Zona Limitans Intrathalamica Research Articles

Development of the serotonergic system in the central nervous system of the sea lamprey

Lampreys belong to the most primitive extant group of vertebrates, the Agnathans, which is considered the sister group of jawed vertebrates. Accordingly, characterization of neuronal groups and their development appears useful for understanding early evolution of the nervous system in vertebrates. Here, the development of the serotonergic system in the central nervous system of the sea lamprey, Petromyzon marinus, was investigated by immunohistochemical analysis of specimens ranging from embryos to adults. The different serotonin-immunoreactive (5-HT-ir) neuronal populations that are found in adults were observed between the embryonic and metamorphic stages. The earliest serotonergic neurons were observed in the basal plate of the isthmus region of late embryos. In prolarvae, progressive appearance of new serotonergic cell groups was observed: firstly in the spinal cord, then in the pineal organ, tuberal region, zona limitans intrathalamica, rostral isthmus, and the caudal part of the rhombencephalon. In early larvae a new group of serotonergic cells was observed in the mammillary region, whereas in the pretectal region and the parapineal organ the first serotonergic cells were seen in the middle and late larval stages, respectively. The first serotonergic fibres appeared in early prolarvae, with fibres that ascend and descend from the isthmic cell group, and the number of immunoreactive fibres increased progressively until the adult stage. The results reveal strong resemblances between lampreys and other vertebrates in the spatio-temporal pattern of development of brainstem populations. This study also reveals a shared pattern of early ascending and descending serotonergic pathways in lampreys and jawed vertebrates.

Patterning the zebrafish diencephalon by the conserved zinc-finger protein Fezl

The forebrain constitutes the most anterior part of the central nervous system, and is functionally crucial and structurally conserved in all vertebrates. It includes the dorsally positioned telencephalon and eyes, the ventrally positioned hypothalamus, and the more caudally located diencephalon [from rostral to caudal: the prethalamus, the zona limitans intrathalamica (ZLI), the thalamus and the pretectum]. Although antagonizing Wnt proteins are known to establish the identity of the telencephalon and eyes, it is unclear how various subdivisions are established within the diencephalon--a complex integration center and relay station of the vertebrate brain. The conserved forebrain-specific zinc-finger-containing protein Fezl plays a crucial role in regulating neuronal differentiation in the vertebrate forebrain. Here, we report a new and essential role of zebrafish Fezl in establishing regional subdivisions within the diencephalon. First, reduced activity of fezl results in a deficit of the prethalamus and a corresponding expansion of the ZLI. Second, Gal4-UAS-mediated fezl overexpression in late gastrula is capable of expanding the prethalamus telencephalon and hypothalamus at the expense of the ZLI and other fore- and/or mid-brain regions. Such altered brain regionalization is preceded by the early downregulation of wnt expression in the prospective diencephalon. Finally, fezl overexpression is able to restore the anterior forebrain and downregulate wnt expression in Headless- and/or Tcf3 (also known as Tcf7l1a)-deficient embryos. Our findings reveal that Fezl is crucial for establishing regional subdivisions within the diencephalon and may also play a role in the development of the telencephalon and hypothalamus.

Cooperation of sonic hedgehog enhancers in midline expression

In zebrafish, as in other vertebrates, the secreted signalling molecule Sonic hedgehog (Shh) is expressed in organiser regions such as the embryonic midline and the zona limitans intrathalamica (zli). To investigate the regulatory mechanisms underlying the pattern of shh expression, we carried out a systematic analysis of the intronic regulatory sequences of zebrafish shh using stable transgenesis. Deletion analysis identified the modules responsible for expression in the embryonic shield, the hypothalamus and the zli and confirmed the activities of previously identified notochord and floor plate enhancers. We detected a strong synergism between regulatory regions. The degree of synergy varied over time in the hypothalamus suggesting different mechanisms for initiation and maintenance of expression. Our data show that the pattern of shh expression in the embryonic central nervous system involves an intricate crosstalk of at least 4 different regulatory regions. When compared to the enhancer activities of the mouse Shh gene, we observed a remarkable divergence of function of structurally conserved enhancer sequences. The activating region ar-C (61% identical to SFPE2 in mouse Shh), for example, mediates floor plate expression in the mouse embryo while it directs expression in the forebrain and the notochord and only weakly in the floor plate in the zebrafish embryo. This raises doubts on the predictive power of phylogenetic footprinting and indicates a stunning divergence of function of structurally conserved regulatory modules during vertebrate evolution.

Sonic hedgehog from the basal plate and the zona limitans intrathalamica exhibits differential activity on diencephalic molecular regionalization and nuclear structure

The diencephalon is the most complex area of the vertebrate brain, being particularly complex in amniotes. It has been suggested that diencephalic regionalization partially depends on local signaling mediated by sonic hedgehog (Shh). However, since the Shh gene is expressed in both the diencephalic basal plate and the zona limitans intrathalamica (ZLI), it is still unclear which of these tissues exerts morphogenetic influence on thalamic regionalization. In the present study using chick and quail embryos, we have found that although Shh from the ZLI and the basal plate induces ectopic expression of diencephalic genes in the posterior prosencephalic alar plate, only Shh originating from the ZLI can induce ectopic gene expression in the anterior alar plate, indicating that the ZLI exerts specific activity in the anterior epithelium. By introducing microbarriers between the diencephalic alar neuroepithelium and either the ZLI or the basal plate, we generated local loss of Shh expression in the ZLI, leading to alterations in molecular regionalization and subsequently, in the nuclear organization of the alar diencephalic derivatives on both sides of the ZLI. We thus demonstrate in vivo that basal signals are required to induce Shh expression in the ZLI and that Shh from the ZLI plays a pivotal role in regionalizing the alar diencephalon. The structural phenotype of Shh abolition in the ZLI consisted of a progressive pattern of alterations in diencephalic organization which were associated with the observed gradient effects in the molecular regionalization of the diencephalon. We conclude that the ZLI is a secondary organizer which exerts its morphogenetic activity through Shh.

Zinc-finger genesFezandFez-likefunction in the establishment of diencephalon subdivisions

Fez and Fez-like (Fezl) are zinc-finger genes that encode transcriptional repressors expressed in overlapping domains of the forebrain. By generating Fez;Fezl-deficient mice we found that a redundant function of Fez and Fezl is required for the formation of diencephalon subdivisions. The caudal forebrain can be divided into three transverse subdivisions: prethalamus (also called ventral thalamus), thalamus (dorsal thalamus) and pretectum. Fez;Fezl-deficient mice showed a complete loss of prethalamus and a strong reduction of the thalamus at late gestation periods. Genetic marker analyses revealed that during early diencephalon patterning in Fez;Fezl-deficient mice, the rostral diencephalon (prospective prethalamus) did not form and the caudal diencephalon (prospective thalamus and pretectum) expanded rostrally. Fez;Fezl-deficient mice also displayed defects in the formation of the zona limitans intrathalamica (ZLI), which is located on the boundary between the prethalamus and thalamus. Fez and Fezl are expressed in the region rostral to the rostral limit of Irx1 expression, which marks the prospective position of the ZLI. Transgene-mediated misexpression of Fezl or Fez caudal to the ZLI repressed the caudal diencephalon fate and affected the formation of the Shh-expressing ZLI. These data indicate that Fez and Fezl repress the caudal diencephalon fate in the rostral diencephalon, and ZLI formation probably depends on Fez/Fezl-mediated formation of diencephalon subdivisions.

Hedgehog signalling from the zona limitans intrathalamica orchestrates patterning of the zebrafish diencephalon

Midway between the anterior neural border and the midbrain-hindbrain boundary, two well-known local signalling centres in the early developing brain, is a further transverse boundary with putative signalling properties -- the zona limitans intrathalamica (ZLI). Here, we describe formation of the ZLI in zebrafish in relation to expression of sonic hedgehog (shh) and tiggy-winkle hedgehog (twhh), and to development of the forebrain regions that flank the ZLI: the prethalamus and thalamus. We find that enhanced Hh signalling increases the size of prethalamic and thalamic gene expression domains, whereas lack of Hh signalling leads to absence of these domains. In addition, we show that shh and twhh display both unique and redundant functions during diencephalic patterning. Genetic ablation of the basal plate shows that Hh expression in the ZLI alone is sufficient for diencephalic differentiation. Furthermore, acquisition of correct prethalamic and thalamic gene expression is dependent on direct Hh signalling. We conclude that proper maturation of the diencephalon requires ZLI-derived Hh signalling.

EphA7 receptor is expressed differentially at chicken prosomeric boundaries

We reexamined tyrosine-kinase receptor EphA7 RNA signal in embryonic chicken forebrain, to clarify its topographic relationships with early regionalization processes, such as establishment of prosomeric boundaries. After neurulation, uniform alar expression appears across prospective prosomeres prosomere 1, prosomere 2 and prosomere 3 (prethalamus, thalamus and pretectum). This pattern soon changes by differential downregulation at or in between some of the prosomeric boundaries in an individual pattern for each limit, and by expansion of expression into the rostral midbrain. The secondary distribution highlights various transversal and longitudinal domains, notably the zona limitans intrathalamica and the pretectum limits, as well as two longitudinal bands in the basal plate, termed paramedian and parabasal. Strong expression of EphA7 appears at the mammillary pouch and a supramammillary tegmental arch from stage Hamburger and Hamilton stages 14–15 onwards. At the end of the developmental period examined, expression of EphA7 in the ventricular zone decreases generally (with some exceptions) and novel expression domains start to appear in the mantle layer, initiating a third phase of differential expression. Thus, while the expression of EphA7 does not show a fixed functional or topographic relationship to prosomeric boundaries, sequential transcription changes during chicken development are consistent with a differential involvement of the diverse interprosomeric boundaries, as well as dorsoventral patterning organizers, in the regulation of EphA7 expression.

Organisation of the lamprey ( Lampetra fluviatilis) embryonic brain: Insights from LIM-homeodomain, Pax and hedgehog genes

To investigate the embryonic development of the central nervous system of the lamprey Lampetra fluviatilis, we have isolated and analysed the expression patterns of members of the LIM-homeodomain, Pax, Hedgehog and Nkx2.1 families. Using degenerate RT-PCR, single representatives of Lhx1/Lhx5, Lhx2/Lhx9, Pax3/Pax7 and Hedgehog families could be isolated in L. fluviatilis. Expression analysis revealed that the lamprey forebrain presents a clear neuromeric pattern. We describe the existence of 4 embryonic diencephalic prosomeres whose boundaries can be identified by the combined and relative expressions of LfPax37, LfLhx15 and LfLhx29. This suggests that the embryonic lamprey and gnathostome forebrain are patterned in a highly similar manner. Moreover, analysis of the LfHh gene, which is expressed in the hypothalamus, zona limitans intrathalamica and floor plate, reveals the possible molecular origin of this neuromeric brain pattern. By contrast, LfHh and LfNkx2.1 expressions suggest major differences in patterning mechanisms of the ventral telencephalon when compared to gnathostomes. In summary, our findings highlight a neuromeric organisation of the embryonic agnathan forebrain and point to the possible origin of this organisation, which is thus a truly vertebrate character. They also suggest that Hh/Shh midline signalling might act as a driving force for forebrain evolution.

Thalamic development induced by Shh in the chick embryo

Patterning of the early neural tube is achieved in part by the inductive signals, which arise from neuroepithelial signaling centers. The zona limitans intrathalamica (ZLI) is a neuroepithelial domain in the alar plate of the diencephalon which separates the prethalamus from the thalamus. The ZLI has recently been considered to be a possible secondary organizer, effecting its inductions via sonic hedgehog (Shh), a signaling molecule which drives morphogenetic information for the thalamus. Using experimental embryological techniques involving the generation of chimeric embryos, we show that the formation of the ZLI in the diencephalic alar plate is due to an interaction between the prechordal and epichordal plate neuroepithelia. We also provide evidence that Shh expression in the ZLI underlies the morphogenetic activity of this putative diencephalic organizer. Ectopic Shh led to the auto-induction of its own gene expression in host cells, as well as to the expression of other genes involved in diencephalic regionalization and histogenesis. Analysis of long-term surviving embryos after Shh ectopic expression demonstrated that Shh was able to induce thalamic structures and local overgrowth. Overall, these results indicate that Shh expressed in the ZLI plays an important role in diencephalic growth and in the development of the thalamus.

The tegmental proliferation region in the sea lamprey

The diencephalic/midbrain tegmental domain of the developing lamprey was characterized by the special features of the ventricular zone and distribution of some postmitotic neuronal populations, using proliferating cell nuclear antigen (PCNA) and HNK-1 immunocytochemistry. In late prolarvae and early larvae, the tegmental ventricular zone differentiated a striking arrangement of thin longitudinal crests with strongly PCNA-immunoreactive cells protruding toward the ventricle and separated by shallow valleys whose cells were faintly or moderately PCNA-immunoreactive. The tegmental ventricular zone was composed of a large caudal region located ventral to the pretectum and the midbrain tectum, and of a smaller rostral wedge-shaped region that extended dorsally between the dorsal and the ventral thalamus, in the last one, ventricular crests coursing in the zona limitans intrathalamica. In other diencephalic and midbrain regions, the ventricular zone lacked this crest-like organization and exhibited different smooth-surfaced domains rather homogeneous in thickness and appearance. The distribution of HNK-1-immunoreactive cell populations in the tegmental domain appears to be closely related with the pattern of proliferation crests. Together, our results reveal a shared basic organization in the tegmental domains of the diencephalon and midbrain of developing lamprey, indicating early appearance of the domain in vertebrate phylogeny.

Shh-dependent formation of the ZLI is opposed by signals from the dorsal diencephalon

The zona limitans intrathalamica (ZLI) is located at the border between the prospective ventral thalamus and dorsal thalamus, and functions as a diencephalic signaling center. Little is known about the mechanism controlling ZLI formation. Using a combination of fate-mapping studies and in vitro assays, I show that the differentiation of the ZLI from progenitor cells in the alar plate is initiated by a Shh-dependent signal from the basal plate. The subsequent dorsal progression of ZLI differentiation requires ongoing Shh signaling, and is constrained by inhibitory factors derived from the dorsal diencephalon. These studies demonstrate that self-organizing signals from the basal plate regulate the formation of a potential patterning center in the ZLI in an orthogonal orientation in the alar plate, and thus create the potential for coordinated thalamic patterning in two dimensions.

Hedgehog signaling from the ZLI regulates diencephalic regional identity

The zona limitans intrathalamica (ZLI), a narrow compartment in the vertebrate forebrain that bisects the diencephalon transversely, expresses the secreted factor sonic hedgehog (Shh). Because genetic disruption of Shh in mouse causes severe early developmental defects, this strategy has not been useful in identifying a ZLI-specific role for this gene. To modulate Shh signaling in a spatiotemporally restricted manner, we carried out gain- and loss-of-function experiments in chick embryos using in ovo electroporation and found that Shh signaling is required for region-specific gene expression in thalamus and prethalamus, the major diencephalic brain areas flanking the ZLI. We further show that differential competence of thalamic and prethalamic primordia in responding to Shh signaling is regulated by the transcription factor Irx3. We show that, through the release of Shh, the ZLI functions as a local signaling center that regulates the acquisition of identity for these important diencephalic regions.

Craniofacial development in the talpid3 chicken mutant

The talpid 3 chicken mutant has a pleiotropic phenotype including polydactyly and craniofacial abnormalities. Limb polydactyly in talpid 3 suggests a gain of Hedgehog (Hh) signaling, whereas, paradoxically, absence of midline facial structures suggests a loss of Hh function. Here we analyze the status of Shh signaling in the talpid 3 mutant head. We show that Shh expression domains are lost from the talpid 3 head—in hindbrain, midbrain, zona limitans intrathalamica, and stomodeal ectoderm—and that direct targets of Hedgehog signaling, Ptc1, Ptc2, and Gli1, are also absent even in areas associated with primary Shh expression. These data suggest that the talpid 3 mutation leads to defective activation of the Shh pathway and, furthermore, that tissue-to-tissue transduction of Shh expression in the developing head depends on Hh pathway activation. Failure to activate the Shh pathway can also explain absence of floor plate and Hnf-3β and Netrin-1 expression in midbrain and hindbrain and absence of Fgf-8 expression in commissural plate. Other aspects of gene expression in the talpid 3 head, however, suggest misspecification, such as maintenance of floor plate-like gene expression in telencephalon. In branchial arches and lower jaw, where Shh is expressed, changes in expression of genes involved in patterning and mesodermal specification suggest both gain and loss of Hedgehog function. Thus, analysis of gene expression in talpid 3 head shows that, as in talpid 3 limb, expression of some genes is lost, while others are ectopically expressed. Unlike the limb, many head regions depend on Hh induction of a secondary domain of Shh expression, and failure of this induction in talpid 3, together with the inability to activate the Shh pathway, explain the loss-of-function head phenotype. This gene expression analysis in the talpid 3 head also confirms and extends knowledge of the importance of Shh signaling and the balance between activation and repression of Shh targets in many aspects of craniofacial morphogenesis.

Fate map of the diencephalon and the zona limitans at the 10-somites stage in chick embryos

The diencephalon is a central area of the vertebrate developing brain, where the thalamic nuclear complex, the pretectum and the anterior tegmental structures are generated. It has been subdivided into prosomeres, which are transversal domains defined by morphological and molecular criteria. The zona limitans intrathalamica is a central boundary in the diencephalon that separates the posterior diencephalon (prosomeres 1 and 2), from the anterior diencephalon (prosomere 3). This intrathalamic limit appears early on in neural tube development, and the molecular pattern that it reveals suggests an important role in the diencephalic histogenesis. We hereby present a fate map of the presumptive territories in the diencephalon of a chick embryo at the 10–11 somite stages (HH9–10), by homotopic and isochronic quail–chick grafts. The anatomical interpretation of chimeric brains was aided by correlative whole-mount in situ hybridization with RNA probes for chicken genes expressed in specific diencephalic territories. The resulting fate map describes the distribution of the presumptive diencephalic prosomeres in the neural tube, and demonstrates their topologically conserved relationships throughout the neural development. Moreover, we show that the presumptive epithelium of ZLI can be localized at early developmental stages in the diencephalic alar plate at the anterior limit of the Wnt8b gene expression domain.

3 dimensional modelling of early human brain development using optical projection tomography

BackgroundAs development proceeds the human embryo attains an ever more complex three dimensional (3D) structure. Analyzing the gene expression patterns that underlie these changes and interpreting their significance depends on identifying the anatomical structures to which they map and following these patterns in developing 3D structures over time. The difficulty of this task greatly increases as more gene expression patterns are added, particularly in organs with complex 3D structures such as the brain. Optical Projection Tomography (OPT) is a new technology which has been developed for rapidly generating digital 3D models of intact specimens. We have assessed the resolution of unstained neuronal structures within a Carnegie Stage (CS)17 OPT model and tested its use as a framework onto which anatomical structures can be defined and gene expression data mapped.ResultsResolution of the OPT models was assessed by comparison of digital sections with physical sections stained, either with haematoxylin and eosin (H&E) or by immunocytochemistry for GAP43 or PAX6, to identify specific anatomical features. Despite the 3D models being of unstained tissue, peripheral nervous system structures from the trigeminal ganglion (~300 μm by ~150 μm) to the rootlets of cranial nerve XII (~20 μm in diameter) were clearly identifiable, as were structures in the developing neural tube such as the zona limitans intrathalamica (core is ~30 μm thick). Fourteen anatomical domains have been identified and visualised within the CS17 model. Two 3D gene expression domains, known to be defined by Pax6 expression in the mouse, were clearly visible when PAX6 data from 2D sections were mapped to the CS17 model. The feasibility of applying the OPT technology to all stages from CS12 to CS23, which encompasses the major period of organogenesis for the human developing central nervous system, was successfully demonstrated.ConclusionIn the CS17 model considerable detail is visible within the developing nervous system at a minimum resolution of ~20 μm and 3D anatomical and gene expression domains can be defined and visualised successfully. The OPT models and accompanying technologies for manipulating them provide a powerful approach to visualising and analysing gene expression and morphology during early human brain development.

Wnt signaling is required at distinct stages of development for the induction of the posterior forebrain.

One of the earliest manifestations of anteroposterior pattering in the developing brain is the restricted expression of Six3 and Irx3 in the anterior and posterior forebrain, respectively. Consistent with the role of Wnts as posteriorizing agents in neural tissue, we found that Wnt signaling was sufficient to induce Irx3 and repress Six3 expression in forebrain explants. The position of the zona limitans intrathalamica (zli), a boundary-cell population that develops between the ventral (vT) and dorsal thalamus (dT), is predicted by the apposition of Six3 and Irx3 expression domains. The expression patterns of several inductive molecules are limited by the zli, including Wnt3, which is expressed posterior to the zli in the dT. Wnt3 and Wnt3a were sufficient to induce the dT marker Gbx2 exclusively in explants isolated posterior to the presumptive zli. Blocking the Wnt response allowed the induction of the vT-specific marker Dlx2 in prospective dT tissue. Misexpression of Six3 in the dT induced Dlx2 expression and inhibited the expression of both Gbx2 and Wnt3. These results demonstrate a dual role for Wnt signaling in forebrain development. First, Wnts directed the initial expression of Irx3 and repression of Six3 in the forebrain, delineating posterior and anterior forebrain domains. Later, continued Wnt signaling resulted in the induction of dT specific markers, but only in tissues that expressed Irx3.

Pitx2 Distinguishes Subtypes of Terminally Differentiated Neurons in the Developing Mouse Neuroepithelium

Pitx2, a homeodomain transcription factor, is essential for normal development of pituitary, eyes, heart, and teeth. In the developing mouse brain, Pitx2 ( Rieg, Ptx2, Otlx2, Brx1) mRNA is expressed in discrete regions of the diencephalon, mesencephalon, and rhombencephalon. While prior reports have provided an overview of the temporal and regional specificity of Pitx2 mRNA expression in the brain, the precise cell types that express PITX2 are not known. In this study, we analyzed Pitx2 mRNA and PITX2 protein expression in individual cells of the developing e10.5–e14.5 mouse CNS using multiple markers of cellular proliferation and differentiation. We identified Pitx2 expression in nestin-positive neural progenitors and in postmitotic, developing neurons. In the diencephalon, PITX2 is expressed in neurons of the zona limitans intrathalamica and mammillary region and in γ-aminobutyric acid (GABA)-producing neurons of the zona incerta. In the mesencephalon, PITX2-labeled nuclei also appear in differentiated neurons, some of which are GABAergic and destined to occupy superior colliculus. Our results suggest that PITX2 expression in postmitotic neurons may contribute to development of GABAergic and other differentiated neuronal phenotypes.

Expression patterns of Wnt8b and Wnt7b in the chicken embryonic brain suggest a correlation with forebrain patterning centers and morphogenesis

The expression patterns of the genes Wnt7b and Wnt8b were analyzed in the brain of chick embryos, having special emphasis in the forebrain. Our results indicated that, at early developmental stages, cWnt8b is expressed in the isthmic organizer and in other areas postulated as forebrain patterning centers, such as the avian cortical hem and the zona limitans intrathalamica (zli). Later in development, cWnt7b becomes expressed in regions neighboring and sometimes overlapping the cWnt8b domains, such as the thalamus on both sides of the zli, or the medial pallium adjacent to the cortical hem. This sequential expression of cWnt8b and cWnt7b is consistent with a role in the patterning and morphogenesis of these forebrain regions.

Mammalian neural tube grafting experiments: an in vitro system for mouse experimental embryology.

We have developed a simple experimental technique which consists of explanting the mouse embryo anterior neural tube--the presumptive brain anlage--onto polycarbonate membranes. The neural epithelium of the explants maintained both its original topology and topography for at least two days in culture. Analysis of cell death in the explants by assaying propidium iodide uptake showed high viability of neuroepithelial cells during the culture period. Both the pattern of gene expression and the initial steps of neural cellular differentiation were well preserved, being similar to those which occur in the normal in vivo situation. We show here two applications of this tissue culture technique which is similar to that which has been previously employed for avian embryo models. The first involves neuroepithelial grafting experiments by heterotopic transplantation of the zona limitans intrathalamica (ZLI) into the mesencephalon and the second consists of fibroblast growth factor (FGF8) protein interaction studies using soaked bead insertions. This in vitro system constitutes a powerful experimental embryological tool which can have other applications including time-lapse imaging and electrophysiology.

P2 An immunocytochemical and in situ hybridisation study of embryonic development of the human diencephalon.

In order to understand gene expression patterns in human embryos for a number of genes known to be of importance to normal brain development and to underlie certain developmental disorders, we have supplemented our in situ hybridisation studies with immunostaining for a number of developmental markers. Here, we present expression patterns for the transcription factors PAX6, PAX3 and OTX2, the ventralising signalling molecule sonic hedgehog (SHH) and immunostaining for nestin (a radial glia marker), β‐tubulin (marker for postmitotic neurons), MAP2 (a marker for differentiating neurons) and GAP43 (a marker for growing axons) in the human diencephalon between Carnegie stages 15 and 21 (33–52 postovulatory days). Human embryos were taken with maternal consent following termination according to Local Ethics Committee Regulations. They were fixed by immersion in paraformaldehyde, embedded in paraffin and oblique transverse serial sections were cut. In situ hybridisation was carried out using radioactively labelled RNA probes, and immunocytochemistry was performed with well characterised, commercially available antibodies using standard methods. SHH expression was confined to the basal plate with the exception of the zona limitans intrathalamica (ZLI) where a band of expression formed a boundary between the alar plates of prosmeres 2 (dorsal thalamus) and 3 (ventral thalamus). The ZLI was bounded by PAX6 and OTX2 hybridisations and was the site of unusually strong β‐tubulin immunoreactivity in the ventricular layer. Nestin immunoreactivity was strong in the ventricular zone and also in alar plate regions where a radial arrangement of glial fibres could be seen. In basal plate regions nestin positive glial fibres were not so evident. However MAP2 immunoreactivity was prevalent in basal regions showing that neuronal development is further advanced compared to alar regions. GAP43 immunostaining indicated that development of longitudinal axon pathways occurs predominantly through the ventral regions of the forebrain at these stages. Ventral thalamus development precedes dorsal thalamus development as shown by its thinner ventricular layer and a thicker intermediate layer that is also strongly MAP2 immunoreactive. The outgrowth of the thalamocortical projection from dorsal thalamus through the ventral thalamus had not begun at this age, judging by the lack of GAP43 immunoreactivity. In addition PAX6 expression was seen in both intermediate and ventricular layers in the ventral thalamus. PAX3 expression was confined to the roofplate region of prosomeres 1 and 2 corresponding to the pretectum and epithalamus. Therefore our results indicate that by CS21, human diencephalon development is less a advanced than the mouse diencephalon at the generally accepted equivalent stage (embryonic day 13–13.5). However development of the human diencephalon was found broadly to conform to the prosomeric model of forebrain development of Puelles & Rubinstein (Puelles, Trends Neurosci.16, 1993).