Topographical organization of mammillary neurogenesis and efferent projections in the mouse brain.

The mammillary body is a hypothalamic nucleus that has important functions in memory and spatial navigation, but its developmental principles remain not well understood. Here, we identify progenitor-specific Fezf2 expression in the developing mammillary body and develop an intersectional fate-mapping approach to demonstrate that Fezf2+ mammillary progenitors generate mammillary neurons in a rostral-dorsal-lateral to caudal-ventral-medial fashion. Axonal tracing from different temporal cohorts of labeled mammillary neurons reveal their topographical organization. Unsupervised hierarchical clustering based on intrinsic properties further identify two distinct neuronal clusters independent of birthdates in the medial nuclei. In addition, we generate Fezf2 knockout mice and observe the smaller mammillary body with largely normal anatomy and mildly affected cellular electrophysiology, in contrast to more severe deficits in neuronal differentiation and projection in many other brain regions. These results indicate that Fezf2 may function differently in the mammillary body. Our results provide important insights for mammillary development and connectivity.

A novel TaulacZ allele reveals a requirement for Pitx2 in formation of the mammillothalamic tract.

The hypothalamic mammillary region is critical for spatial memory and vestibular processing. Pitx2 encodes a paired-like transcription factor that is highly expressed in the developing mammillary region and is required for subthalamic nucleus formation. Here we analyzed a loss of function Pitx2-TaulacZ knock-in allele to study the effects of Pitx2 deficiency on neuronal projections in the embryonic mammillary region. Pitx2-expressing neurons contribute axons to principal mammillary, mammillotegmental and mammillotectal tracts. Embryos with Pitx2 deficiency exhibit axonal fibers in the principal mammillary tract that are improperly bundled and disorganized, yet project caudally toward the tectum and tegmentum. Embryos with Nestin-Cre mediated conditional Pitx2 deficiency exhibit truncated mammillothalamic tracts (mtt) that fail to elongate, and reduced Pax6-positive cells at the branching point of the principal mammillary and mtt. These data suggest that Pitx2 mediates cell-autonomous and nonautonomous guidance cues necessary for mammillary collaterals destined to project to the anterior thalamus.

Perinatal development of the mammillothalamic tract and innervation of the anterior thalamic nuclei.

Axonal projections originating from the mammillary bodies represent important pathways that are essential for spatial information processing. Mammillothalamic tract is one of the main efferent projection systems of the mammillary body belonging to the limbic "Papez circuit". This study was aimed to describe the schedule of the mammillothalamic tract development in the rat using carbocyanine dye tracing. It was shown for the first time that fibers of the mammillothalamic tract being the collaterals of the mammillotegmental tract axons start bifurcating from the mammillotegmental tract on E17. The axons of the mammillothalamic tract grow simultaneously and reach the ventral region of the anterior thalamus where they form first terminal arborizations on E20-E21. Ipsilateral projections from the medial mammillary nucleus to the anteromedial and anteroventral thalamic nuclei develop from E20 to P6. Bilateral projections from the lateral mammillary nucleus to the anterodorsal thalamic nuclei develop later, on P3-P6, after the formation of the thalamic decussation of the mammillary body axons. Unique spatial and temporal pattern of the perinatal development of ascending mammillary body projections to the anterior thalamic nuclei may reflect the importance of these connections within the limbic circuitry.

Aberrant axonal projections from mammillary bodies in Pax6 mutant mice: possible roles of Netrin-1 and Slit 2 in mammillary projections.

Early events in the axonal tract formation from mammillary bodies remain poorly understood. In the present study, we reported an aberrant pattern of axonal projections from mammillary bodies to the dorsal thalamus in mice lacking the transcription factor Pax6. We found that Netrin-1 was ectopically up-regulated and that both Slit1 and Slit2 were down-regulated in the presumptive dorsal thalamus of Pax6 mutant mice. We then examined the effects of Netrin-1 and Slit2 on the mammillary axons by in utero electroporation techniques. Netrin-1 had an attractive action toward the mammillary axons. Moreover, mammillary trajectories were disorganized in Netrin-1-deficient mice. On the other hand, Slit2 had a repulsive effect on the mammillary axons. These findings suggest that the combination of Netrin and Slit may be involved in proper axonal projection from the mammillary bodies and that their misexpression in the diencephalon may cause the misrouting of these axons in Pax6 mutant mice.

[Perinatal development of mammillotegmental connections in rats].

Development of direct axonal connections of the hypothalamic mammillary bodies with ventral and dorsal tegmental nuclei of Gudden was studied on fixed rat brains from day 14 of embryonic development until day 10 of postnatal development using the method of diffusion of the lipophilic fluorescent carbocyanine tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate along the neuronal membranes. The tracer was inserted into the mammillary bodies or into the tegmentum and after incubation in a fixative fluorescent nerve cells and nerve fibers were visualized in the brain tissue. The mammillotegmental tract was found to start developing earlier than other conducting systems of the mammillary bodies. On days 14-15 of embryonic development, it was visualized as a bundle of axons running from the mammillary bodies caudally to the midbrain. A group of neurons in the midbrain tegmentum and their axons going to the mammillary bodies via the mammillary peduncle were first visualized on day 19 of embryonic development. The mammillotegmental tract and mammillary peduncle developed progressively from the moment of birth. Ventral and dorsal tegmental nuclei were formed in the midbrain by day 10 of the postnatal development. Thus, the formation of reciprocal connections of the mammillary bodies with midbrain tegmental nuclei was first described during perinatal development in rats.

Alpha N-catenin deficiency causes defects in axon migration and nuclear organization in restricted regions of the mouse brain.

Alpha N-catenin is a cadherin-binding protein, widely expressed in the nervous system; and it plays a crucial role in cadherin-mediated cell-cell adhesion. Here we report the effects of alpha N-catenin gene deficiency on brain morphogenesis. In addition to the previously reported phenotypes, we found that some of the axon tracts did not normally develop, in particular, axons of the anterior commissure failed to cross the midline, migrating, rather, to ectopic places. In restricted nuclei, a population of neurons was missing or their laminar arrangement was distorted. The ventricular structures were also deformed. These results indicate that alpha N-catenin has diverse roles in the organization of the central nervous system, but only in limited portions of the brain.

Sim1 and Sim2 are required for the correct targeting of mammillary body axons.

The mammillary body (MB), and its axonal projections to the thalamus (mammillothalamic tract, MTT) and the tegmentum (mammillotegmental tract, MTEG), are components of a circuit involved in spatial learning. The bHLH-PAS transcription factors SIM1 and SIM2 are co-expressed in the developing MB. We have found that MB neurons are generated and that they survive at least until E18.5 in embryos lacking both Sim1 and Sim2 (Sim1(-/-);Sim2(-/-)). However, the MTT and MTEG are histologically absent in Sim1(-/-);Sim2(-/-) embryos, and are reduced in embryos lacking Sim1 but bearing one or two copies of Sim2, indicating a contribution of the latter to the development of MB axons. We have generated, by homologous recombination, a null allele of Sim1 (Sim1(tlz)) in which the tau-lacZ fusion gene was introduced, allowing the staining of MB axons. Consistent with the histological studies, lacZ staining showed that the MTT/MTEG is barely detectable in Sim1(tlz/tlz);Sim2(+/-) and Sim1(tlz/tlz);Sim2(-/-) brains. Instead, MB axons are splayed and grow towards the midline. Slit1 and Slit2, which code for secreted molecules that induce the repulsion of ROBO1-producing axons, are expressed in the midline at the level of the MB, whereas Robo1 is expressed in the developing MB. The expression of Rig-1/Robo3, a negative regulator of Slit signalling, is upregulated in the prospective MB of Sim1/Sim2 double mutants, raising the possibility that the growth of mutant MB axons towards the midline is caused by a decreased sensitivity to SLIT. Finally, we found that Sim1 and Sim2 act along compensatory, but not hierarchical, pathways, suggesting that they play similar roles in vivo.

Galanin immunohistochemistry and electron microscopic studies in developing human fetal mammillary bodies.

Development and maturation of nuclear groups in the mammillary complex of second and third trimester human fetal hypothalamus were studied using Nissl stain, galanin immunocytochemistry and transmission electron microscopy. While the identity of the supra and medial mammillary nucleus was established at 24 weeks of gestation (w.g.) in Nissl stained preparation, galanin immunoreactive (Gal-ir) neurons were seen in the supra and medial mammillary nucleus of 27 through 39 w.g. fetuses. Immunoreactive perikarya in the lateral mammillary nucleus appear later at 34 w.g. and show relatively meager population. Gal-ir neurons of the supramammillary nucleus were divisible in dorsal and ventral subgroups. There was a progressive increase in galanin expressing neurons in more and more ventral positions, along the medial margin of either mammillary body so that in term fetal specimens, the ventral subgroup appeared to be continuous with the medial mammillary nucleus. Galanin positive neurons were relatively sparse in the core of the mammillary bodies. Transmission electron micrographs revealed neurons with varying degree of indentation of the nuclear envelope. Vigorous synaptogenesis was seen in the supramammillary region of the mammillary bodies. The height and width of the synaptic complex also showed a progressive increase. Although galanin neurons were reported from the supramammillary nucleus of adult human mammillary complex, no immunoreactivity was detected in the medial and lateral components of the mammillary body. We suggest that expression of galanin in the medial and lateral components may be of transient occurrence and may serve a significant role in the synaptogenesis.

Bsx, an evolutionary conserved Brain Specific homeoboX gene expressed in the septum, epiphysis, mammillary bodies and arcuate nucleus.

Shaping and orchestrating the genetic program involved in embryonic modelling of brain structures is a major function played by homeobox containing genes. Recently, analysis of conditional mouse mutants has pointed out additional roles in supporting adult brain functional activities. During a search for novel homeobox genes in the public released genomic sequences derived by the Human and Mouse genome projects, we were able to identify the mouse homologue of the Drosophila brain specific homeobox gene. We named it Bsx and characterized its expression in embryonic and post-natal mouse brain. Interestingly, Bsx shows an expression pattern restricted to a few specific developing brain structures. Pineal gland, telencephalic septum, hypothalamic pre-mammillary body and arcuate nucleus are the only brain structures where we detected Bsx transcriptional activity, which is maintained also after birth. In particular, Bsx might be considered an important molecular marker for early embryonic stages of epiphysis development, being specifically expressed in this neural structure from E9.5 onwards.

Expression of Foxb1 reveals two strategies for the formation of nuclei in the developing ventral diencephalon.

Fork head b1 (Foxb1; also called Fkh5, HFH-e5.1, Mf3) is a winged helix transcription factor gene whose widespread early expression in the developing neural tube is soon restricted to the ventral and caudal diencephalon. During diencephalic neurogenesis, Foxb1 is expressed in one patch of neuroepithelium comprising a large mammillary portion and a smaller tuberal portion. The labeled cells coming from this patch contribute to nuclear formation by means of two different strategies: (1) caudally, the young neurons aggregate and settle immediately, giving rise to the nuclei of the mammillary body; (2) rostrally, the young neurons separate from the neuroepithelium forming a trail of cells which spans the mantle layer mediolaterally and which will give rise to two separate cell groups (the dorsal premammillary and part of the lateral hypothalamic area). Our results show the elaborate, regionalized histogenetic mechanisms necessary for the differentiation of the caudal diencephalon; moreover, they suggest that specifically labeled populations, arising from specifically labeled neuroepithelial patches and giving place to specific brain nuclei could be a common mechanism to build complex, nonlaminar regions of the forebrain.

Development of the mammillothalamic tract in normal and Pax-6 mutant mice.

The mammillary bodies represent important relay stations for one of the major neuronal circuits in the brain: the limbic circuit. Mammillary projections traveling through the principal mammillary tract are established early during development, forming the mammillotegmental bundle, which appears fully developed by embryonic day 15 (E15). The mammillothalamic tract develops later, around E17-E18, forming a compact system of collateral fibers originating from the principal mammillary tract and reaching the thalamus by E20. The Pax-6 gene is expressed in various regions of the developing brain, among which the border separating the ventral thalamus from the dorsal thalamus, known as the zona limitans intrathalamica, is especially significant. In this report, the development of the efferent mammillary system of fibers was studied in wild type and Pax-6 mutant mice by using carbocyanine tracers and Golgi preparations. In mutant mice, the mammillotegmental bundle developed normally; however, the mammillothalamic tract was missing. By using anti-Pax-6 antibodies in wild type mice, the existence of an immunoreactive cell cluster is described surrounding the bifurcation point of the principal mammillary tract. The results of this study suggest that there is a correlation of these cells with a particular type of Golgi impregnated neuron.

Winged helix transcription factor Foxb1 is essential for access of mammillothalamic axons to the thalamus.

Our aim was to study the mechanisms of brain histogenesis. As a model, we have used the role of winged helix transcription factor gene Foxb1 in the emergence of a very specific morphological trait of the diencephalon, the mammillary axonal complex. Foxb1 is expressed in a large hypothalamic neuronal group (the mammillary body), which gives origin to a major axonal bundle with branches to thalamus, tectum and tegmentum. We have generated mice carrying a targeted mutation of Foxb1 plus the tau-lacZ reporter. In these mutants, a subpopulation of dorsal thalamic ventricular cells "thalamic palisade" show abnormal persistence of Foxb1 transcriptional activity; the thalamic branch of the mammillary axonal complex is not able to grow past these cells and enter the thalamus. The other two branches of the mammillary axonal complex (to tectum and tegmentum) are unaffected by the mutation. Most of the neurons that originate the mammillothalamic axons suffer apoptosis after navigational failure. Analysis of chimeric brains with wild-type and Foxb1 mutant cells suggests that correct expression of Foxb1 in the thalamic palisade is sufficient to rescue the normal phenotype. Our results indicate that Foxb1 is essential for diencephalic histogenesis and that it exerts its effects by controlling access to the target by one particular axonal branch.

Fkh5-deficient mice show dysgenesis in the caudal midbrain and hypothalamic mammillary body.

The murine winged helix gene Fkh5 is specifically expressed in the developing central nervous system (CNS). Early embryonic Fkh5 expression is restricted to the mammiliary body region of the caudal hypothalamus, midbrain, hindbrain and spinal cord. Postnatally, signals persist in specific nuclei of the mammillary body and in the midbrain. We generated Fkh5 deficient mice by homologous recombination to assess its in vivo function. At birth, Fkh5-deficient mice are viable and indistinguishable from wild-type and Fkh5 heterozygous littermates. However, about one third die within the first two days and another fifth before weaning. Surviving Fkh5-deficient mice become growth retarded within the first week and remain smaller throughout their whole life span. Fkh5-deficient females on 129Sv x C57BL/6 genetic background are fertile, but do not nurture their pups. More detailed analysis of Fkh5-deficient brains reveals distinct alterations in the CNS. In the midbrain, mutant mice exhibit reduced inferior colliculi and an overgrown anterior cerebellum. Furthermore, the hypothalamic mammillary body of Fkh5-deficient brains lacks the medial mammillary nucleus. These results suggest that Fkh5 plays a major role during CNS development.

The normal and abnormal genu of the corpus callosum: an evolutionary, embryologic, anatomic, and MR analysis.

PURPOSE: To define the normal and abnormal genu of the corpus callosum by examining its evolution and embryology and by analyzing its normal and abnormal appearance on MR images. METHODS: A reference line was drawn from the mamillary body through the anterior commissure and corpus callosum-the MAC line. This line was used to evaluate the genu in adult mammal brains, in human fetal brains, on MR images of 1800 patients with normal corpora callosi, and on MR images of 113 patients with callosal anomalies. RESULTS: In primates, increased frontal lobe size is associated with an anteriorly shifted genu. In human fetal development, the anterior body of the corpus callosum develops before the definitive genu. The normal human genu always projects in front of the MAC line. In none of the 113 patients with callosal anomalies was there only a normal genu. CONCLUSIONS: The human corpus callosum develops bidirectionally, not from front to back. The MAC line is a useful frame of reference to study the evolution and embryology of the genu and to distinguish the normal from the abnormal genu of the human corpus callosum.

Neurons containing cholecystokinin mRNA in the mammillary region: ontogeny and adult distribution in the rat.

1. The ontogeny and adult distribution of neurons containing cholecystokinin (CCK) mRNA in the premammillary and mammillary nuclei and supramammillary region of the rat brain were studied using hybridization histochemistry. 2. The earliest detection of CCK mRNA in the mammillary region was on E14, followed by a marked increase in transcript levels during the next 4 days, a time during which neurons in this region still divide. During the first 2 weeks of life, few changes in the levels of CCK transcripts were seen, and an adult-like pattern of expression was seen on the twenty-first day of life. 3. Low levels of transcripts were present in numerous neurons located in all divisions of the medial nucleus and in the posterior nucleus known to project ipsilaterally to the anteroventral and anteromedial thalamic nuclei. In contrast, none of the neurons in the lateral nucleus (projecting bilaterally to the anterodorsal thalamic nucleus) had detectable transcripts. 4. Many neurons in the supramammillary nucleus had low, moderate, or high levels of transcripts. Some nearby nuclei (such as the dorsal premammillary nucleus) had smaller numbers of neurons with low levels of CCK mRNA, whereas others (such as the ventral premammillary nucleus) had none.

Development of the diencephalon in the rat. I. Autoradiographic study of the time of origin and settling patterns of neurons of the hypothalamus.

Groups of pregnant rats were injected with two successive daily doses of 3H-thymidine from gestational day 13 (E13 + 14) until the day before birth (E21 + 22). This double labelling procedure was combined with an injection schedule of a single day delay between groups (E13 + 14; E14 + 15; E15 + 16 . .). The two injections assured the comprehensive labelling of practically all neurons of a given structure prior to the onset of their differentiation (comprehensive labelling), whereas the progressive daily delay in injections made it possible to estimate the proportion of neurons formed in various regions of the hypothalamus on a single day. Hypothalamic areas or nuclei were assigned into four classes on the basis of their cytogenetic isochronicity. Structures composed of the earliest arising (class 1) neurons constitute a lateral tier that includes the lateral preoptic and lateral hypothalamic areas, and the lateral mammillary nucleus. Structures composed of early arising (class 2) neurons form a heterogeneous collection of nuclear systems, including the paraventricular, internuclear and supraoptic magnocellular neurons, and several intermediate tier nuclei of the anterior and posterior hypothalamus. The late arising (class 3) and latest arising (class 4) nuclei constitute a periventricular system anteriorly and a more extensive region posteriorly. The latter two nuclear systems may constitute the hypophysiotropic area of the hypothalamus. The nuclei of the mammillary system, which are produced sequentially, are distinguished from other hypothalamic structures by their more rapid generation time. Internuclear labelling gradients were used to infer the neuroepithelial site of origin and settling pattern of neurons. Common sites of origin were indicated for the following structures: the magnocellular neurohypophysial neurons; the neurons of the dorsomedial and ventromedial nuclei; and the neurons of the tubermammillary and arcuate nuclei. The sites of origin of these groups of nuclei were related to specialized ventricular linings in the mature hypothalamus.

Development of the diencephalon in the rat. II. Correlation of the embryonic development of the hypothalamus with the time of origin of its neurons.

The development of the nuclei of the hypothalamus was examined in normal and X-irradiated embryos from day 13 (E13) to the day before birth (E22). The diencephalic neuroepithelium was subdivided into three lobes (dorsal, medial, and ventral) and two lobules (superior and inferior). The hypothalamus is derived from the ventral lobe and the inferior lobule. The ventral neuroepithelial lobe generates the neurons of most of the early arising hypothalamic structures, including those of the lateral tier nuclei associated with the medial forebrain bundle, and the heterogeneous intermediate tier nuclei. A specialized neuroepithelial region lining the diamond shaped ventricle produces the early neurohypophysial magnocellular neurons; the neurons of the paraventricular nucleus remain at this site, whereas the neurons of the supraoptic nucleus could be traced migrating laterally. The neurons of the late arising hypophysiotropic area of the posterior hypothalamus are derived from components of the inferior neuroepithelial lobule: the dorsomedial and ventromedial nuclei apparently from a shared matrix in the main portion of the inferior lobule; the tuberomammillary-arcuate complex from its posteroventral recess. The triple-decked and sequentially produced components of the mammillary system may arise from separate neuroepithelial sites. The autoradiographic results of the previous study (Altman and Bayer, '78a) showed that the structural and functional heterogeneity of the mature hypothalamus is paralleled by cytogenetic heterochronicity; the present embryonic observations indicate that many of the distinguishable components of the hypothalamus arise from a mosaic of heterogeneous neuroepithelial sites.

[Qualitative and quantitative examinations of the corpus mamillare in the ontogenesis of male Tupaia belangeri and SPF cats]. / Qualitative und quantitative Untersuchungen des Corpus mamillare an ontogenetischen Reihen von männlichen Tupaia belangeri und SPF-Katzen.

The cyto-, myelo- and fibrilloarchitectonics of the Corpus mamillare were described in 33 male Tupaia belangeri at an age of 36 to 536 days of ontogenesis and in 37 male SPF-cats at an age of 39 to 584 days of ontogenesis. The fresh volumes of the Nucl. medialis and the Nucl. lateralis corporis mamillaris of Tupaia belangeri were determined and the growth of their fresh volumes described with the 6-parametric growth-function Likewise, the nuclei of SPF-cat were determined and these data were analysed with the logistic growth function The Nucl. medialis corporis mamillaris of Tupaia belangeri has a maximal fresh volume of 2.2 mm3 on the 82nd day of ontogenesis after which it procentually reduces by 16% to 101% of the ideal volume (1.9 mm3) on the 141st day of ontogenesis. The Nucl. lateralis corporis mamillaris reaches its maximum volume of 0.1 mm3 by the 90th day of ontogenesis, procentually reduces by 10% to 101% of the ideal volume (0.09 mm3) on the 169th day of ontogenesis. The half value times are 59 days of ontogenesis for the Nucl. medialis corporismamillaris and 48 days of ontogenesis for the Nucl. lateralis corporis mamillaris. In SPF-cat the Nucl. medialis corporis mamillaris reaches 99% of the ideal volume (11.7 mm3) on the 195th day of ontogenesis and shows a half value time of 86 days of ontogenesis. On the 229th day of ontogenesis the Nucl. lateralis corporis mamillaris reaches 99% of the ideal volume (0.57 mm3y and has a half value time of 83 days of ontogenesis.

[Quantitative growth analysis of limbic nuclei areas fresh volume in diencephalon and mesencephalon of an albino mouse ontogenic series. II. Corpus mammillare]. / Quantitative Analyse des Wachstums der Frischvolumina limbischer Kerngebiet im Diencephalon und Mesencephalon einer ontogenetischen Reihe von Albinomäusen. II. Corpus mammilare

The fresh volumes of the Nucl. medialis and of the Nucl. lateralis corporis mammillaris of 52 white mice aged between 17 and 60 days of ontogenesis have been determined. The fresh volume of the left Nucl. lateralis is significantly larger than that of the right side. The data of the Nucl. laterales have been fitted by a 3parametric logistic function. The data of the Nucl. medialis have been fitted by a sum of logistic functions. This sum has an increasing and a decreasing component and it shows a maximum of the sum of both components near 32 days of ontogenesis. The degree of maturity and the growth-rate of maturity have been discussed. Both nuclei belong to the group of brain regions in white mice with an early development. The growth of different brain regions is asynchronous. There is a need for further quantitative analyses.