Gervasio Martín-Partido

Loss of dioxin-receptor expression accelerates wound healing in vivo by a mechanism involving TGFβ

Delayed wound healing caused by inefficient re-epithelialization underlines chronic skin lesions such as those found in diabetes. The dioxin receptor (AhR) modulates cell plasticity and migration and its activation by occupational polycyclic aromatic hydrocarbons (PAHs) results in severe skin lesions such as contact hypersensitivity, dermatitis and chloracne. Using wild-type (Ahr+/+) and AhR-null (Ahr–/–) mouse primary keratinocyte cultures and tissue explants, we show that lack of AhR increases keratinocyte migration and accelerates skin re-epithelialization without affecting cell proliferation or recruitment of inflammatory cells. Wounds in Ahr–/– animals had elevated numbers of fibroblasts and increased collagen content in their granulation tissue. Importantly, Ahr–/– dermal fibroblasts secreted higher levels of active TGFβ that increased keratinocyte migration in culture and that could account for over-activation of the TGFβ pathway and for faster wound healing in the AhR-null neo-epithelium. Consistently, a TGFβ neutralizing antibody decreased keratinocyte migration in culture and halted re-epithelialization in Ahr–/– mice. Moreover, in vivo treatment with an antisense oligonucleotide for AhR increased TGFβ signaling and improved re-epithelialization in wounds of wild-type mice. These data indicate that AhR is relevant for wound repair and suggest that AhR downmodulation might be a potential new tool for the treatment of chronic, surgical or accidental wounds.

Liver portal fibrosis in dioxin receptor‐null mice that overexpress the latent transforming growth factor‐β‐binding protein‐1

Mice lacking aryl hydrocarbon (dioxin) receptor (AhR) had variable degree of hepatic fibrosis and altered liver architecture. Transforming growth factor‐β (TGF‐β), a major profibrogenic molecule in the liver, is localized to the extracellular matrix by its association to the latent TGF‐β‐binding protein‐1 (LTBP‐1). Very recently, LTBP‐1 has been shown to be negatively regulated by the AhR. Embryonic fibroblasts from AhR‐null (AhR–/–) mice overexpress LTBP‐1 and secrete four times more active TGF‐β than wild‐type fibroblasts. To test whether TGF‐β and LTBP‐1 overexpression colocalize within the fibrotic nodule of AhR–/– liver, we have characterized this hepatic portal fibrosis using collagen protein staining, immunohistochemistry and in situ hybridization. LTBP‐1 mRNA and protein were overexpressed in the fibrotic region and colocalized with other indicators of fibrosis such as collagen and fibronectin and the fibroblast marker proteins α‐actin and vimentin. TGF‐β protein also colocalized with fibrosis, although in contrast, TGF‐β mRNA expression, rather than restricted to the fibrotic compartment, was present throughout the hepatic parenchyma and exhibited similar levels in wild‐type and AhR–/– mice. These results suggest that LTBP‐1 targets TGF‐β to specific areas of the liver and that the AhR could be a negative regulator of liver fibrosis, possibly through the control of LTBP‐1 and TGF‐β activities.

Microglia in the mature and developing quail brain as revealed by a monoclonal antibody recognizing hemopoietic cells

The monoclonal antibody QH1, which recognizes quail endothelial and hemopoietic cells, was found to label microglia in the developing and mature brain of the quail. Forms of microglia similar to those described in mammals were labelled. Ameboid microglia predominated at embryonic stages, became less numerous in late embryonic development, and disappeared completely by day 10 post-hatch (P10). Poorly ramified microglia were present as early as day 5 of incubation (E5), and were progressively replaced by mature ramified microglia from E14 onwards. From P10 onwards, ramified microglia were the only microglial form seen in the quail brain.

Proliferation of glial precursors during the early development of the chick optic nerve

SummaryThe mitotic patterns and cytoarchitecture of the optic stalk were studied in the chick embryo during the period of formation of the optic cup until the elimination of the stalk lumen. Cell proliferation in the superficial regions of the stalk ventral wall is described. Superficial cell proliferation, whose beginning coincides with penetration of the earliest optic fibers, gives rise to an early glioblast plate located internally with respect to the marginal lamina of ganglion cell axon fascicles. The early glioblasts are transformed into marginal glioblasts, which undergo radial mitosis. Radially orented division seems to favour glioblast penetration towards more internal zones of the stalk. Thus the marginal glioblasts are transformed into inner glioblasts, which continue to proliferate. Cell death in the ventral wall of the optic stalk is in close topographical relation with extracellular spaces which loosen up the consistency of the stalk tissue, favouring invasion of the ventral stalk by optic fibers and the addition of new glioblasts by proliferation of preexisting cells.

Spatial and temporal patterns of proliferation and differentiation in the developing turtle eye

Here we show for the first time different aspects of the pattern of neurogenesis in the developing turtle retina by using different morphological and molecular clues. We show the chronotopographical fashion of occurrence of three major aspects of retinal development: (1) morphogenesis of the optic primordia and emergence of the different retinal layers, (2) the temporal progression of neurogenesis by the cessation of proliferative activity, and (3) the apparition and cellular localization of different antigens and neuroactive substances. Retinal cells were generated in a conserved temporal order with ganglion cells born first, followed by amacrine, photoreceptor, horizontal and bipolar/Müller cells. While eventually expressed in many types of retinal neurons, Islet1 was permanently expressed in differentiating and mature ganglion cells. Calbindin-immunoreactive elements were found in the ganglion cell layer and the inner nuclear layer. Interestingly, at later stages the amount of expressing cells in these layers was reduced dramatically. On the contrary, the number of calbindin-immunoreactive photoreceptors increased as development proceeded. In addition, calretinin expressing cells were prominent in the horizontal cell bodies, and their processes extending into the outer plexiform layer were also strongly labeled. Finally, the synthesis of gamma-aminobutyric acid (GABA) was detected in developing and matured horizontal and amacrine cells. All these maturational features began in the dorso-central area, in a region slightly displaced towards the temporal retina.

Differential expression of Otx2, Gbx2, Pax2, and Fgf8 in the developing vestibular and auditory sensory organs.

The vertebrate inner ear is a complex organ with vestibular and auditory sensory functions, which derives from a single ectoderm structure, the otic placode. The development and regional patterning of the otic primordium is determined by the restricted expression of several genes. Here, we show the expression pattern of three transcription factors (Otx2, Gbx2, Pax2) and of a member of the fibroblast growth factor family (Fgf8) in the developing chick inner ear, and we correlate these patterns with the developing sensory and nonsensory elements.

Lateral root initiation by asymmetrical transverse divisions of pericycle cells in adventitious roots ofAllium cepa

SummaryIn onion adventitious roots cellular events have been identified that indicate that lateral root initiation occurs earlier and nearer the apex than previously documented. Lateral roots are not initiated when a pericycle cell divides periclinally but earlier, when a pair of neighbouring pericycle cells in the same column divide transversely and asymmetrically, with both mitoses close to the end towards the neighbouring pericycle cell. Each cell therefore produces two cells of unequal length. The shorter cells produced by the mother pericycle cells are adjacent, while the longer cells are located above and below the shorter cells. This objective morphological criterion allows clear identification of the site of lateral root initiation. Subsequent to these asymmetric divisions, both the longer pericycle cells again divide transversely and asymmetrically producing more short cells adjacent to the previous ones. The first periclinal division occurs in one of these short pericycle cells.

Eye development and retinal differentiation in an altricial fish species, the senegalese sole (Solea senegalensis, Kaup 1858)

We describe the major events in the retinogenesis in an altricial fish species, the Senegalese sole. The major developmental events in the sole retina occurred early after hatching (posthatching day 0, P0). Thus, (1) plexiform layers became recognizable at P1. (2) Proliferative activity disappeared from the central retina at P1, and, as development progressed, became restricted to cells located in the circumferential germinal zone, and to sparse cells dispersed throughout the inner nuclear layer and the outer nuclear layer. (3) Apoptotic cells were sparsely observed, randomly localized in all three nuclear layers of the early posthatching retina from P0 to P4. (4) The first synaptic vesicles were detected at P0 in early postmitotic ganglion cells. However, their appearance in the plexiform layers was delayed until P2. (5) The neurochemical development of most major retinal cell classes occurred between P0 and P5. Thus, although Isl1 immunoreactive ganglion cells were the first to become postmitotic in the vitreal surface of the central retina at P0, the first glutamine synthetase-expressing Müller cells appeared in the central retina by P5. The onset of expression for other retinal markers, such as rod opsin, calretinin, parvalbumin, a-tyrosine hydroxylase, and a-protein kinase C, occurred between P2 and P4. Our results suggest that the most relevant processes involved in Senegalese sole retinogenesis occur during the prolarval and early larval stages (P0–P5). Furthermore, we conclude that altricial fish species may constitute a convenient model organism to address the relationship between the structural and functional development of sensory organs with the acquisition of behavioral repertoires.

Macrophage-like cells in the presumptive optic pathways in the floor of the diencephalon of the chick embryo

SummaryIn the suboptic necrotic centres (SONCs) of the chick embryo diencephalon floor, large numbers of cells die in Hamburger and Hamiltons (HH) developmental stages 14–23. Until recently, it was thought that in these centres, the fragments of dead cells were phagocytosed exclusively by neighboring healthy cells but not by specialized macrophages. We now report morphological evidence of macrophage-like cells within the SONCs of the chick embryo. The distinctive features of these cells are their more or less spherical shape, a nucleus with a thin band of heterochromatin just beneath the nuclear envelope, and cytoplasm showing abundant digestive vacuoles and mitochondria with an electron-lucent matrix. These cells are capable of undergoing mitosis, and selectively stain with the histochemical technique for acid phosphatase. The macrophage-like cells are rare in SONCs in stages HH14-20 and become much more abundant in developmental stages just before the disappearance of these necrotic centres, suggesting that they phagocytose debris from the last cells to die in the SONCs. Acid phosphatase-positive mesenchymal cells with morphological features similar to those of macrophage-like cells are seen in intimate relationship with the basal surface of the SONCs in places where the basal lamina is sometimes missing. These observations suggest that macrophage-like cells in the SONCs arise from the underlying mesenchyme.Free macrophage-like cells with mitotic capacity are also seen in the ventricular lumen adjacent to the apical surface of the diencephalon floor in zones related to the presumptive optic pathways. These cells phagocytose cell debris shed from both the SONCs and from the partially disorganized areas in the neuroepithelium. In these latter we have identified mesenchymal cells with morphological features similar to the macrophage-like cells in the process of traversing the neuroepithelium from the mesenchymal compartment toward the ventricular lumen, thus suggesting that the intraventricular macrophage-like cells arise from the mesenchyme underlying the diencephalon floor.

Spatial and temporal patterns of apoptosis during differentiation of the retina in the turtle

We investigated patterns of cell death in the turtle retina that could potentially be associated with the innervation of the optic tectum, and looked for mechanisms of retinal development that might be common to reptilian and homeotherm vertebrates. We used retinas of turtle embryos between the 23rd day of incubation (E23) (before the first optic fibres reach the optic tectum) and hatching (when all the optic fibres have established synaptic connections). Dying retinal neurons were identified in paraffin sections by the TUNEL technique, which specifically labels fragmented DNA. Apoptotic cells were found in the ganglion cell layer (GCL), the inner nuclear layer (INL), and the outer nuclear layer (ONL). Cell death in the GCL was intense between E29 and E47, and had disappeared by the day of hatching. In the INL, dead and dying cells were most abundant between E31 and E34, and progressively disappeared. The temporal pattern in the ONL was similar to the INL although the density was very low. In all the nuclear layers cell death spread from the dorso-temporal area of the central retina to the periphery. Additional dorsal to ventral and temporal to nasal gradients were distinguishable in a quantitative TUNEL analysis. The patterns of cell death observed in the developing turtle retina were thus similar to those found in birds and mammals. This process could be under the control of differentiation gradients in all the vertebrate classes.