Snapshot: Mouse Primitive Streak Nitya Ramkumar1,2 and Kathryn V

Home , Axial mesoderm, Epiblast, Mesoderm, MESP2, Paraxial mesoderm, Primitive streak

SnapShot: Mouse Primitive Streak Nitya Ramkumar1,2 and Kathryn V. Anderson1 1Sloan-Kettering Institute, New York, NY 10065, USA 2Weill Graduate School of Medical Sciences, Cornell University, New York, NY 10065, USA

A The e7.5 mouse embryo D Maintaining the primitive streak

Axin2 G Tail bud stage Wnt3A Dll1 Brachyury Anterior Posterior FgfR1 Tbx6

Fgf4,8 Sox2

B Initiating the primitive streak E Down-regulating E-Cadherin

A Node Extraembryonic ectoderm (ExE) Mesp2 Furin PACE4 Nodal FgfR1 Tbx6 proBMP4 BMP4 POSTERIOR Nik Eomes Brachyury Dll1 NICD proNodal Wnt 3 p38 Wnt3A E-Cadherin p38IP ANTERIOR T Lefty2 FgfR1 Nodal Snail AVE Mesp1 Mesp2 P Wnt3A FGF8 E-Cadherin Lateral mesoderm F Cytoskeletal reorganization Paraxial mesoderm Visceral Neural ectoderm endoderm Net1 Rac1 Axial mesoderm Primitive streak (tail bud) C The streak and the EMT RhoA Somites

Epb4.1I5

Actin Epiblast cells Cells undergoing EMT at Basement expressing Nanog the streak to give mesoderm membrane

488 Cell 146, August 5, 2011 ©2011 Elsevier Inc. DOI 10.1016/j.cell.2011.07.028 See online version for legend and references. SnapShot: Mouse Primitive Streak Nitya Ramkumar1,2 and Kathryn V. Anderson1 1Sloan-Kettering Institute, New York, NY 10065, USA 2Weill Graduate School of Medical Sciences, Cornell University, New York, NY 10065, USA

Gastrulation is the central event in organizing the body plan of the embryo. It transforms a single layer of epithelial cells, called the epiblast, into the three germ layers—ectoderm, mesoderm, and endoderm—that give rise to all of the tissues and organs of the embryo. In addition to changes in gene expression that define the different germ layers, gastrulation is also an epithelial-to-mesenchymal transition (EMT) that generates migratory cells of the mesoderm and endoderm. The gastrulation EMT in the mouse takes place at a structure called the primitive streak (light blue) at the posterior pole of the embryo. Gastrulation in the mouse initiates at embryonic day 6.25 (e6.25) and continues for several days. Cells adopt different fates depending on when they leave the streak. The first cells to exit from the streak give rise to extraembryonic, cardiac, and cranial mesoderm, as well as definitive endoderm. Later cells give rise to lateral plate and then paraxial mesoderm. Thus, the primitive streak is a stable entity that continually proliferates and generates an ordered series of cell types through an EMT. Recent evidence suggests that, if cells are forced to undergo EMT, they adopt a stem cell-like state and now contribute to a variety of cell fates. This SnapShot depicts the signals and transcription factors, which are required for the establishment, maintenance, and termination of the primitive streak, and illustrates how this process endows stem cell-like fate on the cells that have delaminated from the epiblast.

The e7.5 Mouse Embryo At e7.5, the primitive streak is already established on the posterior side of the embryo (right), regulated by signals from the anterior side of the embryo (left) and extra embryonic region, which is attached to maternal tissue (top). At this stage, two epithelial cell layers, the inner epiblast and the outer visceral endoderm, are already separated by a mesenchymal layer, and the three cell layers are arranged in a simple cup shape.

Signals that Initiate the Primitive Streak Wnt3 and the transforming growth factor β (TGF-β) family member Nodal are responsible for the initiation of primitive streak on the posterior side of the e6.25 embryo (panel B). Inhibitors of Wnt and Nodal define their asymmetric activity. These inhibitors are expressed in the extraembryonic tissue, called anterior visceral endoderm (AVE) (yellow), which restricts the primitive streak to the posterior side of the embryo. Nodal induces autofeedback loops that maintain its levels and induces other pathways in this process. Nodal is derived from its secreted precursor proNodal, which is processed by two secreted proteases of Subtilisin-like proprotein convertase (SPC family), Furin and PACE4. proNodal in the epiblast induces the expression of Furin and PACE4 in the extraembryonic ectoderm (ExE) (pink above the dotted line). proNodal induces the expression of bone morphoge- netic protein 4 (BMP4) in the ExE, which then promotes the expression of Wnt3 in the posterior epiblast, the prospective primitive streak (maroon). Wnt3 mediates the induction of the T box transcription factor Brachyury, a mesodermal marker. Wnt3 positively regulates the levels of proNodal by the proximal epiblast enhancer of Nodal, which initiates the feedback loop. proNodal turns on the expression of BMP4 and the two proteases by binding to and activating the activin receptor complexes. Nodal also induces expression of Lefty2, which negatively regulates the activity of Nodal. This attenuates the initiation loop established by the Nodal signaling, leading to the second phase in which cells move through the streak. At this stage, these cells give rise to extraembryonic mesoderm (blue).

The Streak and the EMT A cross section through the embryo in panel A shows how epiblast cells on the posterior side of the embryo lose their epithelial nature (maroon) and adopt a migratory mesenchymal nature as they move through the streak (blue). The primitive streak includes both the epiblast layer and the cells that are making the transition into the mesenchymal layer. This process, called gastrulation EMT, eventually gives rise to the three germ layers, and it occurs continuously at the streak until e13.5, when gastrulation comes to an end. During this time, the changing nature of signals at the streak temporally regulates the differentiation potentials of the cells exiting the streak. Cells adopt different fates based on when they leave. The pluripotency marker Sox2 is expressed in the entire epiblast, whereas Nanog is restricted to the posterior side (maroon), and they are both turned off as cells to the streak (black arrows). Once in the mesenchymal layer, cells migrate away from the streak (red arrows) and acquire the identity of mesoderm and definitive endoderm cells (purple). The boxed region of the streak is shown in greater detail in panels D–F.

Signals that Maintain the Primitive Streak Positive feedback loops involving the Wnt and fibroblast growth factor (Fgf) pathways maintain the streak (panel D). In the initial stages of the streak, Wnt3 is induced by BMP4 from the ExE. As the streak progresses, Wnt3 is replaced by Wnt3A. Wnt3A turns on the expression of the T box family genes Brachyury and T box6 (Tbx6) in the streak, which in turn maintain the expression of Wnt 3A to establish a feedback loop. Tbx6 negatively regulates the expression of Sox2, inhibiting neural fate. Simultaneously, Fgf4 and Fgf8, expressed transiently in the streak, activate Fgf receptor 1 (FgfR1), which in turn maintains the levels of Brachyury and Tbx6 in the streak. Wnt3A and the T box transcription factors together regulate the expression of Delta-like 1, a ligand of the Notch signaling pathway. During this stage, Wnt3A is positively regulated by Axin2. As cells ingress through the streak (black arrows), they first give rise to cardiac and cranial mesoderm and definitive endoderm and, in the later stages, lateral and paraxial mesoderm.

Downregulation of E-Cadherin at the EMT Downregulation of E-cadherin (red) is a central aspect of the EMT, and it is regulated by several independent inputs (panel E). Active FgfR1 turns on the expression of the zinc finger transcriptional repressor Snail in the streak, which downregulates transcription of E-Cadherin. The basic-helix-loop-helix transcription factors MESoderm Posterior 1 and 2 (Mesp1 and Mesp2) also promote the expression of Snail in ingressing cells, independent of Fgf signaling. The T box family transcription factor Eomesodermin, which is regulated by Nodal, also downregulates E-Cadherin expression at both transcriptional and posttranslational levels as cells exit from the streak. Additionally, p38 map kinase along with p38 interacting protein (p38IP) downregulates E-Cadherin expression posttranslationally at the streak.

Cytoskeletal Reorganization at the EMT Although downregaluting E-Cadherin is central for the cells to undergo EMT at the streak, several E-Cadherin-independent changes are also required for the EMT (panel F). Actin (blue) is expressed apically in the epiblast and becomes relocalized to the entire cell periphery as cells ingress through the streak. Actin reorganization is important for successful EMT, as mutants that fail to reorganize the actin cytoskeleton, such as lulu/Epb4.1l5, also fail to undergo proper EMT. Cells ingressing through the streak (black arrows) have to break down the basement membrane (green) before they migrate away from the streak. Experiments in the chick embryo suggest that this breakdown is mediated by the loss of neuroepithelial cell transforming 1 (Net1), an activator of RhoA, at the streak. Initially, RhoA is activated throughout the cell in the epiblast. As cells ingress through the streak, loss of basal RhoA leads to breakdown of basement membrane. Rac1 is essential for cell adhesion, lamellopodia formation, and cell migration. Epiblast cells in Rac1 mutants have reduced cell adhesion and motility.

Tail Bud Stage At later stages, the primitive streak becomes the tail bud. Fate mapping studies have shown that the generation of mesodermal structures continues late into elongation of the anterior-posterior axis until e13.5 (panel G). At e8.5 (shown here), the cells emerging from the posterior streak give rise to lateral plate mesoderm, whereas cells from the anterior streak mostly give rise to paraxial mesoderm. Cells in the region between the node (i.e., a transient early embryonic organizing structure) (red) and the streak give rise to neu- roectoderm, notochord, and medial somites. At the posterior end of the embryo, the paraxial mesoderm consists of an undifferentiated population of cells called the presomitic mesoderm (PSM). The undifferentiated state is maintained by a combination of Fgf8 and Wnt3A gradients. Production of paraxial mesoderm is coupled to the generation of somites at the late streak stage in that the signals maintaining the PSM also initiate the segmentation clock.

488.e1 Cell 146, August 5, 2011 ©2011 Elsevier Inc. DOI 10.1016/j.cell.2011.07.028 SnapShot: Mouse Primitive Streak Nitya Ramkumar1,2 and Kathryn V. Anderson1 1Sloan-Kettering Institute, New York, NY 10065, USA 2Weill Graduate School of Medical Sciences, Cornell University, New York, NY 10065, USA

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