Lucía Rodríguez-Gallardo

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.

Differential staining of dead and dying embryonic cells with a simple new technique

A new staining technique with toluidine blue and safranin is described which yields satisfactory results in histological sections of chick embryo tissue fixed in a mixture of glutaraldehyde and formaldehyde and embedded in Spurrs resin. In zones of embryonic cell death, this technique allows the blue‐staining of dying cell pyknotic nuclei to be clearly distinguished from red‐staining healthy cells. The possible factors underlying this differential staining of pyknotic nuclei are discussed.

Extra-axonal environment and fibre directionality in the early development of the chick embryo optic chiasm: A light and scanning electron microscopic study

SummaryThe events that occur during the early development of the optic chiasm of the chick embryo have been studied by light and scanning electron microscopy. In developmental stages previous to the arrival of the first optic fibres in the floor of the diencephalon, as well as during the arrival of the leading fibres, extracellular spaces can be seen in the diencephalon ventral wall. These spaces are defined by external cell prolongations which end in a foot-shaped formation. During stages 25 and 26 a prechiasmic degenerative centre appears in the area immediately rostral to the early chiasm, leading to a notable degree of disorganization in the diencephalon wall. This centre appears to be related to the reorganization of the system of external cell processes and extracellular spaces which become progressively more irregularly distributed, coinciding with the arrival of the first optic fibre fascicles to the midline of the floor of the diencephalon. The optic fibre fascicles change their latero-medial directionality in the medial-most regions of the ventral diencephalon, where their course becomes rostrocaudal. This reorientation of the optic fibres seems to be mediated by primitive glial cells which first appear in the ventrorostral region of the early chiasm (previously occupied by the system of external cell processes and extracellular spaces) in stage 26, increasing in number from this stage on. The morphology of the primitive glial cells is laminar in nature and the cells are seen to be densely packed together with no large extracellular spaces between them.

Meis gene expression patterns in the developing chicken inner ear.

We are interested in stable gene network activities operating sequentially during inner ear specification. The implementation of this patterning process is a key event in the generation of functional subdivisions of the otic vesicle during early embryonic development. The vertebrate inner ear is a complex sensory structure that is a good model system for characterization of developmental mechanisms controlling patterning and specification. Meis genes, belonging to the TALE family, encode homodomain‐containing transcription factors remarkably conserved during evolution, which play a role in normal and neoplastic development. To gain understanding of the possible role of homeobox Meis genes in the developing chick inner ear, we comprehensively analyzed their spatiotemporal expression patterns from early otic specification stages onwards. In the invaginating otic placode, Meis1/2 transcripts were observed in the borders of the otic cup, being absent in the portion of otic epithelium closest to the hindbrain. As development proceeds, Meis1 and Meis2 expressions became restricted to the dorsomedial otic epithelium. Both genes were strongly expressed in the entire presumptive domain of the semicircular canals, and more weakly in all associated cristae. The endolymphatic apparatus was labeled in part by Meis1/2. Meis1 was also expressed in the lateral wall of the growing cochlear duct, while Meis2 expression was detected in a few cells of the developing acoustic‐vestibular ganglion. Our results suggest a possible role of Meis assigning regional identity in the morphogenesis, patterning, and specification of the developing inner ear. J. Comp. Neurol. 519:125‐147, 2011.

Morphological and quantitative studies in the otic region of the neural tube in chick embryos suggest a neuroectodermal origin for the otic placode.

Careful histological observation of the development of the anlage of the inner ear in chicken embryos led us to question the traditional view of otic placode (OP) formation. First, morphological studies in the cephalic region carried out on stages preceding the appearance of the placodal epithelium revealed that the medial placodal cells are continuous temporally and spatially with cells belonging to the neural fold (NF). Second, both the formation of the basal lamina between the dorsal region of the neural tube (NT) and ectoderm and the pattern of formation of the neural crest present distinctive characteristics between otic levels and regions located anteriorly and posteriorly. Third, numerical comparisons of parameters for the NT and the OP between different levels of the rhombencephalon allowed us to assign a differential behaviour in the growth pattern of the otic region. These results indicated that the medial part of the OP is not derived from already independent ectoderm that increases in thickness under the influence of the NT (as previously accepted) but that it develops directly from the NFs. Although we do not exclude other possibilities, we propose that at least a proportion of the OP cells originate directly from cells committed to be neural crest. After this incorporation, basal laminal formation would delimit the NT from the OP without transition of the otic cells to ectoderm. This hypothesis would imply that part of the otic cells originate directly from neuroepithelial cells having a neuroectodermal (rather than the previously established ectodermal) origin.

Distinct and redundant expression and transcriptional diversity of MEIS gene paralogs during chicken development

Members of the Meis family of TALE homeobox transcription factors are involved in many processes of vertebrate development and morphogenesis, showing extremely complex transcriptional and spatiotemporal expression patterns. In this work, we performed a comprehensive study of chicken Meis genes using multiple approaches. First, we assessed whether the chicken genome contains a Meis3 ortholog or harbors only two Meis genes; we gathered several lines of evidence pointing to a specific loss of the Meis3 ortholog in an early ancestor of birds. Next, we studied the transcriptional diversity generated from chicken Meis genes through alternative splicing during development. Finally, we performed a detailed analysis of chick Meis1/2 expression patterns during early embryogenesis and organogenesis. We show that the expression of both Meis genes begins at the gastrulation stage in the three embryonic layers, presenting highly dynamic patterns with overlapping as well as distinct expression domains throughout development. Developmental Dynamics 240:1475–1492, 2011.

Macrophages during retina and optic nerve development in the mouse embryo: relationship to cell death and optic fibres

We compared the spatial and temporal patterns of distribution of macrophages, with patterns of naturally occurring cell death and optic fibre growth during early retina and optic nerve development, in the mouse. We used embryos between day 10 of embryogenesis (E10; before the first optic fibres are generated in the retina) and E13 (when the first optic fibres have crossed the chiasmatic anlage). The macrophages and optic axons were identified by immunocytochemistry, and the apoptotic cells were detected by the TUNEL technique, which specifically labels fragmented DNA. Cell death was observed in the retina and the optic stalk long before the first optic axons appeared in either region. Subsequently, specialized F4/80-positive phagocytes were detected in chronological and topographical coincidence with cell death, which disappeared progressively. As development proceeded, the pioneer ganglion cell axons reached the regions where the macrophages were located. As the number of optic fibres increased, the macrophages disappeared. Therefore, cell death, accompanied by macrophages, preceded the growth of fibres in the retina and the optic nerve. Moreover, these macrophages synthesized NGF and the optic axons were p75 neurotrophin receptor (p75NTR)- and TrkA-positive. These findings suggest that macrophages may be involved in optic axon guidance and fasciculation.

Raldh3 gene expression pattern in the developing chicken inner ear.

Retinoic acid (RA), an active metabolite of vitamin A, is a diffusible molecule that regulates the expression of several families of genes, playing a key role in specification processes during chordate development. With the aim of defining its possible role in the developing chick inner ear, we obtained in this work a detailed spatiotemporal distribution of the enzymes involved in its synthesis, the retinaldehyde dehydrogenases (RALH1–4). Our results showed that, in contrast to the mouse inner ear, Raldh3 expression was the only Raldh gene detected in the developing chick inner ear, where it appears as early as stage 18. During inner ear morphogenesis, Raldh3 expression was predominantly observed in the endolymphatic system. The Raldh3 expression pattern delimited totally or partially the Bmp4‐positive presumptive territories of vestibular sensory epithelia by stage 24 and the basilar papilla at stage 34, suggesting a possible involvement of RA in their specification. In addition, several vestibular sensory areas showed some Raldh3‐expressing cells close to the Raldh3‐positive domain. These results suggest that the RA signaling pathway may play a role in the initial patterning of the otic epithelium and cell differentiation therein, providing local positional information. Having in mind this Raldh3 expression pattern, we discuss the regulatory interactions among the RA, bone morphogenetic protein, and fibroblast growth factor signaling pathways in the specification of otic sensory elements. Our investigation may underpin further experimental studies aimed at understanding the possible role of signaling pathways in patterning of the developing chick inner ear. J. Comp. Neurol. 514:49–65, 2009.

Glioblast migration in the optic stalk of the chick embryo.

SummaryLight and electron microscopy was used to study the chick embryo optic stalk from Hamburger and Hamilton stages 21 to 29. Observations of glioblast morphology in different developmental stages suggest that these cells undergo radial migration toward peripheral regions of the stalk. Immediately previous to and during migration, morphological changes were noted in the glioblasts, including the appearance of lateral prolongations which contribute to the subdivision of optic fiber fascicles and the radial elongation of their nucleus, which gives the impression of squeezing itself into the peripheral glioblastic prolongation. These phenomena occur in a retino-diencephalic direction, commencing in the distal optic stalk during stage 23 and continuing in subsequent stages. The significance of glioblastic migration is discussed in relation to possible mechanisms through which optic fiber fascicles, initially located on the surface of the stalk, come to lie in deeper areas of the stalk wall.

A neural plate fate map at stage HH4 in the chick: methodology and preliminary data

This paper centers on the design of a perfected methodology for establishing a fate map of the chick neural plate at stages 3d/4, projected upon the closing neural tube (stages 9-11). The principal aim was to saturate the area of interest with overlapping small isochronic and homotopic grafts (100-300 cells), in order to later derive firmer conclusions from the detailed comparisons thus made possible. We used an ocular grid centered on Hensens node for the localization of the grafts. Chick embryos in New culture were used as donors and hosts, to evade potential differences in intercalation or proliferation behavior between quail and chick cells. Donor tissue was labelled with the non-diffusing fluorescein derivative carboxyfluorescein diacetate succinimidyl ester, later visualized by fluorescence microscopy at various timepoints during survival and by a sensitive whole-mount immunocytochemical protocol after fixation. We present only preliminary data of the ongoing mapping, illustrating well-delimited patches of graft-derived cells which can be identified across the neural/non-neural epiblast continuum, or across the dorsoventral dimension of the neural tube wall.