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Intrinsic determinantsComments

Intrinsic determinants

Found in some eggs are intrinsic (aka ooplasmic, cytoplasmic) determinants, substances localized to particular regions just before or just after fertilizations. These determinants affect the fate of cells that contain them.

The characterized determinants are mRNAs. These mRNAs contain a localization signal in their 3' UTR. Attaching this sequence to a different gene will result in its mRNA having the same localization pattern. In addition, cells containing cytoplasmic determinants (ie, pole cells will differentiate based on their cytoplasmic determinants. Localized mRNAs are transported along microtubules by motor proteins (dynein, kinesin) that move their cargos in different directions along the microtubules.

Consequences of cleavage:
1) Reduction of cytoplasm/nucleus ratio
2) Generation of different cell environments and interactions
3) Segregation of determinants into different cells

Segregation of cytoplasmic determinants:
Different cells will get different cytoplasmic determinants.
Evidence for cytoplasmic determinants?
Remove 4D.

Cell environment and interactions
Extrinsic determinants
Evidnce for extrinsic determinants?

Blastomere isolation/removal experiments on embryos of most species support regulative development; on other species, cytoplasmic determinants. But embryos from same species can give results supporting either extrinsic or cytoplasmic determinants. Many embryos show evidence for both types of determinants.

Cytoplasmic determinants exmaple
P grnaules, which segregate with germline cells.

Identifying Cytoplasmic Determinants

Technique
In Situ HybridizationHybridization of Vg1 cDNA probe to Xenopus egg; Vg1 is localized to the vegetal pole (a microtubule-dependent process). You can see localized mRNAs and gradients of proteins.
GeneticsUse Drosophila and screen for maternal effect mutants affecting cuticular pattern. Bicoid and nanos mRNA are found to be critical for the cuticular pattern. For example nanos mutants lack posterior abdominal segments and bicoid mutants lack anterior thoracic segments. If this only arises when the maternal genotype is homozygously mutated but the progeny's genotype is irrelevant, then you have found a maternal effect mutant.

Hierarchies of cytoplasmic determinants

Cytoplasmic determinants can be hierarchically ordered by phenotype or time of expression.

Phenotype

HierarchyOverview
Gap

Mutants in gap genes cause deletions of large groups of segments.

Examples are: hb, controls G1-T3 and A7 & A8; Kr, controls T1-A5; kni, controls A1-A8. The phenotype of gap gene mutants is the lack of a large group of contiguous segments.

Pair-RuleMutants in pair-rule genes lack part of every pair of segments. Pair-rule genes control the development of alternating segments (for example, ftz on alternating abdominal segments).
1° Pair-RuleExpression of primary pair-rule genes is only altered in embryos also containing gap gene mutation; expression of primary pair-rule genes is not altered in embryos mutant for the other pair-rule genes. Primary pair-rule genes are regulated by different combinations of gap genes.
2° Pair-RuleExpression of secondary gap genes is altered in both gap gene mutant embryos and in embryos carrying mutations in any of the primary pair-rule genes hairy (h), even-skipped (eve) or runt. Secondary pair-rule genes are regulated by the primary pair-rule genes.
The primary pair-rule genes also encode transcription factors (hairy and eve both encode homeobox proteins). By a process of both activation and repression, the primary pair-rule genes generate a seven-striped pattern of expression of the six secondary pair-rule genes (which also encode transcription factors). At each level, i.e., that of the primary pair-rule genes and that of the secondary pair-rule genes, there are auto- and cross-regulatory interactions. An example of autoregulation is that wild type eve function is necessary to maintain its own wild type pattern of expression. An example of a cross-regulatory interaction is that h function is required to maintain eve expression (both are primary pair-rule genes). These interactions refine the borders of the stripes of expression of the pair-rule genes.
Segment Polarity

Mutants in segment polarity genes lack part of every segment.

The segment polarity genes are different from the gap and pair rule genes: as opposed to strictly encoding transcription factors, some segment polarity genes encode signaling peptides; and some segment polarity genes are not expressed in domains corresponding to the portion of the fate map missing in the mutants.

Expression patterns of some of the segment polarity genes corresponds to the regions missing in the mutants. hedgehog (hh), wingless (wg) and engrailed (en) are expressed in 14 stripes, one for each segment (or parasegment), starting during the late blastoderm stage of embryogenesis. The hh stripe (which overlaps with the en stripe) defines the posterior of each segment (or anterior of each parasegment) and the wg stripe is just anterior to it. Most other segment polarity genes are expressed uniformly.

Generation of the 14 hh and wg stripes depends on combinations of pair rule gene activity. Different combinations of pair rule genes (which are expressed in overlapping sets of 7 stripes) promote expression of these genes in odd versus even numbered parasegments (subdivisions of the embryo that are slightly offset from segmental divisions).

The wg and hh expression domains, although initiated by pair rule gene expression, later are required to maintain each other, via cell signaling pathways. wg and hedgehog encode secreted cell signaling proteins; Wg protein is secreted by the cells anterior to the parasegment border, while Hedgehog protein is secreted by the en-expressing cells just posterior to the parasegment border. The other segment polarity genes encode various components of the pathways that transduce these signals.There are distinct receptors for the wg protein and for the hh protein; once each of these receptors has been activated, a kinase is activated (a different one in each case). Ultimately, through a series of intermediary proteins that is different for each pathway, there is activation of transcription of transcription factor genes (these target genes differ depending on whether the cell has received a wg, or a hh signal); this then leads to further transcription of either wg or hh. These series of activations and repressions in the pathways downstream of Hedgehog and Wingless results in very tight regulation of the response to the Wg and Hh signaling molecules.

Gaining a further understanding of these complex pathways has important implications for our understanding of many developmental processes. The Wingless and Hedgehog proteins are both secreted signaling molecules; they appear to establish morphogen gradients. The Hedgehog protein in Drosophila plays a role not only in maintaining parasegment borders, but also in patterning (anterior-posterior and dorsal-ventral) in the wing and the eye (progression of the morphogenetic furrow). Both hh and wg have multiple vertebrate homologs.

In Xenopus (frog), Wnt (Drosophila Wg) signaling plays a role in induction of dorsal mesoderm. A vertebrate Hedgehog homolog, Sonic hedgehog, is an important component of two signaling centers: the floorplate of the neural tube, and the zone of polarizing activity of the limb bud. Not only are these signaling molecules conserved, their receptors and the signal transduction molecules downstream of the receptors are conserved in vertebrates.

Time of expression

HierarchyOverview
Early GenesGap genes are expressed before segment polarity genes.
Late Genes