Signaling in cell differentiation and morphogenesis

Abstract

All the information to make a complete, fully functional living organism is encoded within the genome of the fertilized oocyte. How is this genetic code translated into the vast array of cellular behaviors that unfold during the course of embryonic development, as the zygote slowly morphs into a new organism? Studies over the last 30 years or so have shown that many of these cellular processes are driven by secreted or membrane-bound signaling molecules. Elucidating how the genetic code is translated into instructions or signals during embryogenesis, how signals are generated at the correct time and place and at the appropriate level, and finally, how these instructions are interpreted and put into action, are some of the central questions of developmental biology. Our understanding of the causes of congenital malformations and disease has improved substantially with the rapid advances in our knowledge of signaling pathways and their regulation during development. In this article, I review some of the signaling pathways that play essential roles during embryonic development. These examples show some of the mechanisms used by cells to receive and interpret developmental signals. I also discuss how signaling pathways downstream from these signals are regulated and how they induce specific cellular responses that ultimately affect cell fate and morphogenesis.

Figure 1.

FGF signaling cascades, an example of RTK signaling. The formation of multimeric complexes of FGF ligand, FGF receptors (FGFR), and heparin sulphate proteoglycan (HSPG) results in the transphosphorylation of FGFRs through the action of tyrosine kinase (TK) domains. TK-deficient FGFRL1 molecules may inhibit receptor activation through competition. Several cofactors that can enhance FGFR activation such as anosmin have been identified. Phosphorylated FGFRs recruit adapter molecules such as FRS2 and Shc, which recruits Grb2 and SOS for initiation of the ras/MAPK and Gab1 for the initiation of the PI3K pathways, respectively. The guanine exchange factor SOS activates ras, which allows the signal to be transmitted to Raf1, MEK, and ERK, which translocates to the nucleus where it acts on transcriptional cofactors to alter gene expression. Sprouty genes encode feedback inhibitors of the pathway (broken lines). A key readout of signal strength is c-fos, which is regulated by ERK at both transcriptional and posttranslational levels. Activation of the PI3K pathway is associated with the recruitment and activation of AKT through PIP₃ production and action of PDK1. AKT inhibits cell death pathways mediated by forkhead transcription factors (FKHR) and BAD. ERK can also inhibit cell death through the action of p90RSK on BAD. PLCγ is activated on recruitment to activated RTKs, resulting in the release of second messengers IP₃ and DAG. IP₃ induces the release of intracellular Ca²⁺ and DAG activated-PKC, which can alter gene expression downstream and also feed into the ras/MAPK pathway by activating Raf1. Activated ras can activate other small GTPases such as rho, rac, and cdc42, which activate the p38MAPK or JNK pathways and alter the stability of the cytoskeleton. Noncanonical WNT signaling also feeds into this pathway at the level of rac and rho activation (red arrow) to affect the cytoskeleton and activate JNK.

Figure 2.

Canonical (left) and noncanonical (right) WNT signaling pathways. In the absence of WNT signaling, the effector of canonical WNT signaling, β-catenin is found complexed in the cytoplasm with a destruction complex containing Axin and GSK3β. GSK3β phosphorylates β-catenin, which results in its targeting to and degradation by the proteasome. The binding of canonical WNT ligands to Frizzled (Fz) and LRP5/6 receptors results in the phosphorylation of LRP5/6 through the action of Dishevelled (Dsh). Phosphorylated LRP5/6 recruits Axin away from the destruction complex, liberating β-catenin that accumulates in the cytoplasm and translocates to the nucleus, where it interacts with transcriptional regulators of the TCF/LEF family to alter gene expression. AKT and PKC downstream from RTK signaling may inhibit the activity of the β-catenin destruction complex to promote β-catenin signaling. Noncanonical WNT/Ca²⁺ signaling is initiated by the activation of PLC, probably through trimeric G-proteins such as Gbg. The release of Ca²⁺ results in the activation of NFAT (nuclear factor associated with T cells) transcription factors through calcineurin (Cn). Other downstream transcription factors (TFs) are activated by PKC and CaMKII. A Wnt/planar cell polarity pathway involves Diego (Dgo), Smurf, and Par6, which through cross-inhibitory interactions with Van Gogh-like (Vangl)-Prickle (Pk) complexes regulate planar cell polarity.

Figure 3.

Sonic hedgehog (SHH) signaling pathway. A key first step in the SHH pathway is the posttranslational processing of SHH protein to produce the mature SHH-N ligand, which is lipid (palmitate and cholesterol) modified (Porter et al. 1996a, 1996b). Secretion of SHH is dependent on a transmembrane protein Dispatched (Ma et al. 2002). In the absence of SHH ligand (right-hand side of figure), the 12-span transmembrane protein Patched (Ptch1) prevents a seven-pass transmembrane protein Smoothened (Smo) from entering the primary cilium. Gli3 is proteolytically cleaved with its amino-terminal fragment functioning as a transcriptional repressor of putative repressors (X) to induce the expression of downstream targets (referred to as class I genes in neural development). When SHH binds to Ptch1, the repression of Smo is relieved and Smo moves into the cilium, where it promotes the accumulation of Gli proteins and inhibit the proteolytic cleavage of Gli3. Gli proteins translocate to the nucleus where they activate the transcription of class II genes, Gli1 and Ptch1 (Ruiz i Altaba et al. 2003; Jacob and Lum 2007). The latter produces a negative feedback loop to switch off SHH signaling. Several other regulators of SHH signaling have been identified. The intracellular protein Sufu interacts with Gli3 in a SHH-dependent manner to regulate the efficiency of Gli3 processing. Membrane proteins Gas1, Boc, and Cdo bind to SHH and may regulate the accessibility to SHH ligand to Ptch1. Rab23 promotes the expression of class I while inhibiting the expression of class II genes, and PKA can inhibit the activity of Gli2/3 activator complexes.

Figure 4.

The BMP signaling pathway and regulation of SMAD activity. Binding of BMP ligand to BMPRII results in dimerization with BMPRI and phosphorylation of receptor (R-)Smads (Smad1, 5, or 8) close to the carboxyl terminus. These activated R-Smads complex with a Co-Smad (Smad4) and the Smad complex translocates to the nucleus where they interact with other transcriptional regulators to control gene transcription. Negative feedback mechanisms include the expression of SMAD6,7, which competes with SMAD1,5,8 for binding to BMPRI. Both WNT and RTK signaling pathways have been shown to inhibit BMP signaling at the level of Smad1. ERK phosphorylates the linker region in between the amino- and carboxy-terminal domains of Smad, which primes Smad for a further phosphorylation by GSK3. Dually phosphorylated Smad is targeted for degradation. Secreted BMP antagonists such as Noggin bind to and sequester BMP ligand in the extracellular space.

Figure 5.

The Notch pathway. Binding of the extracellular domain of the Notch receptor to Delta and Serrate/Jagged/LAG-2 (DSL) ligand on an adjacent cell induces a conformational change in Notch that exposes an extracellular ADAM10/TACE cleavage site. A subsequent intramembranous proteolytic cleavage by γ-secretase, releases the Notch intracellular domain (NICD), which translocates to the nucleus. NICD complexes with coactivators (CoA) and Mastermind (Mam) to replace corepressors (CoR) that occupy CBF1/Su(H)/LAG-1 (CSL)-bound Notch target gene promotors. An important group of target genes is the Hairy/enhancer of split (Hes) genes. Hes inhibits the expression of achaete-scute genes such as Ascl1 (Mash1) in cells receiving Notch signals. In cells not receiving Notch signals, Mash1 is expressed, which up-regulates the expression of Notch ligands (DSL).

Figure 6.

Schematic representation of cytoskeletal signals that control cell proliferation and fate. (Reproduced, with permission, from Mammoto and Ingber 2009.) Cell tethering to the ECM through integrins generate tension in the intracellular actin cytoskeleton. These changes can result in the activation of ERK and PI3K pathways, or the small GTPase Rho, through effects on FAK and p190RhoGAP, respectively. p190RhoGAP also modulates transcription by controlling the nuclear translocation of TFII-I and GATA2. Rho activation controls various cell-cycle regulators such as p27 and Cyclin D1. These combined effects on the cytoskeleton can alter the differentiation of multipotent stem/progenitor cells.