Homeobox genes in vertebrate gastrulation

Homeobox genes in vertebrate gastrulation Edoardo Boncinelli and Antonello Mallamaci lstituto

Scientifico

San Raffaele The

formation

layers,

and Institute

and anteroposierior

ectoderm,

ectoderm,

or

for Cellular

mesoderm

epiblast,

What changes from

to system

the nature of the non-epiblast of molecular

markers,

including

goosecoidand

OtxZ,

the underlying

molecular

Current Opinion

mechanisms

structures

Italy

germ

primitive

gastrulation.

implicated.

A number

genes and in particular

and to establish

allow us to explore biologically

relevant

between the various systems.

in Genetics & Development

The emergence of the three embryonic cell layers is probably the most conspicuous event in gastrulation, but by no means the only one. At least two other processes take place at the same time [l]: the establishment of the anteroposterior axis of the embryo and an irreversible change in cell fate of restricted groups of cells as a consequence of the general reorganization of the body pattern. are well described in [l-6], gastrulation still 0 Current

the

theme of vertebrate

a few homeobox

Through gastrulation, an essentially monolayered early vertebrate embryo becomes three-layered. The three definitive layers, namely ectoderm, mesoderm and endoderm, derive from a single cell layer usually called primary ectoderm or epiblast. A slight asymmetry in origin is seen between ectoderm on one side and mesoderm and endoderm on the other. In fact, mesoderm and endoderm originate born cells previously located on the surface and reaching their final destination by ingression through a hole (the blastopore) or a linear slit (the primitive streak). Conversely, the ectoderm derives from those cells that remain on the surface layer and do not ingress. But conceptually, this asymmetry is irrelevant and all three cell layers first originate through gastrulation, as the primitive surface layer, the epiblast, also emerges entirely transformed and reorganized from gastrulation and appropriately receives the new designation of ectoderm. A substantial portion of ectoderm is now committed to differentiate into neuroectoderm. All this is common to several vertebrate systems. What changes from system to system is the geometry of this reorganization as well as the number and nature of non-epiblast cell structures assisting the epiblast in its transformation and reorganization. These structures include, for example, vegetal hemisphere in amphibians, primitive endoderm and extra-embryonic structures in the mouse and hypoblast and marginal zone in the chick.

its major features and review articles

from

Milan,

is the geometry of these events and

transient

Introduction

Although textbooks

starting

are now available that will hopefully

homologies

Pharmacology,

patterning of the three definitive

and endoderm,

is the common

system

and Molecular

Biology

1995,

5:619-627

presents many basic unknowns. A major issue is when gastrulation as a biological process actually starts. In other words, how early do those preparatory cellular and extracellular events begin to take place that subsequently lead to overt gastrulation movements? A second issue arises as to when they are over. There is indeed increasing evidence that, at least in amphibians, gastrulation lasts longer than previously perceived and that tail formation should be considered as being a continuation of gastrulation [7,8]. A third issue concerns the acquisition of inducing properties by specific cell subpopulations. And yet another major issue relates to the origin of axial structures through the generation of anteroposterior regional identities; for example, evidence is accumulating that the prospective head region is specified very early as potentially distinct from the trunk

PI. In this review, we will concentrate on two aspects of gastrulation. The first is what we can learn from expression and functional analyses of a particular class of regulatory genes, namely the homeobox genes, during gastrulation. The second is what we can conclude from the comparison of different vertebrate systems. An aspect of particular interest in this comparative study is the identity of the so-called prechordal plate and its relationship with notochord and other axial structures in connection with the regionalization of the overlying neuroectoderm. Most studies of gastrulation have been conducted in four model systems, two belonging to Anamnia, namely Xenoplrs and zebrafish, and two belonging to Amniota, namely chick and mouse. The comparative analysis of homologous genes in these different systems promises to provide conclusions of general validity. In particular, regulatory genes may not directly mediate inductive effects, as is the case for genes encoding growth factors or secreted proteins [6]; rather, they may control the expression of a number of other genes. In other words, these genes are likely to orchestrate the expression of a number of secreted and non-secreted proteins able to Ltd ISSN 0959-437X

619

620

Differentiation and gene regulation confer positional and histological various regions of the developing

identity to cells of the embryo.

Fate maps Expression analysis of regulatory gene products or other molecular markers would not be possible in the absence of reliable information about cell lineages and fate maps of different cell populations in the gastrulating embryos of various systems. Despite the wealth of data collected on this subject during the past decades, many phenornenological details are yet to be worked out in all these systems. Very recent data accumulated on mouse [lo”,ll’] and chick [12*,13*] embryos have reduced this gap in knowledge. In particular, Rosa Beddington [lO**] has provided the first direct evidence that the mouse node is able to direct body patterning and induce a secondary axis in the mouse gastrulating embryo. Thus, the mouse node can be considered as structurally and functionally homologous to the dorsal lip of the Xenopus blastopore, which contains the organizer. In another study, the neuroectoderm precursors have been mapped at the distal, apical, cap of the mouse epiblast at the beginning of primitive streak formation [l 1.1. A detailed fate map of the epiblast of the early chick embryo [12*] unambiguously shows how the distribution of different prospective cell types in the early chick embryos essentially corresponds, with minor discrepancies, to that found in other vertebrate species. Finally, the properties of different cell populations of the avian Hensen’s node have been investigated by means of quail/chick transplantation experiments [13-l. In particular, the neural-inducing ability of the node is shown to be localized in the medial-anterior sector of the node itself in both the epiblast layer and the mesendoderm, whereas the mesendoderm of the posterolateral part of it does not appear to share this ability. Medial mesendoderm is sufficient to induce neural tissue with midbrain and hindbrain identity, but is unable to confer forebrain specification I)PY se. The epiblast layer of the node is required, in addition to medial mesendoderm, for a complete neural axis to form.

Hox genes Homeobox genes [14] encode nuclear transcription factors containing a DNA-binding domain, termed the homeodomain, which is remarkably conserved in homeobox genes belonging evolution. In particular, to the so-called Hex family [15] have been shown to be structurally and functionally homologous to the Dros@ilu homeotic genes. Their genomic organization in clusters of several HW genes arranged in the same order in vertebrates and in flies has recently been shown to extend to amphioxus [It’?], a cephalochordate possibly related to vertebrate ancestors. Hoh genes collectively control the identity of the various regions along the body axis from the branchial area through to the tail. In several vertebrates, this action occurs in a

collinear way, with 3’ genes controlling anterior regions and progressively more-5’ genes controlling progressively more posterior regions. The exact correspondence between Hex expression domains and morphological structures was compared in both chick and mouse embryos [17*]. In these two taxa, different numbers of segmental elements contribute to the various regions along the anteroposterior axis (cervical vertebrae, thoracic vertebrae and so on). This comparative study reveals that Hux expression boundaries vary between the two species in concert with morphological boundaries, providing support for the class of evolutionary hypothesis inclined to link molecular and morphological homology. Some of the Hex genes, essentially those located at the 3’ extremity of the four homologous Hex loci, are first expressed at late primitive streak stage in chick and mouse embryos [15]. Their expression domain first appears in the posterior region of the primitive streak and is progressively displaced to the fore, up to the branchial area and rhombencephalon. This displacement has now been shown to take place in the chick embryo even across an implanted glass barrier [18-l. Physical continuity is therefore not required for the completion of this programmed anterior spreading. Short-range and long-range cell-cell interactions obviously require physical continuity of the intervening tissue. A developmental clock system could be invoked to account for this observation. Gastrulation lays down the body plan of the future organism, and this process obviously requires positional information. In abstract terms, positional inforrnation within the embryo has first to be created, then transmitted to daughter or neighbouring cells and finally translated into regional identity. Hex genes are believed to be implicated in this last process of translating positional information into regional identity along the rostrocaudal axis of the trunk. Conversely, the two homeobox genes posed and Of&? could be implicated

in the transmission of the anterior positional information during gastrulation. These two genes have both been studied in all four major vertebrate systems.

goosecoid The ~oosemid gene is a vertebrate homeobox gene originally found to be expressed in the dorsal lip of Xerzoppusearly gastrula [19,20] and subsequently cloned in mouse [21], chick [22,23] and zebrafish [24,25]. It encodes a homeoprotein containing a homeodomain of the bicoid class with a characteristic lysine residue at position 50, corresponding to position 9 of the recognition helix. Ectopic expression of goosemid in the ventral half of Xcnop~s embryos leads to the formation of

a secondary embryonic axis. The progeny of the injected cells have been shown to recruit uninjected cells into the secondary axis [26], so mimicking the action of an organizer. All this suggests that ~~oosemid plays a role in preparing and executing the Spemann’s organizer phenomenon [20]. In mice, Cqooscrc)id is expressed at the anterior end of the primitive streak, very early

Homeobox genes in vertebrate gastrulation Boncinelli

in gastrulation and for a very short period of time [21]. This suggests that the anterior tip of primitive streak of the mouse is at least in part homologous to the dorsal lip of the Xenopus blastopore [2,21,27]. Later on, ~oosecoid expression is again detectable in the neural crest of the head of mid-gestation mouse embryos [28]. In zebrafish [24,25], goosecoid is an early gene expressed immediately after the mid-blastula transition in the dorsal side of the blastula. With the onset of involution and the formation of the so-called embryonic shield, the goosecoid-expressing cells are among the first to involute. As gastrulation proceeds, Xoosecoid-expressing cells present in the deep layer, but not those in the epiblast, migrate anteriorly towards the animal pole of the embryo. By the end of gastrulation, goosecoid is expressed in the anterior portion of the deep layer corresponding to the prechordal plate. Particularly interesting for the present discussion is the expression of 8ooSecoid in the chick [22,23,29*]. Briefly, its transcripts are first detected in the hypoblast and in the posterior marginal zone of the blastodisc, in a specific morphological structure termed Keller’s sickle. Cells horn this structure, a crescent-shaped thickening located at the edge of the posterior marginal zone, migrate to form the anterior end of the primitive streak (the medial Hensen’s node) and subsequently the prechordal plate [22]. Cells expressing Xoosecoid appear to proceed across the embryo along its major axis from posterior to anterior. They are first very posterior, at the level of the posterior marginal zone, progress later through the primitive streak to the anterior portion of Hensen’s node and end up in the anteriormost mesendoderm. These three cell populations have been shown to be related to each other by a common lineage [22]. The posterior marginal zone and Hensen’s node of the chick also share the ability to induce a secondary axis upon grafting onto an ectopic site. Therefore, goosecoid is a marker for cells that have organizer activity in the chick and its expression seems to be strictly connected with the functions of a dorsal organizer, as originally proposed in

and Mallamaci

the mouse [31] as one of two vertebrate homologues of orrhodenticle, a regulatory gene controlling the developing head of Drosophila [(i’], it has been studied in mouse [31,32,33**,34*], Xenopus [35”,36*], chick [29’], zebrafish ([37]; E Boncinelli, A Simeone, unpublished data), sea urchin [38], lamprey and planarians (E Boncinelli, A Stornaiuolo, unpublished data). In mouse embryos [31,32], it is already expressed in epiblast and primitive endoderm of the blastocyst at least as early as day 5.5 of development, well before any primitive streak is observable. Between day 7 and 7.5 of mouse development, its expression recedes to a limited anterior region corresponding to presumptive forebrain and midbrain neuroectoderm and anterior mesendoderm. From this stage on, the prevalent expression domain of Otx2 is the rostra1 brain region, including forebrain and midbrain, with a sharp posterior boundary coinciding with the midbraimhindbrain junction. The progressive restriction of Otx2 expression to the anterior portion of the embryo by the headfold stage correlates with the anterior migration of mesendoderm. Experimental manipulation of early mouse embryos provided evidence signal from [33**] that in vivo, a positive inductive anterior mesendoderm is required for a stable expression of Otx2 in anterior regions. On the other hand, a negative inductive signal emanating from posterior mesendoderm represses 0tx2 in the posterior regions of the embryo. Exogenous retinoic acid is able to mimic, in part, the effect of this negative posterior signal [33**,34*]. Early Otx2 expression in the mouse raises the issue of whether the 0tx2 homeodomain protein plays a direct role in specifying anterior structures. Data obtained with Xotx2, the Xenopus homologue of Otx2, seem to support this hypothesis [35**,36*]. The study of Xotx2 expression in normal and experimentally manipulated Xenopus embryos suggests a role for this gene in specifying anterior body structures and their spatial relationship with trunk structures.

otx2

Maternal Xor.rZ transcripts are present in the unfertilized egg and in early blastula in the animal cap and in a few cells of the vegetal hemisphere [35**]. In late blastula, abundant Xotx2 transcripts are first detectable in nuclei of a group of cells located in an internal region of the dorsal marginal zone (Fig. 1). When gastrulation movements start, the major expression site of Xotx2 is in migratory deep zone cells that are fated to give rise to prechordal mesendoderm. A few hours later, Xotx2 expression extends to cells of presumptive anterior neuroectoderm, where the gene is expressed throughout embryogenesis. At late gastrula/early neurula stage, Xotx2 expression appears to be confined to mesendoderm and ectoderm cells of anterior embryonic regions. At this stage, the Xotx2 expression domain is rostrocaudally restricted in a sort of anterior stripe extending across the three germinal layers, reminiscent of the orhoderzkle expression domain in Drosophila blastoderm [c)].

0~~2 is another vertebrate homeobox gene of the class. Originally isolated and characterized in

Microinjection of Xotx2 mRNA into early blastomeres produces shortened embryos with severely reduced

Xenopus.

More recently, a role has been proposed for Xoosecoid in dorsoventral patterning of mesoderm in the Xenopus embryo at the early gastrula stage [30-l. Detailed analyses reveal that ‘qoosecoid transcripts are distributed in the equatorial marginal zone in a graded fashion, with high values in the dorsal side of the marginal zone and lower values in dorsolateral regions. Microinjection of synthetic gooseoid mRNA showed that goosecoid is able to dorsalize the prospective mesoderm in a highly dose-dependent mamrer. Small changes in the amount of microinjected mRNA result in marked changes in mesoderm differentiation. At least three thresholds are observed, which are sufficient to specify four mesodermal cell fates.

bid

621

622

Differentiation and gene regulation

(al Mouse

E7.25

(b) Xenopus

e

@

Fig.

1.

Early

a.5

9.5

10.25

11

mental OtxZ

(c) Chick

expression

Xenopus

mouse,

stages are indicated.

expression

as stippling,

mesendodermal

is

by

indicated

XIII

HH4

HH5-6

OtxZ

in

DevelopVery early

in the epiblast is shown

neuroectodermal

X

of

and chick.

horizontal expression

expression stripes

and

by vertical

stripes.

A notch in the chick diagram in-

dicates

the node.

and XIII,

E, embryonic

Eyal-Ciladi

streak stage; HH,

day; X

and Kochav

Hamburger

pre-

and Hamil-

ton stage.

trunk and tail structures and an expansion of internal head structures. Most of the embryos overexpressing Xotx2 also show the presence of an additional cement gland, observable in both anterior and posterior locations. The cement gland is one of the most anterior structures of the developing Xenoplrs body and induction of a secondary cement gland by ectopic expression of Xotx2 suggests a role for this gene in specifying anterior head structures. The presence of an intact Xotx2 homeodomain with its specific lysine residue at position 9 of the recognition helix is required to produce all of these effects [35”]. It has also been proposed that Xorx2 plays a role in the progressive induction of anterior neuroectoderm during Xenopcrs gastrulation [36*]. In fact, as gastrulation proceeds, Xotx2 expression is induced in the ectoderm by the underlying anterior mesendoderm and this expression domain moves anteriorly in register with it. This expression profile follows a model for neural induction, whereby an anterior neural-inducing signal emanating from the underlying anterior mesendoderm transiently induces anterior neural tissues in the overlying ectoderm. Subsequently, regionalization of anterior neuroectoderm is obtained through the effect of caudalizing signals emanating from more posterior mesoderm. The chick homologue of Otx2, c-otx2, has also been cloned and its expression pattern analyzed during gastrulation [29*]. Transcripts were already detected in the epiblast of the unincubated egg and subsequently also in the ‘forming hypoblast. The chick hypoblast

transiently develops under the epiblast layer. It is not fated to contribute to the embryo proper and is possibly homologous to the primitive endoderm of mammals and to structures of the vegetal pole of Xenopur. Expression of c-otx2 is also seen in a very early population of middle layer cells. These presumptive early mesendoderm, or mesoblast, cells also express the chick homologue of Xoosecoid and have been shown to contribute to Hensen’s node [22]. The presence of these early mesendoderm cells in chick blastoderm is actually difficult to see in the absence of molecular markers such as goosecoid and c-otx2. Nothing is known about the existence of corresponding early mesendoderm cell populations in the mouse, but in Xenopcrs late blastula, Xotx2 is expressed in a restricted cell population in the internal dorsal region of the marginal zone (Fig. 1). These cells are probably fated to give rise to anterior mesendoderm and the so-called prechordal plate. These structures subsequently express both Xotx-2 andgooSe&d. Comparative analyses of Xenopus and chick gastrulation suggest that these early mesendodermal precursors segregate very early from other cell populations. During primitive streak formation and progression, expression of c-otx2 is progressively restricted to anterior regions in all cell layers. When the extension of the streak is maximal, transcripts are found only in the medial-anterior portion of Hensen’s node. This extremely restricted expression lasts for a very short period of time and a second, distinct, phase of c-otx2 expression starts. Transcripts progressively disappear from

Hornet obox genes in vertebrate gastrulation Boncinelli

the regressing node and a strong expression is now detectable in anterior mesendoderm and neuroectoderm. The expression in anterior neuroectoderm becomes predominant with headfold formation and appears to be clearly spatially restricted. Subsequently, c-otx2 expression will remain mainly confined to these areas of the neural tube (forebrain and midbrain) and to anterior mesoderm and endoderm. The expression of c-otx2 in the forming neural plate is most probably a result of reinitiation of expression at the time of streak regression, rather than the maintenance of transcription in anterior cells. This activation might result, at least in part, from inductive signals emanating Tom the underlying prechordal mesendoderm cells, which probably express c-CVXZ in a continual manner from prestreak stages. In conclusion, a strong expression of c-otx2 in the chick embryo is first associated with cells of presumptive anterior mesendoderm Gem their early determination. It remains associated with them during their migration toward anteriormost regions of the embryo and subsequently extends to anterior neuroectoderm. Essentially the same conclusions can be drawn from the study of Ot~2 expression in gastrulating zebrafish embryos [37]. The sea urchin cognate of Otx2 has also been identified [38] as a candidate regulator of the expression of an aboral ectoderm specific gene termed SpccZa [39,40]. Orx2 transcripts are found initially in every cell of the cleaving zebrafish embryo, but they gradually become restricted to oral ectoderm and endoderm.

Evolutionary

considerations

Ofx2 expression clearly represents a precious marker for comparative studies in vertebrate gastrulation (Fig. 1). Careful analyses of the temporal evolution of the expression domains before and during gastrulation in different vertebrate systems provide testable hypotheses for the identification of true homologous structures, such as epiblast, hypoblast and early mesoblast, in different gastrulating vertebrates. Probably even more

and Mallamaci

important is the issue of early determination of anterior mesendoderm and its relation to presumptive anterior neuroectoderm. Ofx2, as well as goosemid [27], expression indicates an origin in space and time for these early committed cells in late blastula vertebrate embryos. A major distinction in metazoan biology is that between protostomes and deuterostomes. These two groups differ, among other things, in the position of the very first opening in gastrulation, the blastopore. Its position corresponds to that of the future mouth in protostomes, but not in deuterostomes; in the latter, the mouth arises from an ectodermal opening away 6om the position of the blastopore. Remarkably, expression of Otx2 and its insect counterpart, orthodenticle, provide a good molecular illustration of this dichotomy (Fig. 2). In vertebrates, in fact, Orx2 expression starts posteriorly and its expression domain includes cells positioned posteriorly but fated to give rise to anterior mesendoderm. Subsequently, at least some of these cells migrate toward their final destination and end up in a very anterior position in anterior mesendoderm, fated to give rise, among other organs and tissues, to the pharynx. Conversely, in DrcmpMa, the only protostome system studied so far, orthodenticle is expressed anteriorly from the very beginning [9]. Thus, in deuterostomes, the anterior migration of expression of Of.r2 parallels the migration of pharynx precursor cells away from blastopore, whereas in insects, the stable anterior location of the expression of orthodenticle marks anterior structures from the beginning. It will be of interest to study the expression of the genes of the orthodenrirle/Orx2 family in systems such as annelids or molluscs with a gastrulation pattern more typical of protostomes.

Other

homeobox

genes in gastrulation

A few homeobox genes other than ~~~>OsecOid and 01x2 have been reported quite recently to play a role in difrerent aspects of gastrulation [41”-43**,44*-470,481. Lirn 1 [41**] is a LIM class [49-511 homeobox gene expressed in the organizer region of mouse embryos. Embryos homozygous for a targeted deletion of this

(a) Xenopus

cl

@-+ ...

0 Anterior

(b) Drosophila

Fig. 2. Ontogenesis

of 0tx2

in a vertebrate embryo W

Posterior

orthodenticle

expression

embryo (expression In vertebrates, pression sively

OK?

starts

expression

Menopus)

shown

by stippling).

(and goosecoid) ex-

posteriorly

and progres-

moves to a very anterior

In Drosophila, anteriorly

orthodenticle

from

Xenopus embryo

and of

in a Drosophila

position.

is expressed

the very beginning.

The

is shown as a represen-

tative of vertebrate gastrulating

embryos.

623

624

Differentiation and gene regulation gene lack anterior head structures, but the remaining body axis develops normally [41**]. More precisely, these embryos lack everything anterior to rhombomere 3. Thus, Lirrr1 appears to be an essential component of the vertebrate ‘head organizer’ or ‘head inductor’, a concept proposed originally by Spemann more than 60 years ago (see [52]). In classical transplantation experiments of the amphibian dorsal blastopore lip by Mangold and Spemann (see [52]), when an early lip was transplanted, a complete axis including head was formed. Conversely, transplanted dorsal lip deriving horn later stage embryos was able to induce only trunk and tail structures. These observations led to the proposal of a distinction between a head and a trunk organizer [52]. Homeobox genes of the LIM family have also been shown to define the topographic organization of embryonic motor neurons in chick [53.*]. Xlirrr 2 [54] is the Xenoplrs cognate of murine Lirrrl and is specifically expressed in the organizer region of the embryo. The gene product contains both a LIM class homeodomain and two cysteine-rich LIM domains. At least under certain conditions, the two LIM domains are able to inhibit the DNA-binding activity of the LIM homeodomain. Overexpression of Xlirrr1 gene products with mutated or deleted LIM domains [42”] elicits neural differentiation in explanted animal caps and enhances muscle formation after co-injection with Xbm. Thus, Xlirrr 1 has latent organizer functions that are tightly regulated. Very recently, another homeobox gene, Siarrroir [43”], has been implicated in Xe~pcrs axis determination. It was cloned using an expression cloning strategy and the functional assay for axis duplication. Its transcripts are first detected shortly after the midblastula transition and are most abundant in the dorsal endoderm of early gastrulae. As little as 5pg of its synthetic RNA are sufficient to induce a complete secondary axis in microinjected embryos. All this suggests that Siarnois plays a role in the formation of the Nieuwkoop centre, the putative early endodermal inducing centre that mediates the induction of the Spemann organizer in overlying mesoderm.

yolk portion of the embryo in zebrafish. A third (middle) cell layer starts to form posteriorly h-om the epiblast and migrates anteriorly. This presumptive mesendoderm, or mesoblast, layer will subsequently give rise to mesoderm and endoderm, discarding and substituting the originary hypoblast. There is increasingly compelling evidence that cellular events leading to the appearance of early mesendoderm take place much earlier than previously thought [22,29*,35**]. Anterior portions of axial and paraxial mesendoderm will generate head structures and induce rostra1 brain regions in the overlying ectoderm. In this action, anterior mesendoderm is assisted by regionalization events taking place in the ectoderm layer itself and spreading in a planar or tangential manner.

Acknowledgements We are indebted to Larry Wrabetz fdr a number of helpful commt‘nts and suggestions throughout the text. We want also to thank various members of our group, and in particular Massimo Gulisano and Antonio Faiella, for their assistance with figures.

References and recommended Papers

of particular

review,

. ..

have been highlighted of special interest of outstanding interest

1.

Stern CD: Vertebrate gastrulation. Curr 0,oin Cenet Dev 1992,

published

within

the annual

period

of

as:

2:55&561. 2.

Niehrs C, De Robertis Opin Gener Dev 1992,

3.

Stern CD: Mesoderm induction and development of the embryonic axis in amniotes. Trends Genet 1992, 2:550-555.

4.

Beddington RSP, Smith JC: Control of vertebrate gastrulation: inducing signals and responding genes. Curr Opin Genet Dev 1993, 3:655-661.

5.

Faust C, Magnuson T: Genetic control of gastrulation mouse. Curr Opin Cenet Dev 1993, 3:491-498.

6.

Kessler DS, Melton DA: Vertebrate embryonic induction: mesodermal and neural patterning. Science 1994, 266:59&604.

7.

Gont LK, Seinbeisser H, Blumberg B, De Robertis EM: Tail formation as a continuation of gastrulation: the multiple cell populations of the Xenopus tail bud derive from the late blastopore lip. Developmenr 1993, 119:991-l 004.

8.

Tucker AS, Slack JMW: The Xenopus /de& Development 1995, 121:249-262.

9.

Finkelstein R, Boncinelli E: From fly head to mammalian forebrain: the story of Otd and Otx. Trends Genet 1994, 10:310-315.

Conclusions From these data on cell lineages and gene excomplemented by recent functional analyses pression, of other major non-homeobox developmental genes [55**,56**,57*,58’], a unitiing picture of gastrulation in several vertebrate systems is slowly emerging. At the beginning, there is an upper cell layer, generally termed the epiblast and possibly corresponding to the Xencjp~s animal cap, from which the entire organism derives. This cell layer may already be potentially specified along an anteroposterior axis or simply committed to a head versus trunk fate. This property usually bears the name of competence. Below the epiblast in amniotes is a transient lower cell layer, the hypoblast (termed hypoblast in chick and primary endoderm in mouse). Functional homologous structures are probably to be found in the vegetal hemisphere in Xerzoplrs and in the

interest,

reading

EM: Vertebrate 2:550-555.

axis formation.

tail-forming

Curr

in the

region.

Beddington RSP: Induction of a second neural axis by the 10 .. mouse node. Developmenl 1994, 120:613-620. This paper provides the tlrst direct evidence that the mouse node IS able to organize body patterns during gastrulation and that the mouse embryonic egg cylinder can be induced to form a secondary axis. Grafts of labelled mid-gastrulation mouse node to a posterolateral location in host embryos of the same developmental stage result In the induction of a second neural axis and the formation of ectopic somites. The graft gives rise predominantly to notochord and endodermal tissue, whereas neuroectoderm and somites are mainly oi host ongin. oi note, the ectopic notochord IS in its entirety derived solely from the’donor node, suggesting that the node may serve as a stem cell population supplying nascent notochord tissue. The author also provides a thoughtful discussion of the peculiarities oi mouse gastrulatlon as compared to that observed in lower vertebrates.

Homeobox genes in vertebrate gastrulation Quinlan GA, Williams EA, Tan S-S, Tam PPL: Neuroectodermal fate of epiblast cells in the distal region of the mouse egg cylinder: implication for body plan organization during early embryogenesis. Development 1995, 121:87-98. It is known that the epiblast of the early primitive streak mouse embryo contains not only the precursors of the ectodermal derivatives, but also of the endoderm and mesoderm. These authors have mapped the location of the neuroectoderm precursors at the distal cap of the epiblast. Cells of the most distal region of the epiblast contribute to all three compartments of the brain, as well as to the spinal cord. Cells just anterior to these colonize the brain and contribute to non-neural ectoderm cells of the amnion and craniofacial ectoderm. Cells at the posterior site of the distal cap mainly contribute to the caudal parts of the neural tube. Thus, the distal cap of the epiblast of the mouse egg cylinder contains the precursor population of the neural tube already endowed with a certain degree of craniocaudal patterning. 11. .

12. Hatada Y, Stern CD: A fate map of the epiblast of the early . chick embryo. Development 1994, 120:2879-2889. A fate map is generated for the epiblast of the very early chick embryo between stage X, corresponding to the unincubated egg, and the beginning of the primitive streak formation. The observed distribution of presumptive cell types reveals considerable overlap of different prospective areas rather than sharp divisions between different territories. Most areas are shown to converge toward the midline and then to move anteriorly along it. Nonetheless, some presumptive cell types, such as the prospective lateral plate mesoderm and the presumptive optic lobe and olfactory areas, move in characteristic ways, sometimes in contrast to the prevailing currents. This suggests that some prospective cell types are already specified at very early stages of chick development, possibly as early as stage X (i.e. the freshly laid egg). 13. .

Storey KG, Selleck MAJ, Stern CD: Neural induction and regionalisation by different subpopulation of cells in Hensen’s node. Development 1995, 121:417-428. The Hensen’s node of the chick embryo, the putative avian organizer, may be subdivided into a number of distinct regions, each comprising specific subpopulations of cells with specific developmental fates. The fate of explants from quail node at definitive primitive streak stage into extra-embryonic sites of a host chick embryo are analyzed to assay the specific inductive ability of the various regions of the node. The ability of. inducing neural tissue in the host is exclusively localized in the medial sector of the anterior node, both in the epiblast layer and in the mesendoderm that resides in the deep portion. This ability is not shared by mesendoderm of the posterolateral part of the node. On the other hand, neural tissue expressing a complete range of anteroposterior axial characteristics is obtained only with grafts that also include the epiblast layer in addition to the medial mesendoderm. Evidence is further provided that prospective prechordal plate cells also play a role in specifying the forebrain. 14.

McGinnis patterning.

W, Krumlauf R: Homeobox Cell 1992, 68:283-302.

15.

Krumlauf R: Hox genes in vertebrate 70:191-201.

genes

development.

and

17. .

18. .

Gaunt SJ, Strachan L: Forward spreading in the establishment of a vertebrate How expression boundary: the expression domain separates into anterior and posterior zones, and the spread occurs across implanted glass barriers. Dev Dyn 1994, 199:229-240. The authors systematically analyze the establishment of the expression pattern of the homeobox gene Hoxd-4 in chick embryos. This expression starts in the posterior part of the primitive streak and then spreads forward. It covers most of the primitive streak by the two-somite stage, covering the entire primitive streak by the five-somite stage and finally reaching the rhombomere 6/7 junction in the hindbrain by the 13-somite stage. This forward spreading does not depend upon cell migration and, most interestingly, does not require tissue continuity. In fact, it is not blocked by glass barriers surgically implanted to divide anterior from posterior embryonic tissues. 19.

Blumberg B, Wright CVE, De Robertis EM, Cho KWY: Organizer-specific homeobox genes in Xenopus laevis embryos. Science 1991, 253:194-l 96.

20.

Cho KWY, Blumberg B, Steinbeisser H, De Robertis EM: Molecular nature of Spemann’s organizer: the role of the Xenopus homeobox gene goosecoid. Cell 1991, 67:lll l-l 120.

21.

Blum M, Gaunt SJ, Cho KWY, Steinbeisser H, Blumberg B, Bittner D, De Robertis EM: Castrulation in the mouse: the role of the homeobox gene goosecoid. Cell 1992, 69:1097-l 106.

22.

Izpislia-Belmonte J-C, De Robertis EM, Storey KG, Stern CD: The homeobox gene goosecoid and the origin of organizer cells in the early chick blastoderm. Cell 1993, 74:645-659.

23.

Hume CR, Dodd J: Cwnt-8C: a novel Wnt gene with a potential role in primitive streak formation and hindbrain organization. Development 1993, 119:1147-1160.

24.

Schulte-Merker S, Hammerschmidt M, Beuchle D, Cho KW, De Robertis EM, NOsslein-Volhard C: Expression of zebrafish goosecoid and no tail gene products in wild-type and mutant no tail embryos. Development 1994, 120:843-852.

25.

Stachel SE, Griinwald DJ, Myers P: Lithium perturbation and goosecoid expression identify a dorsal specification pathway in the pregastrula zebrafish. Development 1993, 117:1261-l 274.

26.

Niehrs C, Keller R, Cho KWY, De Robertis EM: The homeobox gene goosecoid controls cell migration in Xenopus embryos. Cell 1993, 72:491-503.

27.

De Robertis EM, Fainsod A, Gont LK, Steinbeisser H: The evolution of vertebrate gastrulation. Developmenr 1994, Suppl: 117-l 24.

28.

Gaunt SJ, Blum M, De Robertis EM: Expression of the mouse goosecoid gene during mid-embryogenesis may mark mesenchymal cell lineages in the developing head, limbs and body wall. Development 1993, 117:769-778.

Cell 1994,

Garcia-Fernandez J, Holland PWH: Archetypal organization of the amphioxus /fox gene cluster. Nature 1994, 370:563-566. Hex genes are organized in homologous gene clusters. Insects and nematodes have a single gene cluster, whereas mammals have four Hoxgene clusters on four different chromosomes. Changes in the number and genomic organization of Hox genes may play a role in evolution of metazoan body plan - hence the interest in studying the organization of Hex genes in key species. Amphioxus is a cephalochordate, a taxon generally considered to be a sister group of vertebrates. The authors report that the amphioxus genome contains only one Hox gene cluster with a genomic organizationsimilar to that of the gene clusters contained in the mammalian genome. They also conclude that the Hox gene organization present in amphioxus represents a crucial intermediate step in the evolution of this gene family. Burke AC, Nelson CE, Morgan BA, Tabin C: Hex genes and the evolution of vertebrate axial morphology. Development 1995, 121:333-346. Different vertebrates have different numbers of repeated homologous structures along the anteroposterior body axis; for example, the number of cervical vertebrae may vary between 7 and 76 in different vertebrate species. The issue addressed in this paper is whether the anterior boundary of the expression domain of homologous Hox genes is localized in different species in a position that is. structurally and morphologically homblogous despite these changes in the actual number

and Mallamaci

of repeated homologous structures. The answer is to the affirmative, at least in chick and mouse; for example, chick has 14 cervical vertebrae, whereas mouse, like most mammals, has only seven. Despite this difference, Hoxc-5 and Hoxc-6 have an anterior expression boundary at the border between cervical and thoracic regions in both species.

axial

16. ..

Boncinelli

29. .

Bally-Cuif L, Gulisano M, Broccoli V, Boncinelli E: c-otx2 is expressed in two different phases of gastrulation and is sensitive to retinoic acid treatment in chick embryo. Mech Dev 1995, 49:49-63. The expression pattern of the chick homdogue of Otx2, c-otx2, is analyzed in gastrulating chick embryos. This gene is expressed in two distinct phases, separated by the beginning of Hensen’s node regression. The second phase of c-otx2 expression is sensitive to an early treatment with retinoic acid. This treatment abolishes expression in mesendoderm and restricts it to a small region at the anteriormost limit of the forming neural plate. A dominant theme of this study is that c-otx2 expression is associated with cells fated to give rise to anterior mesendoderm from their very first determination. 30. .

Niehrs C, Steinbeisser H, De Robertis EM: Mesodermal patterning by a gradient of the vertebrate homeobox gene goosecoid. Science 1994, 263:817-820. A role is proposed for goosecoid in dorsoventral patterning of Xenopus mesoderm at the early gastrula stage. A dorsoventral gradient of goosecoid transcripts is proposed to act as a patterning system in which the local concentration of goosecoid gene products determines

625

626

Differentiation and aene regulation the fate of cells of the marginal zone. The default state of these prospective mesodermal cells corresponds to blood and mesenchyme, whereas progressive dorsalized fates correspond to pronephros, muscle, notochord and prechordal plate. The authors conclude that goosecoid products are sufficient to to pattern mesoderm cell fates, but it remains to be assessed whether they are also necessary. 31.

32.

Simeone A, Acampora D, Gulisano M, Stornaiuolo A, Boncinelli E: Nested expression domains of four homeobox genes in developing rostra1 brain. Nature 1992, 358:687-690. Simeone A, Acampora D, Mallamaci A, Stornaiuolo A, D’Apice MR, Nigro V, Boncinelli E: A vertebrate gene related to orthodenticle contains a homeodomain of the bicoid class and demarcates anterior neuroectoderm in the gastrulating mouse embryo. EM80 j 1993, 12~2735-2747.

Ang S-L, Conlon RA, Jin 0, Rossant J: Positive and negative signals from mesoderm regulate the expression of mouse Otx2 in ectoderm explants. Development 1994, 120:2979-2989. O&2 expression is analyzed in early mouse embryos and its regulation is investigated by means of explant-recombination assays. The authors focus their attention on the inductive action of early mesendoderm on Otx2 expression during its progressive restriction to the anterior portion of the embryo by the headfold stage. Experimental evidence is provided that in viva, a positive inductive signal from anterior mesendoderm is required for a stable expression of Otx2 in anterior regions of the embryo, whereas a negative inductive signal emanating from later-forming posterior mesendoderm represses Otx2 in the posterior regions. Added exogenous retinoic acid is able to mimic in part the effect of this negative posterior signal and severely restricts the anterior domain of Otx2 expression.

33. ..

Simeone A, Avantaggiato V, Moroni MC, Mavilio F, Arra C, Cotelli F, Nigro V, Acampora D: Retinoic acid affects regionalization of murine CNS at specific developmental stages by altering gene expression in regions involved in CNS polarity. Mech Dev 1995, 51:83-98. This paper reports an extensive study of the effect of exogenous retinoic acid on the early development of the mouse anterior neural tube through the expression analysis of several regulatory genes, including Otx2. Three different classes of anterior phenotypes are observed, depending on the exact timing of retinoic acid administration to pregnant mothers. Treatment at midstreak stage (day 6.8-7.0) results in a mild phenotype with minor defects in the olfactory pit and developing brain. Treatment at late streak stage (day 7.2-7.4) results in complete loss of forebrain morphological and molecular identity and failure in establishing rhombomeric regional identities in the hindbrain. Otxi’ expression is severely repressed in the posterior portion of the headfold. Treatment at first somite formation (day 7.8-8.0) causes a sort of anencephaly associated with neuroepithelial hyperproliferation. The authors suggest that retinoids contribute to the early definition of head versus trunk structures by virtue of a differential action on different sets of regulatory genes. 34. .

Pannese M, Polo C, Andreazzoli M, Vignali R, Kablar B, Barsacchi C, Boncinelli E: The Xenopus homologue of Ofx2 is a maternal homeobox gene that demarcates and specifies anterior body regions. Development 1995, 121:707-720. The study of the expression pattern of Xotx2 in Xenopus embryos suggests that this gene plays a role in events leading to preparation and execution of gastrulation, especially in connection with the specification and possibly patterning of anterior structures. Results of microinjection experiments suggest that Xorx2 gene products are able to respecify the subdivision of the body along the anterior/posterior axis. These hypotheses are supported by the analysis of the general reorganization of %‘otx2 expression upon various treatments of early embryos. In particular, retinoic acid treatment essentially abolishes its expression in neuroectoderm. These embryos lack most of the rostra1 brain including forebrain and midbrain, whereas hindbrain and spinal cord regions are not reduced. The authors speculate on a possible direct correlation between the deletion of these regions and the lack of X0&2 expression in neuroectoderm of embryos subjected to retinoic acid treatment. 35. ..

Blitz IL, Cho KWY: Anterior neurectoderm is progressively induced during gastrulation: the role of the Xenopus homeobox gene 0rHmdenticle. Development 1995, 121:993-1004. A wave of Xotx2 expression moves through the ectoderm of the Xenopus gastrula in parallel with the movement of underlying anterior mesendoderm. The authors suggest that this expression profile is in keeping with the Eyal-Ciladi model for neural induction. According to this model, an anterior neural inducing signal emanating from the 36. .

underlying anterior mesendoderm transiently induces anterior neural tissues in the overlying ectoderm. Graded regionalization of anterior neuroectoderm subsequently requires caudalizing signals from more posterior mesoderm. 37.

Li Y, Allende ML, Finkelstein R, Weinberg ES: Expression of two zebrafish Orthodenticlarelated genes in the embryonic brain. Mech Dev 1994, 48:229-244.

38.

Can L, Mao C-A, Wirkramanayake A, Angerer LM, Angerer RC, Klein WH: An Orthodenticle-related protein from Sbongylocentrotus purpuratus. Dev Biol 1995, 167:517-528.

39.

Mao C-A, Can L, Klein WH: Multiple Otx binding sites required for expression of the Strongybcentrotus purpuratus SpecZA gene. Dev Biol 1994, 165:229-242.

40.

Ca L, Klein WH: A positive cis-regulatory element with a bicoid target site lies within the sea urchin SpecZa enhancer. Dev Bio/ 1993, 157:119-132.

41. ..

Shawlot W, Behringer RR: Requirement for Liml in head-organizer function. Nature 1995, 374:425-430. Liml is a LIM class homeobox gene expressed in the organizer region of mouse embryos. Embryos homozygous for a targeted deletion of the Lim J gene lack anterior head structures, but the remaining body axis develops normally. A partial secondary axis develops anteriorly in some mutant embryos. Close inspection of the mutant phenotype suggests that what is perturbed is the formation and/or the normal patterning of the early involuting axial structures. In fact, the node is not detectable even at day 7.5 of development, but looks normal at day 8.5. Analysis of molecular markers confirms that the early phases of gastrulation, but not the late ones, are affected. Remarkably, early goosecoid expression is reduced and localized in very posterior proximal regions of the developing embryo. 42. ..

Taira M, Otani H, Saint-Jeannet J-P, Dawid IB: Role of the LIM class homeodomain protein Xlim-1 in neural and muscle induction by the Spemann organizer in Xenopus. Nafure 1994, 372:677-679. The function of the vertebrate LIM class homeobox gene XlimI is explored by injecting synthetic transcripts of mutated forms into Xenopus embryos. In particular, the two LIM domains were mutagenized which seems to inhibit the DNA-binding activity of the LIM homeodomain. Whereas the wild-type transcripts exhibit poor activity, the mutated forms display complex, otherwise latent, biological activities essentially in promoting neural induction. Analysis of explanted animal caps reveals induction of anterior markers such as XCC-7, NCAM, En-2 and goosecoid, but not noggin or iollistatin. The same effects were demonstrated in animal caps deriving from uninjected embryos and co-cultivated with caps from injected embryos. Lemaire P, Garrett N, Gurdon JB: Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis. Cell 1995, 81:85-94. Siamois is a Xeno,ous homeobox gene expressed in dorsal vegetal cells of late blastula and able to generate a complete secondary axis, including cephalic structures, when ectopically overexpressed in a ventral blastomere. It encodes a homeodomain protein similar to Mix.1 and HDl and its mRNA can rescue ventralized embryos as efficiently as noggin mRNA, but it is much more potent than the latter at generating complete secondary axes. In this assay, Siamois is certainly more effective than any other known gene encoding a transcription factor.

43. ..

Spyropolous DD, Capecchi MR: Targeted disruption of the even-skipped gene, evxl, causes early postimplantation lethality of the mouse conceptus. Genes Dev 1994, 8: 1949-l 961. The homeobox gene evxl is first expressed in the visceral endoderm of the mouse blastocyst after implantation and prior to gastrulation. Presumptive null mutant blastocysts elicit a decidual response in viva, invade the uterine epithelium and attach to the basement membrane between uterine stroma and epithelium, but fail to differentiate extra-embryonic tissues or to form egg cylinders before resorption. Trophectodermal and inner cell mass tissues of null mutant conceptus grown in vitro proliferate after blastocyst attachment and outgrowth, but then both degenerate rapidly. 44. .

Stein S, Kessel M: A homeobox gene involved in node, notochord and neural plate formation of chick embryos. Mech Dev 1995, 49:37-48. Cnot is a chicken homeobox gene resembling in sequence and expression pattern the Xenopus Xnotl and Xnot2 genes. The early

45. .

Homeobox genes in vertebrate gastrulation Boncinelli transcription domains of Cnot are the node, the notochord and the neural plate fated to become hindbrain and spinal cord. All these cell populations are descendants of the Cnot-expressing cells of the node, suggesting cell lineage relationships. Transcripts of goosecoid and Cnot have complementary distribution in developing axial mesendoderm. Along the midline of early embryos a major distinction is that between prechordal and epichordal axis. On the basis of transplantation studies and of the expression distribution and timing of the two genes, the authors suggest a role for Cnot in the epichordal axis similar to that of goosecoid in the prechordal axis. Spann P, Ginsburg M, Rangini Z, Fainsod A, Eyal-Ciladi H, Gruenbaum y: The spatial and temporal dynamics of Sax1 (CNox3) homeobox gene expression in the chick’s spinal cord. Development 1994, 120:1817-l 828. Sax7 (previously called CHox3) is a chick homeobox gene belonging to the same family as the Drosophila NKI and honeybee HHO homeobox genes. Of particular interest is SaxI expression in the spinal part of the neural plate. The rostra1 border of this expression is always in the transverse plane separating the youngest somite from the yet unsegmented mesodermal plate and regresses with the same dynamics followed by the segregation of the somites from the mesodermal plate. In order to understand the regulation of this mechanism, several manipulation experiments were performed but, surprisingly, in no cases were the spatio-temporal dynamics of Sax1 expression modified. This suggests that Sax1 expression in the spinal cord is autonomously regulated, possibly according to a developmental clock system 46. .

Fjose A, Izpistia-Belmonte J-C, Fromental-Ramain C, Duboule D: Expression of the zebrafish gene h/x-l in the prechordal plate and during CNS development. Developmenf 1994, 120:71-81. The zebrafish h/x-l gene belongs to the H2.0 subfamily of homeobox genes and is closely related to the mouse Dbx gene. In early stages of neurulation, this gene displays a highly dynamic expression pattern in mesendoderm of the head region. Its expression domain rapidly transforms from a circular area into a longitudinal stripe, which is transiently detected in the prechordal plate underlying the rostra1 brain. At later stages, h/x-l is expressed in multiple locations within the central nervous system. In the developing hindbrain, h/x-l transcripts are detected near the inter-rhombomeric boundaries.

47. .

48.

Char BR, Tan H, Maxson R: A POU gene required for early cleavage and protein accumulation in the sea urchin embryo. Developmenr 1994, 120:1929-l 935.

49.

Freyd C, Kim SK, Hovitz HR: Novel cystein-rich motif and homeodomain in the product of the Caenorhabditis e/egans cell lineage gene /in-II. Nature 1990, 344:87&879.

50.

Karlsson 0, Thor S, Norberg T, Ohlsson H, Edlund T: Insulin gene enhancer binding protein IsI-1 is a member of a novel class of proteins containing both a homeo- and a Cys-HisDomain. Nature 1990, 344:879-882.

51.

Way JC, Chalfie M: met-3, a homeobox-containing gene that specifies differentiation of the touch receptor neurons in C. elegans. Cell 1988, 54:5-16.

52.

Spemann H: Embryonic Development and Induction. New York: Hafner Publishing Company; 1938 (reprinted 1962).

Tsuchida T, Ensini M, Morton SB, Baldassare M, Edlund T, Jesse11 TM, Pfaff SL: Topographic organization of embryonic motor neurons defined by expression of L/M homeobox genes. Cell 1994, 79:957-970. The topographic organization of motor projections in the spinal cord depends on the generation of subclasses of motor neurons that select specific paths to their targets. Here, the cloning and characterization of four chick LIM genes is reported, expression of which defines subclasses of motor neurons that segregate into specific structures and select distinct axonal pathways. These data suggest a role for some LIM homeobox genes in the generation of motor neuron diversity and specificity. 53. ..

54.

Taira M, Jamrich M, Good PJ, Dawid IB: The LIM domaincontaining homeo box gene X/h-I is expressed specifically in

and Mallamaci

the organizer region of Xenopus gastrula embryos. Genes Dev 1992, 6:356-366. 55. i:e

Ang S-L, Rossant J: HNF-3fi is essential for node and notochord formation in mouse development. Cell 1994, 78:561-574. annotation [56**1.

Weinstein DC, Ruiz i Altaba A, Chen WS, Hoodless P, Prezioso VR, Jesse11 TM, Darnell JE Jr: The Winged-helix transcription factor HNF-3B is required for notochord development in the mouse embryo. Cell 1994, 78:575-588. HNF-3p, a transcription factor of the HNF-Vfork head winged-helix family, is expressed in embryonic and adult endoderm and in midline structures, including node, notochord, floor plate and gut, in mouse embryos. This paper and [55*-l describe null mutations of this gene leading to embryonic lethality. The primary defect of homozygous HNF-3p null embryos resides in the absence of organized node and notochord formation. This leads to secondary defects in dorsoventral patterning of the neural tube and somites; for example, no motor neurons are formed in the spinal cord. Conversely, patterning along the anteroposterior axis is surprisingly poorly affected. Although HNF-3fl is required for node and notochord formation, some organizer activities persist even in the absence of these axial structures. Remarkably, by day 6.5 of development, exactly as in Liml-null embryos, goosecoid expression is localized not at the rostra1 tip of the forming primitive streak, but in the proximal posterior regions of the egg cylinder, at the junction between embryonic and extra-embryonic ectoderm from which the first wave of cell migration usually moves to subsequently give rise to the streak. Inspection of the expression of this and other molecular markers suggests that the role of HNF-3fl, as well as that of Liml, is crucial for proper axial mesoderm migration. Finally, a definitive endoderm cell sheet normally forms in HNF-3p null embryos, but it fails to fold, and foregut morphogenesis is severely affected. 56. ..

57. .

Conlon FL, Lyons KM, Takaesu N, Barth KS, Kispert A, Herrmann B, Robertson EJ: A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse. Development 1994, 120: 1919-l 928. This paper further explores the role of nodal, a gene encoding a TGFB-related product, in mouse gastrulation. The 413.d insertional mutation provides the opportunity to observe null mutants of this gene and arrests development shortly after gastrulation. Transcripts of nodal are initially detected in the embryonic epiblast. Concomitant with gastrulation, expression becomes restricted to the proximal posterior regions of the embryonic ectoderm as well as to the underlying endoderm. A few hours later, nodal expression is strictly confined to the periphery of the mature node. Null mutant embryos show no morphological evidence of primitive streak formation. On the other hand, random patches of mesodermal cells with different positional characters are detectable in these mutants. The authors conclude that the primary role of nodal is not in mesoderm induction and in its segregation from the ectoderm, but rather in the induction and/or maintenance of the primitive streak. 58. .

Toyama R, O’Connell ML, Wright CVE, Kuehn MR. Dawid IB: Nodal induces ectopic goosecoid and Liml expression and axis duplication in zebrafish. Development 1995, 121:383-391, This paper reports an experimental analysis of the consequences of ectopic expression of mouse nodal in zebrafish embryos. Induction of ectoplc axes that include notochord and somites but lack head is observed, implying that nodal-related factors are involved in axis formation in these embryos. Zebrafish goosecoid and lim7 expression is induced in concomitance with the generation of an ectopic organizing embryonic shield.

E Uoncinelli

and A Mallamaci, IIipardmento

e Tecnologica, 10132 Milano, Author E-mail:

lstituto Italy.

Scientifico

for correspondence: [email protected]

di ILicerca Biologica San I\affaele, Via Olgettina 60,

E Uoncinelli.

627