Temporal variations in early developmental decisions: an engine of forebrain evolution.

Tight control of developmental timing is pivotal to many major processes in developmental biology, such as patterning, fate specification, cell cycle dynamics, cell migration and connectivity. Temporal change in these ontogenetic sequences is known as heterochrony, a major force in the evolution of body plans and organogenesis. In the last 5 years, studies in fish and rodents indicate that heterochrony in signaling during early development generates diversity in forebrain size and complexity. Here, we summarize these findings and propose that, additionally to spatio-temporal tuning of neurogenesis, temporal and quantitative modulation of signaling events drive pivotal changes in shape, size and complexity of the forebrain across evolution, participating to the generation of diversity in animal behavior and emergence of cognition.

Cost-effectiveness of voluntary HIV screening in Russia.

Russia has one of the world's fastest growing HIV epidemics, and HIV screening has been widespread. Whether such screening is an effective use of resources is unclear. We used epidemiologic and economic data from Russia to develop a Markov model to estimate costs, quality of life and survival associated with a voluntary HIV screening programme compared with no screening in Russia. We measured discounted lifetime health-care costs and quality-adjusted life years (QALYs) gained. We varied our inputs in sensitivity analysis. Early identification of HIV through screening provided a substantial benefit to persons with HIV, increasing life expectancy by 2.1 years and 1.7 QALYs. At a base-case prevalence of 1.2%, once-per-lifetime screening cost $13,396 per QALY gained, exclusive of benefit from reduced transmission. Cost-effectiveness of screening remained favourable until prevalence dropped below 0.04%. When HIV-transmission-related costs and benefits were included, once-per-lifetime screening cost $6910 per QALY gained and screening every two years cost $27,696 per QALY gained. An important determinant of the cost-effectiveness of screening was effectiveness of counselling about risk reduction. Early identification of HIV infection through screening in Russia is effective and cost-effective in all but the lowest prevalence groups.

Detailed field pattern is intrinsic to the embryonic mouse hippocampus early in neurogenesis.

There is accumulating evidence that the mammalian cerebral cortex is regionally specified early in neurogenesis. However, the degree and scale of the regional pattern that is intrinsic to different parts of the cortical primordium remains unclear. Here, we show that detailed patterning-the accurate positioning of several areas or fields-is intrinsic to the part of the primordium that generates the hippocampus. A caudomedial portion of the cortical primordium, the site from which the hippocampus arises, was isolated from potential extrinsic patterning cues by maintaining it in explant culture. Explants were prepared at embryonic day (E) 12.5, which is early in hippocampal neurogenesis in the mouse and 3 d before individual fields are seen by differential gene expression. Allowed to develop for 3 d in vitro, E12.5 explants upregulate field-specific patterns of gene expression with striking temporal and spatial accuracy. Possible sources of patterning signals intrinsic to the explants were evaluated by removing the cortical hem or presumptive extrahippocampal cortex from the explants. To expose cells to different local positional cues, explant fragments were grafted into ectopic positions in a larger explant. None of these manipulations altered the development of patterned, field-specific gene expression. Finally, explants harvested at E10.5 also upregulate field-specific gene expression, although less robustly. Some hippocampal patterning information is therefore intrinsic to the caudomedial cortical primordium at the time that the first hippocampal neurons are born at E10.5. By E12.5, hippocampal field patterning appears to be well established and resistant to the manipulation of several potential intrinsic cues.

LIM-homeodomain gene Lhx2 regulates the formation of the cortical hem.

We are interested in the early mechanisms that initiate regional patterning in the dorsal telencephalon, which gives rise to cerebral cortex. Members of the LIM-homeodomain (LIM-HD) family of transcription factors are implicated in patterning and cell fate specification in several systems including the mammalian forebrain. Mice in which Lhx2 is disrupted were reported to have reduced telencephalic development, and the hippocampal primordium appeared to be missing, by morphological observation. We hypothesized that this may be due to a defect in the cortical hem, a Wnt- and Bmp-rich putative signaling center in the medial telencephalon, a source of regulatory signals for hippocampal development. We asked if the expression of any known hem-specific signaling molecule is deficient in Lhx2-/- mice. Our results reveal, unexpectedly, that at embryonic day (E)12.5, what appears to be some spared 'lateral' cortex is instead an expanded cortical hem. Normally restricted to the extreme medial edge of the telencephalon, the hem covers almost the entire dorsal telencephalon in the Lhx2-/- mice. This indicates a role for Lhx2 in the regulation of the extent of the cortical hem. In spite of an expanded, mislocated hem in the Lhx2-/- telencephalon, a potential source of ectopic dorsalizing cues, no hippocampal differentiation is detected in tissue adjacent to the mutant hem, nor does the overall dorsoventral patterning appear perturbed. We propose that Lhx2 is involved at a crucial early step in patterning the telencephalon, where the neuroepithelium is first divided into presumptive cortical tissue, and the cortical hem. The defect in the Lhx2-/- telencephalon appears to be at this step.

Emx2 is required for growth of the hippocampus but not for hippocampal field specification.

The vertebrate Emx genes are expressed in a nested pattern in early embryonic cerebral cortex, such that a medial strip of cortex expresses Emx2 but not Emx1. This pattern suggests that Emx genes could play a role in specifying different areas or fields of the cortex along the mediolateral axis. Such a role has been supported by the observation that in mice lacking functional Emx2 the hippocampus is shrunken and the most medial field of the cortex, the hippocampal dentate gyrus, appears by cytoarchitecture to be missing (Pellegrini et al., 1996; Yoshida et al., 1997). Use of region-specific molecular markers shows, however, that hippocampal fields are specified and correctly positioned in the Emx2 mutant. In particular, a dentate cell population is generated, although it fails to form a morphological gyrus. This failure may be part of a more widespread medial cortical defect in the mutant. Examination of cortical cell proliferation and differentiation indicates a disruption of the maturation of the medial cortex in the absence of Emx2. Thus, Emx2 is required for normal growth and maturation of the hippocampus but not for the specification of cells to particular hippocampal field identities.

Dorsoventral patterning of the telencephalon is disrupted in the mouse mutant extra-toes(J).

Little is known about the mechanisms that control the development of regional identity in the mammalian telencephalon. The Gli family of transcription factor genes is involved in the regulation of pattern at many sites in the embryo and is expressed in the embryonic mouse telencephalon. We have analyzed telencephalic patterning in the extra-toes (J) (Xt(J)) mouse mutant, which carries a deletion in the Gli family member Gli3. We report that dorsoventral patterning of the telencephalon is dramatically disrupted in the Xt(J) mutant. Specific dorsal telencephalic cell types and gene expression patterns are lost in homozygous Xt(J) mutants, and features of ventral telencephalic identity develop ectopically in the dorsal telencephalon. This partial ventralization of the dorsal telencephalon does not appear to be induced by an expansion of Sonic hedgehog expression in the telencephalon, but may be due to a loss of Bmp and Wnt gene expression in a putative dorsal telencephalic signaling center, the cortical hem. Our findings suggest that in dorsal telencephalon Gli3 is needed to repress ventral telencephalic identity.

A local Wnt-3a signal is required for development of the mammalian hippocampus.

The mechanisms that regulate patterning and growth of the developing cerebral cortex remain unclear. Suggesting a role for Wnt signaling in these processes, multiple Wnt genes are expressed in selective patterns in the embryonic cortex. We have examined the role of Wnt-3a signaling at the caudomedial margin of the developing cerebral cortex, the site of hippocampal development. We show that Wnt-3a acts locally to regulate the expansion of the caudomedial cortex, from which the hippocampus develops. In mice lacking Wnt-3a, caudomedial cortical progenitor cells appear to be specified normally, but then underproliferate. By mid-gestation, the hippocampus is missing or represented by tiny populations of residual hippocampal cells. Thus, Wnt-3a signaling is crucial for the normal growth of the hippocampus. We suggest that the coordination of growth with patterning may be a general role for Wnts during vertebrate development.

Patterning events and specification signals in the developing hippocampus.

The mouse hippocampus is an attractive model system in which to study patterning of a cortical structure. Ongoing studies indicate that hippocampal areas or fields are specified many days before birth -- possibly involving signals from within the cortical mantle. Although the hippocampal CA fields are distinguished by cytoarchitecture only after birth, molecular differences between fields appear by late gestation. Moreover, these embryonic fields are already specified to develop additional features that characterize the mature fields. The basic division of the hippocampus into fields may be specified still earlier. Thus, if medial cortical neuroepithelium is isolated in vitro early in hippocampal neurogenesis, it can autonomously generate features of a patterned hippocampus. In vivo, the spatial progression of initial field differentiation suggests that signals regulating growth and patterning could arise from sources close to the hippocampal poles. Observations of mouse mutants indicate that the cortical hem, an embryonic structure close to one pole of the hippocampus, is a source of such regulatory signals.

The hem of the embryonic cerebral cortex is defined by the expression of multiple Wnt genes and is compromised in Gli3-deficient mice.

In the developing vertebrate CNS, members of the Wnt gene family are characteristically expressed at signaling centers that pattern adjacent parts of the neural tube. To identify candidate signaling centers in the telencephalon, we isolated Wnt gene fragments from cDNA derived from embryonic mouse telencephalon. In situ hybridization experiments demonstrate that one of the isolated Wnt genes, Wnt7a, is broadly expressed in the embryonic telencephalon. By contrast, three others, Wnt3a, 5a and a novel mouse Wnt gene, Wnt2b, are expressed only at the medial edge of the telencephalon, defining the hem of the cerebral cortex. The Wnt-rich cortical hem is a transient, neuron-containing, neuroepithelial structure that forms a boundary between the hippocampus and the telencephalic choroid plexus epithelium (CPe) throughout their embryonic development. Indicating a close developmental relationship between the cortical hem and the CPe, Wnt gene expression is upregulated in the cortical hem both before and just as the CPe begins to form, and persists until birth. In addition, although the cortical hem does not show features of differentiated CPe, such as expression of transthyretin mRNA, the CPe and cortical hem are linked by shared expression of members of the Bmp and Msx gene families. In the extra-toesJ (XtJ) mouse mutant, telencephalic CPe fails to develop. We show that Wnt gene expression is deficient at the cortical hem in XtJ/XtJ mice, but that the expression of other telencephalic developmental control genes, including Wnt7a, is maintained. The XtJ mutant carries a deletion in Gli3, a vertebrate homolog of the Drosophila gene cubitus interruptus (ci), which encodes a transcriptional regulator of the Drosophila Wnt gene, wingless. Our observations indicate that Gli3 participates in Wnt gene regulation in the vertebrate telencephalon, and suggest that the loss of telencephalic choroid plexus in XtJ mice is due to defects in the cortical hem that include Wnt gene misregulation.

Early specification and autonomous development of cortical fields in the mouse hippocampus.

Studies of the specification of distinct areas in the developing cerebral cortex have until now focused mainly on neocortex. We demonstrate that the hippocampus, an archicortical structure, offers an elegant, alternative system in which to explore cortical area specification. Individual hippocampal areas, called CA fields, display striking molecular differences in maturity. We use these distinct patterns of gene expression as markers of CA field identity, and show that the two major hippocampal fields, CA1 and CA3, are specified early in hippocampal development, during the period of neurogenesis. Two field-specific markers display consistent patterns of expression from the embryo to the adult. Presumptive CA1 and CA3 fields (Pca1, Pca3) can therefore be identified between embryonic days 14.5 and 15.5 in the mouse, a week before the fields are morphologically distinct. No other individual cortical areas have been detected by gene expression as early in development. Indeed, other features that distinguish between the CA fields appear after birth, indicating that mature CA field identity is acquired over at least 3 weeks. To determine if Pca1 and Pca3 are already specified to acquire mature CA field identities, the embryonic fields were isolated from further potential specification cues by maintaining them in slice culture. CA field development proceeds in slices of the entire embryonic hippocampus. More strikingly, slices restricted to Pca1 or Pca3 alone also develop appropriate mature features of CA1 or CA3. Pca1 and Pca3 are therefore able to develop complex characteristics of mature CA field identity autonomously, that is, without contact or innervation from other fields or other parts of the brain. Because Pca1 and Pca3 can be identified before major afferents grow into the hippocampus, innervation may also be unnecessary for the initial division of the hippocampus into separate fields. Providing a clue to the source of the true specifying signals, the earliest field markers appear first at the poles of the hippocampus, then progress inwards. General hippocampal development does not follow this pronounced pattern. We suggest that the sources of signals that specify hippocampal field identity lie close to the hippocampal poles, and that the signals operate first on cells at the poles, then move inwards.

Regionalization of the developing forebrain: a comparison of FORSE-1, Dlx-2, and BF-1.

The FORSE-1 monoclonal antibody (mAb) was generated using a strategy designed to produce mAbs against neuronal cell surface antigens that might be regulated by regionally restricted transcription factors in the developing CNS. To determine whether FORSE-1 has a labeling pattern similar to that of known transcription factors, the expression of BF-1 and Dlx-2 was examined by in situ hybridization on sections serial to those labeled with FORSE-1. We find a striking overlap between BF-1 and FORSE-1 in the telencephalon; both are expressed in the lateral but not the medial walls of the telencephalon, and the boundaries of expression are apparently identical. FORSE-1 staining is detected prior to BF-1 expression in the neural tube, however. FORSE-1 and Dlx-2 have very different patterns of expression in the forebrain, suggesting that regulation by Dlx-2 cannot by itself explain the distribution of FORSE-1. However, they share some sharp boundaries in the diencephalon. In addition, FORSE-1 identifies some previously unknown boundaries in the developing forebrain. These results indicate that a new cell surface marker can be used to subdivide the embryonic telencephalon and diencephalon into regions smaller than previously described, providing necessary complexity to the developmental patterning in the forebrain.

FORSE-1: a positionally regulated epitope in the developing rat central nervous system.

We designed a protocol to identify cell surface molecules expressed in restricted spatial patterns in the developing central nervous system (CNS) that might be regulated by regionally restricted transcription factors. The immunogen was a membrane fraction from NT2/D1 embryocarcinoma cells that were induced to differentiate into neurons and upregulate Hox gene expression in response to retinoic acid. One monoclonal antibody (mAb), FORSE-1, specifically labels the rostral rat CNS from the earliest stages. Staining is observed in the rostral but not caudal neural folds of the embryo prior to neural tube closure. Staining is enriched in the forebrain as compared to the rest of the CNS, until E18. Between E11.5 and E13.5, only certain areas of the telencephalon and diencephalon are labeled. Later, up to E17.5, FORSE-1 labeling is specifically restricted to the telencephalon, where a correlation with mitotic activity is apparent: the ventricular zone labels with FORSE-1, while the cortical plate is negative. The staining of the neuroepithelium is intensified by acetone fixation, which also reveals, between E11.5 and E13.5, a dorsoventrally restricted, FORSE-1-positive region of the spinal cord. After E18, the entire CNS is labeled, through adulthood. The mAb labels the surfaces of dissociated, living cells. Other, non-CNS areas of FORSE-1 labeling are nasal and otic placodes, nasal epithelium, nasal glands, and early (E9.5-10.5) endoderm. mAb FORSE-1 recognizes an epitope present on both a high-molecular-weight (> 200 kDa) proteoglycan from embryonic and early postnatal brain, and on a 80 kDa doublet that is restricted to the CNS in the adult. These findings suggest the FORSE-1 antigen as a candidate cell surface molecule for mediating regional specification from the earliest stages of CNS development.

Distribution of CD9 in the developing and mature rat nervous system.

CD9 is a cell surface protein implicated in intercellular signaling that has been identified in selected cell types of the hematopoietic system. To begin a study of the role of CD9 in the developing and adult nervous system, we used the anti-rat CD9 monoclonal antibody ROCA2 to determine the distribution of this protein. The identity of the antigen in these tissues was confirmed by immunoblotting and peptide sequencing. Early embryonic sympathetic and dorsal root ganglion sensory neurons and adrenal chromaffin cells all express CD9. ROCA2 also labels the somas, axons, and growth cones of cultured sympathetic and sensory neurons. In the central nervous system (CNS), CD9 is transiently and specifically expressed in embryonic spinal motoneurons. In the adult, central and peripheral glia intensely express CD9. Thus, CD9 is developmentally regulated in a variety of peripheral and central neurons and glia, including proliferating progenitors as well as mature cells. These findings suggest that CD9 may have diverse roles in the nervous system.