Liver organogenesis

Liver function

The liver is a large visceral organ, thought to have around 500 different functions. These functions include bile production (to aid food digestion), blood filtration (to extract nutrients, store vitamins and neutralise pathogens), energy store (large stores of glucose and fat are maintained), xenobiotic neutraliser (including alcohol) and protein producer (including albumin).

Liver structure

Four lobules make up the liver (Figure 1). In the adult liver hepatocytes are arranged in “queues” known as cords (Figure 3). These cords are actually 3-D towers and this structure allows blood to bathe the cells on two sides. Multiple cords gather into lobules (idealised as hexagon structures, Figure 2). Each lobule contain cords centred around a hepatic vein with branches of the hepatic portal vein, hepatic artery and bile ducts at each corner of the hexagon (Figure 2). 

Figure 1: Gross liver morphology

Figure 2: Liver lobule morphologyFigure 3: Detailed hepatocyte morphology

Cells found in liver

Hepatocytes are the main liver cells with other cell types regulating the immune system and lining bile ducts (see table 1).

Table 1: Cell Types of the Adult Liver

Differentiation

Differentiation is the process by which gene expression is changed in order to transform the morphology and function of a cell. While every cell contains the same genetic code, it is possible to “switch” different genes on and off (or indeed turn expression up or down). There are many stages at which this process can be regulated including controlling transcription through histone binding to DNA or DNA methylation.

Induction is the process by which one group of cells cause another set of cells to change their fate. Induction is an important process for organogenesis and is often controlled by paracrine signalling. In paracrine signalling, molecules diffuse from one cell to interact with another cell via a receptor. Often enzymatic activity will be triggered in the receptor, which in turn can cause a signalling cascade, eventually leading to activation of a transcription factor (see Figure 4). The transcription factor can then alter expression of genes in the nucleus and affect cell fate. There are 4 main groups of paracrine signal; Fibroblast growth factors (FGFs), Hedgehog proteins, WNT proteins and TGF-β superfamily. 

Figure 4: paracrine signalling

Developmental directions

Anterior = head side

Posterior = tail side (or spinal cord end)

Dorsal = back (spinal side) 

Ventral = front side

Proximal = close to the body

Distal = far from the body

(see diagram 5.)

Diagram 5: Developmental directions

Embryo development

Development starts after fertilisation. At this point several round of mitosis are triggered as the fertilised egg splits into 2, 4 then 8 cell blastomeres onto a 16 cell morula and then a blastocyst stage (see figure 6). Gastrulation is the next most important process where embryo generates 3 germ layers; endoderm, mesoderm and ectoderm. Inside the endoderm layer the primitive gut will form by ventral folding of the lateral body wall. While the primitive gut is still closing a retinoic acid gradient causes different transcription factor expression along the gut segregating it into foregut, midgut and hindgut. Eventually the endoderm closes to form continuous endoderm and visceral mesoderm layers.

Figure 6: mitosis rounds after fertilisation

Development of the liver

The liver develops from foregut endoderm. This area will produce everything of endoderm derivative from the mouth down to the duodenum in the small intestine. The liver starts from cells at the anterior portion of the endoderm, close to the lateral plate mesoderm (an area of mesodermal cells, at the edge of the embryo, that will go on to form the circulatory system, gut wall and body wall). The endoderm cells that will form the liver express Foxa proteins, identifying it as “future liver” and labelling foregut as different from midgut and hindgut (Figure 7). 

Figure 7: position of developing liver in embryo

Liver bud development

The lateral plate mesoderm is divided during development and part of it is partitioned into the septum transversum mesenchyme (STM). This is an area of cells and connective tissue that primarily goes on to develop into the thoracic diaphragm (Figure 7). The STM is posterior to the heart in the developing embryo and expresses GATA4 a transcription factor which will in turn causes BMP (bone morphogenetic protein, a member of the TGF-β superfamily) expression. The cardiogenic mesoderm (cells that will later become the heart) secrete growth factors including FGF1 and FGF2 in such a way as to create a gradient.  BMP and FGF signalling are able to direct hepatogenesis triggering liver bud development into the STM. HHEX (hematopoiecally-expressed homeobox protein) expression early in liver development also regulates hepatic proliferation, position and formation of the liver bud. Formation of the liver bud occurs before the primitive gut has finished closing and the initial liver bud is mainly composed of hepatoblasts. Hepatoblasts are a liver stem cell that can later be turned into hepatocytes or cholangiocytes, cells which line bile ducts.

Liver bud expansion

To grow a full liver from the liver bud hepatoblasts must replicate. Signals, including homeobox-like protein (Hlx) and Hepatocyte Growth Factor (HGF), continue to be expressed from the STM, causing liver growth. For the liver bud to invade into the STM the cells need to move the laminin, collagen and fibronectin present and this involves activation of matrix-metalloproteinases. PROX1 Prospero homeobox protein 1 (PROX1) expression by hepatoblasts allows break down of cell-attachment proteins to allow cell migration. Hepatoblast migration is then regulated by HHEX and hepatocyte nuclear factor 6 (HNF6). 

After hepatoblast migration many cells differentiate into hepatocytes which involves the activation of transcription factors; HNF1a, HNF1β, HNF4a1, HNF6, FoxA2 and LRH-1. These factors are able to activate each others promoters causing a positive feedback cycle for hepatocyte differentiation. 

Morphology and function development in the liver

Figure 8: changes in liver structure during organogenesis

After the liver expands in size and hepatocytes are formed it becomes important to regulate morphology of the organ (Figure 8). Liver morphology is controlled by Wnt2/β-catenin signalling, this regulates lobule formation. Normally β-catenin gets sequestered in a protein complex in the cell cytoplasm; however when Wnt binds to its receptor β-catenin is released and is able to translocate to the nucleus and effect gene expression. HGF has been demonstrated to increase β-catenin nuclear translocation. β-catenin translocation is thought to increase hepatocyte expansion and maturation through increasing cell-cell adhesion.

A summary of regulation in liver development is shown in figure 9.

Figure 9: signalling molecules involved in liver differentiation

Figure 9: key regulators for each stage of liver development are labelled with bullet points.

Formation and interaction of other liver cell structures

Different cell types need to be able to interact with each other in the liver in order for correct liver growth. There is an important role for stellate cells in the liver as control of their differentiation is regulated by Wilm’s Tumor 1 protein. When stellate cells are fully differentiated they can correctly store vitamin A and therefore control retinoic acid production. Retinoic acid controls hepatocyte reproduction and has a role in regulating liver size. Stellate cells are also responsible for fibrosis in adult liver damage as these cells become activated after liver damage. Stellate cell activation involves proliferation, increased motility and secretion of collagen, thereby causing cirrhosis and fibrosis.

Similarly, vacuolisation of the liver starts with hepatic progenitor cells interacting with stromal cells causing vascular endothelial growth factor (VEGF) expression. Capillary structures result from VEGF expression. After capillaries are formed they express HGF and interleukin 6 (IL6) which leads to further hepatocyte proliferation. After capillaries are formed hematopoietic cells are found in the liver either from circulation or production by the liver. Hematopoietic cells express Oncostatin M which enhances hepatocyte differentiation.

Recent work has demonstrated that bile duct formation occurs via a unique form of tubulogenesis. Primitive ducts are formed next to the portal vein. Newly differentiated cholangiocytes line ducts closest to the portal vein and hepatoblast initially complete the primitive duct further away from the portal vein. To complete the duct hepatoblasts differentiate into more cholangiocytes, a process controlled by asymmetric binding of TGF-β leading to expression of Sox-9, hepatic nuclear factor 6 (HNF6) and FoxA1/A2 and repression of HNF4, this is shown in Figure 10.

 

Figure 10: cell patterning during bile duct formation

Additional information

Liver function change after birth

After birth the liver has to change function. It stops producing blood cells and becomes responsible for regulating the body’s metabolism. To help prepare for this cortisol causes the liver to store glycogen before birth. During and after birth, cortisol increases the rates of gluconeogenesis.

Liver regeneration

The last important fact about the liver is that it shows a rare trait in the human body of being able to regenerate cells and a functional liver can re-grow from just 25%. While the term regeneration is often used in terms of the liver, it is not a true regeneration as dedifferentiated cells are not produced. It is more a compensatory growth of the remaining tissue. It is thought that cells similar to fetal liver cells are retained by the adult liver and that these react to the same cytokine signals that cause fetal liver development during liver regeneration. Multiple pathways are responsible for liver regeneration, these overlap and are redundant to ensure it can always happen. Signals controlling hepatocyte growth after two thirds of the liver was surgically removed in mice are; tumour necrosis factor-alpha (TNF-α), IL-6, NFκB, STAT-3, β-catenin, transforming growth factor-alpha (TGF-α), epidermal growth factor (EGF) and HGF. Nutrient sensing by the liver is thought to control size as it grows dependant on nutrient availability.

Related reading

Sadler, T. W. (2011). Langman’s Medical Embryology (Vol. 2011). Lippincott Williams & Wilkins. 

Zaret, K. S. (2002). Regulatory phases of early liver development: paradigms of organogenesis. Nature reviews. Genetics, 3(7), 499–512. 

Si-Tayeb, K., Lemaigre, F. P., & Duncan, S. A. (2010). Organogenesis and development of the liver. Developmental cell, 18(2), 175–89. 

Thompson & Monga (2007) WNT/β‐catenin signaling in liver health and disease. Hepatology.

Kung, J., Currie, I., Forbes, S., & Ross, J. (2010). Liver development, regeneration, and carcinogenesis. Journal of Biomedicine and Biotechnology.

Antoniou et al (2009) Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9. Gastroenterology, 136(7), 2325–33. 

Acknowledgments

Picture 1 courtesy of cooldesign/ FreeDigitalPhotos.net

Thanks to Dr. Karen Piper Hanley for help editing.

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