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Proc Natl Acad Sci U S A. May 20, 2008; 105(20): 7212–7217.
Published online May 14, 2008. doi:  10.1073/pnas.0707674105
PMCID: PMC2438229
From the Cover
Developmental Biology

Four-dimensional analysis of vascularization during primary development of an organ, the gonad

Abstract

Time-lapse microscopy has advanced our understanding of yolk sac and early embryonic vascularization. However, it has been difficult to assess endothelial interactions during epithelial morphogenesis of internal organs. To address this issue we have developed the first time-lapse system to study vascularization of a mammalian organ in four dimensions. We show that vascularization of XX and XY gonads is a highly dynamic, sexually dimorphic process. The XX gonad recruits vasculature by a typical angiogenic process. In contrast, the XY gonad recruits and patterns vasculature by a novel remodeling mechanism beginning with breakdown of an existing mesonephric vessel. Subsequently, in XY organs individual endothelial cells migrate and reaggregate in the coelomic domain to form the major testicular artery. Migrating endothelial cells respect domain boundaries well before they are morphologically evident, subdividing the gonad into 10 avascular regions where testis cords form. This model of vascular development in an internal organ has a direct impact on the current dogma of vascular integration during organ development and presents important parallels with mechanisms of tumor vascularization.

Keywords: organogenesis, ovary, testis

Insights into vascular development and patterning in internal organs have historically relied on xenograft models and static analysis (17). However, this approach does not elucidate dynamic interactions between endothelial cells and other cells of developing organs. Advances in time-lapse imaging have improved understanding of vasculogenesis, flow-induced vascular remodeling, the genetic programming of angiogenesis, and many other facets of vascular development in the mouse and chick yolk sac and during establishment of the body plan in zebrafish (817). However, culturing internal organs throughout critical and prolonged periods of development has been difficult. The technical challenges of organ culture, compounded by simultaneous live imaging, is a major hurdle in understanding how endothelial cells remodel and integrate in an internal organ undergoing morphogenesis.

For more than a decade the urogenital ridge (UGR) explant model has provided a unique system to analyze morphogenesis of an internal organ (18, 19). The fact that the UGR can be successfully explanted to culture at the critical sex-determination stage [11.5 days postcoitum (dpc)], using conditions that maintain the normal morphological structure of the gonad, has been the driving force behind its broad adoption in the sex-determination field. However, with respect to internal organ vascularization, the UGR has received little attention as a model system. In the UGR explant model the gonad retains contact with the mesonephros, the source of the endothelium, and vascularization ex vivo occurs similarly to vascularization in vivo (20). During the period of vascularization, development of the testis and ovary diverge morphologically. Whereas the XX gonad shows few morphological changes during this period, the XY gonad undergoes dramatic epithelial morphogenesis as cells reorganize de novo into testis cord structures. However, in the absence of live imaging of these systems, it has not been possible to determine how the process of vascularization actually occurs. We reasoned that four-dimensional analysis of the gonad would provide insight into the mechanisms that incorporate vasculature into an organ at the stage when structural organization of the tissue is initiated. Moreover, the divergent morphological development of the testis and ovary would provide a basis of comparison to correlate vascular development with the sex-specific morphological development of the tissue.

Results and Discussion

Gonads Develop Normally Under ex Vivo Conditions.

The somatic gonad arises through proliferation of the mesothelial layer that lines the coelomic cavity in the region that directly overlies the mesonephric ducts. The arrival of germ cells that migrate from the base of the allantois, and the proliferation of this mesothelial layer, produce the primordial gonad, which is a stratified epithelium that is approximately six to eight cell layers thick by 11.5 dpc. This tissue contains a primitive vascular system, in which small branches from the mesonephric vessels extend into the undifferentiated gonads of both sexes (see Fig. 5). Sex-specific development of the vasculature occurs downstream of the expression of the sex-determining gene Sry. In the XY gonad, vascularization is known to involve the migration of endothelial cells from the mesonephros into the tissue beginning at 11.5 dpc (1921). These cells form a meshwork of smaller branched vessels in the coelomic domain of the XY gonad that resolves into a distinctive coelomic vessel by 13.5 dpc (Fig. 1A). Between 12.5 and 13.5 dpc, vascular branches extending from the coelomic vessel can be identified along the outside of testis cords. This vessel is strongly positive for Notch1 and Notch4 and for the arterial marker ephrinB2, identifying this vessel and its branches as the primordial arterial system of the testis (20). By contrast, the XX gonad lacks a coelomic vessel, and XX vascular development does not involve migration of cells from the mesonephros. Vascular development of the XX gonad appears to be the result of proliferation and extension of the branches of the primordial gonadal vasculature (Fig. 1A) (2022).

Fig. 1.
Normal development of XY gonads during time-lapse imaging. (A) Gross anatomy of the XX and XY gonad (G) and mesonephros (M) between 11.5 dpc and 13.5 dpc. At 11.5 dpc the gonad shows no morphological differences between XX and XY embryos. At 12.5 dpc ...
Fig. 5.
A schematic diagram of gonad vascular development. At 11.5 dpc a large vascular plexus is present in the mesonephros (mvp), and small vascular branches extend across the gonad boundary. Soon after the testis pathway is initiated by expression of Sry in ...

It was unclear whether vascular development in the XY gonad occurs by angiogenic branching or by a vasculogenic process involving the formation of a vessel from migration of individual cells. Moreover, it was unclear how vascularization of the XY gonad is integrated with epithelial morphogenesis of testis cords. To overcome the limitations of static vascular analysis, we developed a live imaging system to compare the process of vascularization in XX and XY gonads between 11.5 and 13.5 dpc [supporting information (SI) Fig. S1A].

The three-dimensional UGR explant culture system previously established (18, 19, 21) was modified for time-lapse analysis using differential interference contrast (DIC) and fluorescent microscopy techniques. To determine whether XY gonadal differentiation occurred normally under these experimental conditions, gonads from 11.5-dpc embryos were initially imaged by using DIC over a 24-h culture period to view gross morphological changes (Fig. 1B and Movie S1). The XY gonad developed dynamically in the time-lapse system, and at the conclusion of the culture period all testicular morphological structures were clearly identifiable, including testis cords and the coelomic vessel. Testis cords formed rapidly, and within 8 h 10 cellular condensations were visible within the XY gonad. In addition to morphological development within the gonad, invagination and extension of the Müllerian duct occurred normally within the mesonephros (Fig. 1B, frames 8 h to 14 h). These gross morphological changes within the XY gonad and mesonephros during the culture period recapitulate those of the in vivo XY gonad between 11.5 dpc and 12.5 dpc, indicating that the culture and microscopy conditions support normal differentiation.

To identify the mechanism of endothelial-specific mesonephric migration and XY vascular development, mice expressing Cre recombinase under the control of endothelial-specific promoters Tie2-Cre (Figs. 2, ,33B, and and44A) or Flk1cre (Fig. 3A) (23, 24) were crossed with Z/EG reporter mice in which EGFP is expressed in cells after recombination (25). Efficient transgenic labeling was determined by colocalizing EGFP and rhodamine in vessels of 12.0-dpc embryos injected intravenously with lectin-rhodamine (Fig. S1B). In addition to endothelial cell expression in both the Tie2-Cre;Z/EG and Flk1cre;Z/EG gonad and mesonephros, EGFP expression was detected in some cells near the mesonephric border that do not express PECAM1 but do express the monocyte- and macrophage-specific marker F4/80 (26, 27) (Fig. S1C). Macrophages have previously been shown to label in Tie2-Cre and Flk-Cre lineage tracing experiments. This may reflect a common myeloid ancestor for macrophage and endothelial lineages (28) or derivation from vascular wall resident progenitor cells shown to give rise to the macrophage and other hematopoietic lineages (29). EGFP-positive macrophage-like cells were easily distinguished from the endothelial cell population by their cell movements and distinctive cell morphology (see stellate cells in Fig. 3B, 10 h time frame, and Movie S2). No additional cell types were labeled by Tie2-Cre or Flk-Cre expression.

Fig. 2.
Comparison of vascularization in XX and XY gonads. (A) Low-magnification 12-h time-lapse series of an 11.5-dpc XX gonad (G) and mesonephros (M) expressing GFP in endothelial cells (Tie2-Cre × Z/EG). Although the XX vasculature (green) remodels ...
Fig. 3.
Higher-resolution views of XY vascular patterning. (A) Low-magnification 24-h time-lapse series of an 11.5-dpc XY gonad (G) and mesonephros (M) expressing GFP in endothelial cells (Flk-Cre × Z/EG). Endothelial cells (green) migrate to the coelomic ...
Fig. 4.
Remodeling of the mesonephric vasculature in XY gonads. (A) A high-magnification 13-h time-lapse series of an 11.5-dpc XY gonad (G) and mesonephros (M) expressing GFP in endothelial cells (Tie2-Cre × Z/EG). Movie S6 is focused on the proximal ...

Vascular Remodeling in the XX and XY Gonad Between 11.5 dpc and 12.5 dpc.

Little is known about how a new inflow/outflow vascular network is initially established in a developing organ. Using these endothelial cell-labeled gonads, vascular cells were traced as they remodeled and migrated in both the XX and XY gonads. At 11.5 dpc, primitive vessels extend a short distance into the gonad from the vascular bed at the mesonephric/gonad border (see Fig. 3A, 0 h, and Fig. 5). Proliferation in the coelomic domain is concurrent with vascular development in the XY gonad, driving the coelomic domain progressively farther from the mesonephric/gonad border. The region of the mesonephros near the gonad border contained an elaborate plexus of microvasculature that was clearly identifiable by EGFP expression (Fig. 2).

In both XX and XY gonads, the vasculature continued to develop from endothelial cells in the initial vascular branches that undergo proliferation during this period (2022). Whether additional endothelial cells arise de novo within the XX or the XY gonad cannot be determined in these assays. However, ex vivo movies revealed different endothelial cell behavior in XX and XY gonads. In XX gonads, individual Tie2-positive cells undergo active movements, pausing briefly to make cell contact with other endothelial cells they encounter but failing to establish any recognizable coherent structure (Fig. 2A and Movie S3). Few endothelial cells crossed the border between the mesonephros and the XX gonad or ventured into the coelomic domain, consistent with previous experimental observations in recombination cultures analyzed at fixed time points (1820). Restricted endothelial cell movement in the XX gonad suggests that cortical and medullary domains of the ovary are already established during this time period, although no morphological boundaries are evident. This finding is consistent with molecular evidence of discrete gene expression domains specified by 12.5 dpc in the ovary (30).

The XY gonad employs a distinct vascularization strategy coincident with the diverging morphology between the developing testis and ovary (Fig. 1A). Endothelial cell migration was directed toward the coelomic domain where cells aggregate by the 6-h time point to form the coelomic vessel (Fig. 2B and Movie S4). At the conclusion of the 12-h culture period, a coelomic vessel was identified as a microvascular network (Fig. 2B), as seen in vivo in the 12.5 dpc XY gonad. In contrast to the XX gonad, endothelial cells of the XY UGR complex crossed the boundary between the mesonephros and the gonad, consistent with the results of previous recombination cultures examined at static time points (1821).

Individual Endothelial Cells Migrate via Specific Paths to the Coelomic Domain of the XY Gonad.

XY vasculature was analyzed at higher resolution by increasing the number of images collected over time and by increasing the magnification of the images. As observed in low-resolution movies, endothelial cells entered the XY gonad from focal sites along the border with the mesonephros with no apparent wave of migration from one pole or position (Fig. 3A and Movie S5). However, endothelial cells that exited the mesonephros at its poles appeared to contact the coelomic domain of the gonad earlier than centrally derived endothelial cells (Fig. 3A, frame 4 h). Once endothelial cells reached the coelomic domain, they migrated across the surface of the gonad, where they aggregated into a vascular complex.

This strongly directed endothelial cell migration suggests the presence of attractive cues activated downstream of Sry and localized in the XY coelomic domain. Genes have been identified that are expressed in the XY coelomic domain and are candidates for a role in divergent XX vs. XY vascular development. For example, Tcf21 is expressed in the coelomic domain in XY but not XX gonads, and null mutants in Tcf21 show disrupted XY vascular development (31). Interestingly, in Wnt4 mutants, where the coelomic vessel forms exogenously in XX gonads, the expression domain of Tcf21 extends to the coelomic surface (32). In addition, the extracellular matrix (ECM) plays an essential role in establishing permissive and restrictive scaffolding for endothelial cell migration (33). Sex-specific expression of ECM molecules has been reported in XY vs. XX expression screens (3436) and may play a role in domain patterning and directed migration.

To understand how individual migrating endothelial cells interact, we analyzed cell migration at higher magnification and by initiating the culture at an earlier developmental stage. At 0 h the primary gonadal vasculature is clearly identifiable, but within 2 h this vascular system begins to remodel (Fig. 3B, frames 0 h to 24 h, and Movie S2). Endothelial cells detached from each other and either migrated toward the coelomic domain of the gonad or returned to the mesonephros. As seen in the lower-magnification time-lapse images, individual endothelial cells with no apparent connections to surrounding endothelial cells migrate from the mesonephros to the coelomic surface of the gonad and within the coelomic domain (Fig. 3B, frame 14 h and thereafter, and Movie S2). Endothelial cells within the coelomic domain continue to migrate as isolated cells, only later aggregating into primitive microvascular networks. It is unknown whether remodeled vascular networks in XY gonadal explants form patent lumens in the absence of blood flow, but because of the high rate of continued remodeling it appears unlikely.

Endothelial cells entering the gonad from the mesonephros extended long filopodia both toward and away from the coelomic surface. Filopodia that extended away were retracted, whereas filopodia that extended toward the coelomic domain persisted, and cell bodies moved in that direction. Once pioneer cells had traversed the gonad, other cells followed along the same paths, subdividing the tissue into ≈10 distinct avascular domains. It is of great interest that XY endothelial cell migration segregates ≈10 domains where the de novo condensation of each testis cord takes place over the next 12 h of development. The first endothelial cells that enter the XY gonad pioneer tracks through the tissue well before testis cord domains are visible or molecularly distinct by any known marker.

It is not yet clear whether endothelial cells play an active role in subdividing the field of cells into cord-forming units or the migratory paths of endothelial cells are restricted by signals from pre-Sertoli cells that have already initiated aggregation into cord-forming groups. No precord aggregation domains have been recognized at 11.5 dpc; however, they may exist at the molecular level. This system could be similar to intersomitic vascular integration. Intersomitic vascular patterning depends on the expression of neuronal guidance molecules and their receptors, which establish both permissive and restrictive domains for vascular branching. Endothelial cells express neuronal guidance receptors Plexind1 and Unc5b, whereas the somites express their repulsive ligands, Sema3E and Netrins, establishing avascular somite domains (3739). Numerous neuronal guidance signaling pathway members, including Robo, Semaphorin, and Netrin, have been identified in embryonic testicular EST libraries (www.ncbi.nlm.nih.gov/UniGene/lbrowse2.cgi?TAXID=10090; dbEST Library Lib.2578, Lib.10027, Lib.2555, and Lib.2594) and general embryonic testicular screens (3436). Their role in patterning of the testis vasculature is under investigation.

Vasculature Remodels Within the XY Mesonephros.

To determine the origin of endothelial cells migrating from the mesonephros, time-lapse imaging was performed between 11.5 and 12.5 dpc focusing on the midplane of the mesonephros. The XY mesonephros at 11.5 dpc contained a vascular plexus consisting of an extensive network of microvessels (Fig. 4A and Movie S6). There were also several larger prominent vessels at the anterior aspect of the mesonephros, surrounding the mesonephric tubules, as well as a major vessel along the lateral aspect of the mesonephros. During time-lapse culture of XY samples, the shape of endothelial cells in these large vessels changed rapidly from an elongated squamous cell body to a spherical cell body. Cells detached from neighbor cells resulting in sex-specific breakdown of the male mesonephric vasculature. Immediately after breakdown of the mesonephric vessel, individual endothelial cells migrated into the XY gonad and contributed to the XY gonadal vasculature. In XX gonads, where endothelial cells do not migrate into the gonad from the mesonephros, we did not detect remodeling of this vessel using live imaging techniques.

Breakdown of this vessel in the organ culture system could be an artifact of ex vivo culture and the absence of blood flow. To determine whether remodeling of this mesonephric vasculature occurs in XY gonads in vivo, live embryos were injected intravenously with lectin-rhodamine at 11.5 and 12.5 dpc. Embryos were killed immediately, and the gonads were removed within 5–8 min, immediately fixed, and immunostained with anti-PECAM1, a marker of endothelium and germ cells (19). Colocalization of rhodamine and PECAM1 in these experiments confirmed the XY-specific reorganization of the mesonephric vessels. The vasculatures of the 11.5-dpc XX and XY mesonephroi were very similar, consisting of a highly convoluted network of microvasculature situated adjacent to the gonad (Fig. 4B). This microvasculature branched and connected to one large vessel situated on the lateral aspect of the mesonephros (Fig. 4B, white arrowheads). The vascular anatomy of the mesonephros by 12.5 dpc was dramatically different between XX and XY samples (Fig. 4B). In XY samples, no large vessel or vascular plexus could be identified near the gonad–mesonephros border. By contrast, the larger vessels remained intact in the 12.5-dpc XX mesonephros, suggesting that breakdown/remodeling of these vessels occurs in vivo specifically in the XY and not the XX urogenital ridge and is not an artifact of the ex vivo system or lack of blood flow. Ex vivo fidelity of in vivo remodeling reveals an underlying genetic program that coordinates vascular remodeling, even in the absence of blood flow.

Thus, both the mechanism and process of vascular development is fundamentally different between XX and XY gonads (Fig. 5). In XX gonads, the vasculature grows from preexisting vascular branches that undergo local reorganization, similar to angiogenic mechanisms that have been described in other systems (40). By contrast, in the XY gonad, preexisting vessels in the mesonephros break down, releasing individual endothelial cells that are recruited into the gonad. This mechanism of vascular remodeling represents a previously undescribed model for vascular integration during organ development. It is not clear why this mechanism is used in the testis but not the ovary, although it correlates with a change in the direction of blood flow into the testis and a period of rapid growth, differentiation, and morphogenesis as compared with the relatively quiescent ovary (20). We do not yet understand what triggers breakdown of the mesonephric vessel and recruitment of its cells into new vessels in the forming testis, but the answer to this puzzle could have important implications for signaling mechanisms during tumor vascularization.

The gonad provides an excellent model for the study of vascularization over real time in an organ undergoing extensive epithelial morphogenesis. The development of an ovary or a testis from a single primordial gonad also presents an opportunity to compare the variations in vascularization strategies in response to diverging morphology and signaling cues. Although remodeling to form cohesive, patent vessels does not appear to occur in the absence of blood flow, endothelial cells migrate along physiologically relevant pathways, reflecting the normal in vivo male and female vascular patterns. Using genetic mutants and chemical inhibitors, this ex vivo imaging system has the potential to illuminate the molecular mechanisms that integrate vascular and morphological development of organs, which may inform the development of authentic vascularization in artificial organs.

Materials and Methods

Animals.

Mice carrying a Tie2-Cre transgene [B6.Cg-Tg(Tek-cre)12Flv] (23) or a Cre insertion into the Flk1 gene [Kdrtm1(cre)Sato] (24) were crossed to the reporter strain Z/EG [(βgeo/GFP) Tg(ACTB-Bgeo/GFP)21Lbe] (25). The gonad and mesonephros of 11.5-dpc embryos expressing EGFP were dissected and processed for time-lapse organ culture.

Time-Lapse Organ Culture.

The gonads of 11.5-dpc or 12.0-dpc XX and XY embryos expressing EGFP in endothelial cells were dissected and cultured at the air–surface interface on a 1.5% agar block to stabilize the gonad and optimize imaging using an inverted microscope (19). Blocks were inverted into disposable 35-mm glass-bottom culture dishes and submerged in DMEM without phenol red supplemented with 10% FBS and 50 μg/ml ampicillin. Cultures were positioned in a stage heater insert (heater P-insert; Zeiss, catalog no. 4118619901) and cultured in a microscope stage incubation chamber (S-chamber; Zeiss, catalog no. 4118609902) heated to 37°C in 5% CO2 and humidified to saturation with distilled water (Fig. S1A). The gonads were imaged over a 24- to 48-h period using the Zeiss LSM 510 META confocal microscope with a nonimmersion ×10 or ×20 lens. Confocal optical Z-sections (512 × 512 pixels) were collected every 10 μm through the entire organ at 15-min intervals. Maximum-intensity projections (MIP) over time were created by using Zeiss 510 META confocal software. All time-lapse images presented in this article are MIPs.

Immunohistochemistry.

Lectin injections and whole-mount immunohistochemistry were performed as previously described (20). Antibodies were rat anti-PECAM1 (Pharmingen; 1:500 dilution) or mouse anti-F4/80 (1:100 dilution). Confocal optical Z-sections (1,024 × 1,024 pixel arrays) were collected every 3 μm through the entire organ on a Zeiss LSM 510Meta Confocal. MIPs were created by using Zeiss 510 META confocal software.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Dr. Tom Sato (Weill Cornell Medical College, New York) for the kind gift of the Flk1Cre mice and Dr. Eric Meyers (Duke University Medical Center) for providing the Tie2-Cre mice. Funding for this project was provided by a Lalor Foundation fellowship (to D.C.) and from National Institutes of Health Grant HL63054 and Grant HD39963.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0707674105/DCSupplemental.

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