Arrested differentiation and epithelial cell degeneration in zebrafish lens mutants.

In a chemical mutagenesis screen, we identified two zebrafish mutants that possessed small pupils. Genetic complementation revealed these two lines are due to mutations in different genes. The phenotypes of the two mutants were characterized using histologic, immunohistochemical, and tissue transplantation techniques. The arrested lens (arl) mutant exhibits a small eye and pupil phenotype at 48 hr postfertilization (hpf) and lacks any histologically identifiable lens structures by 5 days postfertilization (dpf). In contrast, the disrupted lens (dsl) mutants are phenotypically normal until 5 dpf, and then undergo lens disorganization and cell degeneration that is apparent by 7 dpf. Histology reveals the arl mutant terminates lens cell differentiation by 48 hpf, whereas the dsl lens exhibits a defective lens epithelial cell population at 5 dpf. Lens transplantation experiments demonstrate both mutations are autonomous to the lens tissue. Immunohistochemistry reveals the retinal cells may suffer subtle effects, possibly due to the lens abnormalities.     

A genetic screen for zebrafish mutants with hepatic steatosis identifies a locus required for larval growth

In a screen for zebrafish larval mutants with excessive liver lipid accumulation (hepatic steatosis), we identified harvest moon (hmn). Cytoplasmic lipid droplets, surrounded by multivesicular structures and mitochondria whose cristae appeared swollen, are seen in hmn mutant hepatocytes. Whole body triacylglycerol is increased in hmn mutant larvae. When we attempted to raise mutants, which were morphologically normal at the developmental stage that the screen was conducted, to adulthood, we observed that most hmn mutants do not survive to the juvenile period when raised. An arrest in growth occurs in the late larval period without obvious organ defects. Maternal zygotic mutants have no additional defects, suggesting that the mutation affects a late developmental process. The developmental window between embryogenesis and the metamorphosis remains under‐studied, and hmn mutants might be useful for exploring the molecular and anatomic processes occurring during this transition period.     

A gene expression screen in zebrafish embryogenesis.

A screen for developmentally regulated genes was conducted in the zebrafish, a system offering substantial advantages for the study of the molecular genetics of vertebrate embryogenesis. Clones from a normalized cDNA library from early somitogenesis stages were picked randomly and tested by high-throughput in situ hybridization for restricted expression in at least one of four stages of development. Among 2765 clones that were screened, a total of 347 genes with patterns judged to be restricted were selected. These clones were subjected to partial sequence analysis, allowing recognition of functional motifs in 163 among them. In addition, a portion of the clones were mapped with the aid of the LN54 radiation hybrid panel. The usefulness of the in situ hybridization screening approach is illustrated by describing several new markers for the characteristic structure in the fish embryo named the yolk syncytial layer, and for different regions of the developing brain.     

Interordinal chimera formation between medaka and zebrafish for analyzing stem cell differentiation

Chimera formation is a standard test for pluripotency of stem cells in vivo. Interspecific chimera formation between distantly related organisms offers also an attractive approach for propagating endangered species. Parameters influencing interspecies chimera formation have remained poorly elucidated. Here, we report interordinal chimera formation between medaka and zebrafish, which separated ～320 million years ago and exhibit a more than 2-fold difference in developmental speed. We show that, on transplantation into zebrafish blastulae, both noncultivated blastomeres and long-term cultivated embryonic stem (ES) cells of medaka adopted the zebrafish developmental program and differentiated into physiologically functional cell types including pigment cells, blood cells, and cardiomyocytes. We also show that medaka ES cells express differentiation gene markers during chimeric embryogenesis. Therefore, the evolutionary distance and different embryogenesis speeds do not produce donor-host incompatibility to compromise chimera formation between medaka and zebrafish, and molecular markers are valuable for analyzing lineage commitment and cell differentiation in interspecific chimeric embryos.     

Cyclops-independent floor plate differentiation in zebrafish embryos.

In zebrafish, development of the ventral neural tube depends on the Nodal-related signal Cyclops (Cyc). One-day-old cyc mutant embryos lack the medial floor plate (MFP). We show here that cells expressing MFP marker genes differentiate gradually in cyc mutant embryos in a delayed manner during the second day of development. This late differentiation is restricted to the hindbrain and spinal cord and depends on an intact Hedgehog (Hh) signalling pathway. Cells expressing MFP marker genes in cyc mutant embryos appear to be derived from lateral floor plate (LFP) cells as they coexpress LFP and MFP marker genes. This finding suggests that the correct temporal development of the MFP is required for the distinction of LFP and MFP cells in wild-type embryos.     

In situ hybridization screen in zebrafish for the selection of genes encoding secreted proteins.

An in situ hybridization expression screen using a signal sequence trap system has been conducted in zebrafish to isolate cDNAs that encode secreted proteins. Random clones (secreted expressed sequence tags; sESTs) were sequenced from zebrafish embryonic (18-24 hr postfertilization) and adult kidney libraries. From the two RNA sources, 627 random sEST cDNAs were identified as being homologous or identical to known genes and 166 clones encode currently unidentified genes. The sESTs represent a broad range of enzymes and other regulatory molecules. Whole-mount in situ hybridization analysis was carried out by using antisense probes generated from 244 selected sESTs, and a range of expression patterns was obtained. Genetic mapping undertaken with sEST sequences demonstrated that assignment of map position was attainable by using 5' primers. The signal sequence trap system used in this work has yielded a range of cDNAs that encode secreted proteins and, together with analysis of patterns of expression and genetic mapping, has the potential to facilitate analysis of signaling pathways central to development and physiology.     

Behavioral screening for nightblindness mutants in zebrafish reveals three new loci that cause dominant photoreceptor cell degeneration

Here we report three dominant nightblindness mutations in zebrafish: nightblindness e (nbe), nightblindness f (nbf) and nightblindness g (nbg). The mutants were isolated in the F1 generation of N-ethyl-N-nitrosourea (ENU) mutagenized zebrafish using a behavioral assay based on visually mediated escape responses. Subsequently, electroretinographic (ERG) recordings were made, and histological sections were screened for degenerative processes. For each mutant line, correlation analysis between behavioral, ERG and histological parameters was performed, and their relationships were determined by either calculating the Pearson correlation coefficient or by ANOVA. nbe is characterized by severe rod outer segments (ROS) degeneration. The degeneration correlates weakly with behavioral threshold and ERG b-wave amplitude, however, behavioral threshold correlates strongly with ERG b-wave. nbf is characterized by a dual histological pathology: patchy ROS-degeneration and 'gaps' homogeneously distributed over the outer nuclei layer (ONL) and between cone outer segments (COS). The correlations between histological pathology and behavioral threshold, and between behavioral threshold and ERG b-wave amplitude are obvious, but the correlation between histology and b-wave amplitude is less prominent. nbg is characterized by moderate ROS degeneration and moderate correlation between histology and behavioral threshold. Interestingly, behavioral threshold correlated inversely with ERG b-wave amplitude and threshold. Thus, contrary to what is normally seen in other nightblindness mutants, in nbg, the fish with the lowest behavioral threshold had the smallest b-waves amplitudes and the highest b-wave threshold. In our interpretation, the major impairment in nbe is photoreceptor-specific. In nbf, both photoreceptor degeneration and altered post-photoreceptor signaling are responsible for the behavioral deficit. In nbg, we find hypersensitivity at a post-photoreceptoral level concurrently with behavioral impairment.     

A cytological procedure to screen mammalian temperature-sensitive mutants for cell-cycle-related defects

Interphase cells stained with quinacrine dihydrochloride show distinctive fluorescent nuclear patterns according to their position in the cell cycle. These patterns were used to determine whether temperature-sensitive growth mutants of the Syrian hamster cell line BHK-21 are blocked at specific stages of the cell cycle. These cytological studies confirmed the previous conclusion that ts Af8 cell line derived from BHK cells is a GI cell-cycle mutant. The method promises to be of use as an initial probe in screening for cell-cycle mutants.     

Langerhans' cell histiocytosis: expression of leukocyte cellular adhesion molecules suggests abnormal homing and differentiation.

Langerhans' cell histiocytosis (LCH) is characterized by an accumulation of cells with a Langerhans' cell (LC) phenotype. Most patients present with solitary skin or bone lesions, but multi-organ lesions may appear. Twenty-two LCH-tissue sections from 13 children and adolescents, with lesions at different sites, were investigated for the expression of leukocyte cellular adhesion molecules. Surprisingly, the LCH cells showed expression for CD2 in 11 lesions. Staining of LCH cells for CD11a and CD11b was positive in six and three lesions, respectively. Staining for CD11c, CD44, CD54, and CD58 was found consistently positive in all lesions. The strong reactivity for CD54 (intercellular adhesion molecule-1) and CD58 (leukocyte function antigen-3) is in contrast with the epidermal LC. LCs in culture are known to up-regulate the expression of CD54 and CD58. These changes are thought to reflect the in vivo situation during migration of activated LCs from the skin to the draining lymph node. It can be concluded that the abnormal cells in LCH not only share characteristics with the epidermal LC, but have additional characteristics of the activated LC, a cell capable of migration. The presumed immunological dysregulation in LCH may affect the expression of cellular adhesion molecules, reflected by the inconsistent expression of CD11a and CD11b and the unexpected expression of CD2. These features may contribute to migration of LCs to aberrant sites in combination with abnormal persistence and proliferation.     

The pineal gland in wild-type and two zebrafish mutants with retinal defects

Light and transmission electron microscopy were used to characterize the ultrastructural features of the pineal glands of wild-type and two mutant zebrafish strains that have retinal defects. Particular attention was given to the pineal photoreceptors. Photoreceptors in the pineal gland appear quite similar to retinal cone photoreceptors, having many of the same structural characteristics including outer segment disk membranes often confluent with the plasma membrane, calycal processes surrounding the outer segments, and classic connecting cilia. The pineal photoreceptor terminals differ from photoreceptor terminals in the retina in that they have short synaptic ribbons and make dyad synapses which may or may not be invaginated. Pineal photoreceptors in two zebrafish mutants with abnormal retinal photoreceptors were also studied. Pineal photoreceptors in the niezerka (nie) mutant degenerate, as they do in the retina, indicating that pineal and retinal photoreceptors share at least some genes. However, the synaptic terminals of no optokinetic response c (nrc) pineal photoreceptors are normal, suggesting that this mutation is specific to the retina.     

fgf1 is required for normal differentiation of erythrocytes in zebrafish primitive hematopoiesis.

Hematopoiesis in vertebrate development involves an embryonic, primitive wave and a later, definitive wave in which embryonic blood cells are replaced with adult blood cells. We here show that zebrafish fgf1 is involved in vivo in primitive hematopoiesis. Fibroblast growth factor-1 (FGF1) morpholino knockdown leads to abnormal accumulation of blood cells in the posterior intermediate cell mass at 32 hr postfertilization. Expression of the erythroid markers gata1 and ika, normally diminishing in differentiating erythrocytes at this stage, is maintained at abnormally high levels in primitive blood cells. The onset of erythrocyte differentiation as assessed by o-dianisidine staining is severely delayed. Most fgf1 morphants later recover to wild-type appearance, and primitive erythrocytes eventually differentiate. Zebrafish fgf1 is syntenic to human FGF1, which maps to a critically deleted region in human del(5q) syndrome posing an increased risk of leukemia to patients. As its knockdown in zebrafish changes expression of gata1, a gene involved in hematopoietic stem cell decisions, FGF1 should be considered to play a role in the pathogenesis of del(5q) syndrome.     

Escherichia coli hemolysin mutants with altered target cell specificity.

In order to understand the functional significance of HlyC-dependent acylation of the Escherichia coli hemolysin structural protein (HlyA), random as well as site-directed substitutions at the known regions of modification, i.e., those at lysine residues at amino acid positions 563 and 689 (HlyAK563 and HlyAK689, respectively), were isolated. Sixteen random hlyA mutations were identified on the basis of a screen for loss of immunoreactivity to the hemolysin-neutralizing D12 monoclonal antibody that reacts to only HlyC-activated HlyA. These substitutions occurred at the region from HlyAE684 to HlyAY696. A recombinant glutathione S-transferase-hemolysin gene fusion encoding glutathione S-transferase-HlyAS608-T725 residues reacts with monoclonal antibody when HlyC is coexpressed with the fusion protein. Therefore, at most only 12% of the total HlyA primary sequence is needed for HlyC-facilitated acylation at the HlyAK689 position, and this modification can occur in the absence of the proximal HlyAK563 acylation site. The cytolytic activities of these HlyA mutants against sheep erythrocytes and bovine and human lymphocyte cell lines (BL-3 and Raji cells, respectively) were analyzed. HlyAK563 and HlyAK689 substitutions displayed various degrees of loss of cytotoxicity that depended on the particular amino acid replacement. An HlyAK563C variant retained greater than 59 and 21% of its BL-3-lytic and erythrolytic activities, respectively, but was nearly inactive against Raji cells. An HlyA mutant with a K-to-E substitution at amino acid 689 (HlyAK689E) was essentially inactive against all three cell types, whereas an HlyAK689R substitution had a pattern of activity similar to that of the HlyAK563C mutant. Preceding the two in vitro acylated HlyA lysines are glycines that appear to be the only amino acids conserved in alignments of these regions among the RTX toxins. Remarkably, considering the retention of cytotoxic activity by some HlYAK689 mutants, each of three different substitutions at the HlyAG688 position was relatively inactive against all three cell types tested. This suggests that HlyAG688 plays a significant structural role in cytotoxic activity apart from its possible participation in an HlyC activation process which presumably requires recognition of pro-HlyA structures. The related RTX toxin, the Pasteurella haemolytica leukotoxin structural protein (LktA), can be activated in an E. coli recombinant background by HlyC. In amino acid sequence alignments, LktAK554 is equivalent to the HlyAK563 position but it has an asparagine (LktAN684) at the homologous HlyAK689 site. An LktAN684K substitution possesses wild-type leukotoxin activity against BL-3 cells and does not acquire hemolytic or Raji cell cytotoxic activity. Surprisingly, both LktAK554C and LktAK554T substitutions retain considerable BL-3 cytotoxicity (45 and 49%, respectively), indicating that there may be additional lysines within LktA that the HlyC activation mechanism is capable of acylating. Based on these results and a comparison of amino acid sequence alignments of 12 RTX toxins, a putative consensus structure of the RTX residues necessary for HlyC activation is hypothesized.     

Malignant islet cell tumor with sarcomatous differentiation.

Malignant mesenchymal neoplasms of the pancreas are rare and malignant islet cell tumors with sarcomatous dedifferentiation are rarer still. We present a case of malignant islet cell tumor with sarcomatous differentiation, which to our knowledge is only the second reported case showing such a combination of morphologic features. Clinically, the neoplasm was not hormonally active and immunohistochemical staining was negative for gastrin, glucagon, insulin and somatostatin. The sarcomatous component strongly reacted with an antibody directed against vimentin, and a minority of cells stained strongly with antisera directed against desmin and smooth muscle actin. The spindle cell component was nonreactive with antibodies directed against Factor VIII. The myogenous direction of differentiation in the present tumor is similar to that seen in the prior case report of malignant islet cell tumor with rhabdomyosarcomatous differentiation.     

Metastatic Leydig cell tumor with sarcomatoid differentiation.

Leydig cell tumors of the testis are uncommon. Only about 10% of cases have a malignant course. It has been stated that the only definite criterion for malignancy is presence of metastasis. We present a 47-year-old patient with metastatic Leydig cell tumor 17 years after initial diagnosis, to our knowledge the longest reported interval between diagnosis and the development of metastasis. The primary tumor did not exhibit convincing features of malignancy. The initial metastasis in the right perirenal fat tissue showed a biphasic tumor with sarcomatoid differentiation not described previously in a metastatic Leydig cell tumor.     

Systematic follow-up and case management of the abnormal newborn screen can improve acceptance of genetic counseling for sickle cell or other hemoglobinopathy trait.

PURPOSE: Sickle cell or other hemoglobinopathy trait detected on the newborn screen provides an opportunity for genetic counseling of families at risk of having a child with a major hemoglobinopathy. However, follow-up of hemoglobinopathy trait is often fragmented and acceptance of counseling is low. We describe the results of systematic follow-up and case management of abnormal newborn screen and the effect on acceptance of counseling. METHODS: From July 1997 to June 2002, families of a newborn with hemoglobinopathy trait were notified by mail. In April 2003, an intensive trait follow-up protocol including letters, telephone calls, educational videos, and genetic counseling was implemented. Demographic information and follow-up activity were documented and tracked using an electronic database. RESULTS: From July 1997 to June 2002, 3095 families were notified by letter of a newborn with hemoglobinopathy trait and were offered genetic counseling. Of these, 165 (5.3%) received counseling by telephone and 60 (2%) underwent extended family testing. From April to December 2003, 694 families with a newborn with hemoglobinopathy trait were notified by mail. Of these, 362 (52%) families were reached by telephone. Of those contacted by telephone, 92% received genetic counseling via telephone, 57% were interested in family testing, and 12% scheduled an appointment. Additionally, 27% of families were mailed an educational video. Among those declining extended family testing, 26% preferred to consult their pediatrician. CONCLUSIONS: Systematic follow-up and case management of abnormal newborn screen can improve the acceptance of genetic counseling.     

Human cytotrophoblast differentiation/invasion is abnormal in pre-eclampsia.

During human placental development, cytotrophoblast stem cells differentiate and invade the uterus. Simultaneously, the cells modulate their expression of several classes of stage-specific antigens that mark transitions in the differentiation process and play a role in either uterine invasion (integrin cell-extracellular matrix receptors and matrix metalloproteinase-9) or immune interactions (HLA-G). The pregnancy disease pre-eclampsia is associated with shallow cytotrophoblast invasion. Previously we showed, by immunofluorescence localization on placental tissue, that in pre-eclampsia invasive cytotrophoblasts fail to properly modulate their integrin repertoire. This finding suggests possible abnormalities in the differentiation pathway that leads to uterine invasion. Here we used a culture system that supports this differentiation process to compare the differentiative and invasive potential of cytotrophoblasts obtained from control (n = 8, 22 to 38 weeks) and pre-eclamptic (n = 9, 24 to 38 weeks) placentas. In culture, the cells from pre-eclamptic placentas failed to properly modulate alpha1 integrin and matrix metalloproteinase-9 expression at the protein and mRNA levels. Their invasive potential was also greatly reduced. Likewise, the cells failed to up-regulate HLA-G protein and mRNA expression. These results suggest that defective cytotrophoblast differentiation/invasion can have significant consequences to the outcome of human pregnancy (ie, development of pre-eclampsia) and that, by the time delivery becomes necessary, the defect is not reversed by removing the cells from the maternal environment.     

A rapid and efficient method of genotyping zebrafish mutants

In order to facilitate high throughput genotyping of zebrafish, we have developed a novel technique that uses High Resolution Melting Analysis (HRMA) to distinguish wild‐type, heterozygous mutants and homogyzous mutants. This one hour technique removes the need for restriction enzymes and agarose gels. The generated melting curve profiles are sensitive enough to detect non‐specific PCR products. We have been able to reliably genotype three classes of mutations in zebrafish, including point mutants, apc^hu745 (apc^mcr), and p53^zy7 (p53^I166T), a small deletion mutant (bap28^y75) and a retroviral insertion mutant (wdr43^hi821a). This technique can genotype individual zebrafish embryos and adults (by tail‐clip) and is applicable to other model organisms. Developmental Dynamics 238:3168–3174, 2009. © 2009 Wiley‐Liss, Inc.