Zebrafish as a vertebrate model of hematopoiesis

Our understanding of hematopoiesis has largely been obtained through the biochemistry and cell biology of hematopoietic cells. The zebrafish, or Danio rerio, is an ideal genetic system for hematopoietic developmental studies. Zebrafish are easy to raise with a short generation time of three months and the developing embryos are easily studied under a dissecting microscope since they are transparent. Embryos develop rapidly, with a beating heart and visible erythrocytes by 24 hours. The females can lay hundreds of eggs at weekly intervals. The organism maintains the diploid state, an important difference to other fish that can be triploid or tetraploid making genetic analysis difficult.

Hematopoiesis in the vertebrate is characterized by the induction of ventral mesoderm to form hematopoietic stem cells and the eventual differentiation of these progenitors to form the final peripheral blood lineages. Several genes have been implicated in the differentiation and development of hematopoietic and vascular progenitor cells, yet our understanding of the discrete steps involved in the induction of these cells from the ventral mesoderm is still incomplete. The zebrafish (Danio rerio) is an especially robust vertebrate system to both isolate and characterize these processes. One strength of the zebrafish system lies in the ease in which hematopoietic mutants have been generated and studied. These mutants span many of the proposed steps of both the primitive and definitive hematopoietic programs. Cloning of the genes that underlie these mutations should yield valuable details of the hematopoietic cascade has therapeutic implications for bone marrow transplantation and stem cell gene therapy.

Zebrafish hematopoiesis

Teleosts such as the zebrafish lack yolk sac blood islands, instead embryonic hematopoiesis occurs in a relatively dorsal location above the yolk tube. This region is called the intermediate cell mass (ICM). The ICM is formed in two paraxial stripes of mesoderm that arise during gastrulation. This site is similar to the extraembryonic location of the earliest hematopoietic progenitors on the yolk sac of higher vertebrates. To define the normal sites of hematopoiesis, the expression within developing blood cells of transcription factors and globin was studied. We found that lmo2 and gata-2 are expressed in two stripes of presumptive blood progenitors in the ventral mesoderm at the 5-somite stage. Fli1 is expressed in hematopoietic and vascular tissues, and is also expressed early in a similar location. Cmyb and gata-1are detected after the early markers as the two stripes of progenitors converge to form the ICM. Prior to circulation ICM cells begin to express large amounts of globin RNA. The early markers lmo2, gata-2, and fli1 are expressed in the posterior region of the ICM and in several regions of the anterior trunk. In contrast, gata-1, cmyb and globin are not detected in these regions. At 48 hours of development, cmyb is detected in a line of cells in the ventral wall of the dorsal aorta. These cells are likely to be definitive hematopoietic stem cells in keeping with observations of other vertebrates that also have cmyb positive hematopoietic cells located in the dorsal aorta. Whole-embryo in-situ hybridization with scl demonstrates that scl is expressed in developing neural tissues, blood islands and circulating blood. In the peripheral blood, two distinct waves of scl expression in the ICM are seen, the first occurs before the 8th hour and subsides by hour 26. The second wave begins at approximately hour 30 and decreases by hour 36. The transcription factors gata-2, fli1,lmo2 and scl are therefore the earliest defined markers for the vertebrate hematopoietic development program to date.

Zebrafish Genetics

Genetic screens for zebrafish hematopoietic mutants

One spontaneous zebrafish mutant, cloche (clo^m39) has recently been investigated because of a defect in hematopoietic and vascular development. Homozygous clo embryos lack endocardial cells, head and trunk endothelial cells, and blood, but retain some tail endothelial cells. gata-1and gata-2are not expressed in the ICM of clo mutant embryos. In cloche homozygotes, expression of flk1 is delayed compared to wildtype embryos and is expressed only in the lower trunk and tail. Furthermore, these flk1 positive cells do not express another endothelial marker tie-1 suggesting an early block in endothelial differentiation in clo mutants.

As part of a large scale chemical mutagenesis screen of the zebrafish genome achieved in the laboratory of Dr. Nüsslein-Volhard, 39 mutants with defects in hematopoiesis were found comprising 26 complementation groups. Investigations of these mutations in our laboratory have demonstrated that they represent a variety of defects in the cascade of steps for hematopoiesis. Further chemical and radiation mutational screens are underway in many labs and additional hematopoietic developmental mutants are continually being found.

Since developing blood cells have a standard size, morphology, rate of development and hemoglobinization, these factors were used in classifying the mutants isolated from the Nüsslein-Volhard ENU screen. Four broad classifications of mutants were found, bloodless mutants, mutants with decreasing blood counts, hypochromic mutants and photosensitive mutants.

One new bloodless mutant, moonshine (mon) has been described from this screen. There are 8 recessive mon alleles, 7 of which are embryonic lethal. At the start of circulation, there are several dozen blood cells but never more than approximately 100 cells at 4 days of age. Additional phenotypic characteristics include jagged fin edges and increased iridophores. The relative strength of the different mon alleles correlates with the amount of gata-1expression as detected by whole-mount in situ hybridization. These data suggest that the moonshinegene product facilitates development of gata-1expressing hematopoietic precursors.

Ten distinct complementation groups chablis (cha, 2 alleles), frascati (frs), retsina (ret, 2 alleles), thunderbird (tbr, 1 allele) merlot (mot, 2 alleles), riesling (ris, 1 allele) grenache (gre, 1 allele), pinotage (pnt, 1 allele)and cabernet (cab ,1 allele) were described in which mutants initially have normal numbers of eyrthroid cells but then have a rapid deterioration in number beginning on days 2-4. These mutations share normal gata-1expression at 24 hours. Blood collected from these mutants reveal cells blocked at various stages of early erythroid differentiation. It appears based on the decreasing number of cells and block in erythroid maturation demonstrated by these mutants, that these mutations may affect erythroid cell proliferation and differentiation.

Initially, the embryos of 5 mutants, zinfandel (zin,1 allele), chardonnay (cdy, 1 allele), weißherbst (weh, 2 alleles), sauternes (sau, 2 alleles), chianti (cia, 1 allele) have normal numbers of erythroid cells but at about 2-3 days of development show a decreased number of erythroid cells and decreased hemoglobin expression (hypochromic). One of these mutations, zinfandel, is dominant, while the rest are recessive.