Section of Neurobiology and , 1 Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA
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Abstract |
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Introduction |
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PCD may have several functions during normal development of the mammalian brain. First, spatially regulated cell death before neurogenesis has been postulated to contribute to the complex three-dimensional brain morphogenesis by eliminating intervening cell populations and thereby allowing the associated flexures to oppose or separate populations of cells (Glucksmann, 1951; Kallen, 1955
). In addition, even small amounts of morphogenetic cell death during the early exponential growth phase of the progenitor population in the VZ can drastically effect the eventual size of the neuronal population in the adult cerebrum (Rakic, 1988
). Second, histogenetic cell death, which occurs during and after neurogenesis, may be involved in matching the size of neuronal populations to that of their targets during the formation and maintenance of synapses (Burek and Oppenheim, 1996
; Sherrard and Bower, 1998
). Lastly, in addition to the roles of apoptosis during normal development, dysregulated or pathogenetic cell death may underlie the etiology of congenital diseases. For example, abnormally high rates of apoptosis occur in Down syndrome both during and after forebrain neurogenesis (Wisniewski and Kida, 1994
; Busciglio and Yankner, 1995
). Thus, delineating the signaling pathways and temporal controls of apoptosis during forebrain development can provide insights into the mechanisms of both normal growth and the pathogenesis of congenital brain malformations.
The signaling pathways that control these different types of developmental apoptosis can be separated into extracellular and intracellular components. Although little is known concerning the external molecules which initiate these apoptotic events, they most probably act via contact-mediated or trophic mechanisms mediated by neighboring cells or target cells. Conversely, several of the intracellular regulatory pathways have been elucidated recently, including the c-Jun N-terminal kinase (Jnk) and caspase-mediated apoptotic mechanisms. Here we describe how targeted mutations of these intracellular pathways has revealed the importance of developmental PCD in specifying proper forebrain development.
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The Jnk Pathway Regulates Region-specific Cell Death in the Brain |
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Beginning at embryonic day (E) 10, Jnk1/Jnk2-deficient animals exhibited defective neural tube closure in the hindbrain (Fig. 1A,B). Prior to neural tube closure, there is clustering of apoptotic cells in the lateral edge of the hindbrain neural tube in normal individuals. Through analysis of pyknotic cells in serial semithin sections, we found that this clustering is absent in Jnk1/2 double knockouts at E9.0 (Fig. 1C,D
). Therefore, as postulated over 40 years ago (Glucksmann, 1951
; Kallen, 1955
), the correlation between the highly restricted cell death induced by the Jnk signaling pathway and neural tube closure indicates that this regionally specific PCD is crucial for proper cephalic neurulation.
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Caspases Control Forebrain Size by Regulating Early Progenitor Cell Death |
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If the caspase cascade is solely a general effector mechanism for apoptotic cell death, it is possible that the specific regional and temporal activation of this pathway during development is controlled by upstream signaling pathways. An example of such regulation is provided by the Jnk pathway above. However, multiple signaling mechanisms may use the caspase cascade to shape the forebrain in still other regions and at other ages. To test this possibility, we analyzed forebrain growth in mice lacking Casp-3 and Casp-9.
Both of the targeted caspase mutations prevented cell death in many regions of the developing brain. In areas where apoptotic cells are typically found during development, including the dorsomedial wall of the diencephalon, the optic stalk and the lamina terminalis, a reduction of pyknotic figures was seen in casp-3/ and casp-9/ embryos (Kuida et al., 1996, 1998
). In addition, the few TUNEL+ cells found in the neocortical wall during neurogenesis are also absent when the caspase cascade is disrupted (Kuida et al., 1998
).
casp-3 / and casp-9/ mice first exhibit defective closure of the neural tube at E10.5 similar to that found in the compound Jnk-deficient mice. This is most likely due to the mutation-induced absence of the previously mentioned cluster of apoptotic cells in the hindbrain neural tube. However, in contrast to Jnk1//Jnk2/ individuals, as early as E10.5 there is prominent hyperplasia of the forebrain progenitor population in caspase-deficient animals (Fig. 3A,B). This enlargement of the forebrain progenitor population before the onset of neurogenesis eventually leads to heterotopic neuronal populations and invaginations of the neocortical wall (Figs 3C,D
, 4A,B
) as well as exencephaly of the forebrain during neurogenesis (Fig. 3E,F
).
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Importance of Apoptosis for Function and Cortical Evolution |
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The increased forebrain size of caspase-deficient embryos, due to the abnormal survival of a specific subgroup of forebrain progenitors before neurogenesis, suggests that PCD may be a primary and efficient mechanism for generating the diverse numbers of forebrain cells evident in different species. For example, because of the exponential growth in the progenitor population before the onset of telencephalic neurogenesis, three extra founder cells can generate 192 extra progenitor cells after six cell divisions (Rakic, 1995). These additional progenitors can then significantly increase the number of neurons produced during neurogenesis. We do not claim that PCD is the only, or even the major, factor leading to the increase in cortical size during evolution. However, we suggest that the protection of a small number of specific founder cells may significantly effect the number of neurons generated during later stages of development. In addition, while larger numbers of generated cells may result in cortical convolutions such as those seen in the mutant mice presented here, the precise species-specific pattern of convolution depends also on areal specification and connectivity between the cortex and cortical or subcortical targets (Richman et al., 1975
; Goldman-Rakic and Rakic, 1984
; Rakic, 1988
; Van Essen, 1997
).
It is interesting to note that the increase in founder cells present in caspase-deficient embryos translates to a neocortical plate with larger surface area but with normal thickness. This finding suggests that the glial scaffolding present within the neocortical wall constrains the migration and deployment of these additional postmitotic cells into additional radial units (see Fig. 4CF ) as predicted by the radial unit hypothesis (Rakic, 1988
) rather than expanding the depth of existing units. However, in addition, many presumably later generated cells accumulate below the cortical plate (Fig. 4D
) in a manner similar to the double cortex observed in human and rodent congenital malformations (Gleeson et al., 1998
; Lee et al., 1998
).
In contrast to the significant role of PCD in modulating the number of early progenitors before the onset of neurogenesis, the present results suggest that PCD may not play a major role during neurogenesis. The selective forebrain degeneration evident in Jnk1//Jnk2/ embryos (Fig. 2A,D) indicates that the Jnk pathway normally has a protective effect on neural progenitors while they are producing young neurons. While it is possible that other upstream pathways may be used to regulate apoptosis during forebrain neurogenesis, the small amount of TUNEL and CM-1 staining in normal brains at E11.5 and afterwards suggests that PCD is probably not a major mechanism for controlling diversity while postmitotic neurons are being generated.
Since apoptotic pathways may affect normal forebrain growth by regulating the expansion of the progenitor population, it is also possible that disturbances in normal developmental apoptosis may lead to congenital brain malformations, such as the reduced number of neurons in the Down syndrome forebrain (Sylvester, 1983; Ross et al., 1984
; Wisniewski et al., 1984
). For example, increased caspase-mediated cell death in the progenitor population before neurogenesis could abnormally decrease the eventual cortical neuron population. Similarly, reduced protection of neuronal progenitors during neurogenesis by the Jnk pathway could limit eventual cortical growth. Regardless, fully understanding how apoptosis controls cortical growth in individuals and during evolution requires future work delineating the features which make certain progenitors more susceptible to death than others as well as how the various apoptosis pathways may work together.
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Notes |
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Address correspondence to Tarik F. Haydar, Section of Neurobiology, Yale University School of Medicine, SHM, C-319, 333 Cedar Street, New Haven, CT 06510, USA. Email: thaydar{at}kafka.med.yale.edu.
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