Andrew Lumsden

Signals from the notochord and floor plate regulate the region-specific expression of two Pax genes in the developing spinal cord.

Members of the paired box (Pax) gene family are expressed in discrete regions of the developing central nervous system, suggesting a role in neural patterning. In this study, we describe the isolation of the chicken homologues of Pax-3 and Pax-6. Both genes are very highly conserved and share extensive homology with the mouse Pax-3 and Pax-6 genes. Pax-3 is expressed in the primitive streak and in two bands of cells at the lateral extremity of the neural plate. In the spinal cord, Pax-6 is expressed later than Pax-3 with the first detectable expression preceding closure of the neural tube. When the neural tube closes, transcripts of both genes become dorsoventrally restricted in the undifferentiated mitotic neuroepithelium. We show that the removal of the notochord, or implantation of an additional notochord, dramatically alter the dorsoventral (DV) expression patterns of Pax-3 and Pax-6. These manipulations suggest that signals from the notochord and floor plate regulate the establishment of the dorsoventrally restricted expression domains of Pax-3 and Pax-6 in the spinal cord. The rapid changes to Pax gene expression that occur in neural progenitor cells following the grafting of an ectopic notochord suggest that changes to Pax gene expression are an early effect of the notochord on spinal cord patterning.

The cellular basis of segmentation in the developing hindbrain

One of the most challenging problems in developmental biology is to identify the mechanisms that generate the complex arrangement and interconnections of cells in the vertebrate brain. This review surveys the evidence that the hindbrain region is laid down as a series of similar modules, final complexity being founded on this simple ground plan. Morphological studies, using immunohistochemical and axon-labelling techniques, reveal repeat patterns of neuronal differentiation and organization, whereas cell marking experiments have shown that the segments, or rhombomeres, are lineage-restriction units each constructing a defined piece of the hindbrain.

Improved method for chick whole-embryo culture using a filter paper carrier.

We describe a simple method of chick whole‐embryo culture, which uses a filter paper carrier to hold the early blastoderm and vitelline membranes under tension while the embryo grows on a substratum of agar‐albumen. This is a quick and efficient means of setting up cultures of chick embryos beginning at pre‐primitive streak stages to stage 10 (stages X–XIV, Eyal‐Giladi and Kochav [1976] Dev Biol 49:321–337; stages 1–10, Hamburger and Hamilton [ 1951 ] J Morphol 88:49–92). This is an improvement on the original method of New, which used a glass ring and watch glass (New [1955] Exp Morphol 3:320–331). Our modification of News method, which we call EC (Early Chick, pronounced EASY) culture, facilitates several manipulations in early chick embryos, including microsurgery, grafting, bead implantation, microinjection, and electroporation. Using the EC method, embryos at stage 8 and older can be readily cultured either dorsal‐side up (in contrast to News method) or ventral‐side up, as desired; embryos younger than stage 8 can be culture only ventral‐side up (as with News method). We also discuss some alternative methods for setting up these cultures.

Compartments and their boundaries in vertebrate brain development

Fifteen years ago, cell lineage restriction boundaries were discovered in the embryonic vertebrate hindbrain, subdividing it into a series of cell-tight compartments (known as rhombomeres). Compartition, together with segmentally reiterative neuronal architecture and the nested expression of Hox genes, indicates that the hindbrain has a truly metameric organization. This finding initiated a search for compartments in other regions of the developing brain. The results of recent studies have clarified where compartment boundaries exist, have shed light on molecular mechanisms that underlie their formation and have revealed an important function of these boundaries: the positioning and stabilization of local signalling centres.

A role for FGF-8 in the initiation and maintenance of vertebrate limb bud outgrowth

BACKGROUND The outgrowth of the vertebrate limb bud is the result of a reciprocal interaction between the mesenchyme and a specialized region of the ectoderm, the apical ectodermal ridge (AER), which overlies it. Signals emanating from the AER act to maintain the underlying mesenchyme, called the progress zone, in a highly proliferative and undifferentiated state. Removal of the AER results in the cessation of limb bud growth, thus causing limb truncation. The best candidates for this AER-derived signal are members of the fibroblast growth factor (FGF) family, in particular FGF-4, which can maintain limb bud outgrowth following removal of the AER. However, FGF-4 is only expressed after considerable outgrowth has occurred and a well-developed limb bud has formed, and then only in the posterior part of the AER. Likewise, the other FGFs studied to date are not candidates for this activity. RESULTS We report evidence that a recently identified member of this family, FGF-8, is expressed in the ectoderm of the prospective limb territory prior to morphological outgrowth of the limb bud in both mouse and chick. Thereafter, expression is maintained throughout the AER during limb development. We have produced and purified the FGF-8 protein, and shown that it will substitute for the AER in maintaining limb bud outgrowth in mouse embryos from which the AER has been surgically removed. FGF-8 does not, however, maintain expression of the sonic hedgehog gene. CONCLUSIONS These results indicate that FGF-8 is an AER-derived mitogen that stimulates limb bud outgrowth. Moreover, our data suggest that FGF-8 may also be an ectodermally derived mitogen that stimulates the onset of limb bud outgrowth (budding) in the absence of a morphological AER, and indicate the possible involvement of FGF-8 in the establishment of the limb field.

Chemotropic effect of specific target epithelium in the developing mammalian nervous system

Developing nerve fibres are guided to their targets by specific directional cues1,2 which are thought to be expressed in the tissues along the route3–7 and may involve the extracellular matrix8–10. Another possibility, that directional cues emanate from the target itself11, is consistent with the recent demonstration of homing behaviour by ectopic retinal ganglion axons12 and our previous demonstration that early trigeminal neurites grow directly to their virgin peripheral target in vitro13. Here we show that this chemotropic effect is precisely limited to the trigeminal system; trigeminal ganglion neurites grow directly to their own target field but not to the adjoining field, normally innervated by the geniculate ganglion; furthermore, the trigeminal field does not influence the growth of geniculate neurites. Also, when trigeminal ganglia are co-cultured with isolated tissue layers of their target, neurites grow only towards the epithelial and not the mesenchymal component. These findings suggest that trigeminal epithelium is specified to attract correct innervation and that pathway mesenchyme, in which preformed guidance cues have been postulated10,14,15, may provide favourable conditions for nerve fibre growth but not govern its direction.

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.

Rostral optic tectum acquires caudal characteristics following ectopic engrailed expression.

BACKGROUND Expression of the homeobox-containing gene Engrailed (En) in an increasing rostral-to-caudal gradient in the dorsal mesencephalon is the earliest known marker for polarity of the chick optic tectum. In heterotopic transplantation experiments, En protein expression correlates well with the subsequent gradient of cytoarchitecture as well as the pattern of retinotectal projections. The En gradient also correlates with the expression of two putative retinal axon-guidance molecules, RAGS and ELF-1, which are Eph-like receptor tyrosine kinase ligands that may function in the establishment of retinotopic projections by excluding temporal axons from the caudal tectum. RESULTS To examine the function of En in determining tectal polarity, we used the replication-competent retroviral vector RCAS to misexpress mouse En-1 throughout the chick tectal primordium. Our results show that the rostral portion of the tectum adopts a caudal phenotype: the gradient of cytoarchitectonic differentiation is abolished, and the molecular markers RAGS and ELF-1 are strongly expressed rostrally. In addition, cell membranes from rostral tectum of RCAS En-1-infected embryos preferentially repel temporal axons in in vitro membrane stripe assays. CONCLUSIONS These results are consistent with a role for En in determining rostrocaudal polarity of the developing tectum. The demonstration that both RAGS and ELF-1 are upregulated following En misexpression provides a molecular basis for understanding the previous observation, also based on retrovirus-mediated En misexpression, that nasal axons form ectopic connections in rostral tectum, from which temporal axons are excluded.

Neural Crest Apoptosis and the Establishment of Craniofacial Pattern: An Honorable Death

During development of the vertebrate head neural crest cells emigrate from the hindbrain and populate the branchial arches, giving rise to distinct skeletal elements and muscle connective tissues in each arch. The production of neural crest from the hindbrain is discontinuous and crest cells destined for different arches, carrying different positional cues, are separated by regions of apoptosis centered on rhombomeres (r) 3 and r5. This cell death program is under the interactive control of the neighboring hindbrain segments. Both r3 and r5 produce large numbers of crest cells when freed from their flanking rhombomere, but when conjoined with their neighbor the cell death program is restored. Two key components of this program are Bmp 4 and msx-2, both of which are expressed in the apoptotic foci of r3 and r5 and which are also regulated by neighbor interactions. Importantly, the addition of recombinant Bmp 4 to isolated cultures of r3 and r5 induces the expression of Bmp 4 and msx-2 and restores the cell death program. This early neural crest segregation is maintained during development and it has profound effects upon the final craniofacial pattern. Even though crest cells from different axial origins will contribute to compound skeletal elements, these distinct populations do not intermingle. Furthermore head muscle connective tissues are exclusively anchored to skeletal domains arising from neural crest from the same axial level. Thus the discontinuous production of neural crest sculpts the crest into nonmixing streams and consequently ensures the fidelity of patterning.

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.