Molecular Genetics of Development Topics
Drosophila Segmentation: Overview
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Drosophila Segmentation: Introduction
The drosophila egg is about half a millimeter long. It takes about one day after fertilization for the embryo to develop and hatch into a worm-like larva. The insect remains as a larva for about six days, then becomes a pupae and emerges as the full adult form after about 3 more days.
In
the early drosophila embryo, genes interact to start defining three broad areas:
head, thorax and abdomen, then segments (see Figure 1). The bodies of both the
larval and adult insect are divided into 14 segments along the anterior-posterior
axis. In the adult fly, each segment ends with an articulated furrow, and thoracic
segments contain appendages: each has a pair of legs, the segment has a pair
of wings, and the most posterior has a pair of halteres (small wing-like structures,
maintaining equilibrium during flight). (Reviewed by Gilbert 2000)
The early embryo expresses different groups of genes over the first 4 hours of development (about 14 cell divisions). The first three groups – maternal genes, gap genes and pair-rule genes – are expressed in the syncytial blastoderm, when nuclei are dividing inside the same cytoplasm. The last early group of genes, segment polarity genes, is expressed in the cellular blastoderm, after the embryonic cell membrane has invaginated and partitioned to from the cell membranes around individual nuclei. These four groups of genes direct progressive levels of organization in the early embryo, from anterior-posterior and dorso-ventral axis patterning to segment delineation. After these early genes have defined segment location, the stage is set for homeotic genes to define the characteristics of each individual segment.
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Drosophila Segmentation: Maternal Genes
Many of the proteins that play a role in early embryo axis definition are the product of maternal genes. Messenger RNA from the maternal genes is deposited into the oocyte by ovarian nurse and follicle cells and is later expressed in discrete areas the early embryo under the control of other expressed maternal genes.
The maternal gene proteins act within the first 2 hours after the egg is fertilized, which corresponds to about 11 cell divisions. Maternal gene products include the proteins BICOID, HUNCHBACK, CAUDAL, NANOS, TORSO and DORSAL. The first four define the anterior-posterior (A-P) axis of the embryo, while TORSO defines the end structures of the embryo and DORSAL defines the dorso-ventral (D-V) axis.
The expression of each of the maternal genes in the embryo depends on were the maternal mRNA is situated during egg development. In the early embryo, Bicoid mRNA is located in what will become the anterior end of the embryo (Berleth et. al. 1988, Tautz D. 1988). Therefore, Bicoid protein is distributed in a concentration gradient, from higher concentration at the anterior end to lower concentration in the posterior third of the embryo (Driever and Nusslein-Volhard 1988). Maternal Hunchback mRNA is distributed throughout the embryo but its expression is repressed in the posterior area by CAUDAL. In addition to its translation from maternal mRNA, embryonic Hunchback is also induced by BICOID (Driever and Nusslein-Volhard 1989).
Bicoid is the key gene in a group of anterior patterning genes that determines anterior structures in the Drosophila embryo. The Bicoid protein is a homeodomain transcription factor expressed mostly in the anterior of the egg, and at decreasing concentrations towards the posterior end (see Figure 2). Bicoid protein induces head structures at high concentrations and thoracic structures at lower concentrations, as demonstrated experimentally by Frohnhöfer and Nüsslein-Volhard (1986, reviewed by Ephrussi and St. Johnston 2004). When they injected cytoplasm from the anterior into other areas of the embryo, formation of head structures was induced at the site of the injection, while thoracic structures developed at either side of the injection site. In other experiments, Bicoid mutants developed posterior structures in the anterior part of the embryo (Driever et. al. 1989). These experiments suggest that the concentration gradient of BICOID and other maternal gene products is the main determinant of their action.
Hunchback protein is a zinc-finger transcription factor (Stanojevic et. al. 1989) that represses abdominal genes in the early Drosophila embryo and also regulates later expression of pair-rule genes. While the initial source of HUNCHBACK is maternal mRNA transcribed by the embryo, BICOID will induce expression of embryonic Hunchback, thus enabling its action as a gap gene. As a gap gene, Hunchback permits parasegment definition by pair-rule genes.
The activity of Hunchback as a maternal effect gene helps to define mostly the head and thorax. Hunchback mutants lack the labium and all three thoracic segments, as well as the most posterior abdominal segments (Lehmann and Nusslein-Volhard 1987).
The
maternal effect gene Nanos is responsible for repressing Hunchback expression
in the posterior part of the embryo. Nanos is the key maternal gene in a group
of posterior/abdominal patterning genes in the Drosophila embryo (Wang and Lehmann
1991). NANOS binds Hunchback mRNA and prevents its translation. Repression of
Hunchback by Nanos seems to be they main determinant of posterior structures,
since embryos lacking Hunchback and Nanos are viable and can survive as fertile
adults (Irish et al 1989). The interaction between Hunchback mRNA and NANOS
is mediated by a cis-acting element in the 3' untranslated region of the Hunchback
transcript (Wharton and Struhl 1991).
Caudal is another important posterior/abdominal patterning genes of maternal origin (Mlodzik and Gehring 1987). While Caudal mRNA is distributed throughout the egg, its transcription is inhibited by Bicoid protein in the anterior portion (see Figure 1). BICOID binds to a specific cis-acting element in Caudal mRNA by means of its homeodomain (Rivera-Pomar et al 1996). Like Bicoid protein, Caudal protein is a homeodomain transcription factor (Macdonald and Struhl 1986).
The systems that control terminal structures and Dorso-Ventral (D-V) axis patterning behave different from the more simple transcription activation or translation repression systems in play for the A-P axis. While still controlled by maternal genes, the terminal and D-V systems require more complex signaling, involving extracellular communication between oocyte and follicle cells beyond the deposition of maternal mRNA.
The maternal gene Torso is the main determinant of terminal structures, although its activity is modified by BICOID. Activation of TORSO leads to expression of segmentation genes (Klingler et. al. 1988) that cause the differentiation of terminal structures into acron (anterior terminal structure), if BICOID is also present, or telson (posterior terminal structure) if acting alone (Finkelstein and Perrimon 1990).
TORSO
is a receptor tyrosine kinase present throughout the oocyte membrane, since
the maternal mRNA is distributed throughout the egg (Sprenger et. al.1989).
But TORSO is active only at the anterior and posterior ends of the embryo by
interaction with its putative ligand, which is released by follicular cells
only at the ends of the embryo. The most likely candidate for TORSO ligand is
TRUNK. Inactive TRUNK precursor is secreted by follicular cells into the fluid
between embryonic membrane and the inner eggshell membrane and somehow cleaved
into the active ligand (Stevens et. al. 2003). Another protein localized to
the inner eggshell membrane at the ends of the embryo, TORSOLIKE (see Figure
3), is required for TORSO activation (Stevens et. al. 1990) and may mediate
TRUNK activation.
Dorsal is the key gene in a group of anterior patterning genes that determines anterior structures in the Drosophila embryo. The maternal mRNA is distributed and the protein expressed throughout the embryo, but since DORSAL induces ventral genes it is only active in the ventral area (Anderson et. al. 1984; Steward et. al.1984; reviewed by Sánchez et al 1997). Because Dorsal is expressed at a later time than the A-P genes (14th cell division), its regulation must occur through fields of cells rather than nuclei in the same cytoplasm. The regional activity of DORSAL is controlled by complex cell-to-cell signaling: from oocyte to follicular cells and from follicular cells to oocyte. The Cactus protein is bound to DORSAL in the cytoplasm and thus prevents DORSAL from entering the nucleus. Oocyte to follicle signaling through the TOLL protein leads to inactivation of CACTUS only in ventral cells (Anderson et al. 1985). This leads to a gradient of DORSAL activity stronger in the ventral area and weaker in the dorsal area.
DORSAL
is a transcription factor that induces ventral genes like Twist and Snail while
repressing dorsal genes like Tolloid and Zerknullt. The DORSAL gradient determines
early tissue types: from amnioserosa in the dorsal end to mesoderm in the ventral
end (see Figure 4).
Agents like the Bicoid, Hunchback, Nanos, Caudal and Dorsal proteins, which control gene expression in a concentration-dependent manner, are known as morphogens and are an essential component of many developmental models, from Drosophila A-P axis patterning to chick embryo limb development (reviewed by Tickle 1999).
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Drosophila Segmentation: Segmentation Genes
After the maternal genes have defined A-P and D-V axes, the drosophila embryo is organized into segments under the influence of three types of segmentation genes: gap genes, pair-rule genes and segment polarity genes. Gap gene products interact with pair-rule gene products to define parasegments (Carroll and Vavra 1989), areas of the embryo that include the future posterior section of the more anterior segment and the future anterior section of the more posterior segment. Segment polarity genes are define anterior and posterior compartments within each segment
The
gap genes are transcription factors under the control of maternal gene. They
divide the embryo into broad regions, each region eventually developing into
several parasegments. Unlike maternal genes, gap genes are expressed throughout
the embryo, although they are more highly expressed only in defined regions
due to regulation by maternal genes and other gap genes, as illustrated in Figure
5.
While individual gap genes are highly expressed in discrete portions of the embryo, these portions do overlap (Gaul and Jackle 1989). Therefore more than one gap gene may act to properly define a specific anatomical region.
Drosophila gap genes can be grouped by the anatomical areas were they overlap and act together into three groups: anterior, posterior and terminal.
The anterior gap genes include Hunchback, Giant and Krüpple. The maternal gene Bicoid protein induces Hunchback (Driever and Nusslein-Volhard 1989). HUNCHBACK induces Giant and represses Krüpple, at the same time that KRÜPPLE represses Giant, leading to Giant being expressed more anterior than Krüpple (Kraut and Levine 1991). Both Hunchback and Krüpple repress the posterior gap genes.
The posterior gap genes include Knirps and Giant (also an anterior gene). The maternal gene Caudal protein induces Knirps and Giant (Rivera-Pomar et. al. 1995; Schulz and Tautz 1995). Knirps is also induced by Krüpple (Pankratz et. al. 1989) but repressed by Giant (Capovilla et. al. 1992), leading to Giant being expressed more posterior than Knirps, just outside the area of Krüpple influence (see Figure 5).
The terminal gap genes include Tailless and Huckebein. These genes are required for the formation of the unsegmented terminal regions of the larvae: acron at the anterior and telson at the posterior end. Terminal gap genes also repress posterior gap genes (Bronner and Jackle 1991)
Signaling by the maternal gene Torso protein induces Tailless while repressing Huckebein (Weigel et. al. 1990). Expression of Tailless by itself leads to formation of the telson. In the anterior end of the embryo, coexpression of Tailless with Bicoid leads to formation of the acron (Pignoni et. al. 1992). Huckebein is required for the establishment of anterior and posterior midgut primordium (Weigel at al 1990).
Gap gene mutations cause defects in the embryo over a large portion of the embryo, i.e. over several consecutive segments (Nusslein-Volhard and Wieschaus 1980). For example, Krüpple is essential for thorax development and Krüpple mutant larvae lack most of their thorax (a lethal phenotype).
The pair-rule genes subdivide gap gene-defined regions into 14 parasegments, and each pair-rule gene is expressed in seven alternative stripes (Lawrence and Johnston 1989). Pair-rule genes can be grouped into primary or secondary genes.
Primary pair-rule genes include Hairy, Even-Skipped and Runt. Initial expression of primary pair-rule genes is controlled by stripe-specific enhancers recognized by maternal & gap gene proteins (Goto et. al. 1989; Pankratz et. al. 1990; Howard et. al. 1988). For example, Even-Skipped is expressed in the third parasegment because its enhancer in that parasegment responds to BICOID and HUNCHBACK as inducers and to GIANT and KRÜPPLE as repressors (Reviewed by Gilbert 2000). At that same time, the inducer proteins are in high concentration and the repressor proteins are in low concentration in the third parasegment, leading to Even-Skipped expression. Later expression of both primary and secondary pair-rule genes is also controlled by their own gene products (Goto et. al. 1989, Edgar et. al. 1989, reviewed by Pankratz and Jackle 1990).
The secondary pair-rule genes include Fushi Tarazu, Odd-paired, Odd-skipped, Sloppy-paired and Paired, which are initially controlled by the primary pair-rule genes, but later may also be controlled by their own protein products (Howard and Ingham 1986). Secondary paired-rule gene levels of expression in different areas of the drosophila embryo will vary as primary pair-rule gene expression increases and their own expression begins. For example, Fushi Tarazu is initially expressed at low levels throughout the future segmented areas. But as its inducers and repressors start to be expressed, the stripes of high Fushi Tarazu expression become more defined, for example by HAIRY as repressor (Carroll et. al. 1988), until distinct stripes can be identified.
Mutations in the pair-rule genes cause patterning defects in the segment regions (anterior or posterior) defined by the mutant gene. For example, embryos with mutant Odd-Skipped show pattern deletions of less than a segment in width and are associated with mirror-image duplications of adjacent regions (Coulter and Wieschaus 1988)
Segment polarity genes are transcription factors that define anterior and posterior compartments within each segment. A major difference in their mode of action compared to previously expressed genes is that segment polarity genes act on cells after the cellular blastoderm has formed, thus their action requires cell-to-cell communication.
Engrailed, Wingless and Hedgehog are the key segment polarity genes (reviewed by Sanson 2001). Their interaction across adjacent rows of cells defines the posterior boundary of each segment.
Engrailed is a homeodomain transcription factor activated by either Even-Skipped, Fushi Tarazu or Paired, and repressed by either Odd-Skipped, Runt or Sloppy-Paired, leading to Engrailed expression in fourteen, 1-2 cell wide stripes along the A-P axis (Harding et. al. 1986; Macdonald et al 1986; Howard and Ingham 1986; DiNardo and O'Farrell 1987; Ingham et. al. 1988; Mullen and DiNardo 1995; Manoukian and Krause 1992; Cadigan et. al. 1994).
Expression of Wingless in the row of cells directly anterior to cells expressing Engrailed is induced by Sloppy-Paired and repressed by either Even-Skipped or Fushi Tarazu (Ingham et. al. 1988; Cadigan et. al. 1994). WINGLESS is a secreted protein that helps maintain Engrailed expression by lifting constitutive repression of Engrailed transcription (Bejsovec and Martinez-Arias 1991; reviewed by Gilbert 2000).
At the same time that WINGLESS maintains Engrailed expression, ENGRAILED maintains Wingless expression by means of HEDGEHOG signaling. HEDGEHOG is a secreted protein expressed in cells expressing Engrailed (posterior compartment of the segment).
Wingless and Hedgehog interact to determine segment identity. The cells expressing Wingless also express the HEDGEHOG receptor, while the cells expressing Engrailed also express the WINGLESS receptor and release HEDGEHOG ligand. This leads to a mutual activation of Hedgehog and Wingless signaling along the parasegment boundary. Cells farther away from the parasegment boundary do not receive the full effect of the Wingless/Hedgehog signaling exactly as it happens at the parasegment boundary. But a gradient of expression of the different proteins involved in the signaling pathway specify distinct cell fates in a concentration-dependent manner, where HEDGEHOG seems to be the main morphogen (Heemskerk and DiNardo 1994; reviewed by Gerhart and Kirschner 1997 and Gilbert 2000).
Mutations in the segment polarity genes cause the formation of mirror-image compartments in a given segment (Kornberg 1981; García-Bellido and Santamaría 1972).
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Hox Genes: Overview
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Hox Genes: topic
The definition of A-P segments, as well as the D-V axis, allows for the development of “genetic addresses” that define the developmental direction to be taken by a particular group of embryonic cells (Morata and Kerridge 1982). After the segments of the Drosophila embryo have been established, the homeotic genes are responsible for the differentiation of tissues and structures characteristics of each segment.
Homeotic genes are expressed in discrete compartments along the A-P axis, spanning at lest one, but for some homeotic genes several segments. The protein products of homeotic genes are transcription factors that activate or repress numerous target genes, making their expression also compartment-specific. Mutations in homeotic genes cause segments to become similar to each other. For example, if the Antennapedia gene, normally expressed in the second thoracic segment, is also expressed in the head region, legs will grow in the place were antennae are supposed to be. (Reviewed by Gerhart and Kirschner 1997, and Gilbert 2000).
Homeosis is the replacement of one body part with another. Homeotic genes control the nature of a body part but not the numer. Homeotic genes contain a homeobox, ~180 bp DNA sequence (discovered at IU and Switzerland). This sequence encodes a homeodomain, ~60 aa DNA binding motif.
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