Major questions in developmental biology Single genome Diverse cell types Totipotent zygote Fate refinement Diverse cell fates Cell commitments are largely driven by cell positions within a developmental field

Major cellular developmental decisions: Establish basic body plan coordinates (anterior-posterior, dorsal-ventral) Subdivision of anterior-posterior axis (segmentation into metameres, specification of fates for each segment) Subdivision of dorsal-ventral axis (differentiation of primary germ layers: endoderm, mesoderm, ectoderm) Organ/tissue differentiation

Fig Drosophila syncitial stage embryo

Fig Chapter 18: Genetic basis of development *

p. 584 Genes controlling early development were discovered in Drosophia mutant screens (Nϋsslein-Volhard, Wieschaus, Lewis)

Fig A-P axis differentiation by gradients of two proteins

Major morphogens directing A/P axis formation in Drosophila BCD (bcd gene): directs anterior development; transcription factor; mRNA is localized; mutations are tail duplications (bicaudal embryos) HB-M (maternal hb gene): differentiates axial development; transcription factor; mRNA unlocalized NOS (nos gene): directs posterior development; translation repressor; mRNA is localized; mutations are head duplications All three are present in gradients in embryos

Fig bcd & nos mRNAs are tightly localized - BCD and NOS proteins form concentration gradients bcd mutation → double-posterior embryo nos mutation → double-anterior embryo

BCD gradient results from diffusion of localized RNA (NOS gradient is similar) HB-M gradient results from translational repression by NOS protein Net effect: cells along the A-P axis of the embryo have distinctive combinations of concentrations of BCD and HB-M transcription factors (Experimental perturbations of the gradients demonstrate their roles in determining the A-P axis)

Fig bcd mRNA is localized to the anterior pole by sequences within its 3’ UTR

Fig The gradient of BCD protein determines A-P axis cell fates (which cells form cephalic furrow)

D-V axis is specified by cell-cell signalling system in Drosophila DL protein (dl gene): transcription factor; uniform distribution but localization gradient; highest nuclear localization in ventral areas SPZ protein (spz gene): extracellular ligand for TOLL receptor; secreted assymetrically by follicle cells during embryogenesis; gradient most concentrated in ventral area TOLL protein (Tl gene): transmembrane receptor activates signal cascade resulting in phosphorylation of CACT protein; uniform distribution CACT protein (cact gene): cytosolic protein; uniform distribution; unphosphorylated form binds DL; phosphorylated form releases DL (permitting DL nuclear localization)

Fig D-V polarity is determined by distribution of the DL protein (transcription factor) DL quantity is similar in all cells Nuclear localization differs in D-V axis Nuclear DL activates “ventralizing” genes

DL nuclear localization is controlled by a signal transduction cascade Fig Loss-of-function mutations that produce “dorsalized” embryos (nuclear DL nowhere): spz toll dorsal Loss-of-function mutations that produce “ventralized” embryos (nuclear DL everywhere): cact

DL nuclear localization is controlled by a signal transduction cascade Fig

Fig Known types of positional information in embryos

A-P and D-V axes are defined by morphogens (BCD, HB-M, DL) encoded by maternal-acting genes These transcription factors differentially activate a set of zygotic-acting genes – the cardinal genes A-P axis cardinal genes are called gap genes (specify general body regions) Gap genes encode transcription factors and activate the set of pair rule genes (cardinal genes specifying alternating segments – creating segments) Pair rule genes encode transcription factors and activate the set of segment polarity genes (cardinal genes that distinguish anterior/posterior compartments of each segment) Segment polarity genes differentially activate the segment identity genes

Fig Delayed cellularization of the Drosophila embryo compartmentalizes factors and their gradients

Fig Compartmentalized factors direct zone-specific development → segments

Fig Loss-of-function mutations of those factors create segment-specific changes

Fig Gap gene expression determines zonal identity Pair-rule gene expression drive segmentation

Fig Gap gene expression determines zonal identity Pair-rule gene expression drive segmentation ftz and eve expression patterns

A-P and D-V axes are defined by morphogens (BCD, HB-M, DL) encoded by maternal-acting genes These transcription factors differentially activate a set of zygotic-acting genes – the cardinal genes A-P axis cardinal genes are called gap genes (specify general body regions) Gap genes encode transcription factors and activate the set of pair rule genes (cardinal genes specifying alternating segments – creating segments) Pair rule genes encode transcription factors and activate the set of segment polarity genes (cardinal genes that distinguish anterior/posterior compartments of each segment) Segment polarity genes differentially activate the segment identity genes

Fig Segment identity genes are mostly found in the homeotic gene complexes ANT-C (Antennapedia complex): genes for anterior segment identity BX-C (Bithorax complex): genes for posterior segment identity

BX-C mutations can transform the identities of posterior segments wild-type bithorax mutant (T3 T2) Fig

Fig Embryonic development is driven by a hierachical cascade of transcription factors and signalling systems

Fig Hox gene clusters are highly similar to Drosophila HOM-C gene clusters …..but, Hox clusters are repeated

Fig Hox gene clusters are highly similar to Drosophila HOM-C gene clusters …..but, Hox clusters are repeated

Hox and HOM-C genes are expressed in similar patterns during development Fig

Fig Testing the role(s) of Hox genes Hox C8 knockout mice Homeotic transformation of vertebra L1 Animals exhibit other skeletal defects

Sex determination in mammals vs. flies Somatic sex differentiation H. sapiensDrosophila XXfemalefemale XYmalemale

Sex determination in mammals vs. flies Somatic sex differentiation H. sapiensDrosophila XXfemalefemale XYmalemale XOfemalemale (Turner) XXYmalefemale (Klinefelter) Determined by Ydetermined by # of Xs

Sex determination in mammals General biological context Hormonally mediated (androgens) Individual cells do not determine their own sex (no mosaicism) Early gonad indifference (to about two months gestation)

Sex differentiation controlled by Y-linked transcription factor gene Y-linked gene (SRY in humans) directs testosterone production in Leydig cells of indifferent gonad (loss-of-function SRY - develops female) Testosterone activates steroid receptors (e.g., Tfm receptor) that lead to “male” differentiation of target organs/tissues Failure to activate receptors leads to “female” differentiation (default pathway) Translocation of Sry (mouse) to other chromosomes transfers sex determination cue to those chromosomes Binary “switch” is presence/absence of functional SRY gene copy in Leydig cells of the indifferent gonad

Fig Sex determination in humans directed by intra- and extra-cellular gene interactions

How are cell fates “sealed” in development? Fig Models for cellular “memory” (feedback loops)

Recommended problems in Chapter 18: 11, 15, 21, 24, 32