The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development.

The X chromosome-linked transcription factor GATA-1 is expressed specifically in erythroid, mast, megakaryocyte, and eosinophil lineages, as well as in hematopoietic progenitors. Prior studies revealed that gene-disrupted GATA-1- embryonic stem cells give rise to adult (or definitive) erythroid precursors arrested at the proerythroblast stage in vitro and fail to contribute to adult red blood cells in chimeric mice but did not clarify a role in embryonic (or yolk sac derived) erythroid cells. To examine the consequences of GATA-1 loss on embryonic erythropoiesis in vivo, we inactivated the GATA-1 locus in embryonic stem cells by gene targeting and transmitted the mutated allele through the mouse germ line. Male GATA-1- embryos die between embryonic day 10.5 and 11.5 (E10.5-E11.5) of gestation. At E9.5, GATA-1- embryos exhibit extreme pallor yet contain embryonic erythroid cells arrested at an early proerythroblast-like stage of their development. Embryos stain weakly with benzidine reagent, and yolk sac cells express globin RNAs, indicating globin gene activation in the absence of GATA-1. Female heterozygotes (GATA-1+/-) are born pale due to random inactivation of the X chromosome bearing the normal allele. However, these mice recover during the neonatal period, presumably as a result of in vivo selection for progenitors able to express GATA-1. Our findings conclusively establish the essential role for GATA-1 in erythropoiesis within the context of the intact developing mouse and further demonstrate that the block to cellular maturation is similar in GATA-1- embryonic and definitive erythroid precursors. Moreover, the recovery of GATA-1+/- mice from anemia seen at birth provides evidence indicating a role for GATA-1 at the hematopoietic progenitor cell level.

https://www.pnas.org/content/pnas/93/22/12355.full.pdf

Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1

The thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) are products of the thyroid gland in all vertebrates. Their role in early development and metamorphosis is well established in mammals and amphibians, respectively, and recently several studies in fish have highlighted the importance of THs during flatfish metamorphosis. THs are present in high quantities in fish eggs and are presumably of maternal origin. During embryogenesis the concentration of T4 and T3 in the eggs decrease until endogenous production starts. Thyroid hormone receptors (TR) have been isolated from several teleosts and in common with tetrapods two receptor isoforms have been identified, TRα and TRβ. Both the receptors are expressed in early embryos and larvae of the Japanese flounder (Paralichthys olivaceus), zebrafish (Danio rerio) and seabream (Sparus aurata) although a different temporal pattern is apparent. The role of THs and TRs in fish embryogenesis, larval development and during metamorphosis will be discussed.

Thyroid hormones in growth and development of fish

Vertebrate hematopoietic stem cells are derived from vental mesoderm, which is postulated to migrate to both extra- and intraembryonic positions during gastrula and neurula stages. Extraembryonic migration has previously been documented, but the origin and migration of intraembryonic hematopoietic cells have not been visualized. The zebrafish and most other teleosts do not form yolk sac blood islands during early embryogenesis, but instead hematopoiesis occurs solely in a dorsal location known as the intermediate cell mass (IM) or Oellacher. In this report, we have isolated cDNAs encoding zebrafish homologs of the hematopoietic transcription factors GATA-1 and GATA-2 and have used these markers to determine that the IM is formed from mesodermal cells in a posterior-lateral position on the yolk syncytial layer of the gastrula yolk sac. Surprisingly, cells of the IM then migrate anteriorly through most of the body length prior to the onset of active circulation and exit onto the yolk sac. These findings support a hypothesis in which the hematopoietic program of vertebrates is established by variations in homologous migration pathways of extra- and intraembryonic progenitors.

https://www.pnas.org/content/pnas/92/23/10713.full.pdf

Intraembryonic hematopoietic cell migration during vertebrate development

Iron has a fundamental role in many metabolic processes, including electron transport, deoxyribonucleotide synthesis, oxygen transport and many essential redox reactions involving haemoproteins and Fe-S cluster proteins. Defective iron homeostasis results in either iron deficiency or iron overload. Precise regulation of iron transport in mitochondria is essential for haem biosynthesis, haemoglobin production and Fe-S cluster protein assembly during red cell development. Here we describe a zebrafish mutant, frascati (frs), that shows profound hypochromic anaemia and erythroid maturation arrest owing to defects in mitochondrial iron uptake. Through positional cloning, we show that the gene mutated in the frs mutant is a member of the vertebrate mitochondrial solute carrier family (SLC25) that we call mitoferrin (mfrn). mfrn is highly expressed in fetal and adult haematopoietic tissues of zebrafish and mouse. Erythroblasts generated from murine embryonic stem cells null for Mfrn (also known as Slc25a37) show maturation arrest with severely impaired incorporation of 55Fe into haem. Disruption of the yeast mfrn orthologues, MRS3 and MRS4, causes defects in iron metabolism and mitochondrial Fe-S cluster biogenesis. Murine Mfrn rescues the defects in frs zebrafish, and zebrafish mfrn complements the yeast mutant, indicating that the function of the gene may be highly conserved. Our data show that mfrn functions as the principal mitochondrial iron importer essential for haem biosynthesis in vertebrate erythroblasts.

Mitoferrin is essential for erythroid iron assimilation

In the rainbow trout, Salmo gairdneri irideus, round, disc-like erythrocytes in the embryonic circulation (Ery L) are replaced by small, elliptical, disc-like erythrocytes (Ery ImA) after hatching. In the peripheral blood of alevins, Ery ImAs grow into mature, adult erythrocytes (Ery A) of large elliptical, disc-like shape (Iuchi, '73b; Yamamoto and Iuchi, '75). Ery L and Ery A have larval and adult hemoglobins, respectively (Iuchi, '73a). The ontogenetic sequence of hemoglobin switching and erythrocyte replacement during erythropoiesis was examined by o-dianisidine histochemistry as well as immunohistochemistry using FITC-antibody probe specific to adult hemoglobins. The first phase of embryonic erythropoiesis (for Ery L) occurs in the intraembryonic "intermediate cell mass" as well as the extraembryonic blood islands on the yolk sac. This phase of erythropoiesis is transient, continuing during the 7th to 12th day after fertilization and ceasing by the 15th day (5th day before hatching). There is a mixed population of Ery L, ImA, and A in the peripheral blood of posthatching alevins. Ery ImA and A showed immunofluorescence with FITC anti-Hb A antibody but Ery L did not. Erythroid cells, stainable with FITC anti-Hb A antibody, were observed in the kidney and the spleen 1 day before hatching and thereafter, but not in the liver throughout all stages examined. Therefore, we conclude that new erythropoiesis (erythropoiesis for Ery ImA and Ery A) began in the kidney and the spleen 1 day before hatching. These findings indicate that hemoglobin switching during the ontogeny of rainbow trout is based on erythrocyte replacement, correlated with a shift in the site of erythropoiesis from one organ to another.

Erythropoiesis in the developing rainbow trout, Salmo gairdneri irideus: histochemical and immunochemical detection of erythropoietic organs

1. 1. Comparative studies on chemical and physiological characteristics of hemoglobins isolated from newly hatched larvae and adult fish of rainbow trout were carried out. 2. 2. No significant differences in absorption spectrum and in molecular size were observed between them, suggesting that both hemoglobins are tetrameric and contain protoporphyrin IX. 3. 3. The larval hemoglobin was more labile than the adult one upon alkali denaturation. Electrophoretic studies revealed that both hemoglobins and their subunits were highly polymorphic, without having subunit components common to both hemoglobins. 4. 4. Although overall amino acid compositions of both hemoglobins were similar in general, the difference in the amino acid residues responsible for net charges at pH 8·6 was observed. 5. 5. The larval hemoglobin was characterized with higher oxygen affinity and less Bohr effect compared with the adult hemoglobin which loses oxygen affinity sharply at pH's below 7·6.

Chemical and physiological properties of the larval and the adult hemoglobins in rainbow trout, Salmo gairdnerii irideus.

The fine structure of the first erythroid cells in the embryonic circulation of the rainbow trout and new erythroid cells appearing after hatching was examined by scanning and transmission electron microscopy. The first erythroid cells in the embryos, where circulation has just begun, are very immature in ultrastructural characteristics; they are spherical, relatively large, and contain a large nucleus with diffuse chromatin and numerous free ribosomes mostly occurring as polyribosomes. The cells divide in the circulatory system. As development proceeds, the entire population undergoes a synchronous maturation, i.e., transformation into round and flat discs, progressive heterochromatization, reduction in number of cytoplasmic organelles, especially of ribosomes, and a concomitant increase of hemoglobin content. The fully mature first erythroid cells persist until after hatching and are gradually replaced by a new population of erythroid cells which begin to appear in the circulatory system within a few days after hatching. The new cells are at first small and spherical, and show immature ultrastructural features, but no dividing cells were encountered. The cytological changes accompanying maturation are almost parallel to those seen in the first class of erythroid cells. In the circulatory system of fry, where yolk absorption has just been completed, almost all the erythroid cells are mature. These cells are uniformly flat and elliptical discs. The differences in ribosome numbers in electron micrographs of erythroid cells from different stages of development are also consistent with the existence of two series of erythroid cell maturation in the peripheral blood of the developing rainbow trout.

Electron microscopic study of erythrocytes in developing rainbow trout, Salmo gairdnerii irideus, with particular reference to changes in the cell line

Major glycoproteins solubilized from the teleostean egg membrane by the action of the hatching enzyme.

In addition to typical, round disc-like larval type erythrocytes (erythrocyte L) in pre-hatching embryos as well as just-hatched larva and the typical elliptic disc-like adult type erythrocytes (erythrocyte A) in two-year-old fish, a third type of erythrocytes has been observed to appear in the peripheral blood of rainbow trout larvae during a short period (a few weeks) of erythrocyte transition from L to A. Analyses of the erythrocyte population at various stages of the transition have suggested that the third type erythrocytes may not be derived from erythrocyte L, but may be an independent population which will develop into erythrocyte A in the blood and therefore have been referred to as immature type erythrocyte A (erythrocyte ImA). Dynamics of erythrocyte replacement from one to the others during post-hatching development is characterized by the rapid decrease in erythrocyte L and the compensatory appearance of erythrocyte ImA, which then will develop into the mature erythrocyte A in the peripheral blood.



The three types of erythrocytes differ with respect to sensitivity to osmotic shock. A difference has also been observed between erythrocyte L and erythrocyte A including A concerning reactivity with concanavalin A and wheat germ agglutinin.

Ichiro Iuchi

Papers

Erythropoiesis in the developing rainbow trout, Salmo gairdneri irideus: histochemical and immunochemical detection of erythropoietic organs

Chemical and physiological properties of the larval and the adult hemoglobins in rainbow trout, Salmo gairdnerii irideus.

Electron microscopic study of erythrocytes in developing rainbow trout, Salmo gairdnerii irideus, with particular reference to changes in the cell line

Major glycoproteins solubilized from the teleostean egg membrane by the action of the hatching enzyme.

The post-hatching transition of erythrocytes from larval to adult type in the rainbow trout, Salmo gairdnerii irideus.