Trophic Factors and Regulation of Gastrointestinal Tract and Liver Development

Introduction

To understand the role of trophic factors in fetal and neonatal gastrointestinal (GI) tract and liver growth, it is important to consider the elements of tissue growth. The fetal and neonatal period is the most dynamic period of postconceptual growth and includes critical developmental milestones, such as gastrulation, organogenesis, morphogenesis, cellular differentiation, and functional maturation, which are described in detail in other chapters. In the case of the intestine, key developmental steps include formation of the gut tube, the appearance of villi and digestive enzymes, and the development of swallowing and mature motility patterns. Intestinal growth also encompasses different elements at the tissue and cellular level that are characterized by increased cellular numbers (hyperplasia) and size (hypertrophy). Intestinal growth involves expansion of the number and size of mucosal crypt and villus units, as well as submucosal tissues, such as smooth muscle, neural, lymphoid, and immune cells. Moreover, the timing and characteristics of fetal and neonatal GI tract and liver growth are exquisitely coordinated with the events of birth and weaning to ensure survival of the organism. The regulation of the timing and nature of GI tract and liver growth is complex and involves multiple and often redundant factors. Among these factors are intrinsic cell programs or signals arising from gene expression, as well as extracellular signals, such as peptide growth factors, hormones, nutrients, and microbes, which originate from surrounding cells, the blood, and the gut lumen.

A fundamental aspect of growth in the gut is the continual proliferation, migration, and apoptotic loss of epithelial cells along the mucosal surface. In the small intestine this process involves four major cell lineages (absorptive enterocyte, goblet, Paneth, and endocrine cells) that differentiate from one pluripotent stem cell located in the crypt ( Fig. 81.1 ). The application of molecular biologic techniques in model organisms such as the mouse, zebrafish, fruit fly, and nematode has revealed that genetic regulatory factors have a critical influence on early organogenesis and morphologic development of the gut, pancreas, and liver. ^, These studies have identified a group of key homeodomain transcription factor genes, Pdx1 and Cdx2 ; GATA transcription factors, Gata4 and Gata6; signaling pathways, including Wnt/β-catenin, Hedgehog, and Notch; and new extracellular signals, including R-spondins. ^, Recent studies have identified several genes that play a key role in the determination of lineage cell commitment in the intestinal stem cell niche ( Fig. 81.2 ), including ATOH1 , Notch1 , Notch2 , Hes1 , and Neurog3 . The homeodomain transcription factors are also involved in the anterior-posterior pattern formation of demarcations in muscular sphincters and regional epithelial morphology along the length of the gut. ^, Genetic regulation via homeodomain factors also influences functional maturation of the intestine that occurs during the late-gestation and neonatal period.

Development of in vitro models of fetal and neonatal GI physiology in the form of intestinal enteroids and organoids has further illuminated the role of crypt cells and growth factors in the proliferation and maintenance of the intestinal epithelium. Intestinal organoids are specifically representative of fetal tissue and have been used to examine signaling pathways that determine the eventual developmental fate (endoderm, mesoderm, or ectoderm) of pluripotent stem cells, from gastrulation onward. Transforming growth factor β (TGFβ) initiates gastrulation, but Wnt, fibroblast growth factors (FGF), retinoic acid (RA), and bone morphogenic protein (BMP) pathways are essential for the development of the intestinal epithelium. Mesenchyme underlying intestinal epithelium produces laminins, which are also necessary for villus formation. Conversely, enteroids are cultured from LGR5+ stem cells present in the base of intestinal crypts and represent the physiology of the tissue the LGR5+ cells were isolated from. Paneth cells alternate with LGR5+ stem cells and produce Wnt3 (see Fig. 81.2 ), which maintains stemness of LGR5+ pluripotent stem cells. In enteroid culture, the Wnt3 signal is amplified by R-spondin to maximize enteroid proliferation and survivability, while epidermal growth factor (EGF) has a similar, profound mitogenic effect on stem cells. In intestinal tissue, BMP signaling is greatest at the villus tip, and lowest at the crypt base, driving terminal differentiation and repressing stemness; therefore Noggin is employed in enteroid culture to maintain the proliferative environment of the crypt compartment.

Environmental Influences

Normal GI tract growth and development during fetal life are critical to facilitate the successful adaptation from nutritional support via the umbilical circulation to that of oral ingestion of breast milk by the neonate. An increase in circulating fetal glucocorticoid concentration just before and during vaginal birth is an important trigger of gut functional development. In the neonatal period, growth of the GI tract is influenced by multiple physiologic factors that serve to prepare the developing neonate for separation from maternal nutritional support (i.e., weaning). In addition, a number of important environmental cues signal adaptive changes in GI tract function to facilitate postweaning survival. The extent and diversity of microbial colonization of the gut serve to prime intestinal lymphoid cell development for normal innate immune function. During these processes, extracellular signals, such as peptide growth factors, are often considered to be the major trophic factors that influence growth. However, the term trophic actually means “of or pertaining to nutrition,” and in the case of the gut, nutrients present in amniotic fluid and breast milk are a major trophic influence. There are numerous extracellular trophic signals, including foods, nutrients, peptide growth factors, biliary and pancreatic secretions, gut peptide hormones, steroid and thyroid hormones, microbes, and neural inputs. The cells within the fetal and neonatal GI tract and liver are influenced by extracellular signals from multiple sources, including (1) blood-borne factors in the circulation such as hormones that act via endocrine mechanisms; (2) luminal factors derived from amniotic fluid, mammary secretions, or gut microbiota; and (3) local factors secreted via autocrine or paracrine mechanisms from surrounding cells ( Fig. 81.3 ).

Recent studies have revealed novel insights into the role of mesenchymal cells, especially myofibroblasts, and luminal microbes in intestinal epithelial growth and differentiation. Mesenchymal cell interactions are mediated via local secretion of tissue growth factors—for example, FGF, hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), and insulin-like growth factor (IGF)—and basement membrane proteins (laminins, collagens, proteoglycans). The extracellular matrix protein laminin may be particularly critical for expression of Cdx-2 and induction of epithelial differentiation genes, such as lactase. Interaction between the integrin receptors on the basal surface of epithelial cells with basement membrane proteins can also affect differentiation and cell death. Studies have shown that disruption of the integrin-receptor binding via protease degradation leads to detachment of epithelial cells from the basement membrane and activation of anoikis-mediated apoptotic signaling pathways. Symbiotic relationships between commensal microorganisms and their mammalian host, such as ruminants, are well known. Yet in the past 10 years, molecular sequencing techniques and metagenomic analysis have markedly expanded our understanding of the microbiome and how it influences the health and disease of the human and mouse gut. Studies in conventional and germ-free animals demonstrated that the presence of intestinal microbes exerts a trophic effect on the gut, as evidenced by increased epithelial cell proliferation, mucosal thickness, and lymphoid cell density. Studies with germ-free mice monoassociated with specific bacterial species show that microbes can alter particular pathways of intestinal differentiation. The emergence of molecular DNA analysis has led to the suggestion that the fetal gut contains a low abundance of microbes, but increased colonization and microbiome diversity during early postnatal life plays a critical role in development of mucosal immune function. Microbes also produce a wide range of bioactive molecules and substrates, including toxins and short-chain fatty acids (SCFAs) that influence the proliferation and function of mucosal epithelial and immune cells. The aim of this chapter is to provide a brief overview of some of the major trophic factors in these categories and discuss their relevance to the growth and development of the fetal and neonatal gut and liver. Most of the references cited in this chapter are review articles, and the number of original articles listed is limited owing to space restrictions.