Cytokeratin staining for intraoperative evaluation of sentinel lymph nodes in patients with invasive lobular carcinoma.

BACKGROUND: Frozen section and intraoperative imprint cytology (IIC(N)) are 2 methods used for intraoperative pathologic assessment of sentinel lymph nodes (SLNs). The SLN evaluation of patients with invasive lobular carcinoma (ILC) results in a relatively high number of false-negative results using either of these methods. The purpose of this study was to evaluate the added benefits that intraoperative immunohistochemical-cytokeratin staining (I(CK-IHC)) can bring to IIC(N) in the evaluation of SLN in patients with ILC. METHODS: A total of 59 breast cancer patients with ILC underwent an SLN biopsy evaluated by our standard IIC(N) assessment in addition to I(CK-IHC). The results of IIC(N) with I(CK-IHC) were compared with the final histopathologic assessment consisting of standard hematoxylin and eosin staining and additional cytokeratin staining of nodes. RESULTS: Intraoperative evaluation of SLN using IIC(N) and I(CK-IHC) correctly diagnosed the nodal status in 45 of 59 (76.3%) patients. On final histopathologic assessment, 31 of 59 (52.5%) patients were found to have positive nodes. Using I(CK-IHC), 17 of these 31 positive cases (54.8%) were detected. Using IIC(N) alone, without the benefit of I(CK-IHC), only 13 of 31 (41.9%) positive cases were detected intraoperatively. CONCLUSIONS: For patients with ILC, I(CK-IHC) staining in addition to IIC(N) improves accuracy over using IIC(N) alone. In this study, I(CK-IHC) staining demonstrated a 12.9% improvement in the detection of SLN metastases in patients with ILC. Cytopathologists should consider employing I(CK-IHC) staining to evaluate the touch-imprint slides of SLN in ILC patients.

Expression of zTlxA, a Hox11-like gene, in early differentiating embryonic neurons and cranial sensory ganglia of the zebrafish embryo.

We have isolated a cDNA encoding a member of the Tlx/Hox11 family of homeodomain factors from the zebrafish, most closely related to the vertebrate Tlx-1/Hox11 and Tlx-3/Hox11L2 proteins. The gene is expressed in a set of early differentiating neurons that project to a common tract, the lateral longitudinal fascicle. We show that the gene is specifically expressed in spinal cord Rohon Beard neurons, in nucleus of the posterior commissure neurons of the midbrain, in a set of hindbrain neurons that include RoL3 reticulospinal interneurons, and in the trigeminal, statoacoustic, anterior lateral line, glossopharyngeal, and vagal cranial sensory ganglia. Timing of expression of the gene in these neurons correlates with the phase of axonal outgrowth and target innervation. Expression of the gene is also observed in several non-neural tissues, including the pharyngeal arches, budding gill filaments, outgrowing semicircular protrusions in the otic vesicle, and in the pectoral fin buds.

The role of vascular endothelial growth factor (VEGF) in vasculogenesis, angiogenesis, and hematopoiesis in zebrafish development.

Vascular endothelial growth factor (VEGF, VEGF-A), a selective mitogen for endothelial cells is a critical factor for vascular development. Two isoforms that differ in the presence of exons 6 and 7, Vegf(165) and Vegf(121), are the dominant forms expressed in zebrafish embryo. Simultaneous overexpression of both isoforms in the embryo results in increased production of flk1, tie1, scl, and gata1 transcripts, indicating a stimulation of both endothelial and hematopoietic lineages. We also demonstrate that vegf can stimulate hematopoiesis in zebrafish by promoting the formation of terminally differentiated red blood cells. Simultaneous overexpression of both isoforms also causes ectopic vasculature and blood cells in many of the injected embryos as well as pericardial edema in later stage embryos. Overexpression of vegf also resulted in earlier onset of flk1, tie1, scl, and gata1 expression in the embryo, indicating a possible role of vegf in stimulating the differentiation of both vascular and hematopoietic lineages. Co-injection of RNAs for both isoforms results in increased expression of three of these markers over and above that observed when either RNA is singly injected and analysis of vegf expression in the notochord mutants no tail and floating head suggests that the notochord patterns the formation of the dorsal aorta by stimulating adjacent somite cells to express vegf, which in turn functions as a signal in dorsal aorta patterning. Finally, studies of vegf expression in cloche mutant indicate that vegf expression is generally independent of cloche function. These results show that in the zebrafish embryo, vegf can not only stimulate endothelial cell differentiation but also hematopoiesis. Moreover, these effects are most dramatic when both vegf isoforms are co-expressed, indicating a synergistic effect of the expression of the two forms of the VEGF protein.

Caged Q-rhodamine dextran: a new photoactivated fluorescent tracer.

An amine-reactive caged rhodamine was synthesized and conjugated to aminodextran. The resulting tracer was injected into a single cell zebrafish embryo, and a portion of the tracer was photolyzed in a single cell after development. The resulting fluorescent cell was imaged by fluorescence microscopy through a single round of cell division.

Heart and gut chiralities are controlled independently from initial heart position in the developing zebrafish.

A fundamental problem in developmental biology is how left-right (LR) asymmetry is generated, both on the whole organism level and at the level of an individual organ or structure. To investigate the relationship of organ sidedness to organ chirality, we examined 12 zebrafish mutants for initial heart tube position and later heart looping direction (chirality). Anomalous initial heart position was found in seven mutants, which also demonstrated loss of normal LR asymmetry in lateral plate mesoderm (LPM) antivin/lefty-1 and Pitx2 expression. Those with a relatively normal notochord (cyc(b16), din, and spt) displayed a predictive correlation between initial heart position and heart chirality, whereas initial heart position and heart chirality were independently randomized in those with a defective notochord (flh, boz, ntl, and mom). The predictability of heart chirality in spt, din, and b16 embryos, even in the absence of normal antivin/lefty-1 and Pitx2 expression, strongly suggests that heart chirality is controlled by a process distinct from that which controls appropriate left-sided LPM expression of antivin-Pitx2 signaling pathway molecules. In addition, there was correlation of initial heart position with gut chirality (and also between heart chirality and gut chirality) in the first class of mutants with normal notochord, but not in the second class, which appears to model human heterotaxy syndrome.

Maternally controlled (beta)-catenin-mediated signaling is required for organizer formation in the zebrafish.

We have identified and characterized a zebrafish recessive maternal effect mutant, ichabod, that results in severe anterior and dorsal defects during early development. The ichabod mutation is almost completely penetrant, but exhibits variable expressivity. All mutant embryos fail to form a normal embryonic shield; most fail to form a head and notochord and have excessive development of ventral tail fin tissue and blood. Abnormal dorsal patterning can first be observed at 3.5 hpf by the lack of nuclear accumulation of (beta)-catenin in the dorsal yolk syncytial layer, which also fails to express bozozok/dharma/nieuwkoid and znr2/ndr1/squint. At the onset of gastrulation, deficiencies in expression of dorsal markers and expansion of expression of markers of ventral tissues indicate a dramatic alteration of dorsoventral identity. Injection of (beta)-catenin RNA markedly dorsalized ichabod embryos and often completely rescued the phenotype, but no measurable dorsalization was obtained with RNAs encoding upstream Wnt pathway components. In contrast, dorsalization was obtained when RNAs encoding either Bozozok/Dharma/Nieuwkoid or Znr2/Ndr1/Squint were injected. Moreover, injection of (beta)-catenin RNA into ichabod embryos resulted in activation of expression of these two genes, which could also activate each other. RNA injection experiments strongly suggest that the component affected by the ichabod mutation acts on a step affecting (beta)-catenin nuclear localization that is independent of regulation of (beta)-catenin stability. This work demonstrates that a maternal gene controlling localization of (beta)-catenin in dorsal nuclei is necessary for dorsal yolk syncytial layer gene activity and formation of the organizer in the zebrafish.

Expression of Otx homeodomain proteins induces cell aggregation in developing zebrafish embryos.

In the zebrafish embryo, cells fated to give rise to the rostral brain move in a concerted fashion and retain tissue coherence during morphogenesis. We demonstrate here that Otx proteins have a dramatic effect on cell-cell interactions when expressed ectopically in the zebrafish embryo. Injection of zebrafish Otx1 or Drosophila otd RNAs into a single cell at the 16-cell stage results in aggregation of descendants of the injected cell. The Otx/Otd homeodomain is necessary for aggregation and appears to be sufficient for the effect when substituted for the homeodomain of an unrelated homeodomain protein. When cells containing injected zOtx1 RNA are limited to the area that is normally fated to become the anterior brain and neural retina, the induced aggregates contribute to anterior brain and retina tissues. In many other embryonic regions, which do not express endogenous zOtx1, the aggregates appear to be incompatible with normal development and do not integrate into developing tissues. By using an activatable Otx1-glutocorticoid receptor fusion protein that results in the stimulation of cell association, we demonstrate that cell aggregates can form as a result of Otx1 activity even after gastrulation is completed. Time-lapse analysis of cell movements show that cell aggregation occurs with only a slight inhibition of the rate of convergence. These results suggest that promotion of cell adhesion or mediation of cell repulsion may be one of the normal functions of the Otx proteins in the establishment of the anterior brain.

Zebrafish genetics: harnessing horizontal gene transfer.

The promiscuous spread of Tc1/mariner transposons across species implies that host factors are relatively unimportant for their transposition. Heterologous elements can integrate on expression of the corresponding transposases, an approach that should greatly facilitate genetic analysis in the zebrafish.

Cloning and characterization of vascular endothelial growth factor (VEGF) from zebrafish, Danio rerio.

We have cloned and sequenced a zebrafish (Danio rerio) Vascular Endothelial Growth Factor (vegf) cDNA. It encodes a precursor protein of 188 amino acids with a putative 23 amino acids signal peptide. Sequence comparison analysis indicates that the zebrafish vegf cDNA corresponds to the human VEGF165 isoform and shows about 52% identity to human VEGF165 at the amino acid level. A 2.8 kb vegf message RNA was detected in adult zebrafish by Northern blot analysis. Expression of vegf165 is also detected by RT-PCR in adult fish and throughout the zebrafish embryonic development. Whole mount in situ hybridization of zebrafish embryos indicates strong expression in four areas of the 18-19 h post-fertilization (hpf) embryo: within the anterior central nervous system in the prospective optic stalk, in mesoderm overlapping the bilaterally located merging heart fields, in mesoderm underlying and flanking the hindbrain posterior to rhombomere 4, and in medial regions of the somites. The study of vegf function in zebrafish embryonic vascular development will contribute to our understanding of the mechanisms of vertebrate endothelial cell differentiation and vasculature formation.

Relationships among msx gene structure and function in zebrafish and other vertebrates.

The zebrafish genome contains at least five msx homeobox genes, msxA, msxB, msxC, msxD, and the newly isolated msxE. Although these genes share structural features common to all Msx genes, phylogenetic analyses of protein sequences indicate that the msx genes from zebrafish are not orthologous to the Msx1 and Msx2 genes of mammals, birds, and amphibians. The zebrafish msxB and msxC are more closely related to each other and to the mouse Msx3. Similarly, although the combinatorial expression of the zebrafish msx genes in the embryonic dorsal neuroectoderm, visceral arches, fins, and sensory organs suggests functional similarities with the Msx genes of other vertebrates, differences in the expression patterns preclude precise assignment of orthological relationships. Distinct duplication events may have given rise to the msx genes of modern fish and other vertebrate lineages whereas many aspects of msx gene functions during embryonic development have been preserved.

Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein.

Determination of fate maps and cell lineage tracing have previously been carried out in the zebrafish embryo by following the progeny of individual cells injected with fluorescent dyes. We review the information obtained from these experiments and then present an approach to fate mapping and cell movement tracing, utilizing the activation of caged fluorescein-dextran. This method has several advantages over single-cell injections in that it is rapid, allows cells at all depths in the embryo to be marked, can be used to follow cells starting at any time during development, and allows an appreciation of the movements of cells located in a coherent group at the time of uncaging. We demonstrate that the approach is effective in providing additional and complementary information on prospective mesoderm and brain tissues studied previously. We also present, for the first time, a fate map of placodal tissues including the otic vesicle, lateral line, cranial ganglia, lens, and olfactory epithelium. The prospective placodal cells are oriented at the 50% epiboly stage on the ventral side of the embryo with anterior structures close to the animal pole, and posterior structures nearer to the germ ring.

Mutations affecting the formation of the notochord in the zebrafish, Danio rerio.

In a large scale screen for mutants with defects in the embryonic development of the zebrafish we identified mutations in four genes,floating head (flh), momo (mom), no tail (ntl), and doc, that are required for early notochord formation. Mutations in flh and ntl have been described previously, while mom and doc are newly identified genes. Mutant mom embryos lack a notochord in the trunk, and trunk somites from the right and left side of the embryo fuse underneath the neural tube. In this respect mom appears similar to flh. In contrast, notochord precursor cells are present in both ntl and doc embryos. In order to gain a greater understanding of the phenotypes, we have analysed the expression of several axial mesoderm markers in mutant embryos of all four genes. In flh and mom, Ntl expression is normal in the germ ring and tailbud, while the expression of Ntl and other notochord markers in the axial mesodermal region is disrupted. Ntl expression is normal in doc embryos until early somitic stages, when there is a reduction in expression which is first seen in anterior regions of the embryo. This suggests a function for doc in the maintenance of ntl expression. Other notochord markers such as twist, sonic hedgehog and axial are not expressed in the axial mesoderm of ntl embryos, their expression parallels the expression of ntl in the axial mesoderm of mutant doc, flh and mom embryos, indicating that ntl is required for the expression of these markers. The role of doc in the expression of the notochord markers appears indirect via ntl. Floor plate formation is disrupted in most regions in flh and mom mutant embryos but is present in mutant ntl and doc embryos. In mutant embryos with strong ntl alleles the band of cells expressing floor plate markers is broadened. A similar broadening is also observed in the axial mesoderm underlying the floor plate of ntl embryos, suggesting a direct involvement of the notochord precursor cells in floor plate induction. Mutations in all of these four genes result in embryos lacking a horizontal myoseptum and muscle pioneer cells, both of which are thought to be induced by the notochord. These somite defects can be traced back to an impairment of the specification of the adaxial cells during early stages of development. Transplantation of wild-type cells into mutant doc embryos reveals that wild-type notochord cells are sufficient to induce horizontal myoseptum formation in the flanking mutant tissue. Thus doc, like flh and ntl, acts cell autonomously in the notochord. In addition to the four mutants with defects in early notochord formation, we have isolated 84 mutants, defining at least 15 genes, with defects in later stages of notochord development. These are listed in an appendix to this study.

Mutations affecting somite formation and patterning in the zebrafish, Danio rerio.

Somitogenesis is the basis of segmentation of the mesoderm in the trunk and tail of vertebrate embryos. Two groups of mutants with defects in this patterning process have been isolated in our screen for zygotic mutations affecting the embryonic development of the zebrafish (Danio rerio). In mutants of the first group, boundaries between individual somites are invisible early on, although the paraxial mesoderm is present. Later, irregular boundaries between somites are present. Mutations in fused somites (fss) and beamter (bea) affect all somites, whereas mutations in deadly seven (des), after eight (aei) and white tail (wit) only affect the more posterior somites. Mutants of all genes but wit are homozygous viable and fertile. Skeletal stainings and the expression pattern of myoD and snail1 suggest that anteroposterior patterning within individual somites is abnormal. In the second group of mutants, formation of the horizontal myoseptum, which separates the dorsal and ventral part of the myotome, is reduced. Six genes have been defined in this group (you-type genes). you-too mutants show the most severe phenotype; in these the adaxial cells, muscle pioneers and the primary motoneurons are affected, in addition to the horizontal myoseptum. The horizontal myoseptum is also missing in mutants that lack a notochord. The similarity of the somite phenotype in mutants lacking the notochord and in the you-type mutants suggests that the genes mutated in these two groups are involved in a signaling pathway from the notochord, important for patterning of the somites.

Specific localization of zebrafish hsp90 alpha mRNA to myoD-expressing cells suggests a role for hsp90 alpha during normal muscle development.

Members of the eukaryotic hsp90 family function as important molecular chaperones in the assembly, folding and activation of a select group of cellular signalling molecules and transcription factors. Several of the molecules with which hsp90 interacts, such as the bHLH transcription factor myoD, are known to be important regulators of developmental events in vertebrates. However, little information is available in support of any specific role for hsp90 in developing embryos in vivo. In this study, we provide the first in vivo evidence that the hsp90 alpha gene may play a role in the process of myogenesis. We show that constitutive hsp90 alpha mRNA in zebrafish embryos is restricted primarily to a subset of cells within the somites and pectoral fin buds which also express myoD. Furthermore, expression of the hsp90 alpha gene is down-regulated along with myoD in differentiated muscles of the trunk at a time when levels of mRNA encoding the muscle structural protein alpha-tropomyosin remain high. No hsp90 alpha mRNA is detectable within the CNS at control temperatures. In contrast, heat shock-induced expression of the hsp90 alpha gene occurs throughout the embryo at all stages of development examined. The expression patterns strongly suggest that the hsp90 alpha gene plays a specific role in the normal process of myogenesis in addition to providing protection to all cells of the embryo during periods of environmental stress.

Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos.

We describe the isolation of the zebrafish MyoD gene and its expression in wild-type embryos and in two mutants with altered somite development, no tail (ntl) and spadetail (spt). In the wild-type embryo, MyoD expression first occurs in an early phase, extending from mid-gastrula to just prior to somite formation, in which cells directly adjacent to the axial mesoderm express the gene. In subsequent phases, during the anterior-to-posterior wave of somite formation and maturation, expression occurs within particular regions of each somite. In spt embryos, which lack normal paraxial mesoderm due to incorrect cell migration, early MyoD expression is not observed and transcripts are instead first detected in small groups of trunk cells that will develop into aberrant myotomal-like structures. In ntl embryos, which lack notochords and tails, the early phase of MyoD expression is also absent. However, the later phase of expression within the developing somites appears to occur at the normal time in the ntl mutants, indicating that the presomitogenesis and somitogenesis phases of MyoD expression can be uncoupled. In addition, we demonstrate that the entire paraxial mesoderm of wild-type embryos has the potential to express MyoD when Sonic hedgehog is expressed ubiquitously in the embryo, and that this potential is lost in some of the cells of the paraxial mesoderm lineage in no tail and spadetail embryos. We also show that MyoD expression precedes myogenin expression and follows or is coincident with expression of snaill in some regions that express this gene.

Cell-autonomous shift from axial to paraxial mesodermal development in zebrafish floating head mutants.

Zebrafish floating head mutant embryos lack notochord and develop somitic muscle in its place. This may result from incorrect specification of the notochord domain at gastrulation, or from respecification of notochord progenitors to form muscle. In genetic mosaics, floating head acts cell autonomously. Transplanted wild-type cells differentiate into notochord in mutant hosts; however, cells from floating head mutant donors produce muscle rather than notochord in wild-type hosts. Consistent with respecification, markers of axial mesoderm are initially expressed in floating head mutant gastrulas, but expression does not persist. Axial cells also inappropriately express markers of paraxial mesoderm. Thus, single cells in the mutant midline transiently co-express genes that are normally specific to either axial or paraxial mesoderm. Since floating head mutants produce some floor plate in the ventral neural tube, midline mesoderm may also retain early signaling capabilities. Our results suggest that wild-type floating head provides an essential step in maintaining, rather than initiating, development of notochord-forming axial mesoderm.

The expression pattern of two zebrafish achaete-scute homolog (ash) genes is altered in the embryonic brain of the cyclops mutant.

Two achaete-scute homolog sequences, Zash-1a and Zash-1b, were isolated from a zebrafish embryonic cDNA library. The Zash-1a cDNA encodes a protein very similar to rat Mash-1 and Xenopus Xash-1, with over 94% identity in the C-terminal three-fourths of all three polypeptides. The Zash-1b cDNA encodes a more distantly related protein, with 80% identity of amino acids to Mash-1 in this part of the sequence. At 24 hr, the Zash-1a transcripts are found in the hindbrain in two bilaterally symmetrical lines of cells which mark the boundary between the alar and basal plates and in rhombomere 1 in ventral cells near the floorplate. The gene is also expressed in particular regions of the telencephalon and diencephalon, in the epiphysis, the ventral tegmentum, the neural retina, and in specific cells in the spinal cord. Zash-1b transcripts are found in the hindbrain in segmentally arranged fan-like groups of cells which are located close to the anterior and posterior boundaries of each of rhombomeres 2-6 and in ventral cells close to the floor plate of most rhombomeres. The gene is also expressed at sites distinct from cells expressing Zash-1a in the tegmentum, diencephalon, telencephalon, and spinal cord. In the mutant cyclops, Zash-1a transcripts are absent from the ventral region of the tegmentum and in the ventral cells of rhombomere 1, while more dorsal expression regions are unaffected. The effects of the mutation on Zash-1b expression, however, are more complex. In the hindbrain, the ventral expression zone of this gene is absent, the more dorsal segmented expression is disorganized, and ectopic expression in the alar plate is observed. A dramatic ectopic expression is also observed in the anterior tegmentum. The cyclops gene, therefore, has both positive and negative effects on the CNS of the wild-type embryo: it is required for activation of both Zash-1a and -1b in particular ventral cells, but it also restricts the expression of Zash-1b in other ventral cells and in some dorsal regions. Zash-1a and -1b gene probes will be extremely useful in the analysis of additional mutations affecting development of the central nervous system in zebrafish embryos.

Expression of two zebrafish orthodenticle-related genes in the embryonic brain.

To analyze the molecular mechanism of pattern formation in the anteriormost regions of the zebrafish embryo, we isolated two zebrafish sequences, zOtx1 and zOtx2, related to the Drosophila orthodenticle (otd) and two murine Otx genes. zOtx1 and zOtx2 encode predicted gene products which are 82% and 94% identical to the corresponding mouse proteins. Transcripts of both zebrafish genes appear abruptly at high levels in a triangular patch at the animal pole of the mid-gastrula, a region which contains cells fated to become midbrain and forebrain. Between 9 and 14 h of development, zOtx transcripts disappear from forebrain regions in a manner characteristic for each gene, and from 14 to 24 h, particular regions of the forebrain and midbrain express one or both genes. The posterior limit of expression of both genes in 10-30-h embryos forms a sharp boundary at the posterior border of the midbrain. As in the mouse, the early expression patterns of the zOtx genes are consistent with a role in defining midbrain and forebrain territories. However, there are a number of interesting differences between the forebrain and midbrain regions which express the genes in the two species.