Faustino Marín

Morphological Fate of Rhombomeres in Quail/Chick Chimeras: A Segmental Analysis of Hindbrain Nuclei

Quail rhombomeres two to six (r2‐r6) were individually grafted homotopically into the hindbrain of chick embryos at 2 days of incubation. Nine to 10 days after the operation the chimeric embryos were fixed and processed for parallel cytoarchitectural and immunocytochemical study (with an anti‐quail antibody) in order to map the anatomical fate of the grafted tissue. Emphasis was placed on conventionally identified and distinct neuronal populations composing the sensory and motor longitudinal columns. Grafted rhombomeres consistently developed as complete transverse slices of the chimeric hindbrain. Interrhombomeric cell migration was either sparse or restricted to specific nuclei. The cranial nerve motor nuclei showed rhombomeric origins consistent with the patterns described in early embryos. Unexpectedly, alar r2 was found to form the auricular part of the cerebellum. As regards the cochlear nuclei, we found that nucleus angularis derives from r3 to r6, nucleus laminaris from r5 to r6, nucleus magnocellularis from r6 to r7 and nucleus olivaris superior from r5. The nuclei of the lateral lemniscus originated between r1 and r3. We also delimited the respective rhombomeric subdivisions of the sensory vestibular and trigeminal columns, both of which extend from r1 caudalwards throughout the hindbrain. There were consistently some interrhombomeric neuronal migrations inside the vestibular column, some motor nuclei and the reticular formation, involving only one rhombomere length. The pontine nuclei, which extended from r1 to r7, showed neuronal migrations that crossed several rhombomeres. On the whole, these results represent the first anatomical analysis of the mature avian hindbrain in terms of rhombomere‐derived domains.

Snail and E47 repressors of E-cadherin induce distinct invasive and angiogenic properties in vivo

The transcription factors Snail and E47 are direct repressors of E-cadherin, with both inducing a full epithelial-mesenchymal transition and invasive behaviour in vitro when expressed in the prototypic epithelial MDCK cell line. The role of these repressors in the invasive process and in other tumorigenic properties is, nevertheless, still poorly understood. However, organotypic cultures and in vivo transplantation assays indicate that cells expressing MDCK-Snail and MDCK-E47 exhibit significant differences. MDCK-Snail cells have a higher infiltrative potential than MDCK-E47 cells. Interestingly, both cell types induce angiogenesis of the host stromal tissue in transplantation assays, but this property is greatly enhanced in transplants of MDCK-E47 cells. Xenografted tumours induced in nude mice also show signs of strong angiogenic potential, again markedly increased in tumours induced by MDCK-E47 which exhibit a higher vessel density and proliferation rate than those induced by MDCK-Snail cells. These results suggest differential roles for Snail and E47 E-cadherin repressors in tumour progression where Snail is implicated in promoting the initial invasion and E47 plays an active role in tumour cell growth by promoting angiogenesis.

Induction of ectopic engrailed expression and fate change in avian rhombomeres: intersegmental boundaries as barriers

We tested the possibility of inducing ectopic expression of the gene Engrailed-2 (En-2) in the rhombomeres of the 2-days-old chick embryo. The experimental procedure consisted of grafting En-2-expressing neuroepithelium (prospective isthmocerebellum) from a quail or a mouse embryo into different rostrocaudal levels in the hindbrain of a host chick embryo. The graft replaced a given excised host rhombomere, the rostral and caudal limits of which were either also excised or left intact in different experiments. Induction of En-2 occurred in the host hindbrain, but only when the graft did not contact with host interrhombomeric boundaries and only in the alar plate of the rhombomeres immediately contacting the graft. Long survival experimental embryos showed that induced rhombomeres give rise to a cerebellar phenotype in their transformed alar plates. We thus demonstrate here a pluripotential state of the early rhombomeres as well as a possible role of the interrhombomeric limits as barriers to morphogenetic influences.

Multiple origins, migratory paths and molecular profiles of cells populating the avian interpeduncular nucleus

The interpeduncular nucleus (IP) is a key limbic structure, highly conserved evolutionarily among vertebrates. The IP receives indirect input from limbic areas of the telencephalon, relayed by the habenula via the fasciculus retroflexus. The function of the habenulo-IP complex is poorly understood, although there is evidence that in rodents it modulates behaviors such as learning and memory, avoidance, reward and affective states. The IP has been an important subject of interest for neuroscientists, and there are multiple studies about the adult structure, chemoarchitecture and its connectivity, with complex results, due to the presence of multiple cell types across a variety of subnuclei. However, the ontogenetic origins of these populations have not been examined, and there is some controversy about its location in the midbrain-anterior hindbrain area. To address these issues, we first investigated the anteroposterior (AP) origin of the IP complex by fate-mapping its neuromeric origin in the chick, discovering that the IP develops strictly within isthmus and rhombomere 1. Next, we studied the dorsoventral (DV) positional identity of subpopulations of the IP complex. Our results indicate that there are at least four IP progenitor domains along the DV axis. These specific domains give rise to distinct subtypes of cell populations that target the IP with variable subnuclear specificity. Interestingly, these populations can be characterized by differential expression of the transcription factors Pax7, Nkx6.1, Otp, and Otx2. Each of these subpopulations follows a specific route of migration from its source, and all reach the IP roughly at the same stage. Remarkably, IP progenitor domains were found both in the alar and basal plates. Some IP populations showed rostrocaudal restriction in their origins (isthmus versus anterior or posterior r1 regions). A tentative developmental model of the structure of the avian IP is proposed. The IP emerges as a plurisegmental and developmentally heterogeneous formation that forms ventromedially within the isthmus and r1. These findings are relevant since they help to understand the highly complex chemoarchitecture, hodology and functions of this important brainstem structure.

Crypto-rhombomeres of the mouse medulla oblongata, defined by molecular and morphological features

The medulla oblongata is the caudal portion of the vertebrate hindbrain. It contains major ascending and descending fiber tracts as well as several motor and interneuron populations, including neural centers that regulate the visceral functions and the maintenance of bodily homeostasis. In the avian embryo, it has been proposed that the primordium of this region is subdivided into five segments or crypto-rhombomeres (r7–r11), which were defined according to either their parameric position relative to intersomitic boundaries (Cambronero and Puelles, in J Comp Neurol 427:522–545, 2000) or a stepped expression of Hox genes (Marín et al., in Dev Biol 323:230–247, 2008). In the present work, we examine the implied similar segmental organization of the mouse medulla oblongata. To this end, we analyze the expression pattern of Hox genes from groups 3 to 8, comparing them to the expression of given cytoarchitectonic and molecular markers, from mid-gestational to perinatal stages. As a result of this approach, we conclude that the mouse medulla oblongata is segmentally organized, similarly as in avian embryos. Longitudinal structures such as the nucleus of the solitary tract, the dorsal vagal motor nucleus, the hypoglossal motor nucleus, the descending trigeminal and vestibular columns, or the reticular formation appear subdivided into discrete segmental units. Additionally, our analysis identified an internal molecular organization of the migrated pontine nuclei that reflects a differential segmental origin of their neurons as assessed by Hox gene expression.

Molecular and Morphological Changes in Placenta and Embryo Development Associated with the Inhibition of Polyamine Synthesis during Midpregnancy in Mice

Polyamines play an essential role in murine development, as demonstrated by both gene ablation in ornithine decarboxylase (ODC)-deficient embryos and pharmacological treatments of pregnant mice. However, the molecular and cellular mechanisms by which ODC inhibition affects embryonic development during critical periods of pregnancy are mostly unknown. Our present results demonstrate that the contragestational effect of alpha-difluoromethylornithine (DFMO), a suicide inhibitor of ODC, when given at d 7-9 of pregnancy, is associated with embryo growth arrest and marked alterations in the development of yolk sac and placenta. Blood island formation as well as the transcript levels of embryonary globins alpha-like x chain and beta-like y-chain was markedly decreased in the yolk sac. At the placental level, abnormal chorioallantoic attachment, absence of the spongiotrophoblast layer and a deficient development of the labyrinthine zone were evident. Real-time RT-PCR analysis showed that transcript levels of the steroidogenic genes steroidogenic acute regulatory protein, 3beta-hydroxysteroid dehydrogenase VI, and 17alpha-hydroxylase were markedly decreased by DFMO treatment in the developing placenta at d 9 and 10 of pregnancy. Plasma values of progesterone and androstenedione were also decreased by DFMO treatment. Transcriptomic analysis also detected changes in the expression of several genes involved in placentation and the differentiation of trophoblastic lineages. In conclusion, our results indicate that ODC inhibition at d 8 of pregnancy is related to alterations in yolk sac formation and trophoblast differentiation, affecting processes such as vasculogenesis and steroidogenesis.

Developmental Expression Pattern of Hspb8 mRNA in the Mouse Brain: Analysis Through Online Databases

Hspb8 is a member of the Hspb family of chaperone‐like proteins. It is involved in several neural disorders such as Alzheimers disease, amyotrophic lateral sclerosis, hereditary distal motor neuropathy, and Charcot‐Marie‐Tooths disease. In this work, we aimed to characterize its expression pattern in the mouse brain, by using the information available at online databases of high‐throughput in situ hybridization. Therefore, we downloaded and analyzed the image series from these databases showing Hspb8 mRNA expression from embryonic to adult and aging stages. In early gestational embryos, Hspb8 was expressed in the hippocampal anlagen and in the ventricular layer of rhombomere 4. At perinatal stages, there appeared transitory expression in the dentate gyrus and the cerebellar cortex. From perinatal to aging stages, the neurons of the mesencephalic trigeminal nucleus and cranial motor nuclei displayed stable and strong Hspb8 expression. Additionally, along these stages there was moderate and relatively homogenous expression in the anterodorsal thalamic, lateral mammillary, arcuate hypothalamic and medial habenular nuclei, and in the locus coeruleus. In its turn, the basal ganglia, cerebellar inner granular layer and diverse sensory and reticular formation nuclei of the hindbrain contained scattered cells with strong expression. In conclusion, Hspb8 mRNA is constitutively expressed in specific brain structures across ontogeny, so that eventually they could be affected by the malfunction or deregulation of this molecule. Anat Rec, 2012.

In silico identification of new candidate genes for hereditary congenital facial paresis.

Hereditary congenital facial paresis (HCFP) consists of the paralysis or weakness of facial muscles caused by a maldevelopment of the facial branchiomotor (FBM) nucleus and its nerve. Linkage analyses have related this disorder to two loci, HCFP1 and HCFP2, placed respectively in human chromosomes 3q21.2‐q22.1 and 10q21.3‐q22.1, but the causative genes are still unknown. In this work we aimed to identify which genes from these loci are expressed in the developing hindbrain and particularly in the FBM nucleus. To this end, we retrieved from the ENSEMBL genomic database the list of these genes as well as their respective mouse orthologs. Subsequently we examined their respective expression patterns in the mouse embryo by using the GenePaint gene expression database. As a result of this screening, we found a new gene (Mgll) from the HCFP1 locus that has strong and specific expression in the developing FBM nucleus. In its turn, the HCFP2 locus appeared as a large gene‐desert region, flanked by two genes, Reep3, with specific expression in the FBM nucleus, and Lrrtm3, broadly expressed in the brainstem, including the same nucleus. The concurrence of genomic position and neural expression pattern makes these genes new potential candidates for HCFP.

Expression patterns of Irx genes in the developing chick inner ear

The vertebrate inner ear is a complex three-dimensional sensorial structure with auditory and vestibular functions. The molecular patterning of the developing otic epithelium creates various positional identities, consequently leading to the stereotyped specification of each neurosensory and non-sensory element of the membranous labyrinth. The Iroquois (Iro/Irx) genes, clustered in two groups (A: Irx1, Irx2, and Irx4; and B: Irx3, Irx5, and Irx6), encode for transcriptional factors involved directly in numerous patterning processes of embryonic tissues in many phyla. This work presents a detailed study of the expression patterns of these six Irx genes during chick inner ear development, paying particular attention to the axial specification of the otic anlagen. The Irx genes seem to play different roles at different embryonic periods. At the otic vesicle stage (HH18), all the genes of each cluster are expressed identically. Both clusters A and B seem involved in the specification of the lateral and posterior portions of the otic anlagen. Cluster B seems to regulate a larger area than cluster A, including the presumptive territory of the endolymphatic apparatus. Both clusters seem also to be involved in neurogenic events. At stages HH24/25-HH27, combinations of IrxA and IrxB genes participate in the specification of most sensory patches and some non-sensory components of the otic epithelium. At stage HH34, the six Irx genes show divergent patterns of expression, leading to the final specification of the membranous labyrinth, as well as to cell differentiation.

20-P005 Study of the development of the interpeduncular nucleus evidences multiple origins in the rostral hindbrain

ume changes were influencing cell movement. We also observed that the shape of the explant influenced the direction of cell migration. We propose that increased cell volume provides the driving impetus for the start of cell migration. The enlarged cells produce points of dislocations, which are determined by the physical constraints of the tissue, and that foreshadow and dictate the direction of cell movement.