Bias and Evolution of the Mutationally Accessible Phenotypic Space in a Developmental System

Genetic and developmental architecture may bias the mutationally available phenotypic spectrum. Although such asymmetries in the introduction of variation may influence possible evolutionary trajectories, we lack quantitative characterization of biases in mutationally inducible phenotypic variation, their genotype-dependence, and their underlying molecular and developmental causes. Here we quantify the mutationally accessible phenotypic spectrum of the vulval developmental system using mutation accumulation (MA) lines derived from four wild isolates of the nematodes Caenorhabditis elegans and C. briggsae. The results confirm that on average, spontaneous mutations degrade developmental precision, with MA lines showing a low, yet consistently increased, proportion of developmental defects and variants. This result indicates strong purifying selection acting to maintain an invariant vulval phenotype. Both developmental system and genotype significantly bias the spectrum of mutationally inducible phenotypic variants. First, irrespective of genotype, there is a developmental bias, such that certain phenotypic variants are commonly induced by MA, while others are very rarely or never induced. Second, we found that both the degree and spectrum of mutationally accessible phenotypic variation are genotype-dependent. Overall, C. briggsae MA lines exhibited a two-fold higher decline in precision than the C. elegans MA lines. Moreover, the propensity to generate specific developmental variants depended on the genetic background. We show that such genotype-specific developmental biases are likely due to cryptic quantitative variation in activities of underlying molecular cascades. This analysis allowed us to identify the mutationally most sensitive elements of the vulval developmental system, which may indicate axes of potential evolutionary variation. Consistent with this scenario, we found that evolutionary trends in the vulval system concern the phenotypic characters that are most easily affected by mutation. This study provides an empirical assessment of developmental bias and the evolution of mutationally accessible phenotypes and supports the notion that such bias may influence the directions of evolutionary change

Neuroanatomical Study of the A11 Diencephalospinal Pathway in the Non-Human Primate

BACKGROUND: The A11 diencephalospinal pathway is crucial for sensorimotor integration and pain control at the spinal cord level. When disrupted, it is thought to be involved in numerous painful conditions such as restless legs syndrome and migraine. Its anatomical organization, however, remains largely unknown in the non-human primate (NHP). We therefore characterized the anatomy of this pathway in the NHP. METHODS AND FINDINGS: In situ hybridization of spinal dopamine receptors showed that D1 receptor mRNA is absent while D2 and D5 receptor mRNAs are mainly expressed in the dorsal horn and D3 receptor mRNA in both the dorsal and ventral horns. Unilateral injections of the retrograde tracer Fluoro-Gold (FG) into the cervical spinal enlargement labeled A11 hypothalamic neurons quasi-exclusively among dopamine areas. Detailed immunohistochemical analysis suggested that these FG-labeled A11 neurons are tyrosine hydroxylase-positive but dopa-decarboxylase and dopamine transporter-negative, suggestive of a L-DOPAergic nucleus. Stereological cell count of A11 neurons revealed that this group is composed by 4002±501 neurons per side. A 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) intoxication with subsequent development of a parkinsonian syndrome produced a 50% neuronal cell loss in the A11 group. CONCLUSION: The diencephalic A11 area could be the major source of L-DOPA in the NHP spinal cord, where it may play a role in the modulation of sensorimotor integration through D2 and D3 receptors either directly or indirectly via dopamine formation in spinal dopa-decarboxylase-positives cells

Sucrose promotes cytokinin synthesis and auxin export during axillary bud outgrowth

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Effect of the neurotoxin MPTP on hypothalamic A11 neurons.

<p><b>A.</b> Representative microphotograph of TH-IR A11 neurons in a control animal. <b>B.</b> Representative microphotograph of TH-IR A11 neurons in an MPTP-intoxicated animal with full parkinsonism. Note the large cell loss within the A11 area. <b>C.</b> Mean (±SD) percentage of total number of TH-IR neurons remaining following MPTP intoxication within the substantia nigra, ventral tegmental area and A11 group (*<i>P</i><0.0005, comparison between MPTP-treated animals (n = 2) and control animals (n = 4); ^#<i>P</i><0.02 and ^###<i>P</i><0.0003, comparison between dopaminergic groups following MPTP intoxication, two-tailed <i>P</i> value, unpaired <i>t</i>-test). Note the difference in cell loss following MPTP intoxication between the A11 group and the various DA regions. <b>D.</b> TH-IR counted cells were mapped in individual sections from anterior to posterior hypothalamic A11 area with 200 µm section intervals. Note the general cell loss at different levels of the A11 area in MPTP-treated animals (n = 2) compared to controls (n = 4). <b>E–J</b>: Representative micrograph of TH (green) and CALB (red) double fluorescent immunostaining obtained under a confocal laser-scanning microscope in VTA (<b>E–G</b>) and A11 (<b>H–J</b>) sections. The white arrows point to typical double-stained neurons. Note the absence of CALB-positive neurons within the A11 region. Abbreviations: Calb = Calbindin 28 k; TH = Tyrosine Hydroxylase; V3 = Third Ventricle.</p

Macroscopic detection of dopamine receptors within the lumbar cord of non-human primate.

<p>Representative film autoradiograms after radioactive <i>in situ</i> hybridization targeted against the mRNA DA receptors and transporter in lumbar spinal cord transverse sections at different levels (L2, L4 and L5) and in frontal brain sections (positive control). Note that D2 and D3 subtypes are the most expressed DA receptors: D2 receptors were more highly expressed in dorsal horn of the spinal cord whereas D3 receptors showed lower levels of expression with much wider distribution within the gray matter of the spinal cord. Expression of the D1 subtype was not detected whereas the D5 subtype was poorly expressed. Abbreviations: Hcd = Head of Caudate nucleus; Pu = Putamen; IClj = Islands of Calleja; Co = Cortex.</p

Histological analyses of FluoroGold injections into the lumbar spinal cord.

<p><b>A.</b> Visualization of FG labeling by fluorescence microscope providing ultraviolet excitation light. <b>B.</b> Visualization of FG labeling by immunohistochemistry directed against FG. Note that FG was injected into the right dorsal horn of the cervical spinal cord and that there was a slight diffusion of the marker within the contralateral side. Abbreviations: DGH = Dorsal Gray Horn; VGH = Ventral Gray Horn.</p

Characterization of tyrosine hydroxylase-positive neurons within the diencephalon in the non-human primate compared to human.

<p><b>A.</b> At the anterior hypothalamus level (AC-3 mm) where TH-IR regions A12, A14 and A15 are delineated. <b>B.</b> At the medial hypothalamus (AC-4 mm) where region A13 is delineated. <b>C.</b> At the posterior hypothalamus level (AC-5 mm) where the TH-IR A11 region is delineated. Note the remarkable concordance between NHP and human TH-IR distributions. Representative drawings of the human diencephalon are taken from Kitahama et al., 1998 with the permission of Elsevier (License N°2361961459060). Abbreviations: AC = Anterior Commissure; ARH = Arcuate Hypothalamic Nucleus; cp = Cerebral Peduncle; DHA = Dorsal Hypothalamic Area; DMH = Dorsomedial Hypothalamic Nucleus; fx = fornix; LHA = Lateral hypothalamic Area; Ltu = Lateral tuberal nucleus; nsp = nigrostriatal dopaminergic pathway; PaF = Parafornical nucleus; PEH = Periventricular Hypothalamic nucleus; PHA = Posterior Hypothalamic Area; TM = Tuberomammillary nucleus; V3 = Third Ventricle; VMH = Ventral Medial Hypothalamus; ZI = Zona Incerta.</p

Aromatic aminoacid decarboxylase (AADC) expression in posterior hypothalamus and spinal cord.

<p><b>A–B</b>: Immunohistochemistry targeted against TH (<b>A</b>) and AADC (<b>B</b>) processed on adjacent sections of the posterior hypothalamus. Note the lack of AADC labeling in the region of TH-IR A11 neurons. Stars label the same blood vessel profiles in adjacent sections (A and B). <b>C–E</b>: Immunohistochemistry targeted against AADC within the dorsal horn aspect of the spinal cord. The black arrows point to typical AADC positive neurons. <b>F–K</b>: Double-fluorescent immunostaining of TH (green) and AADC (red) obtained under a confocal laser-scanning microscope in a section through the VTA (<b>F–H</b>) and A11 (<b>I–K</b>) groups. The white arrows point to typical double-stained cells in VTA. Note the absence of AADC-positive neurons within the A11 region. Abbreviations: AADC = Aromatic Aminoacid Decarboxylase; DGH = Dorsal Gray Horn; MM = Mammillary nucleus; TH = Tyrosine Hydroxylase; V3 = Third Ventricle.</p

Retrograde labeling of A11 neurons projecting to the spinal cord.

<p><b>A.</b> Schematic representation of the hypothalamic A11 area in which TH-IR neurons were reached by FluoroGold. The dots represent the localization of TH-IR A11 neurons. The red frame represents the area where representative micrographs were taken. <b>B.</b> Representative micrograph showing a reconstructed overview of the TH immunopositive and FG retrograde labeled cells in the A11 group. Immunoreactivity was revealed with Novared kit for TH neurons (Red) and with SG kit for FG neurons (Blue). Double-stained neurons (TH-FG) were labeled in a blue-red combination (i.e. black). The black arrows point to typical double-stained cells. The round heads arrows point to TH-stained neurons. Note that no single FG-stained neurons were found and that the majority of double-stained neurons are located on the ipsilateral side of spinal injections (right) <b>C–H</b>: Representative double-fluorescent immunostaining of TH (green) and FG (red) obtained under a confocal laser-scanning microscope in the A11 posterior hypothalamic group. The white arrows point to typical double-stained cells. Note the colocalization between the TH-positive and FG-positive neurons within the A11 region. Abbreviations: cp = Cerebral Peduncle; FG = FluoroGold; MM = Medial Mammillary nucleus; LHA = Lateral Hypothalamic Area; TH = Tyrosine Hydroxylase; TM = Tuberomammillary nucleus; V3 = Third Ventricle; ZI = Zona Incerta.</p