Jacqueline E. Lee

Basic helix-loop-helix genes in neural development.

Several major advances in the understanding of the regulation of vertebrate neurogenesis by members of the basic helix-loop-helix (bHLH) protein family have been made in the past year. Specifically, a number of bHLH genes have been cloned and shown to convert non-neuronal fate to neuronal fate when expressed ectopically. In particular, studies on NeuroD and Neurogenin suggest a regulatory pathway, providing powerful molecular tools to study vertebrate neurogenesis.

Smaller inner ear sensory epithelia in Neurog1 null mice are related to earlier hair cell cycle exit

We investigated whether co‐expression of Neurog1 and Atoh1 in common neurosensory precursors could explain the loss of hair cells in Neurog1 null mice. Analysis of terminal mitosis, using BrdU, supports previous findings regarding timing of exit from cell cycle. Specifically, we show that cell cycle exit occurs in spiral sensory neurons in a base‐to‐apex progression followed by cell cycle exit of hair cells in the organ of Corti in an apex‐to‐base progression, with some overlap of cell cycle exit in the apex for both hair cells and spiral sensory neurons. Hair cells in Neurog1 null mice show cell cycle exit in an apex‐to‐base progression about 1–2 days earlier. Atoh1 is expressed in an apex‐to‐base progression rather then a base‐to‐apex progression as in wildtype littermates. We tested the possible expression of Atoh1 in neurosensory precursors using two Atoh1‐Cre lines. We show Atoh1‐Cre mediated β‐galactosidase expression in delaminating sensory neuron precursors as well as undifferentiated epithelial cells at E11 and E12.5. PCR analysis shows expression of Atoh1 in the otocyst as early as E10.5, prior to any histology‐based detection techniques. Combined, these data suggest that low levels of Atoh1 exist much earlier in precursors of hair cells and sensory neurons, possibly including neurosensory precursors. Analysis of Atoh1‐Cre expression in E18.5 embryos and P31 mice reveal β‐galactosidase stain in all hair cells but also in vestibular and cochlear sensory neurons and some supporting cells. A similar expression of Atoh1‐LacZ exists in postnatal and adult vestibular and cochlear sensory neurons, and Atoh1 expression in vestibular sensory neurons is confirmed with RT‐PCR. We propose that the absence of NEUROG1 protein leads to loss of sensory neuron formation through a phenotypic switch of cycling neurosensory precursors from sensory neuron to hair cell fate. Neurog1 null mice show a truncation of clonal expansion of hair cell precursors through temporally altered terminal mitosis, thereby resulting in smaller sensory epithelia. Developmental Dynamics 234:633–650, 2005.

IA1 is NGN3-dependent and essential for differentiation of the endocrine pancreas

Neurogenin 3 (Ngn3) is key for endocrine cell specification in the embryonic pancreas and induction of a neuroendocrine cell differentiation program by misexpression in adult pancreatic duct cells. We identify the gene encoding IA1, a zinc‐finger transcription factor, as a direct target of Ngn3 and show that it forms a novel branch in the Ngn3‐dependent endocrinogenic transcription factor network. During embryonic development of the pancreas, IA1 and Ngn3 exhibit nearly identical spatio‐temporal expression patterns. However, embryos lacking Ngn3 fail to express IA1 in the pancreas. Upon ectopic expression in adult pancreatic duct cells Ngn3 binds to chromatin in the IA1 promoter region and activates transcription. Consistent with this direct effect, IA1 expression is normal in embryos mutant for NeuroD1, Arx, Pax4 and Pax6, regulators operating downstream of Ngn3. IA1 is an effector of Ngn3 function as inhibition of IA1 expression in embryonic pancreas decreases the formation of insulin‐ and glucagon‐positive cells by 40%, while its ectopic expression amplifies neuroendocrine cell differentiation by Ngn3 in adult duct cells. IA1 is therefore a novel Ngn3‐regulated factor required for normal differentiation of pancreatic endocrine cells.

Pancreatic β Cells Require NeuroD to Achieve and Maintain Functional Maturity

NeuroD, a transactivator of the insulin gene, is critical for development of the endocrine pancreas, and NeuroD mutations cause MODY6 in humans. To investigate the role of NeuroD in differentiated beta cells, we generated mice in which neuroD is deleted in insulin-expressing cells. These mice exhibit severe glucose intolerance. Islets lacking NeuroD respond poorly to glucose and display a glucose metabolic profile similar to immature beta cells, featuring increased expression of glycolytic genes and LDHA, elevated basal insulin secretion and O2 consumption, and overexpression of NPY. Moreover, the mutant islets appear to have defective K(ATP) channel-mediated insulin secretion. Unexpectedly, virtually all insulin in the mutant mice is derived from ins2, whereas ins1 expression is almost extinguished. Overall, these results indicate that NeuroD is required for beta cell maturation and demonstrate the importance of NeuroD in the acquisition and maintenance of fully functional glucose-responsive beta cells.

Requirement of Multiple Basic Helix-Loop-Helix Genes for Retinal Neuronal Subtype Specification

Retinal precursor cells give rise to six types of neurons and one type of glial cell during development, and this process is controlled by multiple basic helix-loop-helix (bHLH) genes. However, the precise mechanism for specification of retinal neuronal subtypes, particularly horizontal neurons and photoreceptors, remains to be determined. Here, we examined retinas with three different combinations of triple bHLH gene mutations. In retinas lacking the bHLH genes Ngn2, Math3, and NeuroD, horizontal neurons as well as other neurons such as bipolar cells were severely decreased in number. In the retina lacking the bHLH genes Mash1, Ngn2, and Math3, horizontal and other neurons were severely decreased, whereas ganglion cells were increased. In the retina lacking the bHLH genes Mash1, Math3, and NeuroD, photoreceptors were severely decreased, whereas ganglion cells were increased. In all cases, glial cells were increased. The increase and decrease of these cells were the result of cell fate changes and cell death and seem to be partly attributable to the remaining bHLH gene expression, which also changes because of triple bHLH gene mutations. These results indicate that multiple bHLH genes cross-regulate each other, cooperatively specify neuronal subtypes, and regulate neuronal survival in the developing retina.

Dynamic regulation of the proinflammatory cytokine, interleukin-1β: Molecular biology for non-molecular biologists

Interleukin-1beta (IL-1beta) is a key mediator and modulator of a wide array of physiological responses important for survival. It is created by a variety of cell types, including immune cells, glia, and neurons. It is a very potent biological molecule, acting both at the periphery as well as within the central nervous system. The production and release of IL-1beta is tightly regulated by far more complex processes than previously thought. An appreciation of this complexity is necessary for proper interpretation of apparent contradictions in the literature where different aspects of IL-1beta expression are measured. Given that many researchers are not molecular biologists by training, yet need an appreciation of the controls that regulate the function of key proteins such as IL-1beta, this review is aimed at both: (a) clarifying the multiple levels at which IL-1beta production is modulated and (b) using IL-1beta regulation to explain the dynamics of gene regulation to non-molecular biologists. Three major topics will be discussed. First, regulation of IL-1beta production will be examined at every level from extracellular signals that trigger gene activation through release of active protein into the extracellular fluid. Second, regulation of IL-1beta bioavailability and bioactivity will be discussed. This section examines the fact that even after IL-1beta is released, it may or may not be able to exert a biological action due to multiple modulatory factors. Last is the introduction of the idea that IL-1beta regulation is, at times, beyond the direct control of host; that is, when IL-1beta production becomes dysregulated by pathogens.

The NeuroD1/BETA2 Sequences Essential for Insulin Gene Transcription Colocalize with Those Necessary for Neurogenesis and p300/CREB Binding Protein Binding

ABSTRACT NeuroD1/BETA2 is a key regulator of pancreatic islet morphogenesis and insulin hormone gene transcription in islet β cells. This factor also appears to be involved in neurogenic differentiation, because NeuroD1/BETA2 is able to induce premature differentiation of neuronal precursors and convert ectoderm into fully differentiated neurons upon ectopic expression in Xenopus embryos. We have identified amino acid sequences in mammalian and Xenopus NeuroD1/BETA2 that are necessary for insulin gene expression and ectopic neurogenesis. Our results indicate that evolutionarily conserved sequences spanning the basic helix-loop-helix (amino acids [aa] 100 to 155) and C-terminal (aa 156 to 355) regions are important for both of these processes. The transactivation domains (AD1, aa 189 to 299; AD2, aa 300 to 355) were within the carboxy-terminal region, as analyzed by using GAL4:NeuroD1/BETA2 chimeras. Selective activation of mammalian insulin gene enhancer-driven expression and ectopic neurogenesis in Xenopus embryos was regulated by two independent and separable domains of NeuroD1/BETA2, located between aa 156 to 251 and aa 252 to 355. GAL4:NeuroD1/BETA2 constructs spanning these sequences demonstrated that only aa 252 to 355 contained activation domain function, although both aa 156 to 251 and 300 to 355 were found to interact with the p300/CREB binding protein (CBP) coactivator. These results implicate p300/CBP in NeuroD1/BETA2 function and further suggest that comparable mechanisms are utilized to direct target gene transcription during differentiation and in adult islet β cells.

Stimulation of NeuroD activity by huntingtin and huntingtin-associated proteins HAP1 and MLK2

NeuroD (ND) is a basic helix–loop–helix transcription factor important for neuronal development and survival. By using a yeast two-hybrid screen, we identified two proteins that interact with ND, huntingtin-associated protein 1 (HAP1) and mixed-lineage kinase 2 (MLK2), both of which are known to interact with huntingtin (Htt). Htt is a ubiquitous protein important for neuronal transcription, development, and survival, and loss of its function has been implicated in the pathogenesis of Huntingtons disease, a neurodegenerative disorder. However, the mechanism by which Htt exerts its neuron-specific function at the molecular level is unknown. Here we report that Htt interacts with ND via HAP1, and that MLK2 phosphorylates and stimulates the activity of ND. Furthermore, we show that Htt and HAP1 facilitate the activation of ND by MLK2. To our knowledge, ND is the first example of a neuron-specific transcription factor involved in neuronal development and survival whose activity is modulated by Htt. We propose that Htt, together with HAP1, may function as a scaffold for the activation of ND by MLK2.

NEUROD AND NEUROGENESIS

NeuroD is a basic helix-loop-helix (bHLH) transcription factor cloned from a two hybrid screen designed to search for new bHLH proteins. In our previous studies, we showed that NeuroD could convert Xenopus ectoderm into fully differentiated neurons and that it could prematurely differentiate neural precursor cells in the nervous system. Recently, an insulin transcription activator, Beta-2, was cloned from a hamster insulinoma cell line by Naya et al. [Genes Dev (1995)9:1,009-1,019]. Sequence analysis revealed that Beta-2 is the hamster homologue of NeuroD. We are currently investigating the role that NeuroD/Beta-2 plays in vertebrate neurogenesis and pancreatic development.

Dynamic Regulation of Proinflammatory Cytokines

Proinflammatory cytokines, including interleukin-1beta (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α), are proteins created and released by a wide variety of immune cells in response to infection, inflammation, and tissue damage. These proteins have long been recognized as critical communication factors for orchestrating early responses of immune cells to such immune challenges. More recently, it has become accepted that these same proinflammatory cytokines are key mediators of immune-to-brain communication as well. In this role, proinflammatory cytokines communicate to the brain, thereby orchestrating sickness responses including fever, decreased food, and water intake, increased sleep, increased pain responsivity, and so forth (Kent, Bluthe, Kelley, & Dantzer, 1992; Maier & Watkins, 1998). Thus, it is clear that proinflammatory cytokines are importantly involved in the response of both the immune system and the central nervous system in these situations.