Early developmental influences on hepatic organogenesis

Abstract

The liver is the largest of the body's organs, with the greatest number of functions, playing a central role in coordinating metabolic homeostasis, nutrient processing and detoxification. The fetal liver forms during early gestation in response to a sequential array of distinct biological events, regulated by intrinsically programmed mechanisms and extracellular signals which instruct hepatic cells to either proliferate, differentiate or undergo apoptosis. A vast number of genes are involved in the initiation and control of liver development, many of which are sensitive to nutritional and hormonal regulation in utero. Moreover, liver mass is influenced by the gestational environment. Therefore, during periods of hepatic cell proliferation and differentiation, the developing fetal liver is sensitive to damage from both internal and external sources including teratogens, infection and nutritional deficiencies. For example, fetuses exposed to decreased materno-fetal nutrition during late gestation have a reduced liver mass, and/or perturbed liver function, which includes increased plasma LDL cholesterol and fibrinogen concentrations. These occur in conjunction with other risk factors present in the early stages of cardiovascular disease i.e. decreased glucose tolerance and insulin insensitivity in later life. Taken together, these findings suggest that liver mass, and later function, are essentially set in utero during fetal development—a process that is ultimately regulated by the intrauterine environment.

Introduction

Fetal liver development represents an array of distinct biological events i.e. foregut endoderm, liver primordium, mid-fetal liver, neonatal liver and adult liver, dependent upon the molecular and structural properties of liver tissue.¹ The intention of the present review is to focus on how the gestational environment may influence the latter stages of liver development that encompass a functional switch from fetal to adult liver. In order to determine the exact role of adequate placentation, prenatal nutrition and the endocrine environment upon hepatic organogenesis it is essential to understand the role of the liver in both fetal and postnatal life and the cellular and molecular events that regulate its development.

Both the location and structure of the liver are critical to its role within the body as a central regulator of metabolic homeostasis which may be compromized during liver injury.² The liver is a remarkable organ, in that it is responsible for maintaining overall metabolic homeostasis and detoxification. In addition, it performs an important endocrine role through the synthesis and secretion of hormones and growth factors which are essential for the promotion of postnatal growth.^3–5 Consequently, the successful development and functional maturation of the fetal liver is vital to fetal and neonatal survival as well as later health.^4,6

Approximately 78% of the adult liver consists of parenchymal hepatocytes,⁷ whilst the remaining liver is comprised of non-parenchymal cells including sinusoidal endothelial cells (SEC), Kupffer cells (hepatic macrophages) and hepatic stellate cells (HSC; fat storing cells).⁸ Due to their high energy requirements, hepatocytes, unlike other endothelial cells, possess vast numbers of organelles.⁹ Hepatocytes can contain as many as 2000 mitochondria, whereas relatively inactive cells such as lymphocytes have just a few mitochondria.⁴ Hepatocytes are histochemically and metabolically heterogeneous with their primary function varying according to their location within the liver. Such hepatocyte heterogeneity, referred to as metabolic zonation, is determined by the distance of hepatocytes from the terminal portal and arterial branches which determine their nutritional and oxygen environment.^10–12 For example, periportal hepatocytes, supplied by the terminal afferent vessels, function in an environment rich in oxygen, substrates and hormones and are predominantly gluconeogenic. In contrast, perivenous hepatocytes, located around the central vein, are exposed to blood partially depleted of nutrients and oxygen and are, therefore, glycolytic.

Functional maturation of liver tissue is characterized according to the expression of liver- and stage-specific genes.^13,14 Alpha-fetoprotein, for example, decreases in abundance as the fetal liver develops.¹⁵ Further markers of metabolic development include glucogenic proteins: Phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase). The fetus does not produce glucose until the prepartum cortisol surge in late gestation,^16,17 relying prior to that on placental uptake of maternal glucose into the fetal circulation. Coincident with the late gestational prepartum cortisol surge, there is a corresponding increase in hepatic PEPCK and G-6-Pase activity17 and a three-to-four-fold increase in glycogen content, which provides the major source of glucose for the newborn infant. However, when exposed to excess glucocorticoids and/or an inadequate nutritional supply, the compromised fetus can prematurely synthesize glucose in late gestation either by glycogenolysis or gluconeogenesis.^18,19

Liver Regeneration and Hepatic Growth Control Mechanisms

Hepatocytes are long-lived cells with a lifespan of approximately 150 days. In normal livers, parenchymal cells rarely divide.⁴ However, upon resection or severe injury, hepatocytes proliferate rapidly to restore organ mass.²⁰ Liver regeneration studies provide a unique means of investigating, not only the cellular and molecular mechanisms that govern liver regeneration, but also organogenesis. Consequently, growth control mechanisms responsible for hepatocyte proliferation and apoptosis common to organogenesis, maintenance of liver size and regeneration have now been identified²¹ and have been extensively reviewed.^1,22–26 Briefly, cross talk between the cell matrix, hepatocytes, and non-parenchymal cells regulate liver growth and encompasses both pro-mitogenic: Pro-inflammatory cytokines (TNFα and IL-6) and growth factors (HGF, EGF, FGF, TGFa),^27,28 and inhibitory apoptotic (TGF-β1 and Bcl-2) mechanisms.^1,3,29,30 Insulin, glucagon, noradrenalin, GH and thyroid hormones further regulate liver growth by enhancing the proliferative potential of mitogens, despite possessing little intrinsic mitogenic activity.^31,32

Growth factors play a major role in the maturation of organ systems prior to birth.¹⁷ For example, in the fetus, IGFs, synthesized by the placenta and fetal liver,³³ mediate growth³⁴ under the regulation of glucose and insulin.³⁵ The ontogenies of a number of genes that have a critical role in regulating both liver growth and development in utero, as well as postnatal growth and endocrine sensitivity,^36,37 have recently been determined in the sheep.³⁸ Key hepatic growth factors (with the exception of IGF-IIR and IGF-I) were found to increase throughout gestation reaching peak levels either during late gestation (PRLR) or at birth (GHR, IGF-IR and GR) and, thereafter, to decrease, signifying the perinatal period as an important time point in the regulation of liver growth and maturation.³⁸

Developmental Programming of Hepatic Organogenesis

Fetal growth and development is dependent on genetic, placental and maternal factors. In the presence of an adequate gestational environment, the fetus will grow to an appropriate size according to its inherent growth potential. However, in utero perturbations that may be intrinsic or extrinsic (see Table 1) can potentially affect the growth and development of the fetus and may play a critical role in the developmental programming of later disease susceptibility^39–41 and, in severe cases, may lead to intrauterine growth restriction (IUGR) and failure to thrive postnatally.^41,42

Table 1.

Intrinsic and extrinsic factors influencing fetal growth and development

Factor		Reference
	Intrinsic
Genetics		47–48
Ethnicity		49,50
Fetal number		51
Hormones e.g. GH		52
Growth factors e.g. IGFs.		53,54
Infection		55
	Extrinsic
Pre-pregnancy weight		56,57
Maternal (uterine) size		58,59
Maternal age		60–62
Maternal nutrition		63
Maternal parity		64
Maternal body condition		65
Maternal smoking		66–68
Alcohol consumption		69
Socio-economic factors		70,71
Placental insufficiency		72
Placental vascularisation		73
Paternal height		74

Asymmetrically growth retarded fetuses, for example, are disproportionate in appearance and are characterized by a relative preservation of length and head circumference, whilst body weight is low (due mainly to a lower proportion of visceral and adipose tissue). Asymmetric IUGR is sometimes referred to as a ‘wasted’ as opposed to a ‘stunted’ (symmetrical) phenotype and may result from uteroplacental insufficiency and/or compromised nutritional supply during critical periods of development. These IUGR fetuses, if hypoxic, experience shunting of fetal blood flow, via the ductus venosus, to preserve the development of important organs, such as the brain, at the expense of the liver and kidneys which are hypoperfused.⁴³ This adaptation, known as fetal brain-sparing, may account for the disproportionate growth retardation and compromised liver growth leading to thinness/shortness at birth^39,44 and perturbed LDL cholesterol and fibrinogen metabolism in later life.^45,46

Animal Models

Direct human research on potential regulators of fetal liver development is limited by ethical considerations. Consequently, animal models offer an alternative method of investigating the pathophysiologic aspects of the developmental origins of health and disease (DOHaD) hypothesis (for a review see refs. ^75–77). The advantage of animal models over retrospective epidemiological studies, such as the Dutch famine,^39,78 is that the effect of nutrition, for example, can be largely assessed independently of confounding factors such as genetics and social status whilst controlling for fetal number, gender, parity and maternal body condition. Using animal models, it is possible to manipulate a number of aspects of fetal development including placental efficiency, maternal nutrition and hormonal balance at specific stages of gestation (e.g. embryonic, placental and fetal growth), thus allowing critical periods in hepatic organogenesis to be targeted. The most commonly used animal models of fetal programming are rats and sheep. The pregnant rat is an established animal model of fetal programming and is susceptible to long-term effects of maternal nutrient restriction which include altered growth and functional development.^79,80 There are a number of advantages of using rodents as models for fetal programming including, for example, short gestation (rat: 21 days) allowing for a high turnover in, and the opportunity for, generational studies. However, there are also a number of important physiological limitations, which include their high specific growth and metabolic rates and the inability to cannulate the fetal rat, thus hindering fetal physiological analyses. In contrast, sheep are large, relatively domesticated animals that develop bonds with their routine handlers, thus allowing experimental implementations and physiological and biochemical readings to be easily taken in a relatively unstressed state. The major advantage, over the rat, of using the pregnant sheep as an animal model is its close parallels with humans. The sheep is a monotocus species and is of comparable birth weight, fetal to maternal body weight ratio, metabolic rate, organogenesis and growth rate to humans. Due to its long gestational period and tolerance for intrauterine surgery, catheterization and chronic instrumentation without premature delivery, the pregnant ewe is considered the best large animal model to investigate fetal programming and is routinely used to study fetal growth, metabolism and endocrinology despite species differences in maternal and placental metabolism. Furthermore, restriction of nutrient intake to a comparable energy intake to that experienced during the Dutch Hunger Winter in pregnant sheep (i.e. to 50–60% metabolisable energy requirements) has been shown to similarly perturb fetal development, resulting in increased offspring blood pressure and glucose intolerance in later life.^81–84

Numerous sheep studies have reported a link between various maternal constraints upon pregnancy including placentation, body condition, parity, adolescence, ambient temperature, fetal number and nutrition on liver growth, maturation and function in the resultant offspring.^{16,38,62,76,81,85–89} Such constraints upon fetal development may result in an increase or decrease in offspring liver mass depending upon the timing, duration and severity of the gestational insult. That said, hepatic cellular and molecular adaptations also occur in the presence of normal liver growth. Therefore, developmental regulation of hepatic organogenesis and later function is not limited to alterations in liver mass, per se.^86,90,91

Nutritional Regulation of Liver Development

Epidemiological studies in several countries have suggested that size at birth and/or placental development predict adult health.^6,39,92 The Dutch Hunger Winter (1944–1945) provides a prominent example of the importance of adequate nutrition during pregnancy for later health.⁷⁸ Offspring born to mothers exposed to famine during early gestation had a three-fold increase in cardiovascular disease,⁹³ raised fibrinogen and decreased factor VII levels (in response to impaired liver development),⁹⁴ increased risk of obesity (women only)⁹⁵ and a more atherogenic lipid profile.⁹⁶ Moreover, men born with a decreased waist circumference, a proxy for compromised liver growth, have deleterious alterations in LDL cholesterol and fibrinogen metabolism in association with a greater mortality from coronary heart disease.^6,45,46 Taken together, epidemiological data imply that hepatic organogenesis is susceptible to nutritional reprogramming and that impaired liver development in utero can have long lasting functional consequences on disease risk in later life.

In the sheep, fetal liver growth is sensitive to changes in the materno-fetal nutrient supply. However, the magnitude of hepatic adaptation is dependent upon the timing and duration of the nutritional insult and on fetal number. For example, fetal and adult liver growth is markedly impaired by severe⁹⁷ and/or prolonged (encompassing a number of critical windows)¹⁶ periods of nutrient deprivation, respectively. Furthermore, liver mass is reduced in twin compared with singleton offspring and in singleton lambs exposed to maternal nutrient restriction during late gestation.⁸⁸

Nutritional manipulation of liver growth is accompanied by a range of endocrine adaptations including increased plasma GH and decreased plasma IGF-I and IGF-II, glucose and insulin concentrations,^97,98 together with decreased expression of hepatic GH and IGF-II receptors.^16,38,86

In contrast, maternal nutrient restriction between early to mid gestation, coincident with the period of maximal placental growth,⁹⁹ has no effect on liver mass in singleton fetuses,^38,91,100 although an increase in liver weight has been observed when both singleton and twin pregnancies were studied.¹⁰¹ Absolute, but not relative, liver mass is also reduced in lambs born to adolescent mothers that were undernourished during mid-gestation.⁸⁵ Furthermore, maternal nutrient restriction, during early to mid-gestation, increases hepatic glucocorticoid sensitivity¹⁰⁰ which is associated with alterations in the balance between mitogenic (GHR, PRLR and IGF-IIR) and pro-apoptotic (Bax and VDAC) proteins.³⁸ These data, therefore, indicate that exposure to an adverse nutritional environment in early pregnancy has a long term impact on liver growth. However, additional studies are needed to ascertain whether reduced liver mass and expression of growth factors in the nutrient restricted sheep affect its function and, indeed, whether such responses contribute to the developmental programming of poor health in later life.

Maternal Parity and Ambient Temperature Influences Liver Development

Maternal parity influences offspring birth weight and growth trajectories in a range of species.^64,102–107 At birth, offspring born to nulliparous mothers tend to be lighter and of asymmetric body proportion with a low ponderal index and head circumference. Furthermore, firstborn offspring have significantly reduced liver weight possibly in response to in utero fetal brain-sparing.⁸⁶ Importantly, epidemiological studies have shown asymmetric offspring experience ‘catch-up’ growth (for both weight and height) over the first twelve months of life¹⁰⁸ which places them at an increased risk of being overweight in later life and for the development of the metabolic syndrome.^109–112

Placental Insufficiency and Hepatic Organogenesis

The placenta forms an interface between the mother and her fetus providing a number of functions essential for fetal growth and survival. Placental efficiency is determined by a number of factors including surface area, vascularity and permeability, uterine and umbilical flow rates and substrate transfer.^73,113 Placental insufficiency with respect to reduced transfer of oxygen and substrate is a major cause of IUGR and is associated with increased morbidity and mortality in the perinatal period and onset of disease in later life.¹¹⁴

The role of the placenta in fetal development, IUGR and later disease has been extensively investigated in the sheep and recently reviewed.⁷⁶ Placental restriction, induced via carunclectomy, embolization, hyperthermia or sub-optimal nutrition, impairs placental development by decreasing placental surface area, vascularity and permeability and/or by compromising uterine and umbilical flow rates. Placental dysfunction, regardless of the surgical/experimental intervention used, results in altered or reduced fetal growth, which may be accompanied by perturbed hepatic organogenesis and morphology. For example, in the sheep, four days of umbilical-placental embolization during late gestation significantly reduces the weight of the 118 day embolized fetus. Moreover, these fetuses have significantly smaller livers relative to their body weights.⁴⁰

Birth weight, body conformation and liver mass are also susceptible to changes in the thermal environment. For example, in human pregnancies maternal fever or exposure to tropical temperatures are associated with IUGR and premature delivery.^115,116 Chronic exposure of the pregnant ewe to a hyperthermic environment, commencing around 40 days gestation (term = 147 days), reduces placental growth rates resulting in placental insufficiency and asymmetric growth retardation. Resultant late gestational fetuses are lighter and have smaller livers and increased brain: Liver weight ratio.^117,118 As a consequence of a reduced substrate supply, fetuses are also hypoxic and hypoglycaemic in late gestation.^117,119 The primary cause of perturbed fetal development in heat stressed pregnancies is related to the degree of placental restriction and associated reduction in substrate supply to the developing fetus which, in males, results in the premature activation of the hypothalamic-pituitary-adrenal (HPA) axis.¹²⁰ In contrast, chronic cold exposure, induced via late gestation shearing, increases newborn liver mass, in response to a reduction in glycogen utilization and an associated increase in hepatic glycogen content.⁸⁹ Taken together, these studies indicate that extreme variations in the ambient temperature of the environment to which the mother is chronically exposed to during pregnancy is an important regulator of both fetal and placental development and also hepatic organogenesis. Moreover, such adaptations may have resulted from, or indeed caused, a reduction in the number of haematopoietic cell clusters within the livers of placentally restricted fetuses,⁴⁰ which may predispose the offspring to altered liver function in fetal and/or later life.

Summary

The fetal liver forms during early gestation in response to an array of intracellular mechanisms. A vast number of genes are involved in the initiation and control of liver development, many of which are sensitive to nutritional and hormonal regulation in utero. Consequently, liver mass and its later function are essentially set in utero during fetal development, a process that is ultimately regulated by the intrauterine environment. This review paper aimed to demonstrate some of the complexities of exposure to a sub-optimal gestational environment upon the developing fetal liver with respect to its mass and expression of key growth factors. Nevertheless, the precise mechanisms responsible for programming liver size remain unknown, although they may be mediated, for example, by nutritional alterations in both hepatic mitogenic and apoptotic factors.¹⁶ Further studies are needed to confirm whether the livers of nutrient restricted offspring are altered as a consequence of impaired organogenesis (altered cell number) or hepatic hypertrophy (altered cell size). Whether one or both of these final pathways are influenced depends upon the timing and severity of the gestational insult.

Abbreviations

SEC

sinusoidal endothelial cells

HSC

hepatic storage cells

PEPCK

phosphoenolpyruvate carboxykinase

G-6-Pase

glucose-6-phosphatase

HGF

hepatocyte growth factor

IGFs

insulin-like growth factors

TGFβ

transforming growth factor β

TNFα

tumor necrosis factor α

IL-6

interleukin 6

EGF

epidermal growth factor

FGF

fibroblast growth factor

GHR

growth hormone receptor

PRLR

prolactin receptor

GR

glucocorticoid receptor

IUGR

intrauterine growth restriction

LDL

low density lipoprotein

DOHaD

developmental origins of health and disease