Induction, neurogenesis, and synaptogenesis of the olfactory bulb are thought to require interactions with the olfactory epithelium. The Dlx family of homeobox genes is expressed in both the olfactory bulb and olfactory epithelium. In particular, Dlx5 is expressed in the olfactory placode, olfactory epithelium, and local circuit neurons of the olfactory bulb. Here we analyzed mice lacking DLX5 function. The Dlx5-/- mutation reduces the size of the olfactory epithelium. Although some olfactory neurons are formed, they fail to generate olfactory axons that innervate the olfactory bulb. Despite the lack of innervation, the olfactory bulb forms, and neurogenesis of projection and local circuit neurons proceeds. However, the mutation has a cell-autonomous effect on the ability of neural progenitors to produce olfactory bulb local circuit neurons, with granule cells more severely affected than periglomerular cells. In addition, the mutation has a noncell-autonomous effect on the morphogenesis of mitral cells.

GnRH neurons must undergo a complex and precise pattern of neuronal migration to appropriately target their projections to the median eminence to trigger gonadotropin secretion and thereby control reproduction. Using NLT GnRH cells as a model of early GnRH neuronal development, we identified the potential importance of Axl and Tyro3, members of the TAM (Tyro3, Axl, and Mer) family of receptor tyrosine kinases in GnRH neuronal cell survival and migration. Silencing studies evaluated the role of Tyro3 and Axl in NLT GnRH neuronal cells and suggest that both play a role in Gas6 stimulation of GnRH neuronal survival and migration. Analysis of mice null for both Axl and Tyro3 showed normal onset of vaginal opening but delayed first estrus and persistently abnormal estrous cyclicity compared with wild-type controls. Analysis of GnRH neuronal numbers and positioning in the adult revealed a total loss of 24% of the neuronal network that was more striking (34%) when considered within specific anatomical compartments, with the largest deficit surrounding the organum vasculosum of the lamina terminalis. Analysis of GnRH neurons during embryogenesis identified a striking loss of immunoreactive cells within the context of the ventral forebrain compartment (36%) and not more rostrally. Studies using caspase 3 cleavage as a marker of apoptosis showed that Axl(-/-), Tyro3(-/-) double-knockout mice had increased cell death in the nose and dorsal forebrain, supporting the underlying mechanism of cell loss. Together these data suggest that Axl and Tyro3 mediate the survival and appropriate targeting of GnRH neurons to the ventral forebrain, thereby contributing to normal reproductive function and cyclicity in the female.

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GnRH neurons follow a carefully orchestrated journey from their birth in the olfactory placode area. Initially, they migrate along with the vomeronasal nerve into the brain at the cribriform plate, then progress caudally to sites within the hypothalamus where they halt and send projections to the median eminence to activate pituitary gonadotropes. Many factors controlling this precise journey have been elucidated by the silencing or over expression of candidate genes in mouse models. Importantly, a number of these factors may not only play a role in normal physiology of the hypothalamic-pituitary-gonadal axis but also be mis-expressed to cause human disorders of GnRH deficiency, presenting as a failure to undergo normal pubertal development. This review outlines the current cadre of candidates thought to modulate GnRH neuronal migration. The further elucidation and characterization of these factors that impact GnRH neuron development may shed new light on human reproductive disorders and provide potential targets to develop new profertility or contraceptive agents. IntroductionGonadotropin-releasing hormone (GnRH) is a hypothalamic releasing hormone that is synthesized in a small heterogenous neuronal population and secreted in an episodic fashion to control pituitary gonadotropin production and normal reproductive function(1-5). The GnRH neurons are unique among hypothalamic releasing factor neurons in that they originate in the olfactory placode/vomeronasal organ and migrate along vomeronasal nerves to the cribriform plate, the boundary between the peripheral olfactory system and the forebrain(1-5). Migrating GnRH neurons follow a branch of the vomeronasal nerve caudally into the hypothalamus(4). They then extend processes to the median eminence to release GnRH into the capillaries of the median eminence to modulate pituitary gonadotropin (luteinizing hormone, LH and follicle stimulating hormone, FSH) production and secretion. Loss of these neurons or misdirecting along the route results in failure of sexual maturation in mice and man. The underlying mechanisms that regulate the migration of GnRH neurons are incompletely understood. Recent studies have identified multiple factors extrinsic and Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptFront Neuroendocrinol. Author manuscript; available in PMC 2012 January 1. intrinsic to GnRH neurons that control specific steps along the migratory route (discussed in (6-9)). This review updates the list of candidate proteins and pathways involved in GnRH neuronal migration and highlights...

Neurons that synthesize GnRH are critical brain regulators of the reproductive axis, yet they originate outside the brain and must migrate over long distances and varied environments to get to their appropriate positions during development. Many studies, past and present, are providing clues for the types of molecules encountered and movements expected along the migratory route. Recent studies provide real-time views of the behavior of GnRH neurons in the context of in vitro preparations that model those in vivo. Live images provide direct evidence of the changing behavior of GnRH neurons in their different environments, showing that GnRH neurons move with greater frequency and with more alterations in direction after they enter the brain. The heterogeneity of molecular phenotypes for GnRH neurons likely ensures that multiple external factors will be found that regulate the migration of different portions of the GnRH neuronal population at different steps along the route. Molecules distributed in gradients both in the peripheral olfactory system and basal forebrain may be particularly influential in directing the appropriate movement of GnRH neurons along their arduous migration. Molecules that mediate the adhesion of GnRH neurons to changing surfaces may also play critical roles. It is likely that the multiple external factors converge on selective signal transduction pathways to engage the mechanical mechanisms needed to modulate GnRH neuronal movement and ultimately migration.

The ventromedial nucleus of the hypothalamus (VMH) in mice first emerges as a histologically distinct cell cluster around embryonic day 17 (E17). The earliest known marker for cells destined to form the VMH is the orphan nuclear receptor, steroidogenic factor 1 (SF-1), which can be detected in the hypothalamic primordium by E11. Strikingly, the VMH is absent in newborn SF-1 knockout mice, suggesting that SF-1 is essential for the development of VMH neurons. We reported previously that the VMH can be identified before it emerges as a histologically distinct nucleus (i.e., at E13) by the exclusion of cells that are immunoreactive for both gamma-aminobutyric acid (GABA) and the synthetic enzyme, glutamic acid decarboxylase (GAD67). Subsequently, by E15, the developing VMH is demarcated further by cells that are immunoreactive for neuropeptide Y, estrogen receptor alpha (ERalpha), and galanin. It is noteworthy that the normal exclusion of GABA from the developing VMH is not seen in SF-1 knockout mice, and cells that are immunoreactive for neuropeptide Y, ERalpha, and galanin also are distributed aberrantly in this region. Thus, the absence of SF-1 profoundly affects the cellular architecture of the VMH from early stages in its formation. These data suggest that, directly or indirectly, SF-1 plays important roles in determining the distribution of cells in the mediobasal hypothalamus.

Throughout the hypothalamus there are several regions known to contain sex differences in specific cellular, neurochemical, or cell grouping characteristics. The current study examined the potential origin of sex differences in calbindin expression in the preoptic area and hypothalamus as related to sources of nitric oxide. Specific cell populations were defined by immunoreactive (ir) calbindin and neuronal nitric oxide synthase (nNOS) in the preoptic area/anterior hypothalamus (POA/AH), anteroventral periventricular nucleus (AVPv), and ventromedial nucleus of the hypothalamus (VMN). The POA/AH of adult mice was characterized by a striking sex difference in the distribution of cells with ir-calbindin. Examination of the POA/AH of androgen receptor deficient Tfm mice suggests that this pattern was in part androgen receptor dependent, since Tfm males had reduced ir-calbindin compared with wild-type males and more similar to wild-type females. At P0 ir-calbindin was more prevalent than in adulthood, with males having significantly more ir-calbindin and nNOS than have females. Cells that contained either ir-calbindin or ir-nNOS in the POA/AH were in adjacent cell groups, suggesting that NO derived from the enzymatic activity of nNOS may influence the development of ir-calbindin cells. In the region of AVPv, at P0, there was a sex difference with males having more ir-nNOS fibers than have females while ir-calbindin was not detected. In the VMN, at P0, ir-nNOS was greater in females than in males, with no significant difference in ir-calbindin. We suggest that NO as an effector molecule and calbindin as a molecular biomarker illuminate key aspects of sexual differentiation in the developing mouse brain.

There is increasing evidence regarding the importance of the hypothalamus for understanding sex differences in relation to neurological, psychiatric, endocrine and sleep disorders. Although different in histology, physiology, connections and function, multiple hypothalamic nuclei subserve non-voluntary functions and are nodal points for the purpose of maintaining homeostasis of the organism. Thus, given the critical importance of hypothalamic nuclei and their key multiple roles in regulating basic functions, it is important to develop the ability to conduct in vivo human studies of anatomic structure, volume, connectivity, and function of hypothalamic regions represented at the level of its nuclei. The goals of the present study were to develop a novel method of semi-automated volumetric parcellation for the human hypothalamus that could be used to investigate clinical conditions using MRI and to demonstrate its applicability. The proposed new method subdivides the hypothalamus into five parcels based on visible anatomic landmarks associated with specific nuclear groupings and was confirmed using two ex vivo hypothalami that were imaged in a 7 Tesla (7T) scanner and processed histologically. Imaging results were compared with histology from the same brain. Further, the method was applied to 44 healthy adults (26 men; 18 women, comparable on age, handedness, ethnicity, SES) to derive normative volumes and assess sex differences in hypothalamic regions using 1.5 Tesla MRI. Men compared to women had a significantly larger total hypothalamus, relative to cerebrum size, similar for both hemispheres, a difference that was primarily driven by the tuberal region, with the sex effect size being largest in the superior tuberal region and, to a lesser extent, inferior tuberal region. Given the critical role of hypothalamic nuclei in multiple chronic diseases and the importance of sex differences, we argue that the use of the novel methodology presented here will allow for critical investigations of these disorders and further delineation of potential treatments, particularly sex-specific approaches to gene and drug discoveries that involve hypothalamic nuclei.