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Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2622-2636
Gene Duplication of Zebrafish JAK2 Homologs Is Accompanied by
Divergent Embryonic Expression Patterns: Only jak2a Is
Expressed During Erythropoiesis
By
Andrew C. Oates,
Alison Brownlie,
Stephen J. Pratt,
Danielle V. Irvine,
Eric C. Liao,
Barry H. Paw,
Kristen J. Dorian,
Stephen L. Johnson,
John H. Postlethwait,
Leonard I. Zon, and
Andrew F. Wilks
From the Ludwig Institute for Cancer Research, Melbourne Tumour
Biology Branch, Royal Melbourne Hospital, Victoria, Australia; the
Howard Hughes Medical Institute, Children's Hospital, Boston, MA; the
Department of Genetics, Washington University Medical School, St Louis,
MO; and the Institute of Neurosciences, University of Oregon, Eugene,
OR.
 |
ABSTRACT |
Members of the JAK family of protein tyrosine kinase (PTK) proteins
are required for the transmission of signals from a variety of cell
surface receptors, particularly those of the cytokine receptor family.
JAK function has been implicated in hematopoiesis and regulation of the
immune system, and recent data suggest that the vertebrate JAK2
gene may play a role in leukemia. We have isolated and characterized
jak cDNAs from the zebrafish Danio rerio. The
zebrafish genome possesses 2 jak2 genes that occupy paralogous
chromosome segments in the zebrafish genome, and these segments
conserve syntenic relationships with orthologous genes in mammalian
genomes, suggesting an ancient duplication in the zebrafish lineage.
The jak2a gene is expressed at high levels in erythroid
precursors of primitive and definitive waves and at a lower level in
early central nervous system and developing fin buds. jak2b is
expressed in the developing lens and nephritic ducts, but not in
hematopoietic tissue. The expression of jak2a was examined in
hematopoietic mutants and found to be disrupted in cloche
and spadetail, suggesting an early role in hematopoiesis. Taken together with recent gene knockout data in the mouse, we suggest
that jak2a may be functionally equivalent to mammalian Jak2, with a role in early erythropoiesis.
© 1999 by The American Society of Hematology.
 |
INTRODUCTION |
CYTOKINES ARE IMPORTANT regulators of
proliferation, differentiation, and cell function for a wide range of
cells of hematopoietic and other lineages. The JAK/STAT signaling
system is required for the function of a number of cytokines, acting to
transduce signals from cytokine receptors.1,2
JAK tyrosine kinases possess a distinctive structure. Amino acid
sequence comparison of the 4 mammalian JAK family members (JAK1, JAK2,
JAK3, and TYK2) shows the presence of 7 highly conserved domains (named
JH1 through JH7).3 The N-terminal region of JAK kinases
contains structures (JH7-JH3) that confer specific binding activity
toward cytokine receptor cytoplasmic domains.4-9 Further
C-terminal, the kinase-related domain (JH2) exhibits significant similarity to a tyrosine kinase domain yet diverges within several critical catalytic motifs.10 Indeed, this domain appears
not to possess phosphotransferase activity10; rather, it is
required for the stability and binding affinity of associated
receptors.11,12 The C-terminal domain (JH1) contains a
protein tyrosine kinase10 responsible for initiating much
of the signaling activity from cytokine receptors that use this family
of PTKs. Expression of kinase inactive JAK variants abrogate most if
not all aspects of signaling in a dominant negative manner through
receptors for cytokines such as erythropoietin (EPO),13
growth hormone (GH),5 granulocyte-macrophage
colony-stimulating factor (GM-CSF),14 granulocyte
colony-stimulating factor (G-CSF),15 interleukin-6 (IL-6),16 interferon (IFN ),11
IFN ,17 and IL-2.18 Analysis of the effects
of dominant negative JAK variants combined with a series of somatic
cell mutants deficient or defective in 1 of the JAK
kinases8,19,20 demonstrates the critical role of the JAK
kinase family in cell signaling.
Three lines of evidence link JAK function directly to the regulation of
the growth and differentiation of hematopoietic cells in vivo. First,
gene targeting of Jak loci in the mouse yields hematopoietic
phenotypes: Jak1-deficient mice exhibit impaired lymphopoiesis,21 Jak2 deficiency abolishes
definitive erythropoiesis,22,23 and Jak3 null
mutation results in the murine equivalent of human severe combined
immunodeficiency syndrome (SCID).24-27 Second, the
dominant Drosophila melanogaster JAK mutant,
hopscotchTum-l causes a lethal leukemia in fruit
flies.28 The protein products of
hopTum-l and a recently identified allele
hopT42 show increased levels of autophosphorylation
and the capacity to activate Stat92E.29-31 Third,
constitutive JAK2 catalytic activity can be detected in some acute
lymphoblastic leukemia (ALL) cells in humans. Chromosomal translocation
in a human T-cell ALL creates a TEL-JAK2 fusion protein capable of
oligomerization through the TEL fusion partner, resulting in
constitutive activation of JAK2 tyrosine kinase activity.32
Mice expressing this fusion protein in bone marrow developed a fatal
mixed myeloproliferative and T-cell lymphoproliferative
disorder.33 These results indicate that JAK genes
are required for normal hematopoiesis and that the deregulation of JAK
catalytic activity is capable of causing hematopoietic neoplasia.
To further explore the role that the JAK family has in
controlling blood cell growth, we have isolated and characterized
JAK genes from the zebrafish, Danio rerio. The
zebrafish offers many technical advantages for the developmental
analysis of gene function, because embryos are plentiful, develop
externally, and are optically transparent. As a vertebrate experimental
system, the zebrafish allows strong analogies to be drawn to human
biology. For example, in contrast to Drosophila melanogaster
and Caenorhabditis elegans, zebrafish has
circulating blood cells of the erythroid, myeloid, and lymphoid
lineages.34,35 Furthermore, a recent mutant screen performed in the zebrafish led to the isolation of mutations in more
than 20 complementation groups that disrupt
hematopoiesis,36,37 including a model for human congenital
sideroblastic anemia, sauternes.38 Importantly, the
possibility of performing modifier screens in the zebrafish offers the
means of identifying other factors in these complex developmental
processes by a genetic approach.
Our search using degenerate oligonucleotide polymerase chain reaction
(PCR) for homologs of JAK and STAT genes in D
rerio yielded several genes from both JAK (jak1, jak2a,
and jak2b) and STAT (stat1 and
stat3) gene families. The characterization of zebrafish members of
the STAT family will be reported elsewhere.38a We
report here in detail on the structure and evolutionary relationships of the zebrafish JAK2 homologs. We determined the expression of these genes in wild-type and several selected zebrafish mutants and
consequently propose a role for jak2a in erythropoiesis.
 |
MATERIALS AND METHODS |
Isolation of zebrafish jak homologs.
JAK-directed degenerate oligonucleotide primers were designed
after Wilks,39 based around conserved subdomains VIb and
IX40 in the catalytic JH1 domain of Homo
sapiens JAK1,10 Mus
musculus Jak2,3 H sapiens
JAK341 and TYK2,42 and D
melanogaster HOP.43 Wherever complete degeneracy was
required at a nucleotide position in the primers, an inosine residue
was incorporated.44 The sequence of the primers is
CCGAATTCCA(C/T)(C/A)GIGA(C/T)(C/T)TIGCIGCI(C/A)GIAA and
CCGAATTCIACICC(A/G)(A/T)AI(G/C)(A/T)CCAIAC(G/A)TC. cDNA libraries were
plated and screened at high stringency according to standard methods,45 with PCR products generated by
JAK-directed degenerate oligonucleotide PCR as described above,
and specific for the zebrafish jak1, jak2a, and
jak2b genes. Mixed developmental stage D rerio cDNA
libraries in Lambda Zap (Stratagene, La Jolla, CA), both random and poly-A primed, were a gift of J. Campos-Ortega (University of Köln, Köln, Germany). Lambda Zap cDNA
libraries generated from staged embryonic mRNA populations were made by
Bob Riggleman and Kathryn Helde and were a kind gift of D. Grunwald
(Eccles Institute, University of Utah, Salt Lake City,
UT). The "Contig Manager" application of the
DNAstar suite of programs (DNAstar Inc, Madison, WI) was used to create
and monitor contigs from the primary sequence data. cDNA
sequences presented herein corresponding to each of the gene
transcripts under study were sequenced in both directions to a minimum
of 2-fold coverage.
Zebrafish care and strains.
Zebrafish were raised and maintained as described.46
Zebrafish carrying the following mutant alleles of cloche
(clom39),47 spadetail
(sptb104),48 cabernet
(cabtl236), retsina
(rettr217), chianti
(ciatu25f), sauternes
(sauty121),38 chablis
(chatu245/tu242e) (thought to be clonal alleles),
weißherbst (wehth238,
wehtp85c), chardonnay
(cdyte216), frascati
(frstm130d, frstq223),
reisling (ristb237), and merlot
(mottm303c)36 were studied.
Sequence analysis and evolutionary comparison.
Electronic database searches were made by submitting nucleic acid
sequence and putative amino acid sequence to the public search facility
at the Baylor College of Medicine (Houston, TX; http://hgsc.bcm.tmc.edu/SearchLaunches/) using
"WU-BLAST."49 To study the evolutionary relationships
between the PTKs and JAKs identified, the deduced amino acid
sequences of the genes in question were aligned with the
"CLUSTAL" protein alignment program50 of the MegAlign
application (DNAstar suite) and refined by hand using structural
information, where available. These alignments were used to create
maximum parsimony phylogenetic trees and distance matrices using the
options of that program. The topology of the phylogenetic tree shown
was insensitive to the order of sequence addition.
Whole mount in situ hybridization.
Embryos were staged according to Kimmel et al.51 Embryos
raised to time points beyond 24 hours postfertilization (hpf) were transferred to E3 embryo medium with 0.003% phenylthiourea (PTU; Sigma, St Louis, MO) to prevent melanization. Riboprobe
synthesis and in situ hybridization were performed essentially as
described52 with the following modifications. Riboprobes
were purified before use over RNA sephadex G-50 columns (Boehringer
Mannheim, Indianapolis, IN). Using estimates of RNA
synthesis based on 32P-CTP incorporation, probes were
resuspended in HYB+ 52 at a concentration of 1 ng/mL for use. Embryos up to 24 hpf were not proteinase K-digested,
embryos between 24 hpf and 36 hpf were digested for 10 minutes (10 µg/mL), and embryos greater than 36 hpf were digested for between 20 and 30 minutes (20 µg/mL). Hybridization and washing was performed at
temperatures of 65°C to 70°C. Nonspecific antidigoxygenin
Fab-AP binding (Boehringer Mannheim; used at a dilution of 1/5,000) was
blocked by 2% wt/vol Blocking Reagent (Boehringer Mannheim)/10%
heat-inactivated sheep serum/MABT (100 mmol/L Maelic acid, 150 mmol/L
NaCl, 0.1% Tween-20, pH 7.5) for 1 hour at room temperature. Color
detection reactions used BM purple substrate (Boehringer Mannheim) and
were developed for up to 2 days before fixing in 4%
paraformaldehyde/PBT (phosphate-buffered saline/0.1%
Tween 20). Embryos were either cleared in glycerol or benzyl
benzoate:benzyl alcohol (2:1) and photographed using a Leitz Wild T
stereo dissection microscope (Leitz, Wetzlar, Germany) or a Nikon
Microphot AX compound microscope (Nikon Inc, Melville, NY). The entire jak2b cDNA was used to generate a digoxygenin (DIG)-labeled probe, and the jak2a riboprobe
contained the 5'-most 800 bp encoding the JH7 and JH6 domains.
Generation of DNA polymorphisms and genetic mapping.
A bacterial artificial chromosome (BAC) library (Genome Systems, St
Louis, MO) containing large insert zebrafish genomic DNA was screened
by hybridization to oligonucleotide probes derived from jak1,
jak2a, and jak2b cDNA. Clones corresponding to the genomic loci were obtained for jak1 (96 E18, 100 K4, and 143 O15) and jak2a (112 K6), but not jak2b. A P1 artificial
chromosome (PAC) library (C. Amemiya, Boston University, Boston,
MA) was screened by hybridization to an oligonucleotide
probe derived from jak2b and clones obtained (35 F6 and 58 G17). Sequence information from the ends of each of these genomic
clones was determined (data not shown) and, along with the sequence of
3' UTR regions from cDNA of the jak genes, was used to
design PCR primer pairs that amplified products from genomic DNA that
segregated in a C32xSJD mapping cross.53 The primers for
jak1 (100 T7-1 GTAGAAGATACAGTCGCCTG, 100 T7-2
GTAAAGCAATATCAATAGAG) give a codominant size polymorphism54 of 290/270 bp; the jak2a codominant size polymorphism (200/220 bp) is from the primer pair j2A.29 (GATCATCCACAGTTCAGCTCC), j2A.30 (TAATGATGAGAGAACACCCGC); jak2b was mapped with a codominant
sequence polymorphism55 in the PCR product generated by the
j2B.M1 (AAGAAAGTCTGTCCGCTGTCTTCACATGTC), j2B.M4
(CGCGCCAGCACTGCTAGCATAACAGAAACC) primer pair. Linkage was determined by
comparison of a given marker on a C32xSJD haploid panel consisting of
96 individuals that were genotyped against approximately 600 markers
for close correlation of segregation patterns53,56-59 using
the program "MapMaker"60 (Massachusetts Institute of
Technology, Cambridge, MA) and the program
"mapmanager"61 (Roswell Park Cancer
Institute, Buffalo, NY).
Assessment of linkage of jak2a to the cab and
mot mutations.
Homozygous diploid embryos were generated as described46
from a ABxDAR hybrid mother carrying a single mutant allele of cabtl236 and from a ABxDAR hybrid mother
heterozygous for mottm303c and scored for an
erythropoietic phenotype. These embryos were typed for the segregation
of the j2A.29/j2A.03 marker from the jak2a 3' UTR with
the mutant phenotype, based on a size polymorphism evident between the
AB and DAR strains.
 |
RESULTS |
Cloning of zebrafish jak2 genes.
To isolate JAK homologs in zebrafish, PCR was used to amplify
cDNA derived from mixed-stage embryonic mRNA with JAK-directed degenerate primer pairs, yielding 8 distinct PTK fragments
corresponding to members from 3 kinase subfamilies
(Fig 1a). Sequence comparison of the PCR
products to known JAK genes suggested that 2 of the PTKs were
closest in identity to mammalian JAK2; these were termed jak2a (HD-1) and jak2b (HD-71). A third, HD-9,
was most similar to mammalian JAK1 and was designated
jak1. This manuscript will focus on the 2 jak2 genes we
detected in the zebrafish. The embryonic expression of jak1
will be reported elsewhere (Oates and Wilks, manuscript in
preparation).



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| Fig 1.
JAK genes from the zebrafish. (a) Alignment of
deduced amino acid sequences for the PTKs identified by PCR from
zebrafish cDNA. The alignment was generated using
CLUSTAL.50 The clone name is listed as HD on the right of
the sequences, followed by the name of the gene that gave the highest
BLAST score in database similarity searches.49 The
JAK-specific sequence elements in Hanks motif VIII are boxed. (b)
Comparison of the zebrafish jak2a and human JAK2 protein. The amino
acid sequence (single-letter code) of the jak2a protein from zebrafish
and the JAK2 protein from human88 is numbered from the
putative initiation methionine. The JAK homology (JH) domains are
indicated by brackets and are labeled JH1 through JH7 according to
Harpur et al.3 Conserved motifs in the catalytic JH1 domain
are labeled in roman numerals. Conserved motifs in the kinase related
domain (JH2) are labeled in roman numerals with the subscript a. A
series of conserved residues mutated in murine Jak2 without phenotypic
effect in IFN signaling8 are indicated by an asterisk above
the amino acid, and are labeled according to Kohlhuber et
al8 by capital letters. The site of the E665K mutation
within JH2 that hyperactivates the catalytic activity31 of
murine Jak2 and D melanogaster HOP is marked with a solid
arrowhead. The autophosphorylation site in the JH1 domain of murine
Jak2, which is required for catalytic activity,89 is marked
by an open arrowhead. The structure of the variant jak2a
cDNA is marked by an arrow at the site of predicted translational
termination due to alternate splicing.62 (c) Phylogeny of
the JAK gene family. The amino acid sequences of zebrafish
(Danio rerio, Dr) jak2a and jak2b were aligned
across the known region of jak2b, consisting of most of JH2 and all of
JH1, with JAK2 proteins from human (Homo sapiens, Hs),88 pig (Sus scrofa,
Ss),90 mouse (Mus musculus,
Mm),3 and rat (Rattus norvegicus,
Rn)91; all other members of the JAK family
from human (Hs),10,41,42 the zebrafish (Dr)
jak1 sequence (this study), and the sequence of the D
melanogaster (Dm) JAK homolog,42
hopscotch, using the CLUSTAL alignment algorithm.50
This alignment was used to construct a dendrogram with the maximum
parsimony options of the DNAstar MegALIGN application to infer the
likely genealogy of the JAK family. The names of the sequences
are displayed to the right of the dendrogram; zebrafish sequences are
in bold.
|
|
Multiple cDNA clones were recovered from an embryonic cDNA library by
DNA hybridization using the PCR product corresponding to jak2a
as a probe. Conceptual translation of the resulting sequence contig
showed an open reading frame (ORF) of 1,095 amino acids with high
similarity across the entire coding region to mammalian JAK2
genes (65% identity to Mm JAK23; Fig
1b). One cDNA possessed an internal
deletion relative to all other cDNAs, consistent with the omission of
an exon due to an alternate splicing event.62 The longer
form of the transcript was named jak2a and the shorter,
alternately spliced form was termed jak2a , consistent with
the nomenclature for alternately spliced forms of the mammalian
STAT1 and STAT3 genes.63,64 Multiple cDNA
libraries were screened with a jak2b probe and a partial cDNA
was isolated 1,967 nucleotides in length containing an ORF of 498 amino
acids consisting of the C-terminal region of the protein (data not shown).
Structure of the zebrafish jak2 transcripts.
Examination of the conceptual translation of both zebrafish
jak2 genes showed a high degree of sequence conservation. The jak2a protein shows approximately 65% identity to the mammalian JAK2
proteins from mouse, human, rat, and pig. As shown in Fig 1b, by
comparison to human JAK2, all recognized structural elements found in
mammalian JAK2 proteins are present. This high degree of sequence
conservation indicated a strong likelihood of functional conservation.
Comparison of the predicted amino acid sequence of the majority of the
JH2 domain and the entire JH1 domain found in the jak2b cDNA
with other JAK kinases indicated that it, too, is most closely related
to mammalian JAK2 proteins.
The mammalian JAK kinases and Drosophila HOP were aligned with
the amino acid sequence of jak2a and jak2b over the JH1 and JH2
domains, and a phylogenetic reconstruction of JAK gene
evolution was derived, as shown in Fig 1c. The zebrafish jak2 proteins
are approximately as different from each other (75% identity) as they are from the mammalian JAK2 proteins, indicating an ancient paralogy. jak2b is slightly more similar to the mammalian proteins (average of
78% identity) than is jak2a (75% identity). However, when nucleotide similarity is assessed across these domains, the zebrafish genes are
more similar to each other (72% identity) than either is to any of the
mammalian genes (jak2a v mammalian, 67% identity;
jak2b v mammalian, 70% identity). Furthermore, there
are 27 amino acid positions at which the zebrafish jak2 proteins are
identical to each other but different from the mammalian sequence,
suggesting the existence of a fish-specific substitution. A striking
feature of the jak2 paralogs is their extensive divergence,
suggesting that the time since paralog duplication is of similar
magnitude to that since divergence of the zebrafish and mammalian
lineages. This is reflected in the short distances between the nodes
leading to the divergence of the zebrafish and mammalian JAK2 sequences in Fig 1c.
Genetic mapping of the jak genes.
Knowledge of the genetic position of a gene is important in assessing
potential linkage to a particular mutation and the identification of
candidate genes from the collection of hematopoietic mutants. To
generate polymorphic markers for each of the jak genes for genetic mapping, genomic DNA or cDNA sequence was used to design PCR
primers for use in single-stranded conformational polymorphism (SSCP)
and simple sequence-length polymorphism (SSLP) assays.
The C32xSJD mapping panel53 was typed with a polymorphic
marker for each gene. Using the segregation of the 100.T7-1/100.T7-2 SSLP polymorphism derived from physically linked genomic DNA in 72 meioses (Fig 2a), jak1 was mapped
to linkage group 6 (LG6) between 14P.1350.c and her6 (Fig 2d).
jak2a was mapped to distal LG21, 1.9 cM proximal to
z16/14Q.1300.c (Fig 2e), on the basis of the segregation of the
3' UTR-derived j2A.29/j2A.03 SSCP polymorphism in 81 meioses (Fig
2b). Segregation of an SSCP polymorphism derived from the 3' UTR
of jak 2B (j2B.M1/j2B.M4) in 48 meioses (Fig 2c) was used to
place this gene on LG5 approximately equidistant between the z3804 and
her1/z4299 markers (Fig 2f). The chromosome segments on which
the jak genes are found were aligned with their counterparts from mouse and human, showing extensive conservation of synteny (Fig 2g
and h).


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| Fig 2.
Genetic mapping of the jak1,
jak2a, and jak2b genes of zebrafish. (a) Segregation of
the 100.T7-1/100.T7-2 SSLP jak1 polymorphism in the C32xSJD
cross. The C32xSJD mapping cross was typed for a polymorphic marker
derived from genomic DNA associated with the jak1 gene. PCR
products amplified from genomic DNA of the haploid embryos of the
C32xSJD mapping panel using the 100.T7-1/100.T7-2 primer pair were
analyzed for length differences by denaturing polyacrylamide gel
electrophoresis and 8 representative lanes are shown. The size variants
of the products were assigned to the maternal (M) or paternal (P)
genome at random, and segregation in the panel was scored. (b)
Segregation of the j2A.29/j2A.30 SSLP jak2a polymorphism in the
C32xSJD cross. PCR products amplified from genomic DNA of the haploid
embryos of the C32xSJD mapping panel using the j2A.29/j2A.30 primer
pair derived from the jak2a cDNA 3' UTR were analyzed for
length differences as described above and 8 representative lanes are
shown. The size variants of the products were assigned to the maternal
(M) or paternal (P) genome at random, and segregation in the panel was
scored. (c) Segregation of the j2B.M1/j2B.M4 SSCP jak2b
polymorphism in the C32xSJD cross. PCR products from genomic DNA of the
haploid embryos of the C32xSJD mapping panel using the j2B.M1/j2B.M4
primer pair derived from the jak2b cDNA 3' UTR were
analyzed for sequence differences by nondenaturing polyacrylamide gel
electrophoresis and 8 representative lanes are shown. The size variants
of the products were assigned to the maternal (M) or paternal (P)
genome at random, and segregation in the panel was scored. (d) Genetic
map position of the jak1 gene. Analysis of the segregation of
the jak1-associated marker in 72 meiotic events of the C32xSJD
mapping panel using the MapMaker and mapmanager
programs59,60 and further manual refinement places the
zebrafish jak1 locus on LG6. (e) Genetic map position of the
jak2a gene. Analysis of the segregation of a
jak2a-associated marker in 81 meiotic events of the C32xSJD
mapping panel as described above places the zebrafish jak2a
locus on LG21. (f) Genetic map position of the jak2b gene.
Analysis of the segregation of a jak2b-associated marker in 48 meiotic events as described above places the zebrafish jak2b
locus on LG5. (g and h) Synteny of the JAK family loci between
the zebrafish, mouse, and human. A schematic diagram of the syntenic
relationship between segments of the chromosomes of zebrafish (LG5, 6, and 21), human (Hsa 1, 9, and 6), and mouse (Mmu 2, 4, and 17) that
contain members of the JAK gene family. The syntenic segments
containing JAK2 homologs are indicated in (a) and those
containing JAK1 homologs in (b). Note that genes have been
illustrated in the same relative positions on syntenic chromosomes;
however, in situ, local gene order may vary between chromosomes. The
diagram is not to scale.
|
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Expression of jak2a in the developing zebrafish.
Analysis of the developmental expression pattern of jak2a
indicates that it may play a role in hematopoiesis. jak2a
transcripts were first detected at a low level throughout the embryo at
10 hpf (Fig
3a), persisting until 14 hpf. During this period, the intensity of
signal increased in the anterior part of the axis, eventually being
strongest in the eyes. By 14 hpf (Fig 3b and c), cells of the medial
lateral plate mesoderm expressed jak2a in a pattern consistent
with the sites of earliest hematopoietic activity.65 Cells
in this region have earlier expressed scl66 and
gata1 and gata2 at 11 hpf.67 Costaining of
jak2a with gata1 at 14 hpf and thereafter showed that
both genes were expressed in identical regions of the lateral plate
mesoderm (data not shown). This suggests that the first cells of the
primitive wave of erythropoiesis express jak2a and that this
expression is a later event in the commitment to this lineage than
gata1 expression. Cells in this region maintained jak2a
expression as they moved from a lateral position to the midline
and differentiated in the intermediate cell mass (ICM; Fig 3d),
consistent with jak2a expression in proliferating proerythroblasts. By 24 hpf, high level staining was restricted to
cells of the anterior ICM, although a low level of expression was
detected in the brain and eyes (Fig 3e). The distribution of
jak2a transcript at this stage differs from the hematopoietic expression of the vascular and stem cell marker scl in 2 important respects. As shown in Fig 3f, with white arrowheads, cells in a set of bilateral stripes located more rostrally than the ICM are
scl-positive and thought to be persistent hematopoietic
progenitor cells66; however, these cells did not express
jak2a. Furthermore, although both scl and jak2a
transcripts were detected at high levels in the anterior ICM, only
scl expression is detected in the posterior ICM (solid
arrowhead, Fig 3f). In both of these aspects, the expression of
jak2a resembles that of gata1.67 Because
scl is expressed in both vascular and hematopoietic
precursors,66 we wished to establish unambiguously the
identity of the cells that expressed jak2a message. Sectioning
of embryos immediately postcirculation showed that jak2a
expression was confined to cells contained within the vasculature with
a large, rounded morphology (Fig
4). These cells also express gata1 and hemoglobin (data not shown, see Detrich et al67) and the embryonic
globin genes (see Fig 6, below), consistent with an
erythroblast identity.

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| Fig 3.
jak2a expression in the developing zebrafish. The
expression of jak2a in embryos at various developmental stages
was examined by whole mount in situ hybridization. (a) 10 hpf embryo,
dorsal view with anterior to the top. jak2a riboprobe gives a
widespread, low-level signal. Elevated expression is apparent in the
dorsal axis, but this corresponds to the thickest region of the embryo
and does not reflect an increase in the density of transcript. (b) 14 hpf embryo, lateral view with anterior to the top and left. Arrowheads
indicate the jak2a-riboprobe labeled line of cells in the
medial lateral plate mesoderm that gives rise to primitive blood
lineages. (c) 14 hpf dorsal view of dissection of dorsal and posterior
axial structures. Anterior (A) is to the left and posterior (P) is to
the right. jak2a transcript is evident in a narrow ribbon
of cells at the medial edge of the lateral plate mesoderm (arrow) that
extend to a distinct anterior limit (arrowhead). (d) 20 hpf embryos,
lateral view. jak2a riboprobe signal is detected at high level
in the medially converging cells of the ICM (arrowheads) and in the
eye. A lower level of signal is seen in the remainder of the anterior
CNS, but not in the trunk or posterior body. (e) 24 hpf embryo, lateral
view showing the labeling of the mature ICM (bracket) and in the eye
and anterior CNS at a lower level by jak2a probe. (f) 24 hpf embryos, lateral view showing a comparison of jak2a
with scl staining. Both jak2a and scl probes
label the anterior ICM (see [e] for reference); however, scl
is also detected in a dorso-anterior pair of bilateral stripes (open
arrowhead) and in the posterior ICM (solid arrowhead), whereas
jak2a is not. Expression of scl is also seen in cells
of the CNS. (g) 36 hpf embryo, lateral view showing detection of
jak2a message in circulating primitive erythrocytes (ce), cells
can be detected in the axial vessels of the trunk and tail, on
the yolk sac, and in the heart (open arrowhead). Elevated signal is
also seen in the eyes and in the midbrain (solid arrowhead). (h) 2.5 dpf embryo, lateral view showing detection of jak2a transcript
in the finbud (open arrowhead), the midbrain (solid arrowhead), and the
eye. Note that, in contrast to (g), there is no jak2a signal
from circulating blood. (i) 3.5 dpf larva, lateral view showing
jak2a message restricted to the eye and elements of the
pharyngeal arches (arrowhead). (j) 8 dpf larva, lateral view.
jak2a transcript can be detected in the pronephros (arrow) and
in circulating cells lodged in the vasculature of the tail (bracket).
A, anterior; aICM, anterior intermediate cell mass; ce, circulating
erythroblasts; cv, caudal vascular plexus; e, eye; fb, fin bud; ICM,
intermediate cell mass; lpm, lateral plate mesoderm; P, posterior; p,
pronephros; pICM, posterior intermediate cell mass.
|
|

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| Fig 4.
Expression of jak2a in circulating erythroblasts.
The expression of jak2a in circulating cells of the primitive
wave of hematopoiesis was examined in thin sections of animals after
the onset of circulation (26 hpf) after whole mount in situ
hybridization. Preparations are oriented with anterior to the left. (a)
Transverse section of trunk and tail at the level of the dorsal aorta.
Arrowheads demarcate the extent of the vasculature, indicating the
anterior dorsal aorta and the posterior vascular sinus.
jak2a-positive cells are confined within the vasculature
(arrows). (b) Higher magnification of transverse section of the caudal
vein showing large, rounded jak2a-positive cells (arrows)
within the vasculature. pm, paraxial mesoderm. Scale bars: for (a), 100 µm; for (b), 50 µm.
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|
The primitive wave of hematopoiesis consists mainly of erythrocytes.
jak2a transcripts were detected in maturing erythrocytes until
36 hpf (Fig 3g). Thereafter, jak2a expression in circulating cells decreased rapidly until 2.5 days postfertilization (dpf), at
which time jak2a was not present in circulating blood or in any
suspected hematopoietic site (Fig 3h). During the next 4 days of larval
development, jak2a remained undetectable in blood cells, although low-level expression persisted in the dorsal midbrain, eyes,
elements of the jaw, and fin buds (Fig 3h and i). At 8 days, jak2a expression was detected in the pronephros and in blood
cells found lodged in the ventral tail veins (Fig 3j). Expression in the site of adult hematopoiesis, the pronephros,65 and in
circulating erythrocytes indicates that definitive erythropoiesis gives
rise to cells that express jak2a. Although rag1
expression in the thymus68,69 was clearly detected by 5 dpf, jak2a was not expressed in the thymus (data not shown).
Thus, the timing and localization of jak2a expression in
hematopoietic cells suggests that it may be involved in the
transduction of signals into committed erythroblasts of both primitive
and definitive lineages. Expression of jak2a in the brain and
eyes suggests that intracellular signaling in these locations may also use the jak2a protein; however, the precise location of jak2a transcript in these structures was not determined.
Expression of jak2b in the developing zebrafish.
Northern blot analysis using total RNA indicated that jak2b was
expressed at a very low level during embryogenesis (data not shown).
Whole mount in situ hybridization demonstrated that expression at 24 hpf was restricted to the lens and the nephritic duct (Fig 5a and b),
persisting until 48 hpf (Fig 5c). The rostral extent of staining of
jak2b in the nephritic duct (Fig 5b) is equivalent to that seen
at 24 hpf with a probe to the ret gene.70 Low-level expression of jak2b was seen in the fin buds in embryos at 2.5 dpf, coincident with jak2a expression, but by 3.5 dpf, no
jak2b transcript was detectable by this method (data not
shown). At 5 dpf, low-level jak2b expression was seen in the
gill arches, elements of the jaw, and the anterior and posterior
lateral line, persisting until 8 dpf (Fig 5d). Thus, based on
thedistribution of transcript, jak2b might play a role in
signaling during embryonic lens and nephritic duct development and in
signaling in the larval lateral line and gills, but not in the
development of the hematopoietic system.

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| Fig 5.
jak2b expression in the developing
zebrafish. The expression of jak2b in embryos at various
developmental stages was examined by whole mount in situ hybridization.
All embryos and larvae are shown with anterior to the left and dorsal
to the top of the frame. (a) 24 hpf embryo, lateral view showing
detection of jak2b message restricted to the lens of the eye
(arrow) and in the nephritic ducts (arrowhead). (b) 24 hpf embryo,
ventro-lateral view at higher magnification, showing the region of the
nephritic duct stained by the jak2b riboprobe. The
bilaterally symmetric nephritic ducts are visible (arrows) as is the
distal tip of the ducts at the proctodeum (arrowhead). (c) 48 hpf
embryo, lateral view showing expression of jak2b persisting in
the lens of the eye (arrow) and decreasing from earlier levels in
the nephritic ducts (arrowhead). (d) 8 dpf larva, lateral view at
higher magnification, indicating detection of a low level of
jak2b message in elements of the jaw (arrow), in the developing
gills (bracket), and in the anterior lateral line (arrowheads).
Expression of jak2b in the posterior lateral line is evident,
but is not shown in this figure.
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|
Analysis of jak2a expression in the zebrafish hematopoietic
mutants.
Mutations that disrupt hematopoiesis have been identified in
zebrafish.36,37,47,67 The majority of these mutations were discovered by screening for the presence and color of circulating erythroblasts; hence, the majority represents genes required in erythropoiesis. Because the screens were performed at developmental stages up to 5 dpf, it seems likely that the target of the screen was
erythroblasts of the primitive cohort.37 Examination of mutant phenotypes allows mutant genes to be categorized into a scheme
of erythroblast development as outlined by Orkin and Zon.71 Embryos from selected mutant lines were examined for perturbation of
jak2a expression by in situ hybridization at various time
points before and/or after the onset of a visible phenotype. A summary of the results of this investigation is presented in
Table 1, and the results are described
below in detail.
Expression of jak2a in a Hemangioblast mutant,
cloche.
Homozygous cloche (clo) animals fail to produce blood
or vasculature and die as embryos.47,66,72,73 Consistent
with a general failure in clo mutants to produce cells of the
hematopoietic lineage, jak2a expression in the hematopoietic
lateral plate mesoderm was not initiated at 13 hpf in approximately one
quarter of the embryos from a given clutch and neither was it detected
in hematopoietic tissue at any stage examined thereafter (Table 1),
although jak2a central nervous system (CNS) expression appears
normal. The expression of jak2a in the clo mutant
background was compared with the expression of other lineage and stage
specific markers at 24 hpf (Fig 6). Clutches of embryos from
heterozygous incrosses were examined before the onset of circulation by
in situ hybridization for expression of the stem cell marker
scl,66 the immature erythroblast marker gata1,67 jak2a, and the embryonic and
globins e1 globin and e3 globin, markers of
primitive erythrocytic differentiation (Brownlie et al, manuscript in
preparation). Three quarters of the embryos in a given
clutch showed strong signal in the ICM from all gene probes (Fig 6a, c,
e, g, and i). One quarter of embryos in a given clutch displayed a
distinctive phenotype involving the dramatic reduction of signal from
the ICM (Fig 6b, d, f, h, and j). In approximately 50% of the embryos
that displayed a loss of expression in the ICM, persistent expression
of scl, gata1, e1 globin, and e3
globin could be seen in 5 to 10 cells in the caudal part of the
anterior ICM that appear to escape the
clo / hematopoietic block (Fig 6b, d,
h, and j; see Stainier et al47). However, ICM expression of
jak2a was not observed in any embryo that also lacked anterior
ICM expression (n = 53, 4 independent experiments).

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| Fig 6.
Comparison of scl, gata1, jak2a,
e1 globin, and e3 globin
expression in cloche mutant embryos. Clutches of embryos
derived from heterozygous clo parents were raised for 24 hours
and assayed for the expression of various hematopoietic and erythroid
marker genes: scl (a and b); gata1 (c and d);
jak2a (e and f); e1 globin (g and h); and e3
globin (i and j). Embryos are displayed in a lateral position with
anterior to the left and dorsal to the top of each panel. Approximately
three quarters of the embryos in a given clutch had the wild-type
expression pattern of the gene in question (a, c, e, g, and i); note
the prominent staining of the ICM. Approximately one quarter of the
embryos in a given clutch showed a near or total absence of all
hematopoietic marker gene expression in the ICM (b, d, f, h, and j);
presumably, these are clo homozygotes. In approximately one
half of mutant embryos, from 5 to 10 cells in the ICM express the
scl, gata1, e1 globin, and e3
globin marker genes (arrow in b, d, h, and j). No jak2a
expression was observed in this area in any mutant embryo (f), ie, an
embryo also lacking jak2a expression in the rostral part of the
anterior ICM.
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Expression of jak2a in an early onset hematopoietic
mutant, spadetail.
In contrast to clo, embryos homozygous for the
spadetail (spt) mutation specify and differentiate
vasculature correctly.73 However, the hematopoietic program
is severely affected, with few, if any primitive erythroblasts reaching
maturity. Clutches of embryos from spt heterozygous parents
were examined for jak2a expression throughout the first 24 hours of development (Fig 7). Despite the
expression of the stem cell marker scl in spt mutants in the lateral plate mesoderm at 14 somites (Fig 7d), and in contrast to wild-type embryos at this stage (Fig 7a), jak2a transcripts could not be detected in the spt homozygotes in regions of
hematopoietic activity (Fig 7c). As spt homozygotes developed,
scl expression in the lateral plate decreased dramatically,
indicating a failure to maintain a population of early stem cells (Fig
7h). Occasionally in spt homozygotes, isolated scl,
gata1, and embryonic globin-positive cells were visible
in the ICM, as shown by arrowheads in Fig 7h (and data not shown, see
Thompson et al73), indicating the emergence of a more
mature primitive erythroid cell. However, jak2a message does
not accumulate in the corresponding locations or in any hematopoietic tissue at the stages examined (Fig 7g). Combined with the data from
clo mutants, this result indicates that, within the boundaries of the sensitivity of the technique, the expression of erythroid markers in the escaper cells of the caudal part of the anterior ICM is
not accompanied by jak2a expression.

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| Fig 7.
Expression of jak2a is perturbed in embryos with
mutation in the spadetail gene. Clutches of embryos derived
from heterozygous spt parents were raised for 14 and 24 hours
and assayed for the expression of stem cell and erythroid marker genes
scl (b, d, f, and h) and jak2a (a, c, e, and g),
respectively. The spt mutant can be unambiguously scored at the
developmental stages presented here by the loss of trunk somites;
embryos shown in (c), (d), (g), and (h) are spt homozygotes.
Note the loss of jak2a staining at all stages in the
spt mutant embryos. Embryos in (a) through (d) are dissected
and flat mounted, with the anterior to the left; all other embryos (e)
through (h) are displayed in a lateral position with anterior to the
left and dorsal to the top of each panel.
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|
Expression of jak2a in late onset hematopoietic mutant
zebrafish.
Expression of jak2a was examined by in situ hybridization in
zebrafish embryos carrying mutations in the frascati
(frs), chablis (cha), retsina
(ret), weißherbst (weh), cabernet
(cab), sauternes (sau), chardonnay
(cdy), and chianti (cia) genes, which display a
late phenotypic onset.36 In all mutants examined,
jak2a expression was found in the cells of the ICM at 24 hpf
and in circulating erythroblasts until 48 hpf, as seen in wild-type
clutches (Table 1). In summary, jak2a expression is perturbed
only in those mutants (clo, spt) that perturb
erythropoiesis at early stages in development, supporting the
hypothesis that jak2a is expressed in immature primitive
erythroblasts in the zebrafish.
Linkage of the jak2a gene to the hematopoietic mutants.
As described above, jak2a was mapped to the distal tip of LG21
(Fig 2b and e). Comparison of the map position of jak2a with those of the mutants with known positions on the zebrafish genetic map
showed that jak2a was not linked to cha, cdy,
chi, clo, frs, gre, mon,
pin, ris, ret, sau, spt, or
weh (data not shown).Thus, jak2a is not a candidate
gene for any of these mutations. Linkage to additional, currently
unmapped hematopoietic mutants cab and mot was tested
by typing genomic DNA from mutant embryos with the
jak2a-associated marker used to map the jak2a gene, as
described above. The jak2a polymorphism did not segregate with
either the cabtl236 or
mottm303c phenotype (data not shown), indicating
that jak2a is not linked to these mutations.
 |
DISCUSSION |
Recent studies in Drosophila and mouse have shown a role for
members of the JAK gene family in the control of growth and
differentiation of multiple blood cell lineages and in leukemogenesis.
The Jak2 gene is required in mice for successful erythropoiesis
and JAK2 is implicated in ALL in human patients. We show here
that the zebrafish has 2 jak2 genes that are expressed in the
developing embryo and larva; however, only 1, jak2a, is
expressed in the erythropoietic system. Our results in wild-type and
mutant embryos suggest that jak2a may play an early role in
primitive erythropoiesis in the zebrafish and, thus, is likely to
represent the functional homolog of the mouse Jak2 gene with
respect to hematopoiesis.
Hematological implications of zebrafish jak2a expression.
The timing of jak2a expression in wild-type embryos suggests
that the presence of jak2a message defines an intermediate
stage in the lineage of the primitive erythrocyte that occurs between the commitment of progenitors and the expression of the
end-differentiated phenotype. Furthermore, the transience of
jak2a expression indicates that any signaling into primitive
erythroblasts via receptors that use jak2a occurs in a window of time
as the erythroblasts mature from 14 hpf to approximately 2 dpf.
Expression of jak2a in the developing erythrocytes of wild-type
and mutant zebrafish has strong implications for the involvement of
jak2a in cytokine signaling in hematopoiesis.
Analysis of jak2a expression in clom39 and
sptb104 homozygotes showed that jak2a
transcription is not initiated at the normal time, and neither is it
present in hematopoietic tissue at subsequent stages. This result is
consistent with jak2a expression in cells of the hematopoietic
lineage and allows inferences about jak2a function to be made.
In some clo mutant embryos, a restricted number of primitive
erythrocytes (5 to 10) form in a remnant tail vasculature; these cells
are heme-reactive, indicating that a terminally differentiated state
has been reached. Likewise, in spt mutants, isolated mature
primitive erythroblasts can be detected. In contrast to wild-type
erythroblasts, jak2a is not coexpressed with gata1 or
the embryonic globins in these cells (Figs 6 and 7), being
completely absent from the ICM in every embryo examined, indicating
that jak2a expression is not required for the completion of
differentiation to a globin/heme-expressing stage in these cells. It
may instead be required for an aspect of survival or proliferation, as
is the case in the mouse.12,13,22,23,74 Because
jak2b is not expressed in erythropoietic tissue, cells without
jak2a are likely to be without JAK2 function. Thus, the surviving
primitive erythrocytes of clo and spt mutant embryos would not be expected to be receptive to cytokine or other signals that
require jak2. Because it is presently unclear whether these cells
represent a normal stage of erythropoiesis that has been unmasked by
the absence of clo or spt or whether these cells are a
peculiarity of clo and spt mutant embryos, we are
unable to make strong conclusions about the requirement for jak2a
function in the differentiation of wild-type erythrocytes.
Examination of jak2a expression in embryos from late onset
hematopoietic mutants showed that the genetic deficiencies in these fish did not disrupt erythropoietic development before the putative cytokine-receptive stage defined by expression of jak2a. This result is not surprising, because their cell morphology, by analogy to
the mouse, suggests a mature post-EPO-dependent stage.71 The correlation of a failure to generate or maintain blood cell number
with absence or loss of jak2a expression in mutants is consistent with a potential role for this gene in the proliferation or
survival of polychromatic erythroblasts.
It is of interest to compare these results with recent findings
obtained from gene targeting experiments in the mouse. Mice lacking
functional Jak2 exhibit significant defects in primitive and
definitive erythropoiesis, although their development is otherwise overtly normal.22,23 This is consistent with the high-level expression of jak2a seen in the erythroid cells of the
zebrafish embryo and larva. The erythropenic phenotype of the
Jak2 / mice is more severe than mice
carrying an Epo or EpoR null mutation,75,76 with fewer circulating yolk sac-derived primitive erythrocytes and an
earlier block in the progression of definitive erythropoiesis. Committed erythroblasts are present in these animals, but they do not
expand and differentiate. Hemoglobinization of definitive erythroid
cells is almost abolished in Jak2 /
fetal liver cells,23 but the expression of embryonic
globins specific for the primitive cohort appears less
affected.22 This phenotype is consistent with the
expression and regulation of jak2a in wild-type and mutant
zebrafish presented here; thus, the jak2a gene of zebrafish
likely represents the functional homolog of the mouse Jak2 gene
with respect to its role in hematopoiesis.
Evolutionary relationships among the jak genes.
The presence of 4 JAK genes per mammalian genome fits well with
current theories about tetraploidization events early in the vertebrate
lineage that suggest 2 successive duplications giving rise to 4 copies
of an ancestral chromosome complement.77 Genes of the
JAK family in zebrafish map to separate chromosomes, indicating that tandem duplication is not the cause of the extra jak2
genes in the zebrafish. Instead, they map to regions in which syntenies are conserved compared with their homologs in mouse and human, a region
known as the Katsanis paralogy group.78 This finding extends the observation that large portions of the chromosomes of early
vertebrates remain intact, with disturbance mainly from local
rearrangement,59,79 and indicates that the cause of the initial JAK2 duplication seems to have been a large-scale
event, possibly involving 1 or more chromosomes (Fig 2g and h). If the paralogous duplication took place before the lineage of ray fin and
lobe fin fish (ie, tetrapods) diverged, there must have existed a
second JAK2 paralog in the genome of both lineages. In this case, there may still be a second JAK2 in existing tetrapod
genomes. However, examination of mammalian JAK2 cDNA sequences
and ESTs in the databases indicates that all JAK2 proteins from
different mammals reported are more than 95% identical to each other
and that all cDNAs or ESTs from any given species are, in fact, from the same gene (data not shown). Consideration of JAK1,
JAK3, and TYK2 database entries in the same manner
indicates that all listed sequences, ignoring splice variants, are
orthologous or identical. In conclusion, mounting evidence of the
existence of higher numbers of gene family members in zebrafish and
other ray finned fish than in mammalian genomes80,81
combined with the chromosomal localization data presented above favors
the scenario in which duplicate jak2 genes are an innovation
specific to ray finned fish.
Comparison and implications of jak2 expression patterns in
the zebrafish.
The jak genes of the zebrafish are expressed at a high level in
restricted groups of cells in the developing embryo and larva. Of
particular interest is the divergence in expression patterns of the
jak2a and jak2b genes. The only site of coexpression
during development was in the lens of the eye at 18 to 36 hpf (Figs 3e and g and 5a and c). Thus, the regulatory regions of the genes have
diverged profoundly in activity, whereas the coding sequence has
maintained a high conservation in sequence identity, consistent with
the generation of genetic novelty by the alteration of gene expression
patterns without radical changes to the biochemical activity of the
protein product.82
Widespread expression of mammalian JAK mRNA detected in cell
lines and adult tissues by Northern blotting3,10,37,83-85 and the propensity of cytokine receptors to use multiple JAK proteins in signaling1 suggested a near ubiquitous expression of
these intracellular signaling components. In this view, developmental timing and positional cues would be supplied by the restricted expression of both extracellular signaling molecules and their cognate
receptors. However, it is clear from this study that the expression of
the appropriate signal transduction components could equally serve
these timing and positional functions. Potentially, a closer
examination of JAK expression in mammals may show a similar distribution and restriction of transcripts.
Prospects for genetic analysis of vertebrate JAK function
using the jak2a gene in zebrafish.
The ability to screen a vertebrate genome for mutations that modify
JAK function, a task that would not be practical in the mouse,
requires the identification or production of a mutation in a
JAK gene. Linkage analysis presented above indicates that jak2a is not a candidate gene for the 17 hematopoietic mutants examined. The data on the timing of jak2a expression in
wild-type and mutant zebrafish suggest that any role for jak2
in erythropoiesis is confined to stages after the onset of
gata1 expression, implying possible jak2a function in
cells equivalent to progenitor or proerythroblasts. This, along with
the phenotype of a Jak2-deficient mouse, suggests that animals
with a jak2a mutation would initially express markers for
hematopoietic stem cells (HSCs) and progenitor cells such as scl, lmo2,73 and gata2 in an
equivalent manner to wild-type.
Other hematopoietic mutants have not been tested for linkage with
jak2a, namely, bloodless, vlt, vmp,
tbr, stb, paw, and
clb.37,67 Of these, bloodless, vlt,
and vmp show a hematopoietic phenotype that appears to be too
early for the expected role of jak2a, whereas tbr,
stb, paw, and clb appear to act too late.
However, because these phenotypes are incompletely
characterized, and it is not known whether any of these mutations is a
complete loss of function in the gene in question, they cannot be ruled
out as presenting potential jak2a mutant phenotypes. Because
calculations presented at the conclusion of the large-scale screens
indicate that approximately 50% saturation was
approached,86 a mutation in the jak2a gene may not
have been isolated to date. Of course, it is formally possible that
jak2a does not perform an essential function in the development
of the erythrocyte lineage in the zebrafish.
An alternative strategy for the generation of a JAK phenotype
would be to use 1 of the leukemogenic JAK alleles known from mammals and flies29-33 to induce a neoplastic state in the
zebrafish hematopoietic system. Screening the zebrafish genome for
enhancer and suppressor loci of such a phenotype should yield
information on genes controlling the initiation and progression of
vertebrate hematopoietic neoplasia.
Given the parallels between mouse Jak2 and zebrafish
jak2a, it is interesting to speculate on the consequences of
the potential subfunctionalization87 of jak2 in the
zebrafish, a scenario for gene duplication in which complementary
functions (eg, expression domains) can be lost by 2 gene duplicates,
making both essential to survival. The expression of jak2b is
not consistent with any role for this paralog in erythropoiesis; thus,
jak2a appears likely to be the functional homolog of the
mammalian JAK2. Mice that can be rescued from their requirement
for Jak2 function in erythropoiesis by reconstituting with
wild-type hematopoietic stem cells may yield interesting additional
phenotypes. One prediction of the studies presented here is that lens
and kidneys of the rescued mice may show defects. To generalize from
this case, the presence of extra gene copies in the zebrafish would not
necessarily prove a hindrance to the analysis of gene function; rather,
it may allow access to restricted or late onset phenotypes that might
not be observable in the mouse.
In conclusion, the findings of this study underscore the potential use
of the zebrafish to model the cytokine functions of mammals.
Furthermore, the ability to search the genome of the zebrafish for loci
that modify the severity of a hematopoietic phenotype would be
invaluable in the analysis of the complex signaling events underlying
the regulation of blood growth.
 |
ACKNOWLEDGMENT |
The authors thank Jana Stickland for her invaluable help with figures.
Thanks are extended also to Cuong Do and to the members of the Growth
Regulation and Cytokine Biology Laboratories for many discussions. Many
thanks to the members of the Zon lab fish collective for providing
mutant embryos and for their support and encouragement. Thanks to
Ashley Bruce, Graham Lieschke, and Jensen Hjorth for constructive
criticism of the manuscript and to Robert Ho, in whose lab this work
was completed.
 |
FOOTNOTES |
Submitted November 30, 1998; accepted June 8, 1999.
A.C.O. was supported by an Anti Cancer Council of Victoria Postgraduate
Research Scholarship. D.V.I. was supported by an Anti Cancer Council of
Victoria Summer Scholarship. E.C.L. is a Pre-Doctoral Fellow of the
Howard Hughes Medical Institute. B.H.P. was supported in part by a
H.H.M.I. Postdoctoral Fellowship for Physicians. J.H.P. was supported
by Grants No. RO1RR10715 and PHS PO1HD22486.
The sequences reported in this manuscript were submitted to the EMBL
database and have the following accession numbers: jak1, AJ005689; jak2a, AJ005690; and jak2b, AJ005691.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Andrew C. Oates, PhD,
Department of Molecular Biology, Princeton University, Washington Road,
Princeton, NJ 08544; e-mail: aoates{at}molbio.princeton.edu.
 |
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