt2/+} (Osr1^GCE/+) heterozygotes were both viable and fertile (Wang et al., 2005). Whole mount analysis of wild-type and Osr1^GCE/+ E8.5 (Fig. S1B–D) and E9.5 (Fig. S1F–H) embryos demonstrated that GFP and Osr1 mRNA co-localize to IM and dorsal lateral plate mesoderm as expected (Fig. S1E, I). Furthermore, GFP in the Six2⁺ cap mesenchyme at E15.5 (Fig. S1K) mirrors endogenous Osr1 expression (Fig. S1J). Thus, the GCE cassette faithfully recapitulates the endogenous expression pattern of Osr1.

Descendants of Osr1⁺ cells initially contribute to all distinct metanephric progenitor compartments and are restricted to nephron progenitors after E11.5.

To identify the fate of Osr1⁺ cells at specific developmental time points, we intercrossed Osr1^GCE/+ animals with Gt(ROSA)26^tm1Sor (R26R) (Soriano, 1999) animals and dams were injected with Tamoxifen (TM) at 24 hour intervals from E6.5 through E11.5 with a TM dose that enables Cre activity for less than 24 hours (Kobayashi et al., 2008) (see Materials and Methods). This pulse labeling strategy covers time points prior to the onset of Osr1 expression through the first inductive events within the metanephric kidney. The contribution of Osr1⁺ descendants to various renal cell types was determined at E15.5.

Low resolution analysis of X-gal staining demonstrates that Cre activity was dependent upon TM as expected; no β-gal activity was present in Osr1^GCE/+;R26R embryos of dams injected with carrier (oil) alone between E7.5 and E9.5 (Fig. S2A). When TM was injected at E6.5 (early gastrulation), a small number cells within the ureteric epithelium were X-gal⁺ in E15.5 Osr1^GCE/+;R26R kidneys (data not shown). When Osr1^GCE/+;R26R embryos were exposed to TM from E7.5 through E11.5, X-gal⁺ cells a variable contribution was observed to multiple cell types of the E15.5 metanephric kidney, gonad and adrenal gland (Fig. S2B–F).

To identify the exact cell types to which Osr1⁺ descendents contributed, we compiled a set of cell specific markers (Table 1) and performed dual immunofluorescence with antibodies directed against β-galactosidase (β-gal) focusing initially on the progenitor compartments of the nephrogenic zone; the ureteric epithelium, cap mesenchyme and cortical interstitial mesenchyme.

Table 1

Cell type specific markers

Six2 and Foxd1 are present within renal vesicle and putative interstitial progenitors, respectively (Hatini et al., 1996; Self et al., 2006). In order to establish Six2 and Foxd1 as definitive markers of the cap mesenchyme and cortical interstitial mesenchyme, respectively, we generated antibodies against Six2 and Foxd1 and examined their distribution relative to each other and Pax2, a marker of both the nephric duct (Fig. 1B, F, J, N, R, V, dashed lines) and the cap mesenchyme (Fig. 1B, F, J, N, R, V) (Torres et al., 1995). Foxd1 was present in cells of the interstitial mesenchyme progenitors from E10.5 through E15.5 (Fig. 1A, E, I) and Pax2⁺ cells of the nephric duct and the cap mesenchyme were Foxd1⁻ (Fig. 1C, D, G, H, K, L). At all time points examined, the majority of Foxd1⁺ cells surrounded Pax2⁺ cells of the cap mesenchyme and after branching had occurred, Foxd1⁺ cells were found in the cleft of the branching ureteric epithelium (Fig. 1E, I, G, K and data not shown). In contrast, Six2⁺ cells were present within the cap mesenchyme and Six2 was down-regulated as these cells underwent MET (Fig. 1M, Q, U) (Self et al., 2006). Unlike Foxd1, Six2 and Pax2 were present in all cap mesenchymal cells, but Six2 was absent from the Pax2⁺ collecting duct epithelium (Fig. 1O, P, S, T, W, X). Thus, from the onset of metanephric development at E10.5, Six2 and Foxd1 demarcate mutually exclusive progenitor compartments; Six2⁺ cap mesenchyme renal vesicle progenitors and Foxd1⁺ putative interstitial mesenchyme progenitors.

Figure 1

Metanephric progenitor domains are established by E10.5

At E15.5, cytokeratins are present solely within the cells of the presumptive collecting duct epithelium (Fig. 2A, E, I, M, Q) (Fleming and Symes, 1987). If TM was administered at either E7.5 (Fig. S2B and Fig. 2A–D) or E8.5 (Fig. S2C and Fig. 2E–H) β-gal⁺, cytokeratin⁺ cells were detected in E15.5 Osr1^GCE/+;R26R kidneys. Conversely, if TM was administered at E9.5 or later (Fig. S2D–F and Fig. 2I–L, M-P, Q–T), E15.5 cytokeratin⁺ cells were β-gal⁻. Thus, the collecting duct lineage arises from an Osr1⁺ cell-type prior to E9.5.

Figure 2

Osr1⁺ cells give rise to the ureteric epithelium between E7.5 and E9.5

Foxd1⁺, β-gal⁺ cells were detected in E15.5 Osr1^GCE/+;R26R kidneys following GCE induction between E7.5 and E10.5 but not at E11.5 (Fig. S2B–F and Fig. 3A–D, E–H, I–L, Q–T, arrowheads). Thus, Osr1⁺ cells contribute to the Foxd1⁺ compartment up until the onset of nephrogenesis. Finally, Six2⁺, β-gal⁺ cells were detected following TM administration at all time points consistent with the continued expression of Osr1 within Six2⁺ renal vesicle progenitors after nephrogenesis commences (Fig. S1K, S2B–F and Fig. 4A–D, E–H, I–L, J–K, Q–T, arrowheads).

Figure 3

Osr1⁺ cells give rise to cortical interstitial mesenchyme precursors between E7.5 and E11.5

Figure 4

Osr1⁺ cells always give rise to the cap mesenchyme

Though our earlier data indicates that Foxd1 and Six2 are mutually exclusive populations of cells (Fig. 1), descendants of Osr1⁺ cells contributed to both Foxd1⁺ and Six2⁺ compartments upon E10.5 TM administration. This suggests that Osr1 is not restricted to Six2⁺ cap mesenchyme until after E11.5. Indeed, at E10.5, GFP was clearly present in both Foxd1⁺ (Fig. S3A–D, arrowheads) and Six2⁺ cells (Fig. S3E–H, arrowheads); however, the intensity of the GFP signal was considerably higher in Six2⁺ cells. At E11.5, Foxd1⁺ cells were GFP⁻ (Fig. S3I–L, arrowheads); only Six2⁺ cells remained GFP⁺ (Fig. S3M–P, arrowheads).

Taken together, these results demonstrate that Osr1⁺ cells have the potential to give rise to all cortical progenitor compartments of the metanephric kidney. Of note, the Osr1⁺ population exhibits its broadest potential between E7.5 and E9.5. The restriction of the Osr1⁺ population to the cap mesenchyme mirrors the spatial restriction of Osr1 to Six2⁺ cells by E11.5. However, this restriction was delayed relative to the apparent separation of the nephron and interstitial lineages since Six2⁺ cells do not contribute to the Foxd1⁺ compartment (Kobayashi et al., 2008) and Six2⁺ and Foxd1⁺ cells are mutually exclusive (Fig. 1). Thus, Osr1 regulation is likely distinct from the mechanisms that control lineage specification within the mesenchymal progenitor compartments.

Osr1⁺ descendants contribute to renal vasculature, pericytes, mesangium, smooth muscle and the kidney capsule

To examine more broadly the contribution of β-gal⁺ cells in E15.5 Osr1^GCE/+;R26R kidneys, we next focused on the vascular-renal tubule interface within the glomerulus, which consists of Flk1⁺ vascular endothelial cells (Robert et al., 1998), PDGFRb⁺ mesangial support cells (Hellstrom et al., 1999) and WT1⁺ podocytes (Mundlos et al., 1993).

As expected, WT1⁺ podocytes, which are derivatives of the Six2⁺ cap mesenchyme (Boyle et al., 2008; Kobayashi et al., 2008), were β-gal⁺ at all TM pulse time points (Fig. 5E–G, I–K, M–O, Q–S, concave arrowheads, Fig. S4 and data not shown). TM administration at E7.5 (Fig. 5A–D, arrowheads) and E8.5 (Fig. 5E–G, arrowheads) generated β-gal⁺, Flk1⁺ endothelial cells in the glomerulus. In addition, the extraglomerular renal vasculature and the vasculature in other organs, including the neural tube, lung and limbs, was also β-gal⁺ (data not shown). However, no β-gal⁺, Flk1⁺ cells were detected following TM administration at or after E9.5 (Fig. 5I–L, M–O, Q–T, arrowheads). In contrast, Osr1⁺ descendants contributed to PDGFRb⁺ mesangial cells at all but the latest time point (E11.5) (Fig. S4A–D, E–G, I–L, M–O, arrowheads). Furthermore, PDGFRb⁺, β-gal⁺ pericytes were detected adjacent to non-glomerular associated kidney vasculature at the same pulse time points (Fig. S4E–G, I–K, Q–T concave arrowheads and data not shown).

Figure 5

Osr1⁺ cells give rise to the renal vasculature between E7.5 and E9.5

The kidney capsule, a layer of epithelial cells surrounding the cortical surface of the metanephric kidney (Levinson et al., 2005), is recognized by the cell surface antigen Liv2 (Fig. S5A, E, I, M, Q) (Y.Q. Soh and A.P. McMahon, unpublished observation). Like Foxd1⁺ interstitial progenitors, PDGFRb⁺ pericytes and mesangium, Liv2⁺, β-gal⁺ cells were only labeled on TM induction between E7.5 and E10.5 (Fig. S5A–D, E–H, I–L, M–P, Q–T arrowheads). That these diverse cell types show a common labeling property may reflect a similar lineage relationship from Osr1⁺ cells.

In contrast, smooth muscle progenitors displayed a distinct temporal lineage relationship from the Osr1 population. At E15.5, smooth muscle identified by Smooth Muscle α-Actin (SMA) surrounds the ureter (Yu et al., 2002). This population was labeled upon TM administration from E8.5 through E10.5 (Fig. S6E–H, I–L, M–O, arrowheads), but not at E7.5 (Fig. S6A–D, arrowheads) or E11.5 (Fig. S6Q–T). This indicates that the Osr1⁺ population is not be fixed at E7.5. Rather, new cells with smooth muscle generating potential are recruited by E8.5.

Finally, macrophages reside among cells of the interstitial stroma and can be identified by the G-protein coupled receptor F4/80 (Challen et al., 2006). No F4/80⁺, β-gal⁺ macrophages were detected in E15.5 Osr1^GCE/+; R26R kidneys when TM was administered anytime between E7.5 and E11.5 (Fig. S7A–D, E–G, I–L, M–O, Q–T, arrowheads). Taken together, these results demonstrate that descendants of Osr1⁺ cells give rise to a broad array of metanephric cell types including the ureteric epithelium, nephron and interstitial mesenchyme progenitors, vasculature, pericytes, mesangium, the kidney capsule and smooth muscle. Thus the Osr1⁺ population is the early progenitor for most kidney populations. However, descendants of Osr1⁺ cells are not restricted to the kidney; notably, descendants contribute to the vasculature of multiple organs that are not IM-derived.

Distinct Osr1⁺cells establish the Foxd1⁺ and Six2⁺ lineages between E9.5 and E10.5

Although the Osr1⁺ population is multi-potent, the potential of an individual Osr1⁺ cell remains an open question. Development of the metanephric kidney mesenchyme is first evident at E10.5. At this time, Six2⁺ and Foxd1⁺ cells form mutually exclusive compartments, though both cell types express Osr1 (Fig. 1). Thus, a single Osr1⁺ cell prior to E10.5 may generate both progenitor cell types. Alternatively, distinct Osr1⁺ cells may independently generate the Six2⁺ and Foxd1⁺ cell compartments.

Previous reports have demonstrated that low doses of TM can activate a limited number of CreERt2⁺ cells, allowing for clonal analysis (Kobayashi et al., 2008; Zhou et al., 2007). In an attempt to address the potential of a single Osr1⁺ cell, we injected dams with low doses of TM at E9.5 (see Materials and Methods) and performed immunofluorescence for β-gal, Pax2 and Foxd1 at E10.5. If a single Osr1⁺ cell is multipotent, then adjacent Pax2⁺, β-gal⁺ and Foxd1⁺, β-gal⁺ cells may be detected at the interface of the E10.5 interstitial and cap mesenchyme. Alternatively, if the Osr1⁺ population is segregated into Six2⁺, Pax2⁺ and Foxd1⁺ cellular compartments, then adjacent β-gal⁺ cells should both be either Pax2⁺ or Foxd1⁺. In the absence of information regarding cell motility or precise mitotic indices, adjacent cells are defined as two nuclei within a distance no greater than 10µm between the centers of their nuclei.

Most β-gal⁺ cells did not occur as adjacent pairs; single β-gal⁺ cells were generally spaced at distances greater than 15µm from each other. Only 16 pairs of adjacent β-gal⁺ in 5 embryos were identified and most were not located at the border between the stromal and cap mesenchyme. No adjacent β-gal⁺, Foxd1⁺ cells were detected. Adjacent β-gal⁺ cells within the Foxd1⁺ compartment consisted of one β-gal⁺, Foxd1⁺ cell and one β-gal⁺, Foxd1⁻ cell (5/11 pairs in 5 embryos) (Fig. 6A–D, brackets E, arrows) or both cells were β-gal⁺, Foxd1⁻ (4/11 pairs in 5 embryos) (Fig. 6F–I, brackets I, arrows). Within the Pax2⁺ cap mesenchyme, β-gal⁺, Pax2⁺ adjacent pairs were identified; however, in all cases both nuclei were β-gal⁺, Pax2⁺ (5/5 pairs in 5 embryos) (Fig. 6K–N, brackets O, arrowheads). No adjacent β-gal⁺ pairs were detected where one cell was Foxd1⁺ and the other Pax2⁺. Taken together, this data suggests that if a single Osr1⁺ cell has the potential to give rise to both cap mesenchyme and interstitial mesenchyme progenitors, this cell is a rare member of the Osr1⁺ population between E9.5 and E10.5. Thus, interstitial mesenchyme and cap mesenchyme are likely separate lineages prior to the onset of metanephric development.

Figure 6

Distinct Osr1⁺ cells give rise to Foxd1⁺ and Pax2⁺ between E9.5 and E10.5

Osr1 function is not required for the specification of the interstitial mesenchyme

Given these results, we next addressed the requirement for Osr1 in development of the interstitial mesenchyme population. Kidneys do not develop in Osr1 mutants; the absence of a number of genes essential for normal cap mesenchyme development including Pax2, Sall1, Six2, Eya1 and Gdnf indicates that cap mesenchyme progenitors are absent at E10.5 (James et al., 2006; Wang et al., 2005). However, the Osr1 locus is still active in Osr1 mutants (James et al., 2006); consequently, Osr1 mutant IM cells can be identified by the presence of GFP in E10.5 Osr1 mutants. In order to generate embryos lacking all Osr1 activity, we removed the PGK Neo cassette from the GCE allele (GCEN) and intercrossed Osr1^GCE/+ and Osr1^GCEN/+ animals (see Materials and Methods).

In agreement with previously reports (James et al., 2006; Wang et al., 2005), Pax2 was present in the nephric duct epithelium in embryos lacking Osr1 activity (dashed lines in Fig. 7B and L, G and Q), but was absent from the GFP⁺ posterior IM mesenchyme in Osr1^GCE/GCEN mutants (compare Fig. 7B–D, L–N with G–I, Q–S). Six2 was also absent from the GFP⁺ E10.5 posterior IM of Osr1^GCE/GCEN mutants (data not shown). Conversely, Foxd1⁺, GFP⁺ cells were present in the posterior IM of E10.5 Osr1^GCE/+ (Fig. 7A, C, D, arrowheads) and E10.5 Osr1^GCE/GCEN embryos (Fig. 7F, H, I arrowheads).

Figure 7

Osr1 function is only required for the formation of the cap mesenchyme

The Hox11 paralogs play a crucial role in the axial specification and patterning of the posterior IM mesenchyme (Mugford et al., 2008; Patterson et al., 2001; Wellik et al., 2002). To determine whether the metanephric mesenchyme is incorrectly specified in Osr1 mutants, Hoxd11 activity was examined. Hoxd11⁺, GFP⁺ cells were detected throughout the posterior IM of E10.5 Osr1^GCE/+ embryos, including the Pax2⁺ cap mesenchyme and the Pax2⁻ interstitial mesenchyme (Fig. 7K–O, concave arrowheads and arrowheads, respectively). In E10.5 Osr1^GCE/GCEN embryos, Hoxd11 was present within mesenchymal GFP⁺ cells (Fig. 7P–T, arrowheads), but no Hoxd11⁺, Pax2⁺, GFP⁺ cells were detected (Fig. 7P–T). Thus, an appropriate Foxd1⁺, Hoxd11⁺ interstitial compartment appears to form in the absence of Osr1 function, but the cap mesenchyme progenitor compartment was not established.

Discussion

Osr1^⁺descendants give rise to the majority of metanephric cell types at discreet developmental time points

We have demonstrated that descendants of Osr1⁺ cells initially contribute to a variety of IM derived cell types including cells within the adrenal gland, gonad and metanephric kidney (Fig. 8). Ultimately, Osr1 expression and consequently Osr1⁺ descendant cell fates are restricted to metanephric nephron progenitors at the onset of active nephrogenesis; Osr1 function is essential for the establishment of this progenitor pool. Analysis of Osr1⁺ descendant cell fate and restriction gives new insights regarding the specification and cellular behaviors of metanephric progenitor domains.

Figure 8

Osr1⁺ cell fates are restricted during the development of the metanephric kidney

The nephric duct is thought to be the first committed cell population to emerge from the IM mesenchyme (Grote et al., 2006; Pedersen et al., 2005). In agreement with this interpretation, Osr1⁺ descendants only contribute to the ureteric epithelium, an outgrowth of the nephric duct, prior to E9.5 coincident with the initial appearance of this epithelial structure. Thus, it is likely that growth rather than continued cell recruitment from an Osr1⁺ mesenchymal population accounts for posterior extension of the nephric duct.

Interestingly, between E7.5 and E11.5, Osr1⁺ descendants contribute to interstitial mesenchyme precursors, pericytes, mesangial cells and capsule cells; a shared temporal labeling consistent with a possible common lineage amongst these cell types. We also demonstrate that descendants of Osr1⁺ cells give rise to renal smooth muscle lining the ureter. The contribution of Osr1⁺ descendants to smooth muscle did not temporally correlate with their contribution to any other metanephric progenitor population, suggesting that metanephric smooth muscle does not arise from the cap mesenchyme, ureteric epithelium or interstitial mesenchyme progenitors. This is in agreement with past reports demonstrating that descendants of these progenitor populations are excluded from smooth muscle (Boyle et al., 2008; Kobayashi et al., 2008; Yu et al., 2002). Thus, the incorporation of a smooth muscle population likely reflects de novo expression of Osr1 in a new cell population around E8.5. Of note, Osr1 is present in both IM and dorsal lateral plate mesoderm, but the precise timing of expression within these compartments is hard to assess due to the absence of tightly restricted lineage markers.

Nephron and interstitial mesenchyme progenitors are likely separated prior to or at the onset of metanephric development

Previous fate mapping experiments have used Cre driver lines to directly label the Six2⁺ cap mesenchyme and Hoxb7⁺ ureteric epithelial progenitor compartments within the metanephric kidney and have demonstrated that the descendants of these populations are exclusive of each other (Boyle et al., 2008; Kobayashi et al., 2008; Yu et al., 2002). Though these progenitor populations are spatially and molecularly distinct at the onset of metanephric development, they arise from a common Osr1⁺ precursor population. Within the mesenchyme, labeling studies suggest that the Osr1⁺ IM mesenchyme is predominantly a heterogeneous population of cells already committed to either cap mesenchyme or interstitial progenitor cell fates by E10.5. It should be noted that our analysis cannot rule out the possibility of a rare multipotent Osr1⁺ cell, nor can we exclude the possibility of rapidly dividing and/or migrating IM mesenchyme progenitors. How distinct cell fates emerge from an Osr1⁺ progenitor pool remains to be determined, but signaling from adjacent tissues (e.g. somatic mesoderm) may play a significant role.

Descendants of Osr1⁺ cells and the hemangioblast hypothesis

Vascular precursors are thought to arise from the splanchnic mesoderm of the lateral plate (Poole et al., 2001; Saha et al., 2004), while the Aorta-Gonad-Mesonephros (AGM) region of the developing embryo is the origin of definitive heamatopoeitic stem cells (HSCs) (Cumano et al., 2000; Kumaravelu et al., 2002; Medvinsky and Dzierzak, 1996; Minehata et al., 2002; Robin and Dzierzak, 2005; Sanchez et al., 1996a). A progenitor cell, the hemangioblast, has been proposed to give rise to both vasculature and hematopoeitic lineages, though the cells existence is somewhat controversial (Xiong, 2008).

Osr1 is expressed in the presumptive AGM and lateral plate (James et al., 2006; Wang et al., 2005); this study) and we have previously shown that descendants of Osr1⁺ cells contribute to the mesonephros (Mugford et al., 2008). Here, we demonstrate that while Osr1 descendants give rise to the vasculature of many organ systems, they do not contribute to renal macrophage, derivatives of HSCs (Gordon and Taylor, 2005). This suggests that Osr1⁺ cells and their descendants, though located within the developing AGM, are excluded from the heamatopoeitic lineage. Thus, if there is a cell type generating the blood and vascular lineages, Osr1 is only activated after the separation of the vascular lineage. Various studies have suggested an IM source and a non-IM source (Bernstein et al., 1981; Loughna et al., 1997; Loughna et al., 1998; Sariola et al., 1984) of vascular progenitors in the kidney. Osr1 is expressed in both the IM and LPM potentially supporting either view. However, definitive evidence would require an independent analysis of the vascular forming potential of the IM and LPM components of the Osr1 expression domain.

Osr1 functions in the formation and maintenance of nephron progenitors

Functionally, Osr1 is upstream of multiple transcriptional regulators required for the development of the cap mesenchyme (James et al., 2006; this study). However, its function is not required for the formation of the nephric duct or for the specification of Hoxd11⁺, Foxd1⁺ interstitial mesenchyme progenitors (James et al., 2006; this study). Together, this suggests that despite its early broad expression in cells that generate multiple cellular compartments in the adult kidney, Osr1 is not a master regulator of the IM. Rather Osr1 is required specifically for the development of the renal vesicle forming cap mesenchyme lineage.

Recent reports suggest that Osr1 encodes a transcriptional repressor that may recruit Groucho-like co-repressors to promote a mesenchymal precursor state (Goldstein et al., 2005; James et al., 2006; Tena et al., 2007). Once nephron and interstitial progenitors are separated, Osr1 may function to activate a cap mesenchyme specific program, transcriptionally suppressing an interstitial progenitor program. While this interpretation remains speculative in the absence of direct information of Osr1 targets, its critical role in establishing the renal vesicle progenitor pool is clear.

Osr1 repressor function is essential for the specification of the cap mesenchyme. In this, Osr1 is genetically upstream of Pax2, Eya1, Six2 and Gdnf (James et al., 2006; this study). Six2 function is required within the cap mesenchyme, not for its specification, but to maintain this renal vesicle forming progenitor population in a self-renewing mesenchymal state (Kobayashi et al., 2008; Self et al., 2006). Gdnf is required for the initial invasion and continual branching of the ureteric epithelium within the metanephric mesenchyme (Costantini, 2006). Six2 and Gdnf are both synergistically activated by a Pax2-Eya1-Hox11 complex (Gong et al., 2007). Thus, Osr1 may provide a molecular link between progenitor maintenance and ureteric epithelial branching through the regulation of a Pax2-Eya1-Hox11 activation complex and in turn, the regulation of Six2 and Gdnf expression. Furthermore, the maintenance of Osr1 in the cap mesenchyme throughout the period of nephrogenesis may ensure both the continued renewal of nephron progenitors and branching morphogenesis throughout kidney organogenesis.

Materials and Methods

Animals and genotyping

Animal care and research protocols were performed in accordance with Harvard University’s institutional guidelines, following approval by Harvard University’s institutional committee on animal use. For staging of embryos, the morning of vaginal plug was designated as embryonic day 0.5 (E0.5). Gt(ROSA)26^tm1Sor (Soriano, 1999) (Jackson Laboratories) were genotyped as previously described. The Osr1^{eGFPCreERt2/+} line (Mugford et al., 2008) (Osr1^GCE/+) was generated by knocking an eGFPCreERt2 (GCE) construct into the endogenous Osr1 locus. 3’ and 5’ homology arms were PCR amplified from BAC clone RP23-398M9. All primer sequences used to generate homology arms are available upon request. Osr1^GCE/+ heterozygotes were genotyped as previously described (Mugford et al., 2008). Tamoxifen was administered at a dose of either 1mg/40g body weight (general cell fate studies) or 0.05mg/40g body weight (low-level cell labeling studies) of the dam.

The PGK Neo selection cassette was removed by intercrossing Osr1^GCE/+ heterozygotes with the Gt(ROSA)26Sor^tm1(FLP1)Dym line (Farley et al., 2000). PGK Neo removal in the resulting allele Osr1^GCEN/+ was confirmed by PCR using the Osr1ER3’GenoRv reverse primer (Mugford et al., 2008) with primer Osr1NoNeoFw: 5’-CGTGGAGGAGACGGACCAAA-3’. Osr1^GCEN/+;R26R animals demonstrated ectopic Cre activity in the absence of Tamoxifen (data not shown) and were therefore not used for fate mapping studies. Osr1 mutant embryos were generated by intercrossing Osr1^GCE/+ heterozygote animals with Osr1^GCEN/+ heterozygote animals. Embryos were genotyped for the presence of both the GCE and GCEN alleles and Osr1^GCE/GCEN embryos were obtained at expected Mendelian ratios.

Generation of a Foxd1 polyclonal antibody

Rabbits were immunized with a KHL-conjugated peptide RLPSSAQRRRRSYAGEDDLE corresponding to amino acids 48–67 of mouse Foxd1 (Covance Research Products). The antiserum was tested by transiently transfecting (Lipofectamine 2000, Invitrogen) COS7 cells with a mouse Foxd1 expression plasmid followed by immunostaining. Immunofluorescence was also performed on E15.5 metanephric cryosections and compared to section in situ hybridization for Foxd1 transcript.

Whole mount and histological analysis

All tissue preparation, whole mount analysis and immunofluorescence was carried out as previously described (Mugford et al., 2008). Confocal images were acquired on a Zeiss LSM510 META confocal microscope (Zeiss). The following antibodies and dilutions were used: anti-Hoxd11 (1:500) (Mugford et al., 2008), anti-Foxd1 (1:2000), anti-Six2 (1:1000) (Kobayashi et al., 2008), anti-GFP (1:500, AvesLabs), anti-cytokeratin (1:500, Sigma), anti-β-galactosidase (1:500, AbCam), anti-β-gal (1:50, DSHB), anti-Liv2 (1:200, MBL International), anti-F4/80 (1:1000, eBiosciences), anti-Smooth Muscle α-actin (1:2000, Sigma), anti-Flk1 (1:1000, BDPharmigen), anti-PDGFRb (1:100, eBiosciences). Appropriate Cy2, Cy5 (1:500, Jackson Immuno), Alexa488, Alexa568 or Alexa647 (1:500, Invitrogen) conjugated secondary antibodies were used to detect primary antibodies. Nuclei were stained with Hoechst 33342 (Invitrogen). Experiments requiring the simultaneous use of two rabbit antibodies were conducted using Zenon dual labeling kits (Invitrogen).

Supplementary Material

01: Supplemental Figure 1 – Generation and confirmation of Osr1^GCE/+ embryos

(A) Targeting strategy for eGFPCreERt2 into the endogenous Osr1 locus. (B–I) Comparison of Osr1 mRNA expression pattern with eGFP in Osr1^GCE/+ embryos. Whole mount analysis comparing Osr1 mRNA in WT E8.5 (B) and E9.5 (F) embryos with eGFP in Osr1^GCE/+ E8.5 (C, D) and E9.5 (G, H) embryos. Transverse sections of E8.5 (E) and E9.5 (I) Osr1^GCE/+ embryos immunostained for GFP. Section analysis in E15.5 metanephric kidneys of WT embryos for Osr1 mRNA (J) and in Osr1^GCE/+ embryos immunostained for GFP (green), Six2 (red) and Cytokeratin (purple). Scale bars = 100µm.

Click here to view.^{(5.0M, tif)}

02: Supplemental Figure 2 – Osr1⁺ cells give rise to multiple metanephric cell types

(A–F) X-gal staining in frontal sections of Osr1^GCE/+;R26R E15.5 metanephric kidneys injected with Oil alone between E7.5 and E9.5 (A), Tamoxifen at E7.5 (B), E8.5 (C), E9.5 (D), E10.5 (E) and E11.5 (F). Scale bar = 500µm.

Click here to view.^{(5.0M, tif)}

03: Supplemental Figure 3 – Osr1 expression is restricted to the Six2⁺ cap mesenchyme at E11.5

(A–P) Immunofluorescent confocal microscopy of transverse sections of the E10.5 metanephric blastema (A–H) and saggital sections of E11.5 metanephric kidneys (I–P). Samples immunostained for Foxd1 (red in A, E), Six2 (red in I, M), GFP (green in B, F, J, N) and Cytokeratin (blue in A, B, E, F, I, J). Nuclei stained for Hoechst 33258 (D, H, L, P). Merged images (C, G, K, O). Arrowheads (A–C) indicate Foxd1⁺, GFP⁺ cells. Arrowheads (I–K) indicate Foxd1⁺, GFP⁻ cells. Arrowheads (E–G, M–O) indicate Six2⁺, GFP⁺ cells. Scale bars = 50µm.

Click here to view.^{(5.1M, tif)}

04: Supplemental Figure 4 – Osr1⁺ cells give rise to pericytes and mesangium between E7.5 and E11.5

(A–T) Immunofluorescent confocal microscopy of glomeruli within E15.5 Osr1^GCE/+;R26R metanephric kidneys after Cre activation at E7.5 (A–D), E8.5 (E–H), E9.5 (I–L), E10.5 (M–P) and E11.5 (Q–T). Samples immunostained for Flk1 (green in A, E, I, M, Q), WT1 (blue in A, B, E, F, I, J, M, N, Q, R) and β-gal (red in B, F, J, N, R). Nuclei stained for Hoechst 33258 (D, H, L, P, T). Merged images (C, G, K, O, S). Arrowheads (A–C, E–G, I–K, M–O) indicate PDGFRb⁺, β-gal⁺ mesangial cells. Concave arrowheads (E–G, I–K) indicate PDGFRb⁺, β-gal⁺ pericytes. Scale bar = 50µm.

Click here to view.^{(5.1M, tif)}

05: Supplemental Figure 5 – Osr1⁺ cells give rise to the kidney capsule between E7.5 and E11.5

(A–T) Immunofluorescent confocal microscopy of E15.5 Osr1^GCE/+;R26R metanephric kidneys after Cre activation at E7.5 (A–D), E8.5 (E–H), E9.5 (I–L), E10.5 (M–P) and E11.5 (Q–T). Samples immunostained for Liv2 (A, E, I, M, Q) and β-gal (B, F, J, N, R). Nuclei stained for Hoechst 33258 (D, H, L, P, T). Merged images (C, G, K, O, S). Arrowheads (A–C, E–G, I–K, M–O) indicate Liv2⁺, β-gal⁺ cells. Arrowheads (Q–S) indicate Liv2⁻, β-gal⁺ cells. Scale bar = 50µm.

Click here to view.^{(5.3M, tif)}

06: Supplemental Figure 6 – Osr1⁺ cells give rise to smooth muscle between E8.5 and E11.5

(A–T) Immunofluorescent confocal microscopy of E15.5 Osr1^GCE/+;R26R ureters after Cre activation at E7.5 (A–D), E8.5 (E–H), E9.5 (I–L), E10.5 (M–P) and E11.5 (Q–T). Samples immunostained for SMA (A, E, I, M, Q) and β-gal (B, F, J, N, R). Nuclei stained for Hoechst 33258 (D, H, L, P, T). Merged images (C, G, K, O, S). Arrowheads (E–G, I–K, M–O) indicate SMA⁺, β-gal⁺ cells. Arrowheads (A–C) indicate SMA⁻, β-gal⁺ cells. Scale bar = 50µm.

Click here to view.^{(5.2M, tif)}

07: Supplemental Figure 7 – Osr1⁺ cells do not give rise to macrophages

(A–T) Immunofluorescent confocal microscopy of E15.5 Osr1^GCE/+;R26R metanephric kidneys after Cre activation at E7.5 (A–D), E8.5 (E–H), E9.5 (I–L), E10.5 (M–P) and E11.5 (Q–T). Samples immunostained for F4/80 (A, E, I, M, Q) and β-gal (B, F, J, N, R). Nuclei stained for Hoechst 33258 (D, H, L, P, T). Merged images (C, G, K, O, S). Arrowheads (A–C, E–G, I–K, M–O, Q–S) F4/80⁻, β-gal⁺ cells. Scale bar = 50µm

Click here to view.^{(5.0M, tif)}

Acknowledgements

We thank Dr. Akio Kobayashi for help in generating the eGFPCreERt2 cassette and Ying Qi (Shirleen) Soh for communicating her Liv2 data. Work in A.P.M.’s laboratory was supported by a grant from the NIH (DK054364). P.S. was funded by grants from the Finnish Academy (#107827), Helsingin Sanomain 100-vuotis and Alfred Kordelin Foundations.

Footnotes

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References

Bernstein J, Cheng F, Roszka J. Glomerular differentiation in metanephric culture. Lab Invest. 1981;45:183–190. [PubMed]
Boyle S, Misfeldt A, Chandler KJ, Deal KK, Southard-Smith EM, Mortlock DP, Baldwin HS, de Caestecker M. Fate mapping using Cited1-CreERT2 mice demonstrates that the cap mesenchyme contains self-renewing progenitor cells and gives rise exclusively to nephronic epithelia. Dev Biol. 2008;313:234–245. [PMC free article] [PubMed]
Carroll TJ, Park JS, Hayashi S, Majumdar A, McMahon AP. Wnt9b plays a central role in the regulation of mesenchymal to epithelial transitions underlying organogenesis of the mammalian urogenital system. Dev Cell. 2005;9:283–292. [PubMed]
Challen GA, Bertoncello I, Deane JA, Ricardo SD, Little MH. Kidney side population reveals multilineage potential and renal functional capacity but also cellular heterogeneity. J Am Soc Nephrol. 2006;17:1896–1912. [PubMed]
Costantini F. Renal branching morphogenesis: concepts, questions, and recent advances. Differentiation. 2006;74:402–421. [PubMed]
Cullen-McEwen LA, Drago J, Bertram JF. Nephron endowment in glial cell line-derived neurotrophic factor (GDNF) heterozygous mice. Kidney Int. 2001;60:31–36. [PubMed]
Cumano A, Dieterlen-Lievre F, Godin I. The splanchnopleura/AGM region is the prime site for the generation of multipotent hemopoietic precursors, in the mouse embryo. Vaccine. 2000;18:1621–1623. [PubMed]
Dressler G. Tubulogenesis in the developing mammalian kidney. Trends Cell Biol. 2002;12:390–395. [PubMed]
Farley FW, Soriano P, Steffen LS, Dymecki SM. Widespread recombinase expression using FLPeR (flipper) mice. Genesis. 2000;28:106–110. [PubMed]
Fleming S, Symes CE. The distribution of cytokeratin antigens in the kidney and in renal tumours. Histopathology. 1987;11:157–170. [PubMed]
Goldstein RE, Cook O, Dinur T, Pisante A, Karandikar UC, Bidwai A, Paroush Z. An eh1-like motif in odd-skipped mediates recruitment of Groucho and repression in vivo. Mol Cell Biol. 2005;25:10711–10720. [PMC free article] [PubMed]
Gong KQ, Yallowitz AR, Sun H, Dressler GR, Wellik DM. A hox-eya-pax complex regulates early kidney developmental gene expression. Mol Cell Biol. 2007;27:7661–7668. [PMC free article] [PubMed]
Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat Rev Immunol. 2005;5:953–964. [PubMed]
Grieshammer U, Le M, Plump AS, Wang F, Tessier-Lavigne M, Martin GR. SLIT2-mediated ROBO2 signaling restricts kidney induction to a single site. Dev Cell. 2004;6:709–717. [PubMed]
Grote D, Souabni A, Busslinger M, Bouchard M. Pax 2/8-regulated Gata 3 expression is necessary for morphogenesis and guidance of the nephric duct in the developing kidney. Development. 2006;133:53–61. [PubMed]
Hartman HA, Lai HL, Patterson LT. Cessation of renal morphogenesis in mice. Dev Biol. 2007;310:379–387. [PMC free article] [PubMed]
Hatini V, Huh SO, Herzlinger D, Soares VC, Lai E. Essential role of stromal mesenchyme in kidney morphogenesis revealed by targeted disruption of Winged Helix transcription factor BF-2. Genes Dev. 1996;10:1467–1478. [PubMed]
Hellstrom M, Kalen M, Lindahl P, Abramsson A, Betsholtz C. Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development. 1999;126:3047–3055. [PubMed]
James RG, Kamei CN, Wang Q, Jiang R, Schultheiss TM. Odd-skipped related 1 is required for development of the metanephric kidney and regulates formation and differentiation of kidney precursor cells. Development. 2006;133:2995–3004. [PubMed]
Kobayashi A, Valerius MT, Mugford JW, Carroll TJ, Self M, Oliver G, McMahon AP. Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development. Cell Stem Cell. 2008;3:169–181. [PMC free article] [PubMed]
Kumaravelu P, Hook L, Morrison AM, Ure J, Zhao S, Zuyev S, Ansell J, Medvinsky A. Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver. Development. 2002;129:4891–4899. [PubMed]
Kume T, Deng K, Hogan BL. Murine forkhead/winged helix genes Foxc1 (Mf1) and Foxc2 (Mfh1) are required for the early organogenesis of the kidney and urinary tract. Development. 2000;127:1387–1395. [PubMed]
Levinson RS, Batourina E, Choi C, Vorontchikhina M, Kitajewski J, Mendelsohn CL. Foxd1-dependent signals control cellularity in the renal capsule, a structure required for normal renal development. Development. 2005;132:529–539. [PubMed]
Loughna S, Hardman P, Landels E, Jussila L, Alitalo K, Woolf AS. A molecular and genetic analysis of renalglomerular capillary development. Angiogenesis. 1997;1:84–101. [PubMed]
Loughna S, Yuan HT, Woolf AS. Effects of oxygen on vascular patterning in Tie1/LacZ metanephric kidneys in vitro. Biochem Biophys Res Commun. 1998;247:361–366. [PubMed]
Medvinsky A, Dzierzak E. Definitive hematopoiesis is autonomously initiated by the AGM region. Cell. 1996;86:897–906. [PubMed]
Meneton P, Loffing J, Warnock DG. Sodium and potassium handling by the aldosterone-sensitive distal nephron: the pivotal role of the distal and connecting tubule. Am J Physiol Renal Physiol. 2004;287:F593–F601. [PubMed]
Minehata K, Mukouyama YS, Sekiguchi T, Hara T, Miyajima A. Macrophage colony stimulating factor modulates the development of hematopoiesis by stimulating the differentiation of endothelial cells in the AGM region. Blood. 2002;99:2360–2368. [PubMed]
Mugford JW, Sipilä P, Kobayashi A, Behringer RR, McMahon AP. Hoxd11 specifies a program of metanephric kidney development within the intermediate mesoderm of the mouse embryo. Dev Biol. 2008;319:396–405. [PMC free article] [PubMed]
Mundlos S, Pelletier J, Darveau A, Bachmann M, Winterpacht A, Zabel B. Nuclear localization of the protein encoded by the Wilms' tumor gene WT1 in embryonic and adult tissues. Development. 1993;119:1329–1341. [PubMed]
Nyengaard JR, Bendtsen TF. Glomerular number and size in relation to age, kidney weight, and body surface in normal man. Anat Rec. 1992;232:194–201. [PubMed]
Park JS, Valerius MT, McMahon AP. Wnt/beta-catenin signaling regulates nephron induction during mouse kidney development. Development. 2007;134:2533–2539. [PubMed]
Patterson LT, Pembaur M, Potter SS. Hoxa11 and Hoxd11 regulate branching morphogenesis of the ureteric bud in the developing kidney. Development. 2001;128:2153–2161. [PubMed]
Pedersen A, Skjong C, Shawlot W. Lim 1 is required for nephric duct extension and ureteric bud morphogenesis. Dev Biol. 2005;288:571–581. [PubMed]
Pepicelli CV, Kispert A, Rowitch DH, McMahon AP. GDNF induces branching and increased cell proliferation in the ureter of the mouse. Dev Biol. 1997;192:193–198. [PubMed]
Poole TJ, Finkelstein EB, Cox CM. The role of FGF and VEGF in angioblast induction and migration during vascular development. Dev Dyn. 2001;220:1–17. [PubMed]
Reeves WB, Winters CJ, Andreoli TE. Chloride channels in the loop of Henle. Annu Rev Physiol. 2001;63:631–645. [PubMed]
Robert B, St John PL, Abrahamson DR. Direct visualization of renal vascular morphogenesis in Flk1 heterozygous mutant mice. Am J Physiol. 1998;275:F164–F172. [PubMed]
Robin C, Dzierzak E. Hematopoietic stem cell enrichment from the AGM region of the mouse embryo. Methods Mol Med. 2005;105:257–272. [PubMed]
Saha MS, Cox EA, Sipe CW. Mechanisms regulating the origins of the vertebrate vascular system. J Cell Biochem. 2004;93:46–56. [PubMed]
Sanchez MJ, Holmes A, Miles C, Dzierzak E. Characterization of the first definitive hematopoietic stem cells in the AGM and liver of the mouse embryo. Immunity. 1996a;5:513–525. [PubMed]
Sanchez MP, Silos-Santiago I, Frisen J, He B, Lira SA, Barbacid M. Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature. 1996b;382:70–73. [PubMed]
Sariola H, Timpl R, von der Mark K, Mayne R, Fitch JM, Linsenmayer TF, Ekblom P. Dual origin of glomerular basement membrane. Dev Biol. 1984;101:86–96. [PubMed]
Saxén L. Organogenesis of the kidney. Cambridge [Cambridgeshire] ; New York: Cambridge University Press; 1987.
Self M, Lagutin OV, Bowling B, Hendrix J, Cai Y, Dressler GR, Oliver G. Six2 is required for suppression of nephrogenesis and progenitor renewal in the developing kidney. Embo J. 2006;25:5214–5228. [PubMed]
Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999;21:70–71. [PubMed]
Spinelli F. Structure and development of the renal glomerulus as revealed by scanning electron microscopy. Int Rev Cytol. 1974;39:345–378. [PubMed]
Stark K, Vainio S, Vassileva G, McMahon AP. Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4. Nature. 1994;372:679–683. [PubMed]
Tena JJ, Neto A, de la Calle-Mustienes E, Bras-Pereira C, Casares F, Gomez-Skarmeta JL. Odd-skipped genes encode repressors that control kidney development. Dev Biol. 2007;301:518–531. [PubMed]
Torres M, Gomez-Pardo E, Dressler GR, Gruss P. Pax-2 controls multiple steps of urogenital development. Development. 1995;121:4057–4065. [PubMed]
Vize PD, Woolf AS, Bard JBL. The kidney : from normal development to congenital diseases. Amsterdam ; Boston: Academic Press; 2002.
Wang Q, Lan Y, Cho ES, Maltby KM, Jiang R. Odd-skipped related 1 (Odd 1) is an essential regulator of heart and urogenital development. Dev Biol. 2005;288:582–594. [PubMed]
Wellik DM, Hawkes PJ, Capecchi MR. Hox11 paralogous genes are essential for metanephric kidney induction. Genes Dev. 2002;16:1423–1432. [PubMed]
Xiong JW. Molecular and developmental biology of the hemangioblast. Dev Dyn. 2008;237:1218–1231. [PubMed]
Yu J, Carroll TJ, McMahon AP. Sonic hedgehog regulates proliferation and differentiation of mesenchymal cells in the mouse metanephric kidney. Development. 2002;129:5301–5312. [PubMed]
Yu J, McMahon AP, Valerius MT. Recent genetic studies of mouse kidney development. Curr Opin Genet Dev. 2004;14:550–557. [PubMed]
Zhou Q, Law AC, Rajagopal J, Anderson WJ, Gray PA, Melton DA. A multipotent progenitor domain guides pancreatic organogenesis. Dev Cell. 2007;13:103–114. [PubMed]