The Cellular and Molecular Basis of Development

Gap Junctions

Gap junctions are channels that permit ions and small molecules (<1 kD) to directly pass from one cell to another, known as gap junctional intercellular communication (GJIC). However, large proteins and nucleic acids cannot transfer through gap junctions. Gap junctions are made from hemi-channels present on the surface of each cell known as connexons. Each connexon is made up of six connexin molecules that form a hexamer. In early development, gap junctions are usually open, permitting exchange of small molecules in relatively large regions. However, as development proceeds, GJIC is more restricted, with establishment of boundaries, such as in the rhombomeres, transient structures of the developing hindbrain. Gap junctions are particularly important for electrical coupling in the heart and brain. Mutations of specific connexin molecules are associated with human diseases (e.g., mutation of CX43 is associated with atherosclerosis).

Cell Adhesion Molecules

Cell adhesion molecules have large extracellular domains that interact with extracellular matrix components or adhesion molecules on neighboring cells. These molecules often contain a transmembrane segment and a short cytoplasmic domain, which regulates intracellular signaling cascades. Examples of cell adhesion molecules are the cadherins, a family of proteins that have important roles during embryonic development.

Cadherins are critical for embryonic morphogenesis, as they regulate the separation of cell layers (endothelial and epidermal), cell migration, cell sorting, establishment of well-defined boundaries, synaptic connections, and the growth cones of neurons. These properties result from cadherins mediating the interaction between the cell and its extracellular milieu (both neighboring cells and extracellular matrix ). Cadherins were originally classified by their site of expression; for example, E-cadherin is highly expressed in epithelial cells, whereas N-cadherin is highly expressed in neural cells.

A typical cadherin molecule has a large extracellular domain, a transmembrane domain, and an intracellular tail ( Fig. 20.1 ). The extracellular domain contains five extracellular repeats and has four Ca ²⁺ -binding sites. Cadherins form dimers that interact with cadherin dimers in adjacent cells. These complexes are found clustered in adherens junctions, which result in the formation of a tight barrier between epithelial or endothelial cells. Via its intracellular domain, cadherin binds to p120-catenin, β-catenin, and α-catenin. These proteins connect cadherin to the cytoskeleton. E-cadherin expression is lost as epithelial cells transition to mesenchymal cells (this is known as the epithelial-to-mesenchymal transition [EMT]). EMT is required for the formation of neural crest cells during development, and the same process also occurs during tumor development.

Retinoic Acid

The AP axis of the embryo is crucial for determining the correct location for structures such as limbs and for the patterning of the nervous system. For decades, it has been clinically evident that alterations in the level of vitamin A (retinol) in the diet (excessive or insufficient amounts) can lead to the development of congenital malformations (see Chapter 19 ). The bioactive form of vitamin A is retinoic acid, which is formed by enzymatic oxidation by retinol aldehyde dehydrogenase and subsequently retinal aldehyde dehydrogenase. Free levels of retinoic acid can be modulated by cellular retinoic acid–binding proteins that sequester retinoic acid. Retinoic acid can also be actively degraded into inactive metabolites by enzymes such as CYP26 ( Fig. 20.2 ).

Normally, retinoic acid acts to “posteriorize” the body plan, and either excessive retinoic acid or inhibition of its degradation leads to a truncated body axis where structures have a more posterior nature. In contrast, insufficient retinoic acid or defects in the enzymes (e.g., retinal aldehyde dehydrogenase) will lead to a more “anteriorized” structure. At the molecular level, retinoic acid binds to its receptors (transcription factors) inside the cell, and their activation will regulate the expression of downstream genes. Hox genes are crucial targets of retinoic acid receptors in development. Because of their profound influence on early development, retinoids are powerful teratogens, especially during the first trimester.

Transforming Growth Factor-β/Bone Morphogenetic Protein

Members of the TGF-β superfamily include TGF-β, BMPs, and activin. These molecules contribute to the establishment of dorsoventral patterning, cell fate decisions, and formation of specific organs and systems, including the kidneys, nervous system, skeleton, and blood. In humans, there are three different forms of TGF-β (isoforms TGF-β ₁ , TGF-β ₂ , and TGF-β ₃ ).

Binding of these ligands to transmembrane kinase receptors results in the phosphorylation of intracellular receptor-associated Smad proteins (R-Smads) ( Fig. 20.3 ). The Smad proteins are a large family of intercellular proteins that are divided into three classes: receptor-activated (R-Smads), common-partner Smads (co-Smads [e.g., Smad4]), and inhibitory Smads (I-Smads). R-Smad/Smad4 complexes regulate target gene transcription by interacting with other proteins or as transcription factors by directly binding to DNA. The diversity of TGF-β ligand, receptor, and R-Smad combinations contributes to particular developmental and cell-specific processes, often in combination with other signaling pathways.

Sonic Hedgehog

Sonic hedgehog (SHH) was the first mammalian ortholog of the Drosophila gene hedgehog to be identified. SHH and other related proteins, such as desert hedgehog and Indian hedgehog, are secreted morphogens critical for early patterning, cell migration, and differentiation of many cell types and organ systems. Cells have variable thresholds for response to the secreted SHH signal. The primary receptor for SHH is Patched (PTCH in human, PTC family in mouse), a transmembrane domain protein. In the absence of SHH, Patched inhibits the transmembrane domain, G-protein–linked protein called Smoothened (Smo). This results in inhibition of downstream signaling to the nucleus. However, in the presence of SHH, PTC inhibition is blocked and downstream events follow, including transcriptional activation of target genes, such as Ptc-1, Engrailed, and others ( Fig. 20.4 ).

Posttranslational modification of SHH protein affects its association with the cell membrane, formation of SHH multimers, and the movement of SHH, which, in turn, alters its tissue distribution and concentration gradients.

The role of SHH in patterning of the vertebrate ventral neural tube is one of its best-studied activities. SHH is secreted at high levels by the notochord, and therefore the concentration of SHH is highest in the floor plate of the neural tube and lowest in the roof plate, where members of the TGF-β family are highly expressed. The cell fates of ventral interneuron classes and motor neurons are determined by the relative SHH concentrations in the tissue and other factors.

The understanding of the requirement of SHH pathway signaling for many developmental processes has been enhanced by the discovery of human mutations of members of the SHH pathway. In addition, corresponding phenotypes of genetically modified mice, in which members of the SHH pathway are either inactivated (loss of function/knockout) or overexpressed (gain of function), have also added to this knowledge. Mutations of SHH and PTCH have been associated with holoprosencephaly in humans, a common congenital brain defect resulting in the fusion of the two cerebral hemispheres, dorsalization of forebrain structures, and anophthalmia or cyclopia (see Chapter 17 ). In sheep, this same defect has been associated with in utero exposure to the teratogen cyclopamine, which disrupts SHH signaling (see Fig. 20.4 ). Gorlin syndrome, often due to germline PTCH mutations, is a constellation of congenital malformations mostly affecting the epidermis, craniofacial structures, and nervous system. Mutations of the GLI3 gene, encoding a zinc finger that mediates SHH signaling, are associated with autosomal dominant polydactyly syndromes.

In vertebrates, the SHH signaling pathway is closely linked to primary cilia (see Fig. 20.4 , inset) and their constituent intraflagellar transport (IFT) and basal body proteins. Primary cilia are sometimes referred to as nonmotile cilia . IFT proteins act upstream of the GLI activator (GLI-A) and repressor (GLI-R) proteins and are necessary for their production. Mutations involving genes encoding basal body proteins, such as KIAA0586 (formerly TALPID3) and oral-facial-digital syndrome 1 (OFD1), affect SHH signaling in knockout mice. A group of human cilia-related diseases called ciliopathies results from disruption of primary cilia function and includes rare genetic diseases and more common disorders such as autosomal recessive polycystic kidney disease. To date, almost 40 ciliopathies have been described involving up to 200 genes. Although there may be some overlap (as with many congenital heart defects and left-right asymmetries), diseases of primary, nonmotile cilia are usually distinguished from disorders affecting motile cilia (found in sperm and in epithelial cells lining the airways, ventricles of the brain, and oviducts). Manifestations of diseases affecting motile cilia include hydrocephalus, lung infections, and infertility.

Wnt/β-Catenin Pathway

The Wnt-secreted glycoproteins are vertebrate orthologs of the Drosophila gene Wingless. Similar to the other morphogens, the 19 Wnt family members control several processes during development, including establishment of cell polarity, proliferation, apoptosis, cell fate specification, and migration. Wnt signaling is a very complex process; three Wnt signaling pathways have been elucidated to date. Only the classic or “canonical” β-catenin–dependent pathway is discussed here ( Fig. 20.5 ). Specific Wnts bind to 1 of 10 Frizzled (Fzd) seven-transmembrane domain cell surface receptors, and with low-density, lipoprotein receptor–related proteins 5 and 6 (LRP5/LRP6) coreceptors, thereby activating downstream intracellular signaling events. In the absence of Wnt binding, cytoplasmic β-catenin is phosphorylated by glycogen synthase kinase 3 (GSK-3) and targeted for degradation. In the presence of Wnts, GSK-3 is inactivated and β-catenin is not phosphorylated and accumulates in the cytoplasm. The β-catenin translocates to the nucleus, where it activates target gene transcription in a complex with T-cell factor (TCF) transcription factors. β-Catenin/TCF target genes include vascular endothelial growth factor (VEGF) and matrix metalloproteinases.

Dysregulated Wnt signaling is a prominent feature in many developmental disorders, such as Williams-Beuren syndrome (heart, neurodevelopmental, and facial defects), and in cancer. LRP5 mutations are found in the osteoporosis-pseudoglioma syndrome (congenital blindness and juvenile osteoporosis). Similar to the SHH pathway, canonical Wnt pathway mutations have been described in children with medulloblastoma, a common pediatric malignant brain tumor.