Question

In newly formed neural tube, the intermediate region close to the sulcus limitans, has been not exposed to molecules that induce ventral or dorsal characteristics. You perform explant experiments in which you take intermediate neural tube (host tissue) and culture them with donor tissue explants that induce specific spinal cord markers in the host explant.

1. The induced host tissue express Shh following transplant.

a. What donor tissue have you explanted next to the host tissue, what signal is released to induce this marker, and what cell type has been induced? Briefly explain your reasoning.

b. What other cell types do you expect to observe? Briefly explain your reasoning.

2. The induced host neural tube expresses HB9. What donor tissue have you explanted next to the host tissue, what signal is released to induce this marker, and what cell type has been induced? Explain your reasoning.

3. How does the signal in parts A and B generate neuronal diversity in the ventral spinal cord? Use only 3 sentences.

4. The host animal is transgenically modified so that Shh irreversibly binds to the receptor. What do you expect to observe with the experiment in Part A?

5. The host animal is transgenically modified so that Pax6 and Pax7 do not respond to Shh signaling. What do you expect to observe with the experiment in part a?

Answer 1

The neurons of the brain are organized into layers (cortices) and clusters (nuclei), each having different functions and connections. The original neural tube is composed of a germinal neuroepithelium that is one cell layer thick. This is a layer of rapidly dividing neural stem cells. Sauer (1935) and others have shown that all the cells of the germinal epithelium are continuous from the luminal surface of the neural tube to the outside surface, but that the nuclei of these cells are at different heights, thereby giving the superficial impression that the neural tube has numerous cell layers. The nuclei move within their cells as they go through the cell cycle. DNA synthesis (S phase) occurs while the nucleus is at the outside edge of the neural tube, and the nucleus migrates luminally as the cell cycle proceeds (Figure 12.15). Mitosis occurs on the luminal side of the cell layer. If mammalian neural tube cells are labeled with radioactive thymidine during early development, 100% of them will incorporate this base into their DNA (Fujita 1964). Shortly thereafter, certain cells stop incorporating these DNA precursors, thereby indicating that they are no longer participating in DNA synthesis and mitosis. These neuronal and glial cells then migrate and differentiate outside the neural tube

If dividing cells in the germinal neuroepithelium are labeled with radioactive thymidine at a single point in their development, and their progeny are found in the outer cortex in the adult brain, then those neurons must have migrated to their cortical positions from the germinal neuroepithelium. This happens because a neuroepithelial stem cell divides “vertically” instead of “horizontally.” Thus, the daughter cell adjacent to the lumen remains connected to the ventricular surface (and usually remains a stem cell), while the other daughter cell migrates away (Chenn and McConnell 1995). The time of this vertical division is the last time the latter cell will divide, and is called that neuron's birthday. Different types of neurons and glial cells have their birthdays at different times. Labeling at different times during development shows that cells with the earliest birthdays migrate the shortest distances. The cells with later birthdays migrate through these layers to form the more superficial regions of the cortex. Subsequent differentiation depends on the positions these neurons occupy once outside the germinal neuroepithelium

As the cells adjacent to the lumen continue to divide, the migrating cells form a second layer around the original neural tube. This layer becomes progressively thicker as more cells are added to it from the germinal neuroepithelium. This new layer is called the mantle (or intermediate) zone, and the germinal epithelium is now called the ventricular zone (and, later, the ependyma) (Figure 12.16). The mantle zone cells differentiate into both neurons and glia. The neurons make connections among themselves and send forth axons away from the lumen, thereby creating a cell-poor marginal zone. Eventually, glial cells cover many of the axons in the marginal zone in myelin sheaths, giving them a whitish appearance. Hence, the mantle zone, containing the neuronal cell bodies, is often referred to as the gray matter; the axonal, marginal layer is often called the white matter.

In the brain, cell migration, differential neuronal proliferation, and selective cell death produce modifications of the three-zone pattern (Figure 12.16). In the cerebellum, some neuronal precursors enter the marginal zone to form clusters of neurons called nuclei. Each nucleus works as a functional unit, serving as a relay station between the outer layers of the cerebellum and other parts of the brain. In the cerebellum, some neuronal precursors can also migrate away from the germinal epithelium. These precursor cells, called neuroblasts, migrate to the outer surface of the developing cerebellum and form a new germinal zone, the external granule layer, near the outer boundary of the neural tube. At the outer boundary of the external granule layer (which is one to two cells thick), neuroblasts proliferate. The inner compartment of the external granule layer contains postmitotic neuroblasts that are the precursors of the major neurons of the cerebellar cortex, the granule neurons. These granule neurons migrate back into the developing cerebellar white matter to produce a region called the internal granule layer. Meanwhile, the original ependymal layer of the cerebellum generates a wide variety of neurons and glial cells, including the distinctive and large Purkinje neurons. Purkinje neurons are not only critical in the electrical pathway of the cerebellum, they also support the granule neurons. The Purkinje cell secretes Sonic hedgehog, which sustains the division of granule neuron precursors in the external granule layer (Wallace 1999). Each Purkinje neuron has an enormous dendritic arbor, which spreads like a tree above a bulblike cell body. A typical Purkinje neuron may form as many as 100,000 connections (synapses) with other neurons, more than any other neuron studied. Each Purkinje neuron also emits a slender axon, which connects to neurons in the deep cerebellar nuclei.

The development of spatial organization is critical for the proper functioning of the cerebellum. All electrical impulses eventually regulate the activity of the Purkinje cells, which are the only output neurons of the cerebellar cortex. For this to happen, the proper cells must differentiate at the appropriate place and time. How is this accomplished?

One mechanism thought to be important for positioning young neurons within the developing mammalian brain is glial guidance (Rakic 1972; Hatten 1990). Throughout the cortex, neurons are seen to ride “the glial monorail” to their respective destinations. In the cerebellum, the granule cell precursors travel on the long processes of the Bergmann glia (Figure 12.18; Rakic and Sidman 1973; Rakic 1975). This neural-glial interaction is a complex and fascinating series of events, involving reciprocal recognition between glia and neuroblasts (Hatten 1990; Komuro and Rakic 1992). The neuron maintains its adhesion to the glial cell through a number of proteins, one of them an adhesion protein called astrotactin. If the astrotactin on a neuron is masked by antibodies to that protein, the neuron will fail to adhere to the glial processes

The three-zone arrangement of the neural tube is also modified in the cerebrum. The cerebrum is organized in two distinct ways. First, like the cerebellum, it is organized vertically into layers that interact with one another (see Figure 12.16). Certain neuroblasts from the mantle zone migrate on glial processes through the white matter to generate a second zone of neurons at the outer surface of the brain. This new layer of gray matter is called the neocortex. The neocortex eventually stratifies into six layers of neuronal cell bodies; the adult forms of these layers are not completed until the middle of childhood. Each layer of the neocortex differs from the others in its functional properties, the types of neurons found there, and the sets of connections that they make. For instance, neurons in layer 4 receive their major input from the thalamus (a region that forms from the diencephalon), while neurons in layer 6 send their major output back to the thalamus.

Second, the cerebral cortex is organized horizontally into over 40 regions that regulate anatomically and functionally distinct processes. For instance, neurons in cortical layer 6 of the “visual cortex” project axons to the lateral geniculate nucleus of the thalamus (see Chapter 13), while layer 6 neurons of the auditory cortex (located more anteriorly than the visual cortex) project axons to the medial geniculate nucleus of the thalamus (for hearing).

Neither the vertical nor the horizontal organization of the cerebral cortex is clonally specified. Rather, the developing cortex forms from the mixing of cells derived from numerous stem cells. After their final mitosis, most of the neuronal precursors generated in the ventricular (ependymal) zone migrate outward along glial processes to form the cortical plate at the outer surface of the brain. As in the rest of the brain, those neuronal precursors with the earliest “birthdays” form the layer closest to the ventricle. Subsequent neurons travel greater distances to form the more superficial layers of the cortex. This process forms an “inside-out” gradient of development (Figure 12.19; Rakic 1974). A single stem cell in the ventricular layer can give rise to neurons (and glial cells) in any of the cortical layers (Walsh and Cepko 1988).

Until recently, it had been generally thought that once the nervous system was mature, no new neurons were “born.” The neurons we formed in utero and during the first few years of life were all we could ever expect to have. However, the good news from recent studies is that environmental stimulation can increase the number of new neurons in the mammalian brain (Kemperman et al. 1997a,b; Gould et al. 1999a,b; Praag et al. 1999). To do these experiments, researchers injected adult mice, rats, or marmosets with bromodeoxyuridine (BrdU), a nucleoside that resembles thymidine and which will be incorporated into a cell's DNA only if the cell is undergoing DNA replication. Thus, any cell labeled with BrdU must have been undergoing mitosis during the time when it was exposed to BrdU. This technique showed that thousands of new neurons were being made each day in adult mammals. Injecting humans with BrdU is usually unethical, since large doses of BrdU are often lethal. However, in certain cancer patients, the progress of chemotherapy is monitored by transfusing the patient with a small amount of BrdU. Gage and colleagues (Erikkson et al. 1998) took postmortem samples from the brains of five such patients who had died between 16 and 781 days after the BrdU infusion. In all five subjects, they saw new neurons in the granular cell layer of the hippocampal dentate gyrus (a part of the brain where memories may be formed). The BrdU-labeled cells also stained for neuron-specific markers (Figure 12.21). It appears that the stem cells producing these neurons are located in the ependyma, the former ventricular layer in which the embryonic neural stem cells once resided (Doetsch et al. 1999; Johansson et al. 1999). These results are surprising, since the ependyma consists of differentiated glial cells whose ciliated surface keeps the cerebral spinal fluid flowing. Indeed, one author (Barres 1999) described them as “the most boring of all glial subtypes.” It appears that these glial cells (or perhaps only some of them) can dedifferentiate and become neural stem cells. Thus, although the rate of new neuron formation in adulthood may be relatively small, the human brain is not an anatomical fait accompli at birth, or even after childhood

Not all neurons, however, migrate radially. O'Rourke and her colleagues (1992) labeled young ferret neurons with fluorescent dye and followed their migration through the brain. While a great majority of the young neurons migrated radially on glial processes from the ventricular zone into the cortical plate, about 12% of them migrated laterally from one functional region of the cerebral cortex into another. These observations meshed well with those of Walsh and Cepko (1992), who infected ventricular stem cells with a retrovirus and were able to stain these cells and their progeny after birth. They found that the neural descendants of a single ventricular stem cell were dispersed across the functional regions of the cortex. Thus, the specification of the cortical areas into specific functional domains occurs after neurogenesis. Once the cells arrive at their final destination, it is thought that they produce particular adhesion molecules that organize them together as brain nuclei (Matsunami and Takeichi 1995).

The cerebrum is quite plastic. The development of the human neocortex is particularly striking in this regard. The human brain continues to develop at fetal rates even after birth (Holt et al. 1975). Based on morphological and behavioral criteria and on comparisons with other primates, Portmann (1941,1945) suggested that human gestation should really last 21 months instead of 9. However, no woman could deliver a 21-month-old fetus because the head would not pass through the birth canal; thus, humans give birth at the end of 9 months. Montagu (1962) and Gould (1977) have suggested that during our first year of life, we are essentially extrauterine fetuses, and they speculate that much of human intelligence comes from the stimulation of the nervous system as it is forming during that first year