Chemo- and optogenetic activation of hypothalamic Foxb1-expressing neurons and their terminal endings in the rostral-dorsolateral PAG leads to tachypnea, bradycardia, and immobility.

Foxb1 -expressing neurons occur in the dorsal premammillary nucleus (PMd) and further rostrally in the parvafox nucleus, a longitudinal cluster of neurons in the lateral hypothalamus of rodents. The descending projection of these Foxb1+ neurons end in the dorsolateral part of the periaqueductal gray (dlPAG). The functional role of the Foxb1+ neuronal subpopulation in the PMd and the parvafox nucleus remains elusive. In this study, the activity of the Foxb1+ neurons and of their terminal endings in the dlPAG in mice was selectively altered by employing chemo- and optogenetic tools. Our results show that in whole-body barometric plethysmography, hM3Dq-mediated, global Foxb1+ neuron excitation activates respiration. Time-resolved optogenetic gain-of-function manipulation of the terminal endings of Foxb1+ neurons in the rostral third of the dlPAG leads to abrupt immobility and bradycardia. Chemogenetic activation of Foxb1+ cell bodies and ChR2-mediated excitation of their axonal endings in the dlPAG led to a phenotypical presentation congruent with a 'freezing-like' situation during innate defensive behavior.

The distribution of Dlx1-2 and glutamic acid decarboxylase in the embryonic and adult hypothalamus reveals three differentiated LHA subdivisions in rodents.

The lateral hypothalamus (LHA) is still a poorly understood brain region. Based on published Dlx and Gad gene expression patterns in the embryonic and adult hypothalamus respectively, three large areas are identified in the LHA. A central tuberal LHA region is already well described as it contains neurons producing the peptides melanin-concentrating hormone or hypocretin. This region is rich in GABAergic neurons and is specified by Dlx gene expression in the rodent embryo. Rostrally and caudally bordering the tuberal LHA, two Dlx-GAD-GABA poor regions are then easily delineated. The three regions show different organizational schema. The tuberal region is reticularly organized, connected with the cerebral cortex and the spinal cord, and its embryonic development occurs along the tractus postopticus. The region anterior to it is associated with the stria medullaris in both embryonic and adult subjects. The posterior LHA region is made of differentiated nuclei and includes the subthalamic nucleus. Therefore, the LHA is divided into three distinct parts: in addition to the well-known tuberal LHA, caudal and anterior LHA regions exist that have specific anatomical and functional characteristics. The hypothalamus is made up of several dozens of nuclei or areas that are more or less well differentiated and whose boundaries and arrangements are drawn differently according to authors and atlases (Allen Institute, 2004; Paxinos and Franklin, 2019; Paxinos and Watson, 2013; Swanson, 2004). The dominant hypothesis for more than 50 years is that these structures are distributed within three antero-posterior areas (anterior, tuberal, posterior) and more or less three longitudinal zones (lateral, medial and periventricular) (Fig. 1). In addition to these regions, several adjacent territories are often associated to the hypothalamus. The preoptic area is functionally related to the hypothalamus, but it is better seen as a telencephalic structure based on developmental data (Croizier et al., 2015; Puelles and Rubenstein, 2015). Lately, the zona incerta and the subthalamic nucleus (STN) have also been associated to the hypothalamus on the basis of their connections and development for the STN (Altman and Bayer, 1986; Barbier and Risold, 2021; Swaab et al., 2021). However, the zona incerta is still included in the 'pre-thalamus' or "ventral thalamus" in the embryo (Puelles and Rubenstein, 2015). Thus, the boundaries of the hypothalamus remain blurred around what we can call a 'core' made of the anterior to posterior regions (Brooks, 1988). In addition, unlike other large brain regions that are characterized early on by a molecular signature, i.e. by the embryonic expression of specific molecular markers, data illustrating the distribution of dozens of transcription factors involved in brain patterning and cell lineage specification confirmed the extremely heterogeneous and mosaic nature of the anterior and posterior regions of the hypothalamus (Alvarez-Bolado, 2019; Puelles et al., 2013; Puelles and Rubenstein, 2015). The rich nuclear organization of the medial and periventricular zones of the hypothalamus is consistent with the mosaic expression of developmental genes. The LHA, however, is often perceived as much more homogeneous in its cytoarchitectural organization. At the same time, there is little information regarding the expression of developmental genes in the anterior and posterior territories of the LHA. Most studies focus on the tuberal LHA which expresses many of these genes. Admittedly, even in the adult hypothalamus, the internal boundaries of the LHA are difficult to identify and the same is true in the embryo. Developmental data alone are insufficient to achieve a better understanding of the LHA anatomical organization and for this region as for medial and periventricular zones, a coherence must be established between development and adult anatomical organization. Among the most useful neurochemical markers to identify large regions of the forebrain, those involved in the identification of GABAergic and glutamatergic neurons have proven to be particularly efficient. Indeed, GABAergic neurons are not ubiquitously distributed. Large regions of the forebrain are rich in such cells, including the basal telencephalon, but others contain few or no GABAergic cells and are rich in glutamatergic neurons instead (for example the dorsal thalamus that is free of GABA-neurons in rodents). The same applies for the hypothalamus: several structures of the hypothalamus are free of GABAergic neurons, as, for example, the mammillary nuclei (Hahn et al., 2019). Recently, we also identified a GABA-poor posterior LHA territory that includes the (STN), and is localized caudal to the GABA-rich tuberal LHA (Barbier et al., 2020; Barbier and Risold, 2021; Chometton et al., 2016b). Therefore, the LHA seems partitioned into GABA-rich/GABA-poor regions. However, to define or confirm distinct neuroanatomical entities, these regions must have a specific embryological origin, and show specific hodological patterns and functions. Hence, the purpose of this short review is to identify divisions of the LHA based on developmental and neurochemical criteria. Such an analysis seems to us relevant in order to allow later functional studies on regions whose boundaries will be based on objective criteria.

Compromised mammillary body connectivity and psychotic symptoms in mice with di- and mesencephalic ablation of ST8SIA2.

Altered long-range connectivity is a common finding across neurodevelopmental psychiatric disorders, but causes and consequences are not well understood. Genetic variation in ST8SIA2 has been associated with schizophrenia, autism, and bipolar disorder, and St8sia2-/- mice show a number of related neurodevelopmental and behavioral phenotypes. In the present study, we use conditional knockout (cKO) to dissect neurodevelopmental defects and behavioral consequences of St8sia2 deficiency in cortical interneurons, their cortical environment, or in the di- and mesencephalon. Neither separate nor combined cortical and diencephalic ablation of St8sia2 caused the disturbed thalamus-cortex connectivity observed in St8sia2-/- mice. However, cortical ablation reproduced hypoplasia of corpus callosum and fornix and mice with di- and mesencephalic ablation displayed smaller mammillary bodies with a prominent loss of parvalbumin-positive projection neurons and size reductions of the mammillothalamic tract. In addition, the mammillotegmental tract and the mammillary peduncle, forming the reciprocal connections between mammillary bodies and Gudden's tegmental nuclei, as well as the size of Gudden's ventral tegmental nucleus were affected. Only mice with these mammillary deficits displayed enhanced MK-801-induced locomotor activity, exacerbated impairment of prepulse inhibition in response to apomorphine, and hypoanxiety in the elevated plus maze. We therefore propose that compromised mammillary body connectivity, independent from hippocampal input, leads to these psychotic-like responses of St8sia2-deficient mice.

Development of the GABAergic and glutamatergic neurons of the lateral hypothalamus.

In the last few years we assist to an unexpected deluge of genomic data on hypothalamic development and structure. Perhaps most surprisingly, the Lateral Zone has received much attention too. The new information focuses first of all on transcriptional heterogeneity. Many already known and a number of hitherto unknown lateral hypothalamic neurons have been described to an enormous degree of detail. Maybe the most surprising novel discoveries are two: First, some restricted regions of the embryonic forebrain neuroepithelium generate specific LHA neurons, either GABAergic or glutamatergic. Second, evidence is mounting that supports the existence of numerous kinds of "bilingual" lateral hypothalamic neurons, expressing (and releasing) glutamate and GABA both as well as assorted neuropeptides. This is not accepted by all, and it could be that genomic researchers need a common set of rules to interpret their data (sensitivity, significance, age of analysis). In any case, some of the new results appear to confirm hypotheses about the ability of the hypothalamus and in particular its Lateral Zone to achieve physiological flexibility on a fixed connectivity ("biochemical switching"). Furthermore, the results succinctly reviewed here are the basis for future advances, since the transcriptional databases generated can now be mined e.g. for adhesion genes, to figure out the causes of the peculiar histology of the Lateral Zone; or for ion channel genes, to clarify present and future electrophysiological data. And with the specific expression data about small subpopulations of neurons, their connections can now be specifically labeled, revealing novel relations with functional significance.

MiR-9 and the Midbrain-Hindbrain Boundary: A Showcase for the Limited Functional Conservation and Regulatory Complexity of MicroRNAs.

MicroRNAs regulate gene expression at post-transcriptional levels. Some of them appear to regulate brain development and are involved in neurodevelopmental disorders. This has led to the suggestion that the role of microRNAs in neuronal development and function may be more central than previously appreciated. Here, we review the data about miR-9 function to depict the subtlety, complexity, flexibility and limited functional conservation of this essential developmental regulatory system. On this basis we propose that species-specific actions of miR-9 could underlie to a large degree species differences in brain size, shape and function.

CRMP2 mediates Sema3F-dependent axon pruning and dendritic spine remodeling.

Regulation of axon guidance and pruning of inappropriate synapses by class 3 semaphorins are key to the development of neural circuits. Collapsin response mediator protein 2 (CRMP2) has been shown to regulate axon guidance by mediating semaphorin 3A (Sema3A) signaling; however, nothing is known about its role in synapse pruning. Here, using newly generated crmp2-/- mice we demonstrate that CRMP2 has a moderate effect on Sema3A-dependent axon guidance in vivo, and its deficiency leads to a mild defect in axon guidance in peripheral nerves and the corpus callosum. Surprisingly, crmp2-/- mice display prominent defects in stereotyped axon pruning in hippocampus and visual cortex and altered dendritic spine remodeling, which is consistent with impaired Sema3F signaling and with models of autism spectrum disorder (ASD). We demonstrate that CRMP2 mediates Sema3F signaling in primary neurons and that crmp2-/- mice display ASD-related social behavior changes in the early postnatal period as well as in adults. Together, we demonstrate that CRMP2 mediates Sema3F-dependent synapse pruning and its dysfunction shares histological and behavioral features of ASD.

Development of neuroendocrine neurons in the mammalian hypothalamus.

The neuroendocrine system consists of a heterogeneous collection of (mostly) neuropeptidergic neurons found in four hypothalamic nuclei and sharing the ability to secrete neurohormones (all of them neuropeptides except dopamine) into the bloodstream. There are, however, abundant hypothalamic non-neuroendocrine neuropeptidergic neurons developing in parallel with the neuroendocrine system, so that both cannot be entirely disentangled. This heterogeneity results from the workings of a network of transcription factors many of which are already known. Olig2 and Fezf2 expressed in the progenitors, acting through mantle-expressed Otp and Sim1, Sim2 and Pou3f2 (Brn2), regulate production of magnocellular and anterior parvocellular neurons. Nkx2-1, Rax, Ascl1, Neurog3 and Dbx1 expressed in the progenitors, acting through mantle-expressed Isl1, Dlx1, Gsx1, Bsx, Hmx2/3, Ikzf1, Nr5a2 (LH-1) and Nr5a1 (SF-1) are responsible for tuberal parvocellular (arcuate nucleus) and other neuropeptidergic neurons. The existence of multiple progenitor domains whose progeny undergoes intricate tangential migrations as one source of complexity in the neuropeptidergic hypothalamus is the focus of much attention. How neurosecretory cells target axons to the medial eminence and posterior hypophysis is gradually becoming clear and exciting progress has been made on the mechanisms underlying neurovascular interface formation. While rat neuroanatomy and targeted mutations in mice have yielded fundamental knowledge about the neuroendocrine system in mammals, experiments on chick and zebrafish are providing key information about cellular and molecular mechanisms. Looking forward, data from every source will be necessary to unravel the ways in which the environment affects neuroendocrine development with consequences for adult health and disease.

The orbitofrontal cortex projects to the parvafox nucleus of the ventrolateral hypothalamus and to its targets in the ventromedial periaqueductal grey matter.

Although connections between the orbitofrontal cortex (OFC)-the seat of high cognitive functions-the lateral hypothalamus and the periaqueductal grey (PAG) have been recognized in the past, the precise targets of the descending fibres have not been identified. In the present study, viral tracer-transport experiments revealed neurons of the lateral (LO) and the ventrolateral (VLO) OFC (homologous to part of Area 13 in primates) to project to a circumscribed region in the ventrolateral hypothalamus, namely, the horizontally oriented, cylindrical parvalbumin- and Foxb1-expressing (parvafox) nucleus. The fine collaterals stem from coarse axons in the internal capsule and form excitatory synapses specifically with neurons of the parvafox nucleus, avoiding the rest of the hypothalamus. In its further caudal course, this contingent of LO/VLO-axons projects collaterals to the Su3- and the PV2 nuclei, which lie ventral to the aqueduct in the (PAG), where the terminals fields overlap those deriving from the parvafox nucleus itself. The targeting of the parvafox nucleus by the LO/VLO-projections, and the overlapping of their terminal fields within the PAG, suggest that the two cerebral sites interact closely. An involvement of this LO/VLO-driven circuit in the somatic manifestation of behavioural events is conceivable.

Cell-Biological Requirements for the Generation of Dentate Gyrus Granule Neurons.

The dentate gyrus (DG) receives highly processed information from the associative cortices functionally integrated in the trisynaptic hippocampal circuit, which contributes to the formation of new episodic memories and the spontaneous exploration of novel environments. Remarkably, the DG is the only brain region currently known to have high rates of neurogenesis in adults (Andersen et al., 1966, 1971). The DG is involved in several neurodegenerative disorders, including clinical dementia, schizophrenia, depression, bipolar disorder and temporal lobe epilepsy. The principal neurons of the DG are the granule cells. DG granule cells generated in culture would be an ideal model to investigate their normal development and the causes of the pathologies in which they are involved and as well as possible therapies. Essential to establish such in vitro models is the precise definition of the most important cell-biological requirements for the differentiation of DG granule cells. This requires a deeper understanding of the precise molecular and functional attributes of the DG granule cells in vivo as well as the DG cells derived in vitro. In this review we outline the neuroanatomical, molecular and cell-biological components of the granule cell differentiation pathway, including some growth- and transcription factors essential for their development. We summarize the functional characteristics of DG granule neurons, including the electrophysiological features of immature and mature granule cells and the axonal pathfinding characteristics of DG neurons. Additionally, we discuss landmark studies on the generation of dorsal telencephalic precursors from pluripotent stem cells (PSCs) as well as DG neuron differentiation in culture. Finally, we provide an outlook and comment critical aspects.

Foxb1 Regulates Negatively the Proliferation of Oligodendrocyte Progenitors.

Oligodendrocyte precursor cells (OPC), neurons and astrocytes share a neural progenitor cell (NPC) in the early ventricular zone (VZ) of the embryonic neuroepithelium. Both switch to produce either of the three cell types and the generation of the right number of them undergo complex genetic regulation. The components of these regulatory cascades vary between brain regions giving rise to the unique morphological and functional heterogeneity of this organ. Forkhead b1 (Foxb1) is a transcription factor gene expressed by NPCs in specific regions of the embryonic neuroepithelium. We used the mutant mouse line Foxb1-Cre to analyze the cell types derived from Fobx1-expressing NPCs (the Foxb1 cell lineage) from two restricted regions, the medulla oblongata (MO; hindbrain) and the thalamus (forebrain), of normal and Foxb1-deficient mice. Foxb1 cell lineage derivatives appear as clusters in restricted regions, including the MO (hindbrain) and the thalamus (forebrain). Foxb1-expressing NPCs produce mostly oligodendrocytes (OL), some neurons and few astrocytes. Foxb1-deficient NPCs generate mostly OPC and immature OL to the detriment of neurons, astrocytes and mature OL. The axonal G-ratio however is not changed. We reveal Foxb1 as a novel modulator of neuronal and OL generation in certain restricted CNS regions. Foxb1 biases NPCs towards neuronal generation and inhibits OPC proliferation while promoting their differentiation.

The Foxb1-expressing neurons of the ventrolateral hypothalamic parvafox nucleus project to defensive circuits.

The parvafox nucleus is an elongated structure that is lodged within the ventrolateral hypothalamus and lies along the optic tract. It comprises axially located parvalbumin (Parv)-positive neurons and a peripheral cuff of Foxb1-expressing ones. In the present study, injections of Cre-dependent adenoviral constructs were targeted to the ventrolateral hypothalamus of Foxb1/Cre mice to label specifically and map the efferent connections of the Foxb1-expressing subpopulation of neurons of the parvafox nucleus. These neurons project more widely than do the Parv-positive ones and implicate a part of the axons known to emanate from the lateral hypothalamus. High labeling densities were found in the dorsolateral and the upper lateral portion of the periaqueductal gray (PAG), the Su3 and PV2 nuclei of the ventrolateral PAG, the cuneiform nucleus, the mesencephalic reticular formation, and the superior colliculus. Intermediate densities of terminals were encountered in the septum, bed nucleus of the stria terminalis, substantia innominata, various thalamic and hypothalamic nuclei, pedunculopontine nucleus, Barrington's nucleus, retrofacial nucleus, and retroambigual nucleus. Scattered terminals were observed in the olfactory bulbs, the prefrontal cortex and the lamina X of the cervical spinal cord. Because the terminals were demonstrated to express the glutamate transporter VGlut2, the projections are presumed to be excitatory. A common denominator of the main target sites of the Foxb1-positive axons of the parvafox nucleus appears to be an involvement in the defensive reactions to life-threatening situations. The hypothalamic parvafox nucleus may contribute to the autonomic manifestations that accompany the expression of emotions. J. Comp. Neurol. 524:2955-2981, 2016. © 2016 Wiley Periodicals, Inc.

Birthdate of parvalbumin-neurons in the Parvafox-nucleus of the lateral hypothalamus.

The Parvafox-nucleus in the lateral hypothalamus is characterized by the presence of two distinct neural populations, the Parvalbumin (Parv) and the Foxb1-expressing ones. Foxb1-neurons are born at day 10 in the subventricular zone of the mouse mammillary region. It would be interesting to know if the subpopulation of Parv- neurons develop independently at different times and then meet the Foxb1- expressing neurons in the lateral hypothalamus, their final settling place. The aim of this study was to define the period of birth of the Parv-positive neurons using an in-vivo Bromodeoxyuridine-based method in rats. Parv-neurons are generated from embryonic day 10 to day 13, with a peak at day 12. Thus, it appears that the birthdates of the two subpopulations in these two species is similar, perhaps suggesting that they are born from the same neuroepithelial region.

Differential developmental strategies by Sonic hedgehog in thalamus and hypothalamus.

The traditional concept of diencephalon (thalamus plus hypothalamus) and with it the entire traditional subdivision of the developing neural tube are being challenged by novel insights obtained by mapping the expression of key developmental genes. A model in which the hypothalamus is placed in the most rostral portion of the neural tube, followed caudally by a diencephalon formed by prethalamus, thalamus and pretectum has been proposed. The adult thalamus and hypothalamus are quite unlike each other in connectivity and functions. Here we review work on the role of the secreted morphogen protein Sonic hedgehog (Shh) in the developing diencephalon and hypothalamic region to show how different these two regions are also from this point of view. Shh from the prechordal plate (PCP) induces and patterns the hypothalamus but there is no evidence that this role is fulfilled by a morphogen gradient. Later, the hypothalamic primordium itself expresses Shh and a large part of the hypothalamus belongs to the Shh lineage, including the ventral domains. Neural Shh is necessary to complete the specification (lateral hypothalamus), differentiation and growth of the hypothalamus. Although Gli2A is the major effector of Shh in this region, hypothalamic specification also depends on the suppression of Gli3R by Shh secreted by the PCP as well as the neuroepithelium. The thalamus is patterned by an Shh morphogen gradient originated in the ZLI following similar mechanisms to those in the spinal cord. The thalamus itself does not belong to the Shh lineage. Gli2A is necessary for appropriate growth and specification of the thalamic nuclei, to the exception of the medial and intralaminar groups (limbic-related), whose development depends on Gli3R. Beyond specification and patterning, the scarce data available about cell sorting and aggregation in these two regions shows key differences between them as well. In summary, not only expression patterns but also developmental mechanisms support a separation of the traditional thalamus and hypothalamus into different prosomeric domains.

The ventrolateral hypothalamic area and the parvafox nucleus: Role in the expression of (positive) emotions?

The lateral hypothalamus has been long suspected of triggering the expression of positive emotions, because stimulations of its tuberal portion provoke bursts of laughter. Electrophysiological studies in various species have indeed confirmed that the lateral hypothalamus contributes to reward mechanisms. However, only the rudiments of the neural circuit underlying the expression of positive emotions are known. The prefrontal cortex, the lateral hypothalamus, and the periaqueductal gray matter (PAG) are involved in these circuits; so, too, are the brainstem nuclei that control the laryngeal muscles and subserve mimicry, as well as the cardiovascular and respiratory systems. The implicated populations of hypothalamic neurons have not been defined either anatomically or molecularly. One promising candidate is the novel parvafox nucleus, which we recently described, in the murine medial forebrain bundle (mfb), which specifically expresses parvalbumin and Foxb1. With the molecularly defined parvafox nucleus as a centerpiece, the inputs from the prefrontal cortex and the projections to the PAG and brainstem can be studied with precision. By drawing on genetic approaches, it will be possible to manipulate the circuitry selectively with spatial and temporal exactitude and to evaluate the concomitant autonomic changes. These data will serve as a basis for imaging studies in humans using various paradigms to provoke the expression of positive emotions. In conclusion, studies of the hypothalamic parvafox nucleus will reveal whether this entity represents the fulcrum for positive emotions, as is the amygdala for fear and the insula for disgust.

LIM homeobox protein 5 (Lhx5) is essential for mamillary body development.

The mamillary body (MM) is a group of hypothalamic nuclei related to memory and spatial navigation that interconnects hippocampal, thalamic, and tegmental regions. Here we demonstrate that Lhx5, a LIM-HD domain transcription factor expressed early in the developing posterior hypothalamus, is required for the generation of the MM and its derived tracts. The MM markers Foxb1, Sim2, and Lhx1 are absent in Lhx5 knock-out mice from early embryonic stages, suggesting abnormal specification of this region. This was supported by the absence of Nkx2.1 and expansion of Shh in the prospective mamillary area. Interestingly, we also found an ectopic domain expressing Lhx2 and Lhx9 along the anterio-posterior hypothalamic axis. Our results suggest that Lhx5 controls early aspects of hypothalamic development by regulating gene expression and cellular specification in the prospective MM.

Lhx5 controls mamillary differentiation in the developing hypothalamus of the mouse.

Acquisition of specific neuronal identity by individual brain nuclei is a key step in brain development. However, how the mechanisms that confer neuronal identity are integrated with upstream regional specification networks is still mysterious. Expression of Sonic hedgehog (Shh), is required for hypothalamic specification and is later downregulated by Tbx3 to allow for the differentiation of the tubero-mamillary region. In this region, the mamillary body (MBO), is a large neuronal aggregate essential for memory formation. To clarify how MBO identity is acquired after regional specification, we investigated Lhx5, a transcription factor with restricted MBO expression. We first generated a hypomorph allele of Lhx5-in homozygotes, the MBO disappears after initial specification. Intriguingly, in these mutants, Tbx3 was downregulated and the Shh expression domain abnormally extended. Microarray analysis and chromatin immunoprecipitation indicated that Lhx5 appears to be involved in Shh downregulation through Tbx3 and activates several MBO-specific regulator and effector genes. Finally, by tracing the caudal hypothalamic cell lineage we show that, in the Lhx5 mutant, at least some MBO cells are present but lack characteristic marker expression. Our work shows how the Lhx5 locus contributes to integrate regional specification pathways with downstream acquisition of neuronal identity in the MBO.

Cadherins mediate sequential roles through a hierarchy of mechanisms in the developing mammillary body.

Expression of intricate combinations of cadherins (a family of adhesive membrane proteins) is common in the developing central nervous system. On this basis, a combinatorial cadherin code has long been proposed to underlie neuronal sorting and to be ultimately responsible for the layers, columns and nuclei of the brain. However, experimental proof of this particular function of cadherins has proven difficult to obtain and the question is still not clear. Alternatively, non-specific, non-combinatorial, purely quantitative adhesive differentials have been proposed to explain neuronal sorting in the brain. Do cadherin combinations underlie brain cytoarchitecture? We approached this question using as model a well-defined forebrain nucleus, the mammillary body (MBO), which shows strong, homogeneous expression of one single cadherin (Cdh11) and patterned, combinatorial expression of Cdh6, -8 and -10. We found that, besides the known combinatorial Cdh pattern, MBO cells are organized into a second, non-overlapping pattern grouping neurons with the same date of neurogenesis. We report that, in the Foxb1 mouse mutant, Cdh11 expression fails to be maintained during MBO development. This disrupted the combination-based as well as the birthdate-based sorting in the mutant MBO. In utero RNA interference (RNAi) experiments knocking down Cdh11 in MBO-fated migrating neurons at one specific age showed that Cdh11 expression is required for chronological entrance in the MBO. Our results suggest that neuronal sorting in the developing MBO is caused by adhesion-based, non-combinatorial mechanisms that keep neurons sorted according to birthdate information (possibly matching them to target neurons chronologically sorted in the same manner). Non-specific adhesion mechanisms would also prevent cadherin combinations from altering the birthdate-based sorting. Cadherin combinations would presumably act later to support specific synaptogenesis through specific axonal fasciculation and final target recognition.

Differential requirements for Gli2 and Gli3 in the regional specification of the mouse hypothalamus.

Secreted protein Sonic hedgehog (Shh) ventralizes the neural tube by modulating the crucial balance between activating and repressing functions (GliA, GliR) of transcription factors Gli2 and Gli3. This balance-the Shh-Gli code-is species- and context-dependent and has been elucidated for the mouse spinal cord. The hypothalamus, a forebrain region regulating vital functions like homeostasis and hormone secretion, shows dynamic and intricate Shh expression as well as complex regional differentiation. Here we asked if particular combinations of Gli2 and Gli3 and of GliA and GliR functions contribute to the variety of hypothalamic regions, i.e., we wanted to approach the question of a possible hypothalamic version of the Shh-Gli code. Based on mouse mutant analysis, we show that: (1) hypothalamic regional heterogeneity is based in part on differentially stringent requirements for Gli2 or Gli3; (2) another source of diversity are differential requirements for Shh of neural vs. non-neural origin; (3) the medial progenitor domain known to depend on Gli2 for its development generates several essential hypothalamic nuclei plus the pituitary and median eminence; (4) the suppression of Gli3R by neural and non-neural Shh is essential for hypothalamic specification. Finally, we have mapped our results on a recent model which considers the hypothalamus as a transverse region with alar and basal portions. Our data confirm the model and are explained by it.