The use of venous anastomotic flow couplers to monitor buried free DIEP flap reconstructions following nipple-sparing mastectomy.

Nipple sparing mastectomy with free tissue transfer for breast reconstruction offers excellent aesthetic outcomes but poses a challenge in monitoring the buried flap. Venous anastomotic flow couplers directly monitor buried flaps without the need for monitoring skin paddles. In a two year period we used the Synovis GEM™ flow coupler on 24 DIEP flaps. In our practice, flow couplers are effective in monitoring buried free flaps for breast reconstruction. The avoidance of a second procedure to remove a skin paddle improves patient experience and nullifies the additional flow coupler cost. One patient needed return to theatre when a Doppler wire became dislodged early in the series. There were no other issues with flap monitoring and no flap failures. We offer our tips to optimise flow coupler use.

Midline signals regulate retinal neurogenesis in zebrafish.

In zebrafish, neuronal differentiation progresses across the retina in a pattern that is reminiscent of the neurogenic wave that sweeps across the developing eye in Drosophila. We show that expression of a zebrafish homolog of Drosophila atonal, ath5, sweeps across the eye predicting the wave of neuronal differentiation. By analyzing the regulation of ath5 expression, we have elucidated the mechanisms that regulate initiation and spread of neurogenesis in the retina. ath5 expression is lost in Nodal pathway mutant embryos lacking axial tissues that include the prechordal plate. A likely role for axial tissue is to induce optic stalk cells that subsequently regulate ath5 expression. Our results suggest that a series of inductive events, initiated from the prechordal plate and progressing from the optic stalks, regulates the spread of neuronal differentiation across the zebrafish retina.

A nodal signaling pathway regulates the laterality of neuroanatomical asymmetries in the zebrafish forebrain.

Animals show behavioral asymmetries that are mediated by differences between the left and right sides of the brain. We report that the laterality of asymmetric development of the diencephalic habenular nuclei and the photoreceptive pineal complex is regulated by the Nodal signaling pathway and by midline tissue. Analysis of zebrafish embryos with compromised Nodal signaling reveals an early role for this pathway in the repression of asymmetrically expressed genes in the diencephalon. Later signaling mediated by the EGF-CFC protein One-eyed pinhead and the forkhead transcription factor Schmalspur is required to overcome this repression. When expression of Nodal pathway genes is either absent or symmetrical, neuroanatomical asymmetries are still established but are randomized. This indicates that Nodal signaling is not required for asymmetric development per se but is essential to determine the laterality of the asymmetry.

The nodal pathway acts upstream of hedgehog signaling to specify ventral telencephalic identity.

The Nodal and Hedgehog signaling pathways influence dorsoventral patterning at all axial levels of the CNS, but it remains largely unclear how these pathways interact to mediate patterning. Here we show that, in zebrafish, Nodal signaling is required for induction of the homeobox genes nk2.1a in the ventral diencephalon and nk2.1b in the ventral telencephalon. Hedgehog signaling is also required for telencephalic nk2.1b expression but may not be essential to establish diencephalic nk2.1a expression. Furthermore, Shh does not restore ventral diencephalic development in embryos lacking Nodal activity. In contrast, Shh does restore telencephalic nk2.1b expression in the absence of Nodal activity, suggesting that Hedgehog signaling acts downstream of Nodal activity to pattern the ventral telencephalon. Thus, the Nodal pathway regulates ventral forebrain patterning through both Hedgehog signaling-dependent and -independent mechanisms.

Regulatory gene expression boundaries demarcate sites of neuronal differentiation in the embryonic zebrafish forebrain.

During development of the zebrafish forebrain, a simple scaffold of axon pathways is pioneered by a small number of neurons. We show that boundaries of expression domains of members of the eph, forkhead, pax, and wnt gene families correlate with the positions at which these neurons differentiate and extend axons. Analysis of genetically or experimentally altered forebrains indicates that if a boundary is maintained, there is appropriate neural differentiation with respect to the boundary. Conversely, in the absence of a boundary, there is concomitant disruption of neural patterning. We also show that a strip of cells within the dorsal diencephalon shares features with ventral midline cells. This strip of cells fails to develop in mutant fish in which specification of the ventral CNS is disrupted, suggesting that its development may be regulated by the same inductive pathways that pattern the ventral midline.

floating head and masterblind regulate neuronal patterning in the roof of the forebrain.

The epiphysial region of the dorsal diencephalon is the first site at which neurogenesis occurs in the roof of the zebrafish forebrain. We show that the homeobox containing gene floating head (flh) is required for neurogenesis to proceed in the epiphysis. In flh- embryos, the first few epiphysial neurons are generated, but beyond the 18 somite stage, neuronal production ceases. In contrast, in masterblind- (mbl-) embryos, epiphysial neurons are generated throughout the dorsal forebrain. We show that mbl is required to prevent the expression of flh in dorsal forebrain cells rostral to the epiphysis. Furthermore, epiphysial neurons are not ectopically induced in mbl-/flh- embryos, demonstrating that the epiphysial phenotype of mbl- embryos is mediated by ectopic Flh activity. We propose a role for Flh in linking the signaling pathways that regulate regional patterning to the signaling pathways that regulate neurogenesis.

Development of noradrenergic neurons in the zebrafish hindbrain requires BMP, FGF8, and the homeodomain protein soulless/Phox2a.

We report that the zebrafish mutation soulless, in which the development of locus coeruleus (LC) noradrenergic (NA) neurons failed to occur, disrupts the homeodomain protein Phox2a. Phox2a is not only necessary but also sufficient to induce Phox2b+ dopamine-beta-hydroxylase+ and tyrosine hydroxylase+ NA neurons in ectopic locations. Phox2a is first detected in LC progenitors in the dorsal anterior hindbrain, and its expression there is dependent on FGF8 from the mid/hindbrain boundary and on optimal concentrations of BMP signal from the epidermal ectoderm/future dorsal neural plate junction. These findings suggest that Phox2a coordinates the specification of LC in part through the induction of Phox2b and in response to cooperating signals that operate along the mediolateral and anteroposterior axes of the neural plate.

A role for the extraembryonic yolk syncytial layer in patterning the zebrafish embryo suggested by properties of the hex gene.

Recent studies in mouse suggest that the extraembryonic endoderm has an important role in early embryonic patterning [1]. To analyze whether similar mechanisms operate in other vertebrates, we cloned the zebrafish homologue of Hex, a homeobox gene that is expressed asymmetrically in the mouse visceral endoderm [2]. Early expression of zebrafish hex is restricted to the dorsal portion of the yolk syncytial layer (YSL), an extraembryonic tissue. By the onset of gastrulation, hex is expressed in the entire dorsal half of the YSL, which directly underlies the cells fated to form the neural plate. We show that hex expression is initially regulated by the maternal Wnt pathway and later by a Bmp-mediated pathway. Overexpression experiments of wild-type and chimeric Hex constructs indicate that Hex functions as a transcriptional repressor and its overexpression led to the downregulation of bmp2b and wnt8 expression and the expansion of chordin expression. These findings provide further evidence that the zebrafish YSL is the functional equivalent of the mouse visceral endoderm and that extraembryonic structures may regulate early embryonic patterning in many vertebrates.

Border disputes: do boundaries play a role in growth-cone guidance?

One of the earliest indications of regional patterning in the CNS is the spatially restricted expression of regulatory genes within the neuroepithelium. Many of these genes encode transcription factors and, although little is known of their downstream targets, it seems likely that they control the identity of cells in different regions of the CNS. This review discusses how the expression of these patterning genes might influence the location at which the first axon pathways in the CNS are pioneered. Evidence is described that suggests that the boundary regions between adjacent domains of regulatory gene expression influence where the first axons will extend.

Acquisition of regional and cellular identities in the developing zebrafish nervous system.

In the past year, several new techniques have been used with great success in the study of nervous system development in the zebrafish. Perhaps the most exciting results have come from experiments in which single identified cells or small groups of cells have been transplanted between embryos in order to examine cell determination and the site of action of genetic mutations.

Pax proteins and eye development.

Homologous members of the Pax gene family are required for eye development in Drosophila and vertebrates. Despite superficial similarities in the phenotypes of vertebrates with mutations in pax-6 and Drosophila eyeless mutants, it remains uncertain whether the two proteins encoded by these genes have comparable functions. The genetic cascade triggered by eyeless leads to eye formation, whereas pax-6 is not necessary for optic vesicle formation, but is required at other stages of eye development. A second vertebrate Pax gene, pax-2, is also required during eye development and appears to play a role during closure of the choroid fissure.

The zebrafish homologue of the ret receptor and its pattern of expression during embryogenesis.

The c-ret proto-oncogene, a member of the receptor tyrosine kinase gene superfamily, plays a critical role in the development of the excretory system and the enteric and autonomic nervous systems of mammalian embryos. To study the potential function of the c-ret locus in lower vertebrates, we have isolated its zebrafish homologue, ret1 and established its expression pattern during embryogenesis. Ret1 mRNA first appears during early somitogenesis in the presumptive brain, spinal cord and excretory system. Within the CNS, expression of ret1 is detected in primary motor and sensory (Rohon-Beard) neurons. Ret1 transcripts are also expressed in subsets of neural crest cells and cranial ganglia as well as in the enteric nervous system. In the excretory system, expression is detected in the developing nephric duct and the pronephros. Our findings reveal a remarkable similarity in the expression pattern of c-ret between higher and lower vertebrates, suggesting that the function of this locus has been conserved throughout vertebrate evolution. Furthermore, the conservation of ret1 expression in cell types which remain unaffected by the mammalian c-ret mutations, such as motor and sensory neurons, suggests a function of this receptor in these cell lineages.

Isolation, expression and regulation of a zebrafish paraxis homologue.

The formation of somites involves the subdivision of segmented presomitic mesoderm into segmentally arranged somite blocks. In mice and chicks, the basic-helix-loop-helix (bHLH) gene, paraxis, is involved in this process. Here, we report the isolation of a zebrafish homologue of paraxis, par1. par1 is expressed in presomitic paraxial mesoderm from late gastrula stages, and expression is maintained in ventrolateral cells after somite formation. In spt- embryos, par1 expression is both delayed and severely reduced whereas in flh- embryos, ectopic transcripts are detected in axial mesoderm. Spatial regulation of par1 expression within the somites is affected in several mutants with defects in axial midline tissues.

Expression of zebrafish GATA 3 (gta3) during gastrulation and neurulation suggests a role in the specification of cell fate.

In order to understand the role of the transcription factor GATA 3 in vertebrate development, we have examined its expression and some aspects of its regulation during gastrulation and neurulation in the zebrafish. The complete coding sequence of the cDNA encoding the zebrafish GATA 3 homologue, termed gta3, is described. Analysis of expression patterns by in situ hybridisation shows the gene to be expressed during gastrulation in the ventral region of the embryo which includes tissue fated to form the non-neural ectoderm. By the end of gastrulation, there is a clear border to the gta3 expression domain that is close to the edge of the neural plate. Subsequently, gta3 expresses in the pronephric duct and in defined regions of the central nervous system which include specific cells in each segment of the spinal cord and nuclei in the brain. Double labelling embryos with a probe for gta3 and antibodies which identify differentiated neurons suggest that gta3 is dynamically expressed during the early differentiation phase of a subset of neurons but not in the terminal phase. Analysis of gta3 expression in dorsalised embryos and in cyc and spt mutant embryos indicates that the neural expression of the gene is subject to control by signals from the mesoderm, including both the notochord and the somites, which influence the segmental organisation of expression in the spinal cord.

Distribution of Pax6 protein during eye development suggests discrete roles in proliferative and differentiated visual cells.

Although Pax6 is required during eye development in rodents and humans, little is known about the precise role of the protein in this process. To aid in the interpretation of functional studies, we have determined the precise spatial and temporal distributions of the Pax6 protein in the eye. We find that Pax6 is initially distributed contiguously throughout a large domain of the anterior neural plate of zebrafish, including the presumptive eye fields and the dorsal diencephalon. After evagination of the optic vesicle, Pax6 becomes restricted to all proliferating cells of the pigment epithelial and neural layers of the retina. Pax6 is downregulated in most cells concomitant with differentiation. However, it remains present in several mature cell types of the eye including amacrine cells and the lens and corneal epithelia. This expression is conserved across diverse vertebrate species and suggests that Pax6 has additional conserved functions in the mature eye.

Anf: a novel class of vertebrate homeobox genes expressed at the anterior end of the main embryonic axis.

Five novel genes homologous to the homeobox-containing genes Xanf-1 and Xanf-2 of Xenopus and Hesx-1/Rpx of mouse have been identified as a result of a PCR survey of cDNA in sturgeon, zebrafish, newt, chicken and human. Comparative analysis of the homeodomain primary structure of these genes revealed that they belong to a novel class of homeobox genes, which we name Anf. All genes of this class investigated so far have similar patterns of expression during early embryogenesis, characterized by maximal transcript levels being present at the anterior extremity of the main embryonic body axis. The data obtained also suggest that, despite considerable high structural divergence between their homeodomains, all known Anf genes may be orthologues, and thus represent one of the most quickly evolving classes of vertebrate homeobox genes.

Axonal trajectories and distribution of GABAergic spinal neurons in wildtype and mutant zebrafish lacking floor plate cells.

The role of the midline floor plate cells in the neuronal differentiation of the spinal cord was examined by comparing putative GABAergic neurons in wildtype zebrafish embryos with those in cyc-1 mutant embryos. The mutation produces a pleiotropic recessive lethal phenotype and is severe in rostral brain regions, but its direct effect in the caudal hindbrain and the spinal cord is apparently restricted to the depletion of the midline floor plate cells. In wildtype embryos, an antibody against the neurotransmitter GABA labeled the cell bodies, axons, and growth cones of three classes of previously identified neurons; dorsal longitudinal neurons (DoLA), commissural secondary ascending neurons (CoSA), and ventral longitudinal neurons (VeLD). A novel ventral cell type, Kolmer-Agduhr (KA) neurons, was also labeled. In the cyc-1 mutant, abnormalities were observed in some, but not all, of the GABAreactive CoSA, VeLD, and KA axons, while the axonal trajectories of DoLA neurons were not affected. Furthermore, the number of KA cells was reduced in the mutant while the numbers of the other GABAreactive cells were unperturbed. These observations corroborate our earlier hypothesis that the floor plate cells are one of several guidance cues that direct axonal outgrowth near the ventral midline of the spinal cord. They also suggest that the floor plate cells may play a role in the cellular differentiation of the spinal cord of zebrafish embryos.

Continuous growth of the motor system in the axolotl.

During growth of the axolotl, motor neurons, and muscle fibres are added to the motor system. By double labelling neurons with tritiated thymidine and retrogradely transported HRP, we show that some motor neurons are born at postembryonic stages. Further analysis of motor neurons with the aid of HRP reveals this population of newly born cells relatively frequently in small (5-7 cm long) axolotls, but only rarely in large (7-13 cm long) axolotls. Evidence is presented that suggests that these immature cells are in the process of migrating from close to the ependyma out to the ventral horn. HRP transport also reveals growth cones of advancing axons within spinal nerves in animals up to 6 cm in length. Cell counts by light and electron microscopic methods show that muscle fibres are generated throughout larval life in the iliotibialis, a typical limb muscle. This analysis provides data consistent with the notion that new muscle fibres are added from a localised growth zone situated at the superficial edge of the muscle. These results are discussed in terms of the correlation between continuous growth of the motor system and the ability of the axolotl to functionally repair lesions to the peripheral nervous system.

Non-heart-beating donors: a model of thoracic allograft injury.

4ACKGROUND. Non-heart-beating donors (NHBDs) have been proposed for the critical shortage of donors for cardiac and pulmonary transplantation. We determined the effects of prearrest hypoxia and postarrest warm ischemia on cardiac and pulmonary allografts procured from NHBDs undergoing hypoxic arrest. METHODS. Rabbit hearts and lungs were procured from separate donors and placed on isolated blood perfusion circuits. Controls were excised and perfused without ischemia. Heart from NHBDs underwent either prearrest hypoxic perfusion alone or consecutive periods of prearrest hypoxic perfusion and 20 minutes of postarrest warm ischemia. A third group of hearts underwent 30 minutes of warm, global ischemia alone. Two groups of pulmonary allografts were studied using similar hypoxic perfusion/20-minute ischemia and 30-minute ischemia donors. RESULTS. Prearrest hypoxic perfusion clearly causes significant dysfunction of cardiac allografts from NHBDs compared with nonischemic controls. Prearrest hypoxic perfusion combined with postarrest ischemia results in an additive degree of dysfunction more severe than a similar period of warm ischemia alone. Both groups of experimental lungs displayed function similar to that of nonischemic controls in terms of pulmonary hemodynamics, airway resistance, and oxygenation potential. CONCLUSIONS. We conclude that prearrest hypoxic perfusion significantly contributes to the dysfunction of NHBD cardiac allografts. Pulmonary allografts may be more amenable to procurement of NHBDs.

Controlled reperfusion of cardiac grafts from non-heart-beating donors.

BACKGROUND: Hearts harvested from non-heart-beating donors sustain severe injury during procurement and implantation, mandating interventions to preserve their function. We tested the hypothesis that limiting oxygen delivery during initial reperfusion of such hearts would reduce free-radical injury. METHODS: Rabbits sustained hypoxic arrest after ventilatory withdrawal, followed by 20 minutes of in vivo ischemia. Hearts were excised and reperfused with blood under conditions of high arterial oxygen tension (PaO2) (approximately 400 mm Hg), low PaO2 (approximately 60 to 70 mm Hg), high pressure (80 mm Hg), and low pressure (40 mm Hg), with or without free-radical scavenger infusion. Non-heart-beating donor groups were defined by the initial reperfusion conditions: high PaO2/ high pressure (n = 8), low PaO2/high pressure (n = 7), high PaO2/low pressure (n = 8), low PaO2/low pressure (n = 7), and high PaO2/high pressure/free-radical scavenger infusion (n = 7). RESULTS: After 45 minutes of reperfusion, low PaO2/ high pressure and high PaO2/low pressure had a significantly higher left ventricular developed pressure (63.6 +/- 5.6 and 63.1 +/- 5.6 mm Hg, respectively) than high PaO2/high pressure (40.9 +/- 4.5 mm Hg; p < 0.0000001 versus both). However, high PaO2/high pressure/free-radical scavenger infusion displayed only a trend toward improved ventricular recovery compared with high PaO2/ high pressure. CONCLUSIONS: Initially reperfusing nonbeating cardiac grafts at low PaO2 or low pressure improves recovery, but may involve mechanisms other than decreased free-radical injury.