Small Groups Of Cells Research Articles

Microfilariae of Wucheria bancrofti in bone marrow.

We report an incidental fi nding of microfi lariae in the bone marrow aspirate of a patient who was under investigation of back pain with destruction of fi rst lumbar vertebrae. Filaria-sis is a major health problem in tropical countries including India. Filaria is a nematode belonging to the order spirurida and superfamily fi lariorida [1]. Microfi lariae may inhabit the blood vessels, lymphatic system, connective tissue and serous cavities of man and animals.A 55-year-old female, resident of village Lakhimpur, District- Budayun of Uttar Pradesh, presented with lower backache for 1 yr. On examination tenderness was found at 1st and 2nd lumbar vertebral region. Radiographs showed destruction of fi rst lumbar vertebrae. The differential diag-nosis of tuberculosis spine, metastasis to spine and multiple myeloma were put forth.To rule out these, a bone marrow aspiration was done and it was sent to pathology department for evaluation. Routine hematological investigation was within normal range. On peripheral smear the red cells were normocytic and normochromic. Platelets were adequate. No hemo-parasite or immature cells were noted in the smear. Bone marrow showed contamination with peripheral blood. The red cell precursors showed normoblastic reaction. Myeloid to erythroid ratio and myelopoisis was normal. Megakaryo-cytes also showed a normal picture. There was increase in plasma cell population. They comprised 14% of the mar-row cellularity. A small group of metastatic columnar cells (glandular origin) were seen (Fig. 1). They were supposed to be of metastatic tumor origin. One of the submitted smears showed microfi lariae of Wucheria bancrofti (Fig. 2). Based on these fi ndings the diagnosis of metastatic lesion of bone with Wucheria bancrofti microfi lariae was given.The clinical manifestations of lymphatic fi lariasis may range from asymptomatic microfi larisis to hydro-cele, lymphangitis, lymphadenitis with high grade fever(fi larial fever) and lymphatic obstruction. Our patient was clinically asymptomatic. The presence of microfi lariaehas been documented in the fi ne needle aspirationcytology of various organs other than lymphoid in past. The fi rst documentation of microfi lariae in bone marrow aspirate, available in English literature was by Pradhanet al. [2] in 1976. They showed 7 cases with peripheral

Enzyme-containing hydrogel micropatterns serving a dual purpose of cell sequestration and metabolite detection

The integration of sensing elements with small groups of cells is a critical step towards miniaturization of cell cultivation and analysis. This paper describes the development of an optical, enzyme-based biosensor for local detection of hydrogen peroxide (H2O2) secreted by stimulated macrophages. Photolithographic patterning of horseradish peroxidase (HRP)—containing poly (ethylene glycol) (PEG) hydrogel microstructures was used to create sensing structures on the glass surface. Importantly, enzyme-entrapping hydrogel micropatterns did not support protein or cell deposition and allowed to guide attachment of macrophages next to the sensing elements. Amplex Red, an organic molecule that becomes fluorescent in the presence of H2O2 and HRP, was either immobilized inside hydrogel elements alongside enzyme molecules or added into the cell culture media during cell activation. The production of H2O2 after mitogenic stimulation of macrophages resulted in appearance of fluorescence in the HRP-containing hydrogel microstructures, with fluorescence intensity being a strong function of analyte concentration. The novel cell culture system with integrated sensing elements described here may be enhanced in the future by incorporating additional biorecognition elements to enable multi-metabolite detection at the site of a cell.

Functional Consequences of Neuronal Divergence Within the Retinogeniculate Pathway

The neuronal connections from the retina to the dorsal lateral geniculate nucleus (dLGN) are characterized by a high specificity. Each retinal ganglion cell diverges to connect to a small group of geniculate cells and each geniculate cell receives input from a small number of retinal ganglion cells. Consistent with the high specificity of the connections, geniculate cells sharing input from the same retinal afferent are thought to have very similar receptive fields. However, the magnitude of the receptive-field mismatches, which has not been systematically measured across the different cell types in dLGN, seems to be in contradiction with the functional anatomy of the Y visual pathway: Y retinal afferents in the cat diverge into two geniculate layers (A and C) that have Y geniculate cells (Y(A) and Y(C)) with different receptive-field sizes, response latencies, nonlinearity of spatial summation, and contrast sensitivity. To better understand the functional consequences of retinogeniculate divergence, we recorded from pairs of geniculate cells that shared input from a common retinal afferent across layers and within the same layer in dLGN. We found that nearly all cell pairs that shared retinal input across layers had Y-type receptive fields of the same sign (i.e., both on-center) that overlapped by >70%, but frequently differed in size and response latency. The receptive-field mismatches were relatively small in value (receptive-field size ratio <5; difference in peak response <5 ms), but were robustly correlated with the strength of the synchronous firing generated by the shared retinal connections (R(2) = 0.75). On average, the percentage of geniculate spikes that could be attributed to shared retinal inputs was about 10% for all cell-pair combinations studied. These results are used to provide new estimates of retinogeniculate divergence for different cell classes.

Ectopic expression of LEAFY COTYLEDON1-LIKE gene and localized auxin accumulation mark embryogenic competence in epiphyllous plants of Helianthus annuus × H. tuberosus

The clone EMB-2 of the interspecific hybrid Helianthus annuus x H. tuberosus provides an interesting system to study molecular and physiological aspects of somatic embryogenesis. Namely, in addition to non-epiphyllous (NEP) leaves that expand normally, EMB-2 produces epiphyllous (EP) leaves bearing embryos on the adaxial surface. This clone was used to investigate if the ectopic expression of H. annuus LEAFY COTYLEDON1-LIKE (Ha-L1L) gene and auxin activity are correlated with the establishment of embryogenic competence. Ha-L1L expression was evaluated by semi-quantitative RT-PCR and in situ hybridization. The endogenous level and spatial distribution of free indole-3-acetic acid (IAA) were estimated by a capillary gas chromatography-mass spectrometry-selected ion monitoring method and an immuno-cytochemical approach. Ectopic expression of Ha-L1L was detected in specific cell domains of the adaxial epidermis of EP leaves prior to the development of ectopic embryos. Ha-L1L was expressed rapidly when NEP leaves were induced to regenerate somatic embryos by in vitro culture. Differences in auxin distribution pattern rather than in absolute level were observed between EP and A-2 leaves. More precisely, a strong IAA immuno-signal was detected in single cells or in small groups of cells along the epidermis of EP leaves and accompanied the early stages of embryo development. Changes in auxin level and distribution were observed in NEP leaves induced to regenerate by in vitro culture. Exogenous auxin treatments lightly influenced Ha-L1L transcript levels in spite of an enhancement of the regeneration frequency. In EP leaves, Ha-L1L activity marks the putative founder cells of ectopic embryos. Although the ectopic expression of Ha-L1L seems to be not directly mediated by auxin levels per se, it was demonstrated that localized Ha-L1L expression and IAA accumulation in leaf epidermis domains represent early events of somatic embryogenesis displayed by the epiphyllous EMB-2 clone.

Multifocality in Testicular Germ Cell Tumors

Standard treatment for testicular germ cell tumor is radical orchiectomy. Several groups have suggested an organ sparing approach in patients with bilateral tumors or tumor in a solitary testis. We determined the prevalence of multifocality in testicular germ cell tumor cases. Orchiectomy specimens from 145 consecutive patients treated for testicular germ cell tumor between 1995 and 2006 were included in the study. In the current series slides were reviewed by a single dedicated uropathologist. Multifocality was defined as 1 of 4 distinct pathological entities, including 1) distinct tumor focus conspicuously separable from the main tumor mass, 2) microinvasive tumor characterized by a single or small groups of malignant germ cells scattered within the normal interstitial parenchyma, 3) extra tumor vascular invasion and 4) rete testis invasion by pagetoid tumor spread. Multifocality was identified in 48 patients (33%), of whom 17 (12%) had an additional distinct tumor focus, 21 (14%) had microinvasive tumor, 17 had extra tumor vascular invasion and 2 had rete testis invasion by pagetoid tumor spread. Multifocality was more prevalent in men with smaller tumors and seminomatous histology (pure seminoma or as part of a mixed germ cell tumor). Multifocality was present in 63% of men with an index mass of 2 cm or less in diameter. Study limitations include potential pathological sampling errors resulting from the retrospective design. Multifocality is a frequent finding in testicular germ cell tumor cases that is associated with small mass size and seminomatous histology. Data suggest that additional invasive tumor outside the index mass may be present in up to 63% of men considered potential candidates for organ sparing surgery.

Simulation of advanced microfluidic systems with dissipative particle dynamics

Computational Fluid Dynamics (CFD) is widely and successfully used in standard design processes for microfluidic μTAS devices. But for an increasing number of advanced applications involving the dynamics of small groups of beads, blood cells or biopolymers in microcapillaries or sorting devices, novel simulation techniques are called for. Representing moving rigid or flexible extended dispersed objects poses serious difficulties for traditional CFD schemes. Meshless, particle-based simulation approaches, such as Dissipative Particle Dynamics (DPD) are suited for addressing these complicated flow problems with sufficient numerical efficiency. Objects can conveniently be represented as compound objects embedded seamlessly within an explicit model for the solvent. However, the application of DPD and related methods to realistic problems, in particular the design of microfluidics systems, is not well developed in general. With this work, we demonstrate how the method appears when used in practice, in the process of designing and simulating a specific microfluidic device, a microfluidic chamber representing a prototypical bead-based immunoassay developed in our laboratory (Glatzel et al. 2006a, b; Riegger et al. 2006).

A microfluidic cell trap device for automated measurement of quantal catecholamine release from cells

Neurons and endocrine cells secrete neurotransmitter and hormones in discrete packets in a process called quantal exocytosis. Electrochemical microelectrodes can detect spikes in current resulting from the oxidation of individual quanta of transmitter only if the electrodes are small and directly adjacent to release sites on the cell. Here we report development of a microchip device that uses microfluidic traps to automatically target individual or small groups of cells to small electrochemical electrodes. Microfluidic channels and traps were fabricated by multi-step wet etch of a silicon wafer whereas Pt electrodes were patterned in register with the trap sites. We demonstrate high-resolution amperometric measurement of quantal exocytosis of catecholamines from chromaffin cells on the device. This reusable device is a step towards developing high-throughput lab-on-a-chip instruments for recording quantal exocytosis to increase the pace of basic neuroscience research and to enable screening of drugs that target exocytosis.

Network Dynamics Underlying the Formation of Sparse, Informative Representations in the Hippocampus

During development, activity-dependent processes increase the specificity of neural responses to stimuli, but the role that this type of process plays in adult plasticity is unclear. We examined the dynamics of hippocampal activity as animals learned about new environments to understand how neural selectivity changes with experience. Hippocampal principal neurons fire when the animal is located in a particular subregion of its environment, and in any given environment the hippocampal representation is sparse: less than half of the neurons in areas CA1 and CA3 are active whereas the rest are essentially silent. Here we show that different dynamics govern the evolution of this sparsity in CA1 and upstream area CA3. CA1, but not CA3, produces twice as many spikes in novel compared with familiar environments. This high rate firing continues during sharp wave ripple events in a subsequent rest period. The overall CA1 population rate declines and the number of active cells decreases as the environment becomes familiar and task performance improves, but the decline in rate is not uniform across neurons. Instead, the activity of cells with initial peak spatial rates above approximately 12 Hz is enhanced, whereas the activity of cells with lower initial peak rates is suppressed. The result of these changes is that the active CA1 population comes to consist of a relatively small group of cells with strong spatial tuning. This process is not evident in CA3, indicating that a region-specific and long timescale process operates in CA1 to create a sparse, spatially informative population of neurons.

Molecular markers in the surgical margin of oral carcinomas

Local or regional lymph node recurrence is the most common pattern of treatment failure in oral squamous cell carcinoma (SCC). The local recurrence rate is 30% even when the surgical resection margin is diagnosed as tumour free. Accumulation of genetic changes in histologically normal epithelium in the surgical resection margin may explain the local recurrence rate. The purpose of this study is to investigate the presence of senescence markers, which may represent early malignant changes in the margin that in routine pathological evaluations are classified as histologically normal. Formalin-fixed, paraffin-embedded surgical specimens from 16 consecutive patients with oral SCC and a clear surgical margin were obtained. The margin was analysed by immunohistochemistry for p53, p16, Chk2, Laminin-5 and glycosylated oncofetal fibronectin. Two patterns of p53 expression were found in the histologically normal epithelium in the surgical resection margin. One was characterized by no protein expression in the majority of cells, except for small clusters of basal and parabasal cells with nuclear staining. The other was characterized by p53 expression in the nuclei of most basal cells. The expression of p16 was confined to small groups of cells in the basal cell layer whereas Chk2 was only seen in one case. Upregulation of the stromal proteins, Laminin-5 or glycosylated oncofetal fibronectin, was only seen at regions of invasion. Small groups of cells expressing p53 and p16 were found in the surgical resection margin that appeared to be histologically normal and may represent early malignant changes.

The Neurovascular Unit in Health and Disease

Over the past 10 years, study of the blood vessel in cerebrovascular disease has expanded from consideration of only endothelial cells to include interactions with neurons, astrocytes, pericytes, and extracellular matrix. The role of other cells in the pathobiology of cerebral blood vessels has been encompassed under the term “neurovascular unit (NVU).” This session brings together a series of papers on the role of the NVU in stroke and neurodegenerative diseases. The endothelial cell remains central to the NVU, but its function is regulated by input from adjacent pericytes, astrocytes, and neurons.1 The extracellular matrix and the matrix-degrading enzymes and inhibitors play a key role at the basal lamina and the cell surface in the regulation of cell signaling.2 Much has been learned about the function of astrocytes, which rapidly transduce information between the microenvironment and other brain cells and the macrophage-like pericytes in effecting changes at the cerebral capillary level. The presentations were arranged to emphasize recent advances in our understanding of the anatomy and physiology of the NVU (Lo), recent studies on the astrocytes (Nedergaard), and pericytes (Fisher). The final 2 presentations describe the role of extracellular matrix enzymes in amyloid metabolism as it relates to Alzheimer disease (Lee) and vascular cognitive impairment (Rosenberg). The goal of the session was to provide an overview on the role of the separate cell types composing the NVU and to show how they interact in acute and chronic cerebrovascular diseases. Transgenic …

A microwell array device capable of measuring single-cell oxygen consumption rates

Due to interest in cell population heterogeneity, the development of new technology and methodologies for studying single cells has dramatically increased in recent years. The ideal single cell measurement system would be high throughput for statistical relevance, would measure the most important cellular parameters, and minimize disruption of normal cell function. We have developed a microwell array device capable of measuring single cell oxygen consumption rates (OCR). This OCR device is able to diffusionally isolate single cells and enables the quantitative measurement of oxygen consumed by a single cell with fmol/min resolution in a non-invasive and relatively high throughput manner. A glass microwell array format containing fixed luminescent sensors allows for future incorporation of additional cellular parameter sensing capabilities. To demonstrate the utility of the OCR device, we determined the oxygen consumption rates of a small group of single cells (12 to 18) for three different cells lines: murine macrophage cell line RAW264.7, human epithelial lung cancer cell line A549, and human Barrett's esophagus cell line CP-D.

Frequency Clustering in Spontaneous Otoacoustic Emissions from a Lizard's Ear

Spontaneous otoacoustic emissions (SOAEs) are indicators of an active process in the inner ear that enhances the sensitivity and frequency selectivity of hearing. They are particularly regular and robust in certain lizards, so these animals are good model organisms for studying how SOAEs are generated. We show that the published properties of SOAEs in the bobtail lizard are wholly consistent with a mathematical model in which active oscillators, with exponentially varying characteristic frequencies, are coupled together in a chain by visco-elastic elements. Physically, each oscillator corresponds to a small group of hair cells, covered by a tectorial sallet, so our theoretical analysis directly links SOAEs to the micromechanics of active hair bundles.

Parasite eggs in urine cytology: Fact or artifact?

Urine sediment cytology should be done for three main reasons: initial evaluation in symptomatic patients, pursuit of patients with tumoral pathology, and risk population screening. Nevertheless, in the course of microscopic observation it is possible to observe several elements or structures that initially seem unrelated to what we are looking for. Schistosomiasis is an endemic disease affecting humans and animals in tropical countries and the Middle and Far East, but infrequent in the developed countries. Typical eggs from Schistosoma are nonoperculate, embryonated when passed in feces and with transparent shell. The eggs are elongate and large in S. mansoni (114–175 3 40– 70 lm in size), with a prominent lateral spine, and in S. haematobium (112–170 3 40–70 lm) and S. intercalatum (140–240 3 50–85 lm) with a terminal spine. In the cases of S. japonicum (70–100 3 55–65 lm) and S. mekongi (51–78 3 39.66 lm) the eggs are round with a small inconspicuous spine. The presence of miracidium inside the eggs could be a method of differentiation of cytological artefacts in certain clinical settings. Eggs of S. haematobium may also be found in biopsies of the urinary bladder and rectum, and occasionally they may found in feces. Here, we show some structures observed in urinary sediment that because of their morphology, were initially mistaken for parasitic eggs (Schistosoma spp.). The images correspond to voided urine sediment from a 70year-old man presenting hematuria as the only symptom. Biochemical analysis was within normal limits, except for high level of uric acid. Presence of eosinophils in urine and/or eosinophilia was not appreciated. The patient does not report a travel to an area where schistosomiasis is endemic. Figure C-1 shows, against a clear background, and surrounding a small group of urothelial cells, several oval structures (true acid uric crystals) with a spine-like appearance at the ends (thick arrows). The similar unstained and transparent crystals are also observed (thin arrows). In Figure C-2, corresponding to the same sample, three acid uric crystals showed as structures of granular appearance are observed, being the two at the margins with oval shape, 80 3 25 microns in size, and with two spine-like projections at the ends. Figure C-3 shows a true egg of S. haematobium found in human feces (the miracidium can be observed inside of the structure). Because there are few publications relating the presence of S. haematobium eggs in urine sediment, we think that it is easy to mistake or ignore or to fall in the temptation of believing or not. Although schistosomiasis of the urinary bladder is an unusual parasitic infection in our environment, because of the increase in immigration and travel to exotic countries it may be wise to include this disease in asymptomatic hematuria differential diagnosis. The majority of crystals found in urine, except a few such as cystine, tyrosine, and leucine, are of limited clinical value. In many cases, crystals found in urine sediment are not present in the freshly voided specimen, although pH changes and refrigeration are promoters of crystals formation. Uric acid crystals present a variety of shapes such as oval with more or less eroded ends (‘‘lemon-like’’ shape), polygonal, in needles and/or rosettes, etc. Coloration may oscillate between brown-yellow or uncolored, and under polarized light the color variation is quite typical. Nevertheless, because of unusual morphology it may cause false interpretations as it is shown here in inexperienced hands. CF Anatomia Patologica y Citologia, Instituto de Piedras BlancasAsturias, Spain Faculty of Pharmacy, Departament of Parasitology, Universidad de Valencia, Spain Infectious Diseases, Medical Faculty, Ondokuz Mayis University, Samsun, Turkey *Correspondence to: Rafael Martinez-Giron, M.D., Ph.D., Marques de San Esteban, 22-2 B, Gijon 33206, Spain. E-mail: rmartinezgiron@hotmail.com Received 16 January 2008; Accepted 19 July 2008 DOI 10.1002/dc.20940 Published online 30 October 2008 in Wiley InterScience (www. interscience.wiley.com).

Immunohistochemical detection of estrogen receptors in fish scales

Calcium mobilization from internal stores, such as scales, induced by 17β-estradiol during sexual maturation in salmonids is well documented. This calcium mobilization from scales is proposed to be mediated by the estrogen receptor (ER). However, the ER subtypes involved and signaling mechanisms responsible for this effect remain to be fully characterized. In the present study, we have localized ERα, ERβa and ERβb proteins in juvenile and adult sea bream ( Sparus auratus) and Mozambique tilapia ( Oreochromis mossambicus) scales by immunohistochemistry with sea bream ER subtype specific antibodies. The three ERs were detected in isolated or small groups of round cells, in the basal layer of the scales of both juvenile and adult fish and the localization and signal intensity varied with the species and age of the animals. The ERs may be co-localized in cells of the scale posterior region that expressed tartrate-resistant acid phosphatase (TRAP), a marker for osteoclasts. These results suggest that the calcium mobilizing action of 17β-estradiol on fish scales is via its direct action on ERs localized in osteoclasts.

A possible role of ectopic action potentials in the in vitro hippocampal sharp wave–ripple complexes

Sharp wave–ripple (SPW-R) complexes are physiological pattern of network activity in the hippocampus thought to play important role in memory consolidation. During SPW-R activity the excitability of both pyramidal cells and certain types of interneurons in the CA1 region is transiently increased. As a result pyramidal cells receive inhibitory input during network oscillation, yet a relatively small group of pyramidal cells transmit their output to CA1 targets. However, the exact nature of CA1 output during SPW-R activity is not clear. In this study, using simultaneous intracellular and field recordings from rat ventral hippocampal slices maintained at 32 °C and spontaneously generating SPW-R complexes we show that 20% of CA1 pyramidal cells fired putative ectopic action potentials (e-APs) phase-related to SPW-Rs. The highest probability of ectopic discharge occurred at the maximal amplitude of the ripple oscillation and always during the period of SPW-R-associated inhibitory postsynaptic potentials (IPSPs) in pyramidal cells. Both e-APs and IPSPs were abolished under blockade of GABAA receptor-mediated synaptic transmission by bicuculline. Ectopic APs phase-locked to SPW-R events were also evoked by Schaffer collateral stimulation subthreshold for and with longer latency than monosynaptic orthodromic APs. A fraction of CA1 pyramidal cells (25.7%), most of them distinct from the cells firing e-APs, fired orthodromic APs with highest probability before the onset of SPW-Rs. We hypothesize that putative ectopic spikes in pyramidal cells, presumably triggered by GABAergic synaptic mechanisms, by serving as output of the CA1 region might provide a reliable mechanism for optimized information transfer between hippocampus and its cortical targets during SPW-R activity. On the other hand, orthodromic APs might contribute to the initiation and synchronization of the population activity.

Tracing retinal fiber trajectory with a method of transposon-mediated genomic integration in chick embryo

We report convenient retinal fiber tracing by transfecting the tracer cDNA by in ovo electroporation. Long-term and stable expression of tracer proteins such as green fluorescent protein is achieved by transposon-mediated genome integration of the tracer protein expression cassette. We carried out coelectroporation of a plasmid containing CAGGS-tracer cDNA flanked by the Tol2 transposable element along with a transposase expression vector to the optic vesicle of chick embryos at stage 11. By selecting electrodes, we can label a large group of retinal ganglion cells, or a small group of retinal ganglion cells; parallel electrodes assure transfection of large areas of the retina, and needle type electrodes label small areas of the retina. The retinal fiber trajectory and terminal zone (TZ) could be detected in the precise retinotopic manner on the contra-lateral side of the optic tectum. The method has advantage in that we can show the retinal fiber trajectory in relation to the molecules that are responsible for pathfinding for the retinal fibers in the same specimen.

P.303 A review of FNA/core biopsies in head and neck lumps

We report 4 cases of pseudomesotheliomatous carcinoma of the lung, which has clinical and microscopic features similar to malignant mesothelioma, but with ultrastructural, immunohistochemical, and molecular characteristics suggestive of a histogenesis from type II pneumocytes. Neoplasm grows as a diffuse or solid pattern of large polygonal cells with sharply defined borders. Hale's colloidal iron is positive in the cytoplasm of small groups of cells and, focally, in some intercellular spaces. Ultrastructure showed short microvilli in the surface. Immunohistochemically, tumor cells were positive for thyroid transcription factor–1, podoplanin, mesothelin, pan-cytokeratin, CK-7, CK-19, Ber-EP4, epithelial membrane antigen, apoprotein surfactant A, epidermal growth factor receptor, Leu-M1, carcinoembryonic antigen, E-cadherin, and CD-44 and negative for mesothelioma markers thrombomodulin and calretinin. In some areas, there were small cysts which contained a concentric fibrilar basophilic material apoprotein surfactant A positive. Chromosomal imbalances with comparative genomic hybridization technique were identified with a median of 15 abnormalities per case (range, 1-26): 51 gains, 6 losses, and 1 high-level amplification. The most frequent aberrations among the cases were gains on chromosomes regions 1q, 3q, 5p, 8q, 16p, and 18q and losses in 17p11-13 and 17q 22-q25. High-level amplifications were detected on 7p13-p21. In all cases, there was a characteristic association between the gains on 16p and those on 18q. The 4 cases resulted in death in less than 14 months, in spite of complete surgery and chemotherapy in 2 cases. Our aim is to complement the current understanding of this pseudomesotheliomatous “pneumocytic” carcinoma and alert pathologists to this rare entity to avoid misdiagnosis.

The molecular code involved in midbrain dopaminergic neuron development and maintenance

Midbrain dopaminergic neurons comprise a small group of cells that regulate important functions in the mammalian brain. Their degeneration or dysfunction is involved in many common neurological disorders such as Parkinson’s disease, depression, schizophrenia, attention deficit hyperactive disorder and drug addiction. Here we review recent studies that unravel the molecular events involved in various steps required for the embryonic development and maturation of midbrain dopaminergic neurons, with emphasis on the role of epigenetic cues and transcription factors acting during the late differentiation steps.

Formation of the zebrafish midbrain–hindbrain boundary constriction requires laminin-dependent basal constriction

The midbrain–hindbrain boundary (MHB) is a highly conserved fold in the vertebrate embryonic brain. We have termed the deepest point of this fold the MHB constriction (MHBC) and have begun to define the mechanisms by which it develops. In the zebrafish, the MHBC is formed soon after neural tube closure, concomitant with inflation of the brain ventricles. The MHBC is unusual, as it forms by bending the basal side of the neuroepithelium. At single cell resolution, we show that zebrafish MHBC formation involves two steps. The first is a shortening of MHB cells to approximately 75% of the length of surrounding cells. The second is basal constriction, and apical expansion, of a small group of cells that contribute to the MHBC. In the absence of inflated brain ventricles, basal constriction still occurs, indicating that the MHBC is not formed as a passive consequence of ventricle inflation. In laminin mutants, basal constriction does not occur, indicating an active role for the basement membrane in this process. Apical expansion also fails to occur in laminin mutants, suggesting that apical expansion may be dependent on basal constriction. This study demonstrates laminin-dependent basal constriction as a previously undescribed molecular mechanism for brain morphogenesis.

Making Animals the Right Size

Classical experiments with amphibians revealed that bisected embryos develop into properly proportioned tadpoles and that transplantation of a small group of dorsal cells called "Spemann’s organizer" into the ventral side of an early embryo leads to the formation of a properly proportioned second axis. Thus, the morphogen activation gradients that drive patterning in the early embryo appear to scale with size. Two theoretical models (an inhibition-based model and the shuttling-based model) have been proposed to explain how the bone morphogenetic protein (BMP) activity gradient that drives axis formation is established. Ben-Zvi et al . provide evidence for the shuttling-based model, which previously had only experimental support in fly embryos, in frog ( Xenopus laevis ) embryos and suggest that the additional BMP ligand antidorsalizing morphogenic protein (ADMP), which is produced in the dorsal pole and which is not present in Drosophila , allows the Xenopus embryos to scale patterning with size. First, they created a mathematical model for the core patterning network containing the BMP ligands BMP (which represented BMP2, 4, and 7) and ADMP, the BMP antagonist chordin, and the protease Xlr, which degrades chordin. In this model, chordin and ADMP were produced at the dorsal pole, and BMP signaling repressed ADMP. Systematic screening for parameters that allowed the activation gradient of BMP to be robust and to scale with embryo size resulted in the identification of 21 networks that scaled properly out of the 26,000 possible networks. In each of the networks that scaled, the ligands were concentrated at the ventral pole, suggesting that activation gradients were generated by shuttling of the ligands from their site of production to the ventral pole and not simply by localized production of an inhibitor. Further, the models were consistent with experimentally proven properties of the core patterning components: Properly scaling networks required that chordin binds BMP with higher affinity than it binds ADMP, that binding of chordin facilitates diffusion of BMP ligands, and that chordin is degraded primarily when complexed with ligand. A shuttling model that relied solely on BMP and chordin was insufficient to provide scaling; however, the addition of ADMP with its BMP-mediated repression was sufficient. Experiments in which Xenopus embryos were injected with a tagged BMP4 with or without coinjection of morpholino oligonucleotides (MOs) against chordin showed that BMP4 only concentrated at the ventral pole in the presence of chordin. Axis inversion, which was also consistent with a shuttling mechanism, occurred when chordin was depleted from the organizer at the dorsal side with MOs and a second organizer was induced on the ventral side. Induction of a second organizer on the ventral side in conjunction with MOs for ADMP resulted in the expression of a ventrally expressed gene between the original dorsal organizer and the ventral organizer, which is also consistent with shuttling of BMP ligands. Thus, as in fly embryos, vertebrates also appear to use a shuttling mechanism for robust patterning that scales with embryo size. D. Ben-Zvi, B.-Z. Shilo, A. Fainsod, N. Barkai, Scaling of the BMP activation gradient in Xenopus embryos. Nature 453 , 1205-1211 (2008). [PubMed]