Editors' ChoiceDevelopmental Biology

Confining Hippo for Fate Specification

Sci. Signal.  16 Jul 2013:
Vol. 6, Issue 284, pp. ec163
DOI: 10.1126/scisignal.2004511

Prior to implantation, mammalian blastocysts consist of two cell layers: the outer trophectoderm (TE), which are polarized cells that will give rise to the placenta, and the inner cell mass (ICM), which are nonpolarized cells that will form the embryo. In TE cells, the transcriptional cofactor Yap is localized to nuclei, where it cooperates with the transcription factor Tead4 to promote expression of the TE identity marker Cdx2. In the ICM, Yap is excluded from nuclei, thus repressing the TE fate. Nuclear accumulation of Yap is prevented by direct phosphorylation of Yap by the kinases Lats1 and -2 (Lats1/2) downstream of the kinase Hippo. However, Lats1/2 are present in both the TE and ICM, so how Yap nuclear exclusion is restricted to the ICM has been unclear. Likewise, it has also been unclear whether Hippo signaling is required for specifying ICM fate or merely for repressing the TE fate. Lorthongpanich et al. report that Lats1/2 are required for proper fate specification of the ICM. When blastocysts from mouse zygotes that had been injected with small interfering RNAs targeting Lats1/2 were cultured, no embryonic stem cells were recovered, indicating that ICM self-renewal had been lost. In the knockdown blastocysts, the inner cells expressed the TE marker Cdx2 and became polarized but did not express ICM fate markers, suggesting their transformation into TE cells. Cockburn et al. demonstrated that expression of a dominant-negative form of the FERM domain protein Merlin (also known as Nf2), which promotes Hippo signaling, resulted in nuclear localization of Yap and expression of Cdx2 in inner blastocyst cells. The inner cells of late blastocysts lacking both zygotic and maternal Merlin expressed other TE fate markers in addition to Cdx2, exhibited TE-like distribution of cell polarity proteins, and did not express ICM fate markers. These two studies demonstrated that Hippo signaling is required not just for repressing TE fate but also for promoting ICM fate.

Hirate et al. addressed the mechanism by which Hippo signaling is restricted to ICM cells. Restriction of Hippo signaling to the ICM depended upon both the polarity of TE cells and the adherens junctions of ICM cells. Disrupting the apical polarity protein complex in blastocysts resulted in increased accumulation of phosphorylated, nuclear-excluded Yap and reduced expression of Cdx2 in outer cells. Consistent with the nonpolarized morphology of ICM cells, disrupting the apical polarity complex had no effect on the accumulation of phosphorylated, nuclear-excluded Yap in the inner cells. However, disrupting cell adhesions by dissociating embryos caused nuclear accumulation of Yap in all cells, implying that cell adhesion was required for Hippo-mediated exclusion of Yap from nuclei. The adhesion junction–associated protein angiomotin (Amot) promotes Hippo signaling by binding to several Hippo signaling components, including Lats1/2 and Merlin. Amot was localized to the apical membrane, facing the outside of the blastocyst, of TE cells and uniformly distributed along the plasma membrane in ICM cells. Apical localization of Amot in TE cells required the apical polarity complex, and Amot was required for Hippo pathway activation and proper fate specification in the ICM. The N-terminal domain of Amot was required for the Merlin-mediated association of Amot with the adherens junctions between adjacent ICM cells and for Amot to bind to F-actin, an inhibitor of Hippo signaling. Lats phosphorylated Amot on Ser176, and this phosphorylation event promoted the Amot-Lats interaction and the release of Amot from F-actin, thus enhancing Hippo pathway activation. Cockburn et al. noted that, whereas Merlin was uniformly distributed along the membrane in ICM and TE cells, Lats1/2 were enriched in the apical regions of TE cells and evenly distributed throughout the cytoplasm in ICM cells. These results suggest a model (see commentary by Manzanares and Rodriguez) in which Amot and Lats1/2 are recruited to adherens junctions in ICM cells through the direct association of Amot with cortical F-actin and the Merlin-mediated association of Amot with the adherens junction component E-cadherin. Phosphorylation of Amot by Lats causes Amot to dissociate from F-actin and enhances the interaction between Amot and Lats, thus promoting Hippo signaling. In the polarized TE cells, restriction of Amot to the apical membrane prevents accumulation of Amot at the more basally located adherens junctions, thus preventing Lats activation and nuclear exclusion of Yap. Although the Hippo pathway is best known for controlling cell growth and organ size, these studies demonstrate that the pathway can also function in cell fate specification and describe a mechanism for spatially restricting Hippo signaling.

C. Lorthongpanich, D. M. Messerschmidt, S. W. Chan, W. Hong, B. B. Knowles, D. Solter, Temporal reduction of LATS kinases in the early preimplantation embryo prevents ICM lineage differentiation. Genes Dev. 27, 1441–1446 (2013). [Abstract] [Full Text]

Y. Hirate, S. Hirahara, K.-i. Inoue, A. Suzuki, V. B. Alarcon, K. Akimoto, T. Hirai, T. Hara, M. Adachi, K. Chida, S. Ohno, Y. Marikawa, K. Nakao, A. Shimono, H. Sasaki, Polarity-dependent distribution of angiomotin localizes Hippo signaling in preimplantation embryos. Curr. Biol. 23, 1181–1194 (2013). [PubMed]

K. Cockburn, S. Biechele, J. Garner, J. Rossant, The Hippo pathway member Nf2 is required for inner cell mass specification. Curr. Biol. 23, 1195–1201 (2013). [PubMed]

M. Manzanares, T. A. Rodriguez, Development: Hippo signalling turns the embryo inside out. Curr. Biol. 23, R559–R561 (2013). [PubMed]