Neurogenetic Heterochrony in Chick, Lizard, and Rat Mapped with Wholemount Acetylcholinesterase and the Prosomeric Model.

In the developing brain, the phenomenon of neurogenesis is manifested heterotopically, that is, much the same neurogenetic steps occur at different places with a different timetable. This is due apparently to early molecular regionalization of the neural tube wall in the anteroposterior and dorsoventral dimensions, in a checkerboard pattern of more or less deformed quadrangular histogenetic areas. Their respective fate is apparently specified by a locally specific combination of active/repressed genes known as "molecular profile." This leads to position-dependent differential control of proliferation, neurogenesis, differentiation, and other aspects, eventually in a heterochronic manner across adjacent areal units with sufficiently different molecular profiles. It is not known how fixed these heterochronic patterns are. We reexamined here comparatively early patterns of forebrain and hindbrain neurogenesis in a lizard (Lacerta gallotia galloti), a bird (the chick), and a mammal (the rat), as demonstrated by activation of acetylcholinesterase (AChE). This is an early marker of postmitotic neurons, which leaves unlabeled the neuroepithelial ventricular cells, so that we can examine cleared wholemounts of the reacted brains to have a birds-eye view of the emergent neuronal pattern at each stage. There is overall heterochrony between the basal and alar plates of the brain, a known fact, but, remarkably, heterochrony occurs even within the precocious basal plate among its final anteroposterior neuromeric subdivisions and their internal microzonal subdivisions. Some neuromeric units or microzones are precocious, while others follow suit without any specific spatial order or gradient; other similar neuromeric units remain retarded in the midst of quite advanced neighbors, though they do produce similar neurogenetic patterns at later stages. It was found that some details of such neuromeric heterochrony are species-specific, possibly related to differential morphogenetic properties. Given the molecular causal underpinning of the updated prosomeric model used here for interpretation, we comment on the close correlation between some genetic patterns and the observed AChE differentiation patterns.

Quail-chick grafting experiments corroborate that Tbr1-positive eminential prethalamic neurons migrate along three streams into hypothalamus, subpallium and septocommissural areas.

The prethalamic eminence (PThE), a diencephalic caudal neighbor of the telencephalon and alar hypothalamus, is frequently described in mammals and birds as a transient embryonic structure, undetectable in the adult brain. Based on descriptive developmental analysis of Tbr1 gene brain expression in chick embryos, we previously reported that three migratory cellular streams exit the PThE rostralward, targeting multiple sites in the hypothalamus, subpallium and septocommissural area, where eminential cells form distinct nuclei or disperse populations. These conclusions needed experimental corroboration. In this work, we used the homotopic quail-chick chimeric grafting procedure at stages HH10/HH11 to demonstrate by fate-mapping the three predicted tangential migration streams. Some chimeric brains were processed for Tbr1 in situ hybridization, for correlation with our previous approach. Evidence supporting all three postulated migration streams is presented. The results suggested a slight heterochrony among the juxtapeduncular (first), the peripeduncular (next), and the eminentio-septal (last) streams, each of which followed differential routes. A possible effect of such heterochrony on the differential selection of medial to lateral habenular hodologic targets by the migrated neurons is discussed.

Longitudinal developmental analysis of prethalamic eminence derivatives in the chick by mapping of Tbr1 in situ expression.

The prethalamic eminence (PThE) is the most dorsal subdomain of the prethalamus, which corresponds to prosomere 3 (p3) in the prosomeric model for vertebrate forebrain development. In mammalian and avian embryos, the PThE can be delimited from other prethalamic areas by its lack of Dlx gene expression, as well as by its expression of glutamatergic-related genes such as Pax6, Tbr2 and Tbr1. Several studies in mouse embryos postulate the PThE as a source of migratory neurons that populate given telencephalic centers. Concerning the avian PThE, it is visible at early embryonic stages as a compact primordium, but its morphology becomes cryptic at perinatal stages, so that its developmental course and fate are largely unknown. In this report, we characterize in detail the ontogeny of the chicken PThE from 5 to 15 days of development, according to morphological criteria, and using Tbr1 as a molecular marker for this structure and its migratory cells. We show that initially the PThE contacts rostrally the medial pallium, the pallial amygdala and the paraventricular hypothalamic alar domain. Approximately from embryonic day 6 onwards, the PThE becomes progressively reduced in size and cell content due to massive tangential migration of many of its neuronal derivatives towards nearby subpallial and hypothalamic regions. Our analysis supports that these migratory neurons from the avian PThE target telencephalic centers such as the commissural septal nuclei, as previously described in mammals, but also the diagonal band and preoptic areas, and hypothalamic structures in the paraventricular hypothalamic area.

The rostral and caudal boundaries of the diencephalon.

Knowledge of nature and features of the boundaries between the main neural regions seems to be essential to understand the rules of brain regionalization. On the light of several current and classical criteria used to define cerebral boundaries, we examine the features of the places recognized as rostral and caudal boundaries in the developing diencephalon and provide new images about the glial features of these boundaries. One demonstrated property of some embryonic boundaries is the prevention of the crossing cells in the early ventricular zone (clonal restriction), while the intermediate zone seems to lack it. Data available so far indicate that the early boundary between diencephalon and mesencephalon (d/m) is a clonal restriction limit, but not between diencephalon and telencephalon (d/t). Later, while diencephalic nuclei form, cellular dispersion does not occur through the alar part of d/m, but it achieves in the corresponding d/t alar portion. The relationship between origin, migration, and cell-type specification of neural cells is being the object of special attention in the telencephalon, where specific cellular fenotipes can migrate to distant regions following non-radial routes. Such is the case of most GABAergic interneurons of avian and mammalian pallium and oligodendrocytes of the forebrain. In this regard, little attention has been devoted to the diencephalon, where this type of migration, specially those through the rostral boundary, has been reported by different authors. We introduce increasing evidence about non-conventional neuronal migration in the developing diencephalon and compare the reported migratory behavior with respect to both boundaries.

Neuronal typology of the thalamic area triangularis of Gallotia galloti (reptilia, sauria).

In a Golgi study of the area triangularis (AT), a rostral nucleus of the ventral thalamus of Gallotia galloti, we have identified four major neuronal types on the basis of their morphological characteristics: medium-sized fusiforms with two processes, medium-sized fusiforms with three or four processes, small bipolars, and small and medium-sized multipolars. These neurons are characterized by a simple morphology and radial arrangement. Cell size varies from small to medium, and all axons project laterally. These characteristics distinguish AT neurons from those of neighboring nuclei. In addition, we found some evidence of differential topographic distribution of each neuronal type within the nucleus. Medium-sized fusiform neurons with two processes are located in the most ventral part, where they constitute the ventral nuclear limit. Small multipolar neurons prevail in the dorsal and ventromedial parts, and in the rest of the nucleus medium-sized neurons, including both fusiform with three or four processes and multipolar types, are normally found. Finally, we discuss a putative homology of the reptilian AT with a part of the mammalian zona incerta.

Neuronal differentiation in the thalamic area triangularis of a lizard.

Development of neurons in the area triangularis of Gallotia galloti was investigated in Golgi-impregnated brain tissue. Four major neuronal types present in adults were found to originate from two migratory neuroblast types, which were followed from embryonic stage S.32. One type has a thick main medial process, whereas the second type has a long main lateral process. As they migrate toward the periphery of the nucleus, morphological characteristics of maturation appear, including growth cones, filopodia, and outgrowth of axons. Neuroblasts with a main lateral process differentiate into two immature neuronal types, bipolars and pyramidals, observed at S.33 and thereafter. The neuroblasts with a main medial process undergo some somatic translocation through a transitory tangential shaft. Then they develop into monopolar immature forms with a long varicose medial, process, appearing from S.36. onward. Immature bipolar neurons do not experience great changes in their dendritic arborization during development to the adult stage, but pyramidals and monopolars undergo a rapid development of the dendritic tree after S.36. By S.38 archetypes of adult neuronal forms are established. Hairlike appendages first appear on neurons at S.36 They decrease suddenly in S.38 and then proliferate in S.39 when spines first appear. Around the time of hatching, the hairlike appendages begin to disappear and spines become established. Reduction of spines occurs after hatching and continues to the adult stage. Possible influences of several external factors on neuronal maturation are discussed.