Emx1 and Emx2 cooperate in initial phase of archipallium development

Emx1 and Emx2 are mouse cognates of the Drosophila head gap gene, ems. Previously we have reported that the dentate gyrus is affected in Emx2 single mutants, and defects are subtle in Emx1 single mutants. In most of the cortical region Emx1 and Emx2 functions would be redundant. To test this assumption here we examined the Emx1 and Emx2 double mutant phenotype. In the double mutants the archipallium was transformed into the roof without establishing the signaling center at the cortical hem and without developing the choroid plexus. We propose that Emx1 and Emx2 cooperate in generation of the boundary between the roof and archipallium; these genes develop the archipallium against the roof. This process probably occurs immediately after the neural tube closure concomitant with the Emx1 expression.     

Gli3 is required for Emx gene expression during dorsal telencephalon development.

Dentate gyrus and hippocampus as centers for spatial learning, memory and emotional behaviour have been the focus of much interest in recent years. The molecular information on its development, however, has been relatively poor. To date, only Emx genes were known to be required for dorsal telencephalon development. Here, we report on forebrain development in the extra toes (Xt(J)) mouse mutant which carries a null mutation of the Gli3 gene. This defect leads to a failure to establish the dorsal di-telencephalic junction and finally results in a severe size reduction of the neocortex. In addition, Xt(J)/Xt(J) mice show absence of the hippocampus (Ammon's horn plus dentate gyrus) and the choroid plexus in the lateral ventricle. The medial wall of the telencephalon, which gives rise to these structures, fails to invaginate during embryonic development. On a molecular level, disruption of dorsal telencephalon development in Xt(J)/Xt(J) embryos correlates with a loss of Emx1 and Emx2 expression. Furthermore, the expression of Fgf8 and Bmp4 in the dorsal midline of the telencephalon is altered. However, expression of Shh, which is negatively regulated by Gli3 in the spinal cord, is not affected in the Xt(J)/Xt(J) forebrain. This study therefore implicates Gli3 as a key regulator for the development of the dorsal telencephalon and implies Gli3 to be upstream of Emx genes in a genetic cascade controlling dorsal telencephalic development.     

Emx2 directs the development of diencephalon in cooperation with Otx2

The vertebrate brain is among the most complex biological structures of which the organization remains unclear. Increasing numbers of studies have accumulated on the molecular basis of midbrain/hindbrain development, yet relatively little is known about forebrain organization. Nested expression among Otx and Emx genes has implicated their roles in rostral brain regionalization, but single mutant phenotypes of these genes have not provided sufficient information. In order to genetically determine the interaction between Emx and Otx genes in forebrain development, we have examined Emx2(-/-)Otx2(+/-) double mutants and Emx2 knock-in mutants into the Otx2 locus (Otx2(+/Emx2)). Emx2(-/-)Otx2(+/-) double mutants did not develop diencephalic structures such as ventral thalamus, dorsal thalamus/epithalamus and anterior pretectum. The defects were attributed to the loss of the Emx2-positive region at the three- to four-somite stage, when its expression occurs in the laterocaudal forebrain primordia. Ventral structures such as the hypothalamus, mammillary region and tegmentum developed normally. Moreover, dorsally the posterior pretectum and posterior commissure were also present in the double mutants. In contrast, Otx2(+/Emx2) knock-in mutants displayed the majority of these diencephalic structures; however, the posterior pretectum and posterior commissure were specifically absent. Consequently, development of the dorsal and ventral thalamus and anterior pretectum requires cooperation between Emx2 and Otx2, whereas Emx2 expression is incompatible with development of the commissural region of the pretectum.     

The role of Emx2 during scapula formation

The scapula is subdivided into head, collum, and blade. Due to the expression pattern of Emx2 and the absence of the scapula blade in Emx2 knockout mice, it has been suggested that Emx2 is involved in the formation of the scapula. Micromanipulation experiments revealed that ectoderm ablation over the somites does not affect Emx2 expression but inhibits the formation of the scapula blade indicating that Emx2 is not sufficient to induce scapula blade formation. Furthermore, we show that the formation of the scapula head is dependent, scapula blade formation independent of FGFR-1-mediated signaling. Overexpression of Emx2 does not influence scapula blade formation but leads to the development of an additional posterior digit in the anterior border of the limb. Taken together, the data presented implicate that Emx2 expression is necessary but not sufficient for the development of the scapula blade. It is not a marker for scapula development but rather provides positional information along the proximodistal and anterior-posterior limb axes, whereas the specificity of the developing skeletal elements is determined by the concerted interaction of Emx2 with other factors.     

Mouse forebrain development. The role of Emx2 homeobox gene

Over the last few years great progress has been made in the understanding of the formation of the mouse forebrain. Among the genes involved in this process, the mouse Emx homeobox genes Emx1 and particularly Emx2 play a primary role. Here we describe the mRNA and protein expression related to Emx2 in the developing mouse telencephalon, as well as the results obtained studying the corresponding knock-out mice. Our findings indicate a role for this gene in the specification of the forebrain via the control of cell proliferation, as well as in guiding neuronal migration during development through the cortical plate. These studies will hopefully enable us to better understand the molecular mechanisms underlying the formation of the mouse cerebral cortex as well as to establish relevant interactions between the various proteins present in this region of the brain.     

Patterns and consequences of vertebrate Emx gene duplications

SUMMARY We have cloned and analyzed two Emx genes from the lamprey Petromyzon marinus and our findings provide insight into the patterns and developmental consequences of gene duplications during early vertebrate evolution. The Emx gene family presents an excellent case for addressing these issues as gnathostome vertebrates possess two or three Emx paralogs that are highly pleiotropic, functioning in or being expressed during the development of several vertebrate synapomorphies. Lampreys are the most primitive extant vertebrates and characterization of their development and genomic organization is critical for understanding vertebrate origins. We identified two Emx genes from P. marinus and analyzed their phylogeny and their embryological expression relative to other chordate Emx genes. Our phylogenetic analysis shows that the two lamprey Emx genes group independently from the gnathostome Emx1, Emx2 , and Emx3 paralogy groups. Our expression analysis shows that the two lamprey Emx genes are expressed in distinct spatial and temporal patterns that together broadly encompass the combined sites of expression of all gnathostome Emx genes. Our data support a model wherein large-scale regulatory evolution of a single Emx gene occurred after the protochordate/vertebrate divergence, but before the vertebrate radiation. Both the lamprey and gnathostome lineages then underwent independent gene duplications followed by extensive paralog subfunctionalization. Emx subfunctionalization in the telencephalon is remarkably convergent and refines our understanding of lamprey forebrain patterning. We also identify lamprey-specific sites of expression that indicate either neofunctionalization in lampreys or sites-specific nonfunctionalization of all gnathostome Emx genes. Overall, we see only very limited correlation between Emx gene duplications and the acquisition of novel expression domains.     

Choroid plexus and paraphysis in lower vertebrates

Cytoarchitecture of the choroid plexus of the third ventricle and the paraphysis was investigated in some lower vertebrates to compare the histologic characteristics of these organs. Both epithelia are similar in appearance in the same class. Minor microscopic variations exist in the different classes of vertebrates, but do not provide a fundamental distinction between the two organs. The epithelia, moreover, have similar staining properties, contain mucicarmine- and PAS-reactive materials, and are derived from a common neuroepithelium. Tubules are identified in the choroid plexus and in the paraphysis; all are similarly formed by simple folding of epithelium on the surface into the stroma. The paraphyses in all vertebrates studied contain villi similar to those seen in the choroid plexus. Cilia are identified in both choroidal and paraphyseal epithelia, and are not an indication of degree of epithelial differentiation. Many types of epithelium are noted in both organs during histologic differentiation as well as in the mature stage. Functionally, the choroid plexus is active in both secretion and absorption. Accumulation of particulate material within the epithelial cytoplasm may indicate phagocytic as well as absorptive activity of cells. Based on a common neuroepithelial origin and similar histochemical properties, we conclude that the paraphysis is a modified choroid plexus. The velum transversum is an arbitrary boundary between diencephalon and telencephalon, and is itself formed of choroid plexus. The medial telencephalic ventricle is the rostral portion of the third ventricle. All neuroepithelial infoldings at the rostral end of the diencephalic roof including the velum transversum are intraventricular choroid plexuses; the neuroepithelial outpouchings in this region are the extraventricular choroid plexuses (paraphysis) of the diencephalon.     

Emx2 and early hair cell development in the mouse inner ear

Emx2 is a homeodomain protein that plays a critical role in inner ear development. Homozygous null mice die at birth with a range of defects in the CNS, renal system and skeleton. The cochlea is shorter than normal with about 60% fewer auditory hair cells. It appears to lack outer hair cells and some supporting cells are either absent or fail to differentiate. Many of the hair cells differentiate in pairs and although their hair bundles develop normally their planar cell polarity is compromised. Measurements of cell polarity suggest that classic planar cell polarity molecules are not directly influenced by Emx2 and that polarity is compromised by developmental defects in the sensory precursor population or by defects in epithelial cues for cell alignment. Planar cell polarity is normal in the vestibular epithelia although polarity reversal across the striola is absent in both the utricular and saccular maculae. In contrast, cochlear hair cell polarity is disorganized. The expression domain for Bmp4 is expanded and Fgfr1 and Prox1 are expressed in fewer cells in the cochlear sensory epithelium of Emx2 null mice. We conclude that Emx2 regulates early developmental events that balance cell proliferation and differentiation in the sensory precursor population.     

Hes genes and neurogenin regulate non-neural versus neural fate specification in the dorsal telencephalic midline

The choroid plexus in the brain is unique because it is a non-neural secretory tissue. It secretes the cerebrospinal fluid and functions as a blood-brain barrier, but the precise mechanism of specification of this non-neural tissue has not yet been determined. Using mouse embryos and lineage-tracing analysis, we found that the prospective choroid plexus region initially gives rise to Cajal-Retzius cells, specialized neurons that guide neuronal migration. Inactivation of the bHLH repressor genes Hes1, Hes3 and Hes5 upregulated expression of the proneural gene neurogenin 2 (Ngn2) and prematurely depleted Bmp-expressing progenitor cells, leading to enhanced formation of Cajal-Retzius cells and complete loss of choroid plexus epithelial cells. Overexpression of Ngn2 had similar effects. These data indicate that Hes genes promote specification of the fate of choroid plexus epithelial cells rather than the fate of Cajal-Retzius cells by antagonizing Ngn2 in the dorsal telencephalic midline region, and thus this study has identified a novel role for bHLH genes in the process of deciding which cells will have a non-neural versus a neural fate.