We present a correlative fate map of the nonsegmented caudal hindbrain down to the medullospinal boundary (medulla oblongata), as a companion to a previous fate mapping study of the hindbrain rhombomeres r2-r6 in quail chick chimeras at stages HH10/11 [Marín and Puelles (1995) Eur J Neurosci 7:1714-1738]. For reproducibility and equivalent precision of analysis, successive portions of the medulla-called pseudorhombomeres "r7" to "r11"-were delimited by transverse planes through the center of adjacent somites at stages HH10/11. These units were each grafted homotopically and isochronically from quail donors into chick hosts. The chimeric specimens were fixed at stages HH35/36 and alternate Nissl-stained sagittal sections were compared to adjacent sections in which quail cells were detected immunocytochemically. This analysis in general showed that there is little intermixing between adjacent pseudorhombomeric domains, although some neuronal populations in the vestibular and trigeminal columns, as well as in the reticular formation and pontine nuclei, do migrate selectively into the host hindbrain. Contralateral migration was scarce up to the stages examined. Several motor nuclei, i.e., the vagal motor complex, or sensory nuclei, i.e., the medial vestibular nucleus, show cytoarchitectonic limits that coincide with pseudorhombomeric ones; however, most conventional grisea were found to originate across several pseudorhombomeres. The inferior olivary complex originated between "r8" and "r11" (between the centers of somites 1 and 5). The medullospinal boundary coincided precisely with the center of the fifth somite, slightly caudal to the obex and the end of the choroidal roof, and correlated with the end of many medullary cytoarchitectonic units. In contrast, the dorsal column nuclei and the caudal subnucleus of the descending trigeminal column fell within the spinal cord. On the whole, the patterns observed were very similar to those found before within the overtly segmented part of the hindbrain, suggesting that some underlying common mechanism may account for the transverse cytoarchitectonic boundaries.

The Hoxb1 autoregulatory enhancer directs segmental expression in vertebrate hindbrain. Three conserved repeats (R1, R2, and R3) in the enhancer have been described as Pbx-Hoxb1 (PH) binding sites, and one Pbx-Meinox (PM) binding site has also been characterized. We have investigated the importance and relative roles of PH and PM binding sites with respect to protein interactions and in vivo regulatory activity. We have identified a new PM site (PM2) and found that it cooperates with the R3 PH site to form ternary Prep1-Pbx1-Hoxb1 complexes. In vivo, the combination of the R3 and PM2 sites is sufficient to mediate transgenic reporter activity in the developing chick hindbrain. In both chicken and mouse transgenic embryos, mutations of the PM1 and PM2 sites reveal that they cooperate to modulate in vivo regulatory activity of the Hoxb1 enhancer. Furthermore, we have shown that the R2 motif functions as a strong PM site, with a high binding affinity for Prep1-Pbx1 dimers, and renamed this site R2/PM3. In vitro R2/PM3, when combined with the PM1 and R3 motifs, inhibits ternary complex formation mediated by these elements and in vivo reduces and restricts reporter expression in transgenic embryos. These inhibitory effects appear to be a consequence of the high PM binding activity of the R2/PM3 site. Taken together, our results demonstrate that the activity of the Hoxb1 autoregulatory enhancer depends upon multiple Prep1-Pbx1 (PM1, PM2, and PM3) and Pbx1-Hoxb1 (R1 and R3) binding sites that cooperate to modulate and spatially restrict the expression of Hoxb1 in r4 rhombomere.Hox proteins belong to a large family of transcription factors that control cell identity, differentiation, and patterning in embryonic development. The ordered expression of these genes along the body axis is a critical aspect of regional identity (24, 37). For example, the functional identity of segmental units, rhombomeres (r), in the vertebrate hindbrain depends on the combinatorial activity of Hox proteins along the anteroposterior axis (reviewed in references 22, 24, 27, and 38). Members of the Hox protein family participate in a cascade that involves regulation of their spatial and temporal expression by direct auto-, para-, and/or cross-regulatory mechanisms. Initial expression of Hox genes in the hindbrain is regulated by a variety of transient inputs, including retinoic acid (RA) and fibroblast growth factors (FGFs) (2,21,35,36,48,51) along with several transcription factors, such as retinoic acid receptors, Kreisler, and Krox20 (33,34,44,54). Following this initial phase, direct cross talk and feedback circuits between the Hox genes are one of the important mechanisms that serve to maintain restricted segmental expression once the early cues are no longer functioning (17,29,32,40,49,50).

The Hoxa2 gene is an important component of regulatory events during hindbrain segmentation and head development in vertebrates. In this study we have used sequenced comparisons of the Hoxa2 locus from 12 vertebrate species in combination with detailed regulatory analyses in mouse and chicken embryos to characterize the mechanistic basis for the regulation of Hoxa2 in rhombomere (r) 4. A highly conserved region in the Hoxa2 intron functions as an r4 enhancer. In vitro binding studies demonstrate that within the conserved region three bipartite Hox/Pbx binding sites (PH1-PH3) in combination with a single binding site for Pbx-Prep/Meis (PM) heterodimers co-operate to regulate enhancer activity in r4. Mutational analysis reveals that these sites are required for activity of the enhancer, suggesting that the r4 enhancer from Hoxa2 functions in vivo as a Hox-response module in combination with the Hox cofactors, Pbx and Prep/Meis. Furthermore, this r4 enhancer is capable of mediating a response to ectopic HOXB1 expression in the hindbrain. These findings reveal that Hoxa2 is a target gene of Hoxb1 and permit us to develop a gene regulatory network for r4, whereby Hoxa2, along with Hoxb1, Hoxb2 and Hoxa1, is integrated into a series of auto- and cross-regulatory loops between Hox genes. These data highlight the important role played by direct cross-talk between Hox genes in regulating hindbrain patterning.

Sequence divergence in cis-regulatory elements is an important mechanism contributing to functional diversity of genes during evolution. Gene duplication and divergence provide an opportunity for selectively preserving initial functions and evolving new activities. Many vertebrates have 39 Hox genes organized into four clusters (Hoxa-Hoxd); however, some ray-finned fishes have extra Hox clusters. There is a single Hoxa2 gene in most vertebrates, whereas fugu (Takifugu rubripes) and medaka (Oryzias latipes) have two coparalogous genes [Hoxa2(a) and Hoxa2(b)]. In the hindbrain, both genes are expressed in rhombomere (r) 2, but only Hoxa2(b) is expressed in r3, r4, and r5. Multiple regulatory modules directing segmental expression of chicken and mouse Hoxa2 genes have been identified, and each module is composed of a series of discrete elements. We used these modules to investigate the basis of differential expression of duplicated Hoxa2 genes, as a model for understanding the divergence of cis-regulatory elements. Therefore, we cloned putative regulatory regions of the fugu and medaka Hoxa2(a) and -(b) genes and assayed their activity. We found that these modules direct reporter expression in a chicken assay, in a manner corresponding to their endogenous expression pattern in fugu. Although sequence comparisons reveal many differences between the two coparalogous genes, specific subtle changes in seven cis elements of the Hoxa2(a) gene restore segmental regulatory activity. Therefore, drift in subsets of the elements in the regulatory modules is responsible for the differential expression of the two coparalogous genes, thus providing insight into the evolution of cis elements.Hox gene regulation ͉ hindbrain ͉ vertebrate development ͉ fugu

Here, we define a gene regulatory network for Hoxa2, responsible for temporal and spatial expression in hindbrain development. Hoxa2 plays an important role in regulating the regional identity of rhombomere 2 (r2) and is the only Hox gene expressed in this segment. In this study, we found that a Hoxa2 cis-regulatory module consists of five elements that direct expression in r2 of the developing hindbrain. Surprisingly, the module is imbedded in the second coding exon of Hoxa2 and therefore may be constrained by both protein coding and gene regulatory requirements. This highly conserved enhancer consists of two consensus Sox binding sites and several additional elements that act in concert to direct strong r2 specific expression. Our findings provide important insight into the regulation of segmental identity in the anterior hindbrain. Furthermore, they have broader implications in designing arrays and interpreting data from global analyses of gene regulation because regulatory input from coding regions needs to be considered.cis elements ͉ hindbrain segmentation ͉ Hox genes ͉ regulatory network