Repulsive cues combined with physical barriers and cell-cell adhesion determine progenitor cell positioning during organogenesis.

The precise positioning of organ progenitor cells constitutes an essential, yet poorly understood step during organogenesis. Using primordial germ cells that participate in gonad formation, we present the developmental mechanisms maintaining a motile progenitor cell population at the site where the organ develops. Employing high-resolution live-cell microscopy, we find that repulsive cues coupled with physical barriers confine the cells to the correct bilateral positions. This analysis revealed that cell polarity changes on interaction with the physical barrier and that the establishment of compact clusters involves increased cell-cell interaction time. Using particle-based simulations, we demonstrate the role of reflecting barriers, from which cells turn away on contact, and the importance of proper cell-cell adhesion level for maintaining the tight cell clusters and their correct positioning at the target region. The combination of these developmental and cellular mechanisms prevents organ fusion, controls organ positioning and is thus critical for its proper function.

Control of chemokine-guided cell migration by ligand sequestration.

Primordial germ cell (PGC) migration in zebrafish is directed by the chemokine SDF-1a that activates its receptor CXCR4b. Little is known about the molecular mechanisms controlling the distribution of this chemoattractant in vivo. We demonstrate that the activity of a second SDF-1/CXCL12 receptor, CXCR7, is crucial for proper migration of PGCs toward their targets. We show that CXCR7 functions primarily in the somatic environment rather than within the migrating cells. In CXCR7 knocked-down embryos, the PGCs exhibit a phenotype that signifies defects in SDF-1a gradient formation as the cells fail to polarize effectively and to migrate toward their targets. Indeed, somatic cells expressing CXCR7 show enhanced internalization of the chemokine suggesting that CXCR7 acts as a sink for SDF-1a, thus allowing the dynamic changes in the transcription of sdf-1a to be mirrored by similar dynamics at the protein level.

Control of receptor internalization, signaling level, and precise arrival at the target in guided cell migration.

Activation of the chemokine receptor CXCR4 by SDF1 controls a variety of biological processes in development, immune response, and disease [1-5]. The carboxyl-terminal region of CXCR4 is subject to phosphorylation that allows binding of regulatory proteins [5]; this results in downregulation of CXCR4 signaling and receptor internalization [6]. Notably, truncations of this part of CXCR4 have been implicated in WHIM syndrome, a dominantly inherited immunodeficiency disorder [7, 8]. Despite its importance in receptor signaling and the clinical relevance of its regulation, the precise function of regulating signaling level and internalization in controlling cell behavior is not known. Whereas a number of in vitro studies suggested that the carboxyl terminus of CXCR4 positively regulates chemotaxis (e.g., [9]), others reached the opposite conclusion [8, 10, 11]. These conflicting results highlight the importance of investigating this process under physiological conditions in the live animal. In this study, we demonstrate the significance of internalization and of controlling receptor signaling level for SDF-1-guided migration. We found that whereas internalization and the control over signaling intensity are dispensable for cell motility and directional sensing, they are essential for fine-tuning of migration in vivo, allowing precise arrival of zebrafish PGCs at their target, the region where the gonad develops.

Autonomous modes of behavior in primordial germ cell migration.

Zebrafish primordial germ cells (PGCs) are guided toward their targets by the chemokine SDF-1a. PGCs were followed during three phases of their migration: when migrating as individual cells, while remaining in a clustered configuration, and when moving as a cell cluster within the embryo. We found that individually migrating PGCs alternate between migratory and pausing modes. Pausing intervals are characterized by loss of cell polarity and correlate with subsequent changes in the direction of migration. These properties constitute an intrinsic behavior of PGCs, enabling erasure of prior polarity and re-sampling of the environment. Following migration arrest at a site of high SDF-1a levels, PGCs resume migration as a cluster. The seemingly coordinated cluster migration is a result of single-cell movement in response to local variations in SDF-1a distribution. Together, these behavioral modes allow the cells to arrive at specific destinations with high fidelity and remain at their target site.