Development

Development broadly refers, in the case of humans and other mammals, to the process of developing from a single cell into a collection of trillions of cells. This collection spans many different cell types that are organized into distinct tissues and organs. Coordinated by a complex interplay of intrinsic and extrinsic signals, developmental processes generally drive cell division, formation of the body axis, tissue and organ development and cell differentiation. 

Despite the vast array of cell behaviors and fates that arise during development, a relatively small number of core developmental pathways are relied upon. These pathways act repeatedly at different time points throughout embryogenesis and in different cellular environments to orchestrate different cellular outcomes.

Here we highlight a few key pathways: WNT, SMAD and NF-kB.

WNT regulation of epithelial-mesenchymal transition and planar cell polarity

Early in embryogenesis, a subset of epithelial cells undergoes epithelial-mesenchymal transition (EMT). During this process the epithelial cells transition into mesenchymal stem cells, losing their cell polarity and cell-cell adhesion and acquiring motility. The canonical WNT pathway is a key regulator of EMT.

In this context, WNT acts through frizzled receptors (FZD) to free ß-catenin (CTNNB1) from an inhibitory complex of glycogen synthase kinase 3 (GSK3), casein kinase 1 (CK1), adenomatous polyposis coli (APC) and Axin (1). Freed ß-catenin translocates to the nucleus where it complexes with transcription factor TCF/LEF (T-cell factor/lymphoid enhancing factor) activating expression of critical EMT regulators including SNAIL1/2 and TWIST.

Mutation of canonical WNT pathway members is associated with improper initiation of EMT, uncontrolled cell proliferation and metastasis (2). As a result, the WNT pathway plays a complex role in many cancers and other diseases.

Beyond WNT, additional factors contribute to EMT including TGF-ß, Notch and growth factors EGF, HGF and FGF which act through their own signaling pathways, including the SMAD, JAK/STAT, PI3K/Akt and Ras/Raf/MEK/ERK signaling pathways, to activate expression of the same EMT regulators as well as others including ZEB1/2 (1).

In contrast, the non-canonical WNT-PCP pathway is the main driver of planar cell polarity (PCP). A specialized process that occurs later in development, PCP sets the direction of cellular structures across a tissue layer to ensure proper function. As an example, during development of the inner ear, PCP guides the orientation of hair cells responsible for detecting sound and establishing balance. Without the proper orientation, hearing and balance would be impaired.

At a cellular level, PCP is accomplished by asymmetric segregation of the relevant proteins within the cell. The segregation pattern is repeated throughout individual cells across the entire tissue.

On one side of the cell, WNT acts through the FZD receptor, scaffold protein Disheveled (DVL), DAAM1 (Dvl-associated activator of morphogenesis) and RHOA to promote actin polymerization (3). It also acts through Rac1 and JNK to activate expression of cell polarity and cell migration genes via c-Jun. CELSR contributes by helping to establish asymmetrical localization of FZD.

On the other side of the cell, VANGL2 (Vnag-like 2) and PRICKLE antagonize DVL, inhibiting WNT-PCP signaling. This asymmetric segregation establishes and ultimately maintains cell polarity throughout the tissue ensuring proper function.

A second non-canonical WNT-Ca²⁺ pathway also contributes to cell fate, with WNT acting through downstream PKC, calcium calmodulin mediated kinase II (CAMKII) and calcineurin (3).

SMAD signaling in cardiomyocyte differentiation and cardiogenesis

Cardiogenesis, beginning with cardiomyocyte differentiation, provides an example of employing the SMAD pathway for establishing a unique tissue and organ.

Multiple pathways are involved in cardiomyocyte differentiation including the previously discussed WNT pathway, as well as the Notch, sonic hedgehog, Hippo and BMP pathways (4). In the latter, bone morphogenic proteins (BMPs) acting on mesoderm cells signal through type I (ALK3 and ALK6) and II (BMPR2) membrane receptors to phosphorylate SMAD1, SMAD5 and SMAD8. The SMADs in turn bind to SMAD4, translocating to the nucleus and activating expression of cardiomyocyte-specific genes directly, including the miRNA 17-92 cluster that promotes outflow tract myocardial development.

In addition to direct transcriptional effects, the SMADs also activate other key transcriptional regulators including ATF2, NKX2.5, GATA4 and ISL1 which in turn increase expression of additional cardiomyocyte-specific genes including β-MHC, NPPA and NPPB. In the case of ISL1, activation is accomplished through crosstalk with the MAPK pathway. Phosphorylated SMADs activate p38 which in turn phosphorylates ISL1, preventing its degradation and allowing for translocation to the nucleus.

Many of these same transcriptional regulators are critical for promoting cardiogenesis. As described above, SMAD4 downstream of BMP receptors activates ATF2, NKX2.5 and GATA4. These regulators of cardiogenesis are joined by ß-catenin and TCF/LEF downstream of the canonical WNT/FZD/DVL pathway as well as MEF2C and TBX5.

LTβR signaling in secondary lymphoid organ development

Secondary lymphoid organ development via the lymphotoxin β receptor (LTβR) provides an example of use of the NF-kB pathway for unique differentiation.

A member of the tumor necrosis factor receptor (TNFR) family, LTβR is activated by membrane-bound ligands LTα1β2 and LIGHT (5,6). Their binding leads to aggregation of TRAF proteins including TRAF2, TRAF3 and TRAF5 which activate NF-kB transcription factors via canonical and non-canonical pathways. Canonical activation of NF-kB form RelA/p50 occurs immediately via IκB kinase (IKK) phosphorylation of Inhibitor of IκB (IκB), resulting in IκB degradation and subsequent freeing of RelA/p50. The unhindered complex translocates to the nucleus and activates expression of pro-inflammatory cytokines/chemokines and cell adhesion molecules including CXCL1 and VCAM1.

The non-canonical activation of NF-kB form RelB/p52 occurs over a longer time frame via increased proteolytic processing of p100, the inactive form of p52. RelB/p52 activates expression of homeostatic chemokines and genes important for secondary lymphoid organ development. Both pathways are dependent on NFκB-inducing kinase (NIK). Although the mechanism is not well understood, LTβR can also activate the JNK pathway.

Important for immune development and host defense, aberrant LTβR signaling is associated with autoimmune and inflammatory disorders as well as some cancers.