David J. Chambers

Expression of sprouty2 during early development of the chick embryo is coincident with known sites of FGF signalling

The Drosophila sprouty protein is a recently-identified intracellular modulator of FGF and EGF receptor tyrosine kinase activity which antagonises ras/MAP kinase signalling. In a differential display analysis to identify genes involved in patterning the mid/hindbrain region of the chick neural tube, we have identified a sprouty orthologue, sprouty2. Here we report expression of sprouty2 transcripts in the developing chick embryo. We find a close correlation with known sites of FGF activity but little correlation with expression patterns of members of the EGF family. Initially, transcripts are associated with the primitive streak. During the period of neural tube patterning expression is detected in the anterior neuropore, in the isthmic region and in neural plate and posterior spinal cord. Transcripts are also detected in the otic placode, tail bud, mesoderm of the branchial arches, somitic myotome, retina, limb buds and gut mesenchyme; all known sites of FGF action.

Alpha-stat acid-base regulation during cardiopulmonary bypass improves neuropsychologic outcome in patients undergoing coronary artery bypass grafting

Neuropsychologic impairment in patients undergoing cardiopulmonary bypass may be associated with cerebral blood flow changes arising from different management protocols for carbon dioxide tension during bypass. Seventy patients having coronary artery bypass grafting were randomized to either pH-stat or alpha-stat acid-base management during cardiopulmonary bypass with a membrane oxygenator. In each patient, cerebral blood flow (xenon 133 clearance), middle cerebral artery blood flow velocity (transcranial Doppler sonography), and cerebral oxygen metabolism (cerebral metabolic rate and cerebral extraction ratio) were measured during four phases of the operation: before bypass, during bypass (at hypothermia and at normothermia), and after bypass. A battery, of neuropsychologic tests were also conducted before and 6 weeks after the operation. During hypothermic (28 degrees C) bypass, cerebral blood flow was significantly (p < 0.001) greater in the pH-stat group (41 mlx100 gm(-1)xmin(-1); 95% confidence interval 39 to 43 mlx100 gm(-1)xmin(-1)) than in the alpha-stat group (24 mlx100 gm(-1)xmin(-1); confidence interval 22 to 26 mlx100 gm(-1)xmin(-1)) at constant pressure and How. Arterial carbon dioxide tensions were 41 mm Hg (40 to 41 mm Hg) and 26 mm Hg (25 to 27 mm Hg), respectively; pH was 7.36 (7.34 to 7.38) and 7.53 (7.51 to 7.55), respectively. Middle cerebral artery flow velocity was significantly (p < 0.05) reduced in the alpha-stat group to 87% (77% to 96%) of the prebypass value, whereas it was significantly (p < 0.05) increased (152%; 141% to 162%) in the pH-stat group. Cerebral extraction ratio for oxygen demonstrated relative cerebral hyperemia during hypothermic (28 degrees C) bypass in both the pH-stat and alpha-stat groups (0.12 [0.11 to 0.14] and 0.25 [0.22 to 0.28], respectively); however, hyperemia was significantly more pronounced in the pH-stat group, indicating greater disruption in cerebral autoregulation. Neuropsychologic impairment criteria of deterioration in results of three or more tests revealed that a significantly (Fishers exact test, p = 0.02) higher proportion of patients in the pH-stat group fared poorly than in the alpha-stat group at 6 weeks (17/35, 48.6% [32% to 65.1%], and 7/35, 20% [6.7% to 33.2.2%], respectively). In conclusion, patients receiving alpha-stat management had less disruption of cerebral autoregulation during cardiopulmonary bypass, accompanied by a reduced incidence of postoperative cerebral dysfunction.

RALDH-independent generation of retinoic acid during vertebrate embryogenesis by CYP1B1

Several independent lines of evidence have revealed an instructive role for retinoic acid (RA) signalling in the establishment of normal pattern and cellular specification of the vertebrate embryo. Molecular analyses have previously identified the major RA-synthesising (RALDH1-3) and RA-degrading (CYP26A-C1) enzymes as well as other components involved in RA processing (e.g. CRABP). Although the majority of the early effects of RA can be attributed to the activity of RALDH2, many other effects are suggestive of the presence of an as yet unidentified RA source. Here we describe the identification, expression, biochemistry and functional analysis of CYP1B1, a member of the cytochrome p450 family of mono-oxygenases, and provide evidence that it contributes to RA synthesis during embryonic patterning. We present in vitro biochemical data demonstrating that this enzyme can generate both all-trans-retinal (t-RAL) and all-trans-retinoic acid (t-RA) from the precursor all-trans-retinol (t-ROH), but unlike the CYP26s, CYP1B1 cannot degrade t-RA. In particular, we focussed on the capacity of CYP1B1 to regulate the molecular mechanisms associated with dorsoventral patterning of the neural tube and acquisition of motor neuron progenitor domain identity. Concordant with its sites of expression and biochemistry, data are presented demonstrating that CYP1B1 is capable of eliciting responses that are consistent with the production of RA. Taken together, we propose that these data provide strong support for CYP1B1 being one of the RALDH-independent components by which embryos direct RA-mediated patterning.

The endocannabinoid receptor, CB1, is required for normal axonal growth and fasciculation

Endocannabinoids are retrograde neurotransmitters, which act upon the presynaptically located, G-protein coupled receptor CB1, to modulate synaptic transmission in the adult brain. Recently, however, a number of lines of evidence have suggested that endocannabinoid signalling may play an important role in early neuronal development. In this study, we show that the CB1 receptor has a wide expression pattern in the developing nervous system and that its expression follows neuronal differentiation in the embryo from the earliest stages. We also show that the enzymes involved in 2-AG synthesis are expressed in an overlapping manner at these stages. We further show that interfering with CB1 function using a pharmacological inhibitor causes problems in axon pathfinding and fasciculation. Similarly, CB1 gene knock down in the zebrafish by morpholino injection results in defects in axonal growth and fasciculation in these embryos. Thus CB1 function is required in the early embryo for axonal growth and fasciculation.

Differential Display of Genes Expressed at the Midbrain – Hindbrain Junction Identifies sprouty2: An FGF8-Inducible Member of a Family of Intracellular FGF Antagonists

Specification and polarization of the midbrain and anterior hindbrain involve planar signals originating from the isthmus. Current evidence suggests that FGF8, expressed at the isthmus, provides this patterning influence. In this study, we have sought to identify novel genes which are involved in the process by which regional identity is imparted to midbrain and anterior hindbrain (rhombomere 1). An enhanced differential display reverse transcription method was used to clone cDNAs derived from transcripts expressed specifically in either rhombomere 1 or midbrain during the period of isthmic patterning activity. This gene expression screen identified 28 differentially expressed cDNAs. A clone upregulated in cDNA derived from rhombomere 1 tissue showed a 91% identity at the nucleotide level to the putative human receptor tyrosine kinase antagonist: sprouty2. In situ hybridization on whole chick embryos showed chick sprouty2 to be expressed initially within the isthmus and rhombomere 1, spatially and temporally coincident with Fgf8 expression. However, at later stages this domain was more extensive than that of Fgf8. Introduction of ligand-coated beads into either midbrain or hindbrain region revealed that sprouty2 could be rapidly induced by FGF8. These data suggest that sprouty2 participates in a negative feedback regulatory loop to modulate the patterning activity of FGF8 at the isthmus.

Cardiopulmonary bypass: Perioperative cerebral blood flow and postoperative cognitive deficit

Increased cerebral blood flow occurring during cardiopulmonary bypass as a result of changes in arterial carbon dioxide tension during acid-base regulation is thought to increase postoperative cognitive dysfunction. We studied 70 patients undergoing coronary artery bypass procedures who were randomized to two different acid-base protocols: pH-stat or alpha-stat regulation. Cerebral blood flow, cerebral blood flow velocity, and cerebral oxygen metabolism were measured before bypass, during bypass (hypothermic [28 degrees C] and normothermic phases), and after bypass. Detailed cognitive tests were conducted before operation and 6 weeks after operation. During 28 degrees C bypass, cerebral blood flow was significantly (p < 0.05) higher in the pH-stat group than in the alpha-stat group (41 +/- 2 versus 24 +/- 2 mL.100 g-1.min-1), and cerebral blood flow velocity was significantly increased in the pH-stat group and significantly decreased in the alpha-stat group (152% +/- 10% versus 78% +/- 7%). Cerebral extraction ratio of oxygen demonstrated a relatively greater disruption of autoregulation in the pH-stat group than in the alpha-stat group with relative hyperemia of 0.12 +/- 0.02 versus 0.26 +/- 0.03, respectively, during 28 degrees C bypass. Using the criterion of deterioration in three or more neuropsychologic tests, a significantly higher proportion of patients in the pH-stat group fared less well than in the alpha-stat group (49% +/- 17% versus 20% +/- 13%). Patients in the alpha-stat group experienced less disruption of cerebral autoregulation during hypothermic cardiopulmonary bypass, and this was accompanied by a reduction in postoperative cognitive dysfunction.

Relative Changes in Cerebral Blood Flow During Cardiac Operations Using Xenon-133 Clearance Versus Transcranial Doppler Sonography

BACKGROUND Changes in cerebral blood flow (CBF) during cardiac operations have implications in terms of postoperative neurologic and neuropsychological dysfunction. Current techniques of CBF measurement are cumbersome and invasive. Transcranial Doppler sonography offers a noninvasive means of assessing changes in CBF. The aim of this study was validation of this technique with existing methods of CBF measurement during cardiac operations. METHODS We compared the changes in CBF using xenon-133 clearance with changes in middle cerebral artery velocity by transcranial Doppler sonography (VMCA) using pH-stat and alpha-stat acid-base management during cardiopulmonary bypass. Measurements were taken (1) before bypass, (2) at 28 degrees C on bypass, (3) at 37 degrees C on bypass, and (4) after bypass. Relative changes in CBF and VMCA, calculated as the percent change from the prebypass baseline value normalized to 100%, were used in this analysis. RESULTS During the hypothermic phase of cardiopulmonary bypass, CBF and VMCA increased by 45.9% and 51.8%, respectively (p < 0.001), during pH-stat acid-base management but decreased by only 26.4% and 22.4%, respectively (p < 0.0001), during alpha-stat acid-base management. Linear regression analysis of the absolute changes in CBF (mL . 100 g-1 . min-1) and VMCA (cm/s) showed a significant correlation (r = 0.60; r2 = 0.36; p < 0.0001), but a better correlation was obtained when relative changes in CBF and VMCA were compared (r = 0.89; r2 = 0.79; p < 0.0001). CONCLUSIONS Measurements of VMCA, expressed as relative changes of a pre-cardiopulmonary bypass level (using the noninvasive transcranial Doppler sonographic technique), can be used to examine CBF changes during cardiopulmonary bypass.

Rhombomere-specific analysis reveals the repertoire of genetic cues expressed across the developing hindbrain

BackgroundThe Hox family of homeodomain transcription factors comprises pivotal regulators of cell specification and identity during animal development. However, despite their well-defined roles in the establishment of anteroposterior pattern and considerable research into their mechanism of action, relatively few target genes have been identified in the downstream regulatory network. We have sought to investigate this issue, focussing on the developing hindbrain and the cranial motor neurons that arise from this region. The reiterated anteroposterior compartments of the developing hindbrain (rhombomeres (r)) are normally patterned by the combinatorial action of distinct Hox genes. Alteration in the normal pattern of Hox cues in this region results in a transformation of cellular identity to match the remaining Hox profile, similar to that observed in Drosophila homeotic transformations.ResultsTo define the repertoire of genes regulated in each rhombomere, we have analysed the transcriptome of each rhombomere from wild-type mouse embryos and not those where pattern is perturbed by gain or loss of Hox gene function. Using microarray and bioinformatic methodologies in conjunction with other confirmatory techniques, we report here a detailed and comprehensive set of potential Hox target genes in r2, r3, r4 and r5. We have demonstrated that the data produced are both fully reflective and predictive of rhombomere identity and, thus, may represent some the of Hox targets. These data have been interrogated to generate a list of candidate genes whose function may contribute to the generation of neuronal subtypes characteristic of each rhombomere. Interestingly, the data can also be classified into genetic motifs that are predicted by the specific combinations of Hox genes and other regulators of hindbrain anteroposterior identity. The sets of genes described in each or combinations of rhombomeres span a wide functional range and suggest that the Hox genes, as well as other regulatory inputs, exert their influence across the full spectrum of molecular machinery.ConclusionWe have performed a systematic survey of the transcriptional status of individual segments of the developing mouse hindbrain and identified hundreds of previously undescribed genes expressed in this region. The functional range of the potential candidate effectors or upstream modulators of Hox activity suggest multiple unexplored mechanisms. In particular, we present evidence of a potential new retinoic acid signalling system in ventral r4 and propose a model for the refinement of identity in this region. Furthermore, the rhombomeres demonstrate a molecular relationship to each other that is consistent with known observations about neurogenesis in the hindbrain. These findings give the first genome-wide insight into the complexity of gene expression during patterning of the developing hindbrain.

Regulation and function of FGF8 in patterning of midbrain and anterior hindbrain

In this article, an adjunct to a platform presentation at the Winternational 2000 Symposium, we summarize the recent findings of this group concerning the regulation and functions of FGF8 expressed at the isthmus of the developing brain. We show that several different FGF8 isoforms, ectopically expressed in midbrain or posterior forebrain, are able to mimic the proliferative and patterning functions previously attributed to the isthmus in tissue grafting studies. Moreover, we also show that FGF8 protein is sufficient to induce an ectopic isthmic organiser (Fgf-8+, Gbx2+) in anterior midbrain. We also provide evidence that isthmic FGF8 patterns anterior hindbrain, repressing Hox-a2 expression and setting aside a territory of the brain that includes the cerebellar anlage. We show that these effects of FGF8 are likely to be mediated via FGFR1 and be modulated by the putative FGF antagonist, Sprouty2, identified using a differential display screen. Finally, we provide evidence that the onset of Fgf8 expression is regulated by En1 and that its expression at the isthmus is subsequently maintained by a specific and direct interaction between rhombomere 1 and midbrain.

Doublesex and mab-3–related transcription factor 5 promotes midbrain dopaminergic identity in pluripotent stem cells by enforcing a ventral-medial progenitor fate

Understanding the control of cell-fate choices during embryonic stem cell (ESC) differentiation is crucial for harnessing strategies for efficient production of desired cell types for pharmaceutical drug screening and cell transplantation. Here we report the identification of the zinc finger-like doublesex and mab-3–related transcription factor 5 (Dmrt5) as a marker for mammalian ventral-medial mesencephalic neuroepithelium that give rise to dopamine neurons. Gain- and loss-of-function studies in ESC demonstrate that Dmrt5 is critically involved in the specification of ventral-medial neural progenitor cell fate and the subsequent generation of dopamine neurons expressing essential midbrain characteristics. Genome-wide analysis of Dmrt5-mediated transcriptome changes and expression profiling of ventral-medial and ventral-lateral mesencephalic neuroepithelium revealed suppressive and inductive regulatory roles for Dmrt5 in the transcription program associated with the ventral-medial neural progenitor fates. Together, these data identify Dmrt5 as an important player in ventral mesencephalic neural fate specification.