Introduction

Blood cell development begins as early as the seventh day of embryonic life.[1] Red blood cells are essential in delivering oxygen to tissues and the development of vascular channels during embryogenesis. The ontogeny and maturation of these blood cell lineages is a complex process that involves two critical developmental steps: the production of primitive erythroid cells (EryP) followed by an expanding population of definitive erythroid cells (EryD) that predominate subsequently.[2] Failure of primitive erythropoiesis in toto can prove fatal to the embryo.

Development

The process of gastrulation begins with the epiblast, a single epithelial cell layer, transforming into three embryonic germ layers (ectoderm, mesoderm, and endoderm).[2] The first wave of primitive hematopoietic and endothelial cell development occurs via signals to the extraembryonic, endodermal yolk sac within the first two weeks of gestation, which results primarily in the formation of EryP, megakaryocytes, macrophages, and the endothelium.[2] These EryP cells are distinct from their erythroid progenitors in that they are larger, nucleated, have embryonic globins and are detected only in the yolk sac. EryP help in the formation of structures called blood islands in which the entrally placed cells give rise to erythroid and myeloid cells while peripherally placed cells form endothelial cells that form these channels. These blood islands fuse to form vascular channels that span throughout the yolk sac. Through these vascular channels, oscillatory plasma flows containing EryP cells and various other primitive cell types, which is stimulated by the developing heart.[2] Once in circulation, the EryP cells are enucleated by the fetal liver and macrophages clear the nuclei. EryP cells continue to form only for a short period once vascular channels develop in the yolk sac, while the remaining progenitor cells continue to mature from proerythroblast to orthochromatic erythroblast to reticulocytes and remain in the bloodstream until at least birth.[3]

Shortly after the development of primitive hematopoietic cells (EryP), a group of cells called highly proliferative, multipotent progenitor colony forming cells (HPP-CFC) arise in the yolk sac. These cells initiate the first wave of definitive hematopoiesis and produce cells of the definitive erythroid lineage. These cells are often called erythroid/myeloid progenitors, and they bridge the transition between primitive and hematopoietic stem cell (HSC) derived erythropoiesis.[2] These cells will migrate and begin to colonize the liver, which is the next definitive site of hematopoiesis during gestation.

The second wave of definitive hematopoiesis replaces primitive hematopoiesis and the first wave of definitive hematopoiesis. Hematopoietic stem cells (HSC) emerge from a specialized hemogenic endothelium within a limited region of the developing aorta's ventral wall called the para-aortic splanchnopleure. The aorta-gonad-mesonephros (AGM) region develops from the para-aortic splanchnopleure and produces HSC.[4] These cells colonize the fetal liver by the 7th week of gestation, where they cycle at a continuous pace and begin to differentiate. At this point, the liver becomes a significant source of hematopoietic stem cell production. HSC cells also colonize the spleen around week 20 and produce red cells for a brief period. A vital organ that HSC starts colonizing around this time is the bone marrow. HSC seeding in the marrow is critical because it is the bone marrow that will predominate in erythropoiesis as gestation advances. The fetal liver provides the microenvironment needed for expansion and differentiation of definitive HSCs, from which definitive erythroid cells will differentiate from a hierarchy of progenitors. HSC in the fetal liver and spleen produces enucleated erythrocytes (EryD) that rapidly outnumber EryP cells in circulation.

EryD cells express fetal hemoglobin (HbF) and are composed of two γ-globin chains and two adult alpha-globin chains. HbF remains the predominant hemoglobin for most of gestation.[5] A switch from the HbF to adult hemoglobin (HbA) occurs at about 32 weeks and continues after birth. There is a transcriptional change from gamma- to beta globin, marking the end of erythroid ontogeny.

Toward the third trimester of development, as skeletal components begin ossification and bone marrow is developing inside bony cavities, the marrow of specific bones will become the essential hematopoietic organ. Both the liver and spleen at this point cease erythropoiesis as the bone marrow predominates in hematopoietic cell production. In postnatal life, definitive erythropoiesis originates from the marrow (BM) that occurs under normal physiologic conditions. In infants, all spongy bone and trabecular bone produce RBC. However, in adults, RBC production is limited to the vertebra, sternum, ribs, and proximal ends of long bones. HSCs in the BM give rise to all mature hematopoietic cells through a series of intermediate progenitors that will be discussed further in the next section.[6]

Cellular

Many models have been studied to explain the development of the definitive erythroid lineage. HSC lack lineage-specific cell markers and instead express markers called lineage negative (lin-), Sca1+, and c-Kit+ (LSK).[7] There are three main groups of cells expressing LSK: long term HSCs (CD34- Flt3-), short term HSCs (Cd34+ and Flt3), and multipotent progenitor cells (MPP) (CD34+ Flt3+). In one model, MPP is proposed to give rise to standard lymphoid (CLP) and myeloid (CMP) progenitors.[8] From CMP, a megakaryocytic erythroid progenitor (MEP) (c-kit +, CD34-, CD71 low, CD16/32 - in mice and CD45+, GPA-, IL-3R-, CD34+, CD36-, and CD7 low) will give rise to erythroid progenitors. In another model, there is a debate on whether hematopoiesis does not undergo a hierarchal progression through MPP, long term, or short term HSC. Instead, megakaryocytes and erythrocytes arise directly from HSCs.[9] Other additional models have been described earlier; however, for this activity, the former will be discussed in further detail.

The earliest erythroid progenitors arise from MEPs and can form a burst-forming unit (BFU-E), which are large red colonies. BFU-E is a large colony that has thousands of hemoglobinized cells. They have the same immunohistochemistry as MEP, which makes it difficult to distinguish between the two. These cells respond to erythropoietin, stem cell factor, interleukin 3 and 6, corticosteroids, and insulin growth factor 1 and divide to form colony forming units erythroid (CFU-E). It is here that CFU-E is committed to erythroid differentiation and require EPO for survival (4). These cells display the phenotype of CD45+, GPA -, IL-3R-, CD34-, CD36+, and CD71 high in humans, and IL-3R-, c-Kit+ and CD71 high in mice.[10]

The transition from CFU-E to proerythroblast involves the loss of c-Kit to gain Ter119 expression. EPO engages cell division, the expression of erythroid-specific genes, and prevents apoptosis. Proerythroblast undergoes three mitoses, resulting in 2 basophilic, four polychromatic, eight orthochromatic erythroblasts and ending in 16 reticulocytes. After each division, the maturing erythroblast gets smaller, its nucleus will condense, and hemoglobin gathers in the cytoplasm. By the orthochromatophilic stage of growth, the erythroid cell will exit the cell cycle resulting in a condensed nucleus polarized to one side of the cytoplasm. The nucleus gets extruded and forms the reticulocyte. Reticulocytes express high amounts of CD71, and as they progress to become fully mature red blood cells, they lose most CD71 but gain CD235a+ expression.[11]

Molecular Level

Induction of HbF is a heavily regulated process that depends on various gene expressions. Three genetic loci that influence HbF levels include a region in chromosome 2 within the BCL11A gene, an area that is intergenic between HBSIL, MYB genes located on chromosome 6, and also variants that are within the beta-globin locus on chromosome 11.[5] Researchers have conducted extensive research with the BCL11A gene and its role as a critical mediator from HbF to HbA. This gene is involved in silencing gamma-globin gene expression by communicating with transcription factors, such as SOX6 that will bind to chromatin at the proximal site of gamma-globin promoters. An erythroid transcription factor called KLF1 controls BCL11A, and it works by silencing the expression of this gene.[12] Variants on chromosome six that are near the MYB gene region have also been shown to influence HbF through an unknown mechanism.[13] Understanding which genes control the transition of HbF to HbA predominance is critical in developing treatments for various inherited hemoglobinopathies affecting HbA by reducing the amount of mutated adult beta-globin and up-regulating gamma-globin to minimize symptoms. 

Clinical Significance

Errors in the development of HbA can lead to significant consequences for the newborn infant to adulthood. Beta-thalassemia is a genetic mutation where there is a reduced amount or absence of beta-globin chain synthesis. A reduced amount of beta-globin chains results in a decrease in hemoglobin in red blood cells, decreased red blood cell production, and anemia. There are more than 200 mutations reported with the majority of these caused by point mutations in the functionally essential gene regions for beta-globin chain production.[14] With a reduced amount of beta-globin chains, there will be an excessive amount of alpha-globin chains that cluster within the bone marrow. The conglomeration of alpha-globin chains leads to the premature death of erythroid precursors and ineffective erythropoiesis. Anemia stimulates the production of erythropoietin, and this drive to produce more red blood cells results in extramedullary hematopoiesis.[14] There are many different types of beta-thalassemia, and the severity can range from asymptomatic to severely symptomatic.

Sickle cell anemia is the result of a non-conservative missense mutation, a change of one nucleotide in the beta-globin gene region. This mutation will cause red blood cells to distort into a "sickle" shape when deoxygenated. These sickle cell RBC (HbS) can continuously change their form, which results in their premature destruction intravascularly.[15] In their sickled shape, HbS tend to entrap themselves in the capillaries, called vaso-occlusion. Excessive vaso-occlusion can result in a sickle cell pain crisis.

In both diseases, hydroxyurea has been shown to improve the quality of life for patients.[14][15] Hydroxyurea helps to increase the amount of HbF in the bloodstream. Since HbF is not composed of beta-globin chains, this hemoglobin type would not be affected by the mutation form these diseases.